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https://openalex.org/W2150088027
https://dash.harvard.edu/bitstream/1/4553340/1/2857650.pdf
English
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Alternative Strategies to Reduce Maternal Mortality in India: A Cost-Effectiveness Analysis
PLoS medicine
2,010
cc-by
15,907
Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:4553340 Published Version doi:10.1371/journal.pmed.1000264 Published Version doi:10.1371/journal.pmed.1000264 Abstract Background: Approximately one-quarter of all pregnancy- and delivery-related maternal deaths worldwide occur in India. Taking into account the costs, feasibility, and operational complexity of alternative interventions, we estimate the clinical and population-level benefits associated with strategies to improve the safety of pregnancy and childbirth in India. Methods and Findings: Country- and region-specific data were synthesized using a computer-based model that simulates the natural history of pregnancy (both planned and unintended) and pregnancy- and childbirth-associated complications in individual women; and considers delivery location, attendant, and facility level. Model outcomes included clinical events, population measures, costs, and cost-effectiveness ratios. Separate models were adapted to urban and rural India using survey-based data (e.g., unmet need for birth spacing/limiting, facility births, skilled birth attendants). Model validation compared projected maternal indicators with empiric data. Strategies consisted of improving coverage of effective interventions that could be provided individually or packaged as integrated services, could reduce the incidence of a complication or its case fatality rate, and could include improved logistics such as reliable transport to an appropriate referral facility as well as recognition of referral need and quality of care. Increasing family planning was the most effective individual intervention to reduce pregnancy-related mortality. If over the next 5 y the unmet need for spacing and limiting births was met, more than 150,000 maternal deaths would be prevented; more than US$1 billion saved; and at least one of every two abortion-related deaths averted. Still, reductions in maternal mortality reached a threshold (,23%–35%) without including strategies that ensured reliable access to intrapartum and emergency obstetrical care (EmOC). An integrated and stepwise approach was identified that would ultimately prevent four of five maternal deaths; this approach coupled stepwise improvements in family planning and safe abortion with consecutively implemented strategies that incrementally increased skilled attendants, improved antenatal/postpartum care, shifted births away from home, and improved recognition of referral need, transport, and availability/quality of EmOC. The strategies in this approach ranged from being cost-saving to having incremental cost-effectiveness ratios less than US$500 per year of life saved (YLS), well below India’s per capita gross domestic product (GDP), a common benchmark for cost-effectiveness. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility eve Sweet1, Natalie Carvalho1, Uma Chandra Mouli Natchu2,4, Delphine Hu1,3 1 Center for Health Decision Science, Harvard School of Public Health, Boston, Massachusetts, United States of America, 2 Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts, United States of America, 3 Department of Health Policy and Management, Harvard School of Public Health, Boston, Massachusetts, United States of America, 4 Pediatric Biology Centre, Translational Health Science and Technology Institute, Delhi, India Citation: Goldie SJ, Sweet S, Carvalho N, Natchu UCM, Hu D (2010) Alternative Strategies to Reduce Maternal Mortality in India: A Cost-Effectiveness Analysis. PLoS Med 7(4): e1000264. doi:10.1371/journal.pmed.1000264 Academic Editor: Zulfiqar A. Bhutta, Aga Khan University, Pakistan Received September 14, 2009; Accepted March 12, 2010; Published April 20, 2010 Copyright:  2010 Goldie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: John D. and Catherine T. MacArthur Foundation #07-89007-0 00GSS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: bEmOC, basic emergency obstetrical care; cEmOC, comprehensive emergency obstetrical care; EmOC, emergency obstetrical care; GDP, gross domestic product; MDG, Millennium Development Goal; MMR, maternal mortality ratio; PPH, postpartum hemorrhage; SBA, skilled birth attendant; TFR, total fertility rate; YLS, year of life saved * E-mail: sue_goldie@harvard.edu Sue J. Goldie1,3*, Steve Sweet1, Natalie Carvalho1, Uma Chandra Mouli Natchu2,4, D Sue J. Goldie1,3*, Steve Sweet1, Natalie Carvalho1, Uma Chandra Mouli Natchu2,4, Delphine Hu1,3 Introduction relevant comorbidities, aggregates individual outcomes to the population level, and reflects setting-specific epidemiology. Separate models were adapted to urban and rural India using data on antenatal care, family planning, facility births, and skilled birth attendants (SBAs), and information about access to transport, referral facilities, and quality of care. Model outcomes include clinical events (e.g., pregnancies, live births, maternal complica- tions), measures of maternal mortality (e.g., MMR, proportionate mortality ratio [i.e., proportion of deaths that are pregnancy- related among women aged 15–45 y], and lifetime risk of maternal death), population outcomes (e.g., life expectancy), and economic costs. Approximately one-quarter of all pregnancy- and delivery-related maternal deaths worldwide occur in India, which has the highest burden of maternal mortality for any single country [1,2]. Although the inclusion of maternal mortality reduction in the United Nations’ Millennium Development Goals (MDGs) reflects the importance of improving maternal health as a key mechanism in reducing poverty and promoting social and economic growth, global progress has been suboptimal [2–4]. Several factors may be changing the landscape for maternal health in India in particular [5]. These factors include more information on maternal mortality measures [6,7], an increasing number of studies evaluating interventions [8], renewed determination on the part of the maternal health and public health communities [9], and, most importantly, the emergence of maternal mortality reduction as a clear priority on the Indian national political agenda [5,10–12]. We evaluated alternative approaches to reducing maternal mortality in settings in India that differ according to underlying maternal risk, health, and socioeconomic status. Interventions can be provided individually or packaged into integrated services. Following standard recommendations for economic evaluation [25], strategies are first ranked in terms of increasing costs and benefits; those that are less effective and more costly than an alternative strategy are considered inefficient, and those that cost less than the status quo are considered ‘‘cost saving.’’ For all other strategies, we calculate an incremental cost-effectiveness ratio, defined as the additional cost of a specific strategy divided by its additional clinical benefit, compared with the next least expensive strategy. We considered interventions with cost-effectiveness ratios of less than the per capita gross domestic product (GDP) (US$1,068) to be very cost-effective as suggested by the Commission on Macroeconomics and Health. Sensitivity analyses are conducted to assess the impact of parameter uncertainty. The Model The computer-based Global Maternal Health Policy Model simulates the natural history of pregnancy (both planned and unintended) and pregnancy- and childbirth-associated complica- tions (Figure 1). This model defines health states to reflect important characteristics that affect prognosis, quality of life, and resource use. The time horizon incorporates a woman’s lifetime and is divided into equal time increments during which women transition from one health state to another. Nonpregnant girls enter the model and in each time period may become pregnant depending on age, use of contraception, and clinical history (Figure 1, upper panel). Once pregnant, women have a chance of spontaneous abortion (i.e., miscarriage), induced abortion, or continued pregnancy. A proportion of induced abortions will be unsafe (i.e., surgical or medical abortion conducted by untrained personnel). Labor and delivery may be associated with a direct complication of pregnancy (e.g., hypertensive disorders of pregnancy, obstructed labor, hemorrhage, sepsis). Case fatality rates are conditional on the type and severity of complication (e.g., moderate sepsis requiring antibiotics versus severe hemorrhage requiring blood transfusion) and underlying comorbidity (e.g., anemia). Nonfatal complications include neurological sequelae, rectovaginal fistula, severe anemia, and infertility (Figure 1, upper panel). In addition to death from maternal complications, women face an annual risk of death from age-specific all-cause mortality. Previous model-based studies have provided important insights into the potential high public health value of reducing maternal deaths, however, many of these have not considered the full range of interventions to reduce maternal mortality, such as family planning, safe abortion, and intrapartum care [7,17,18]. Some have only focused on single interventions [19–21], others have not included costs [22], and recent analyses that did assess multiple strategies did not explicitly model critical barriers to life-saving referral, such as recognition of referral need and accessible transport [23,24]. Taking into account the costs, feasibility, and operational complexity of alternative interven- tions, we extend this body of work to estimate the clinical and population-level benefits associated with a comprehensive set of strategies to improve the safety of pregnancy and childbirth in India. Strategies in the model to reduce maternal mortality consist of improving coverage of effective interventions, which may be provided individually or packaged as integrated services. Introduction Despite consensus on the need for universal access to high-quality intrapartum and emergency obstetrical care (EmOC), uncertainties remain about how to adapt ‘‘ideal recommendations’’ to specific situations [8,13]. The need for an adequate supply of skilled providers, functional referral and transport, and well-equipped facilities for EmOC will prove a formidable barrier in the near-term for countries with weak health systems, as well as for states with inadequate health delivery infrastructure and for communities in predominantly rural areas [13]. This challenge will be particularly relevant for India, with its largely rural population, and striking disparities between states. For example, while Kerala reports a maternal mortality ratio (MMR) of fewer than 100 maternal deaths per 100,000 live births, rural Uttar Pradesh and Rajasthan report MMRs of more than 400 [14,15]. To most effectively leverage India’s national commitment to reducing maternal mortality, identifying evidence-based strategies that consider the local context is imperative. Our analysis is motivated by questions that include: What are the fundamental drivers of the effectiveness, cost-effectiveness, and affordability of a package of interventions to reduce maternal mortality? Because adequate facilities, health infrastructure, and skilled human resources will not be readily available in all settings, can we provide interim guidance to policy makers? While no single empirical study can provide clear answers to these questions, a modeling approach within a decision-analytic framework can extend empiric informa- tion by extrapolating outcomes beyond the time horizon of a single study, can facilitate synthesis of multiple data sources in an internally consistent and epidemiologically plausible way [16], and may be adapted to specific settings so that existing infrastructure, resources, and political realities can be considered. Abstract Conclusions: Early intensive efforts to improve family planning and control of fertility choices and to provide safe abortion, accompanied by a paced systematic and stepwise effort to scale up capacity for integrated maternal health services over several years, is as cost-effective as childhood immunization or treatment of malaria, tuberculosis, or HIV. In just 5 y, more than 150,000 maternal deaths would be averted through increasing contraception rates to meet women’s needs for spacing and limiting births; nearly US$1.5 billion would be saved by coupling safe abortion to aggressive family planning efforts; and with stepwise investments to improve access to pregnancy-related health services and to high-quality facility-based intrapartum care, more than 75% of maternal deaths could be prevented. If accomplished over the next decade, the lives of more than one million women would be saved. Please see later in the article for the Editors’ Summary. Citation: Goldie SJ, Sweet S, Carvalho N, Natchu UCM, Hu D (2010) Alternative Strategies to Reduce Maternal Mortality in India: A Cost-Effectiveness Analysis. PLoS Med 7(4): e1000264. doi:10.1371/journal.pmed.1000264 Academic Editor: Zulfiqar A. Bhutta, Aga Khan University, Pakistan Received September 14, 2009; Accepted March 12, 2010; Published April 20, 2010 Copyright:  2010 Goldie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: John D. and Catherine T. MacArthur Foundation #07-89007-0 00GSS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abbreviations: bEmOC, basic emergency obstetrical care; cEmOC, comprehensive emergency obstetrical care; EmOC, emergency obstetrical care; GDP, gross domestic product; MDG, Millennium Development Goal; MMR, maternal mortality ratio; PPH, postpartum hemorrhage; SBA, skilled birth attendant; TFR, total fertility rate; YLS, year of life saved * E-mail: sue_goldie@harvard.edu PLoS Medicine | www.plosmedicine.org 1 April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 PLoS Medicine | www.plosmedicine.org Reducing Maternal Mortality in India The Model In addition to family planning, antenatal care (i.e., prenatal care) and treatment of anemia, safe abortion, and postpartum care, the model includes both intrapartum interventions that reduce the incidence of a complication (e.g., misoprostol for postpartum hemorrhage [PPH], clean delivery for sepsis), as well as those that PLoS Medicine | www.plosmedicine.org Analytic Overview The effectiveness of interventions to reduce the incidence of complications (e.g., active management of labor) was estimated from published studies using methods detailed in Text S1. The effectiveness of interventions to reduce case fatality rates was from published studies and assumed treatment in an appropriate facility; a wide plausible range was used for sensitivity analyses (Table 1; Text S1). Data on facility births, SBAs, family planning for spacing or limiting births, and antenatal care were from country- specific surveys [28]. The effectiveness of interventions to either reduce the incidence of complications or to reduce case fatality rates associated with complications depends, in part, on access to specific services (e.g., trained SBA) and to specific levels of facilities (e.g., comprehensive EmOC [cEmOC] with capacity for blood transfusion). Accord- ingly, the ultimate impact of interventions depends on several setting-specific factors. These include delivery site, presence of birth attendant, quality and type of referral facility, as well as successful referral when necessary. The model therefore explicitly considers the location of delivery, type of assistance, access to basic or comprehensive obstetrical care, and the ability to overcome a series of barriers around the timing of delivery (e.g., recognition of referral need, reliable transport, timely treatment at an appropri- ate referral facility); these factors collectively determine the health services a woman can access and the specific interventions that would be included (Figure 1, lower panel). For women delivering at home or in a birthing center, the probability of successful referral depended on overcoming three potential barriers [74]: (1) delay in recognizing referral need; (2) delay in transfer to referral facility (means of transport and interim care en route); (3) delay in receiving appropriate care at the appropriate EmOC facility such as inadequate staffing and supplies, inexpedient attention (e.g., delay to collect fees), and/or non-evidence-based or substandard care. Assumptions about the latter two delays were based on survey data (e.g., National Family Health Survey [NFHS-3] [28,75], District Level Household Survey [DLHS] [76], and Facility Survey [26]), state-level facility surveys [77,78], government reports [27], and published studies (Text S1) [14,15,79–83]. ( g p ) Delivery setting is differentiated by provider (e.g., family member, traditional birth attendant [TBA], or SBA) and by site (e.g., home versus facility). Analytic Overview The best available data were synthesized using a computer- based model that simulates the natural history of pregnancy and April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 2 Reducing Maternal Mortality in India Figure 1. Schematic of the model. Upper panel: Model simulates the natural history of pregnancy (both planned and unintende pregnancy- and childbirth-associated complications. Case fatality rates for complications depend on severity and comorbidity. General interv categories (open red boxes) include family planning for spacing or limiting births, antenatal or prenatal care (and treatment of anemia), safe ab Reducing Maternal Mortality i Figure 1. Schematic of the model. Upper panel: Model simulates the natural history of pregnancy (both planned and unintended) and pregnancy- and childbirth-associated complications. Case fatality rates for complications depend on severity and comorbidity. General intervention categories (open red boxes) include family planning for spacing or limiting births, antenatal or prenatal care (and treatment of anemia), safe abortion, intrapartum care (e.g., active management of labor), basic and comprehensive EmOC, and postpartum care. Interventions can reduce the incidence or severity of a complication or can reduce the case fatality rate through appropriate treatment. Lower panel: Model reflects the intervention pathway during labor and delivery, including location (home, birthing or health center, bEmOC, cEmOC), attendant (family member, traditional birth attendant [TBA], or SBA), and three potential barriers to effective treatment in the event of a complication, including recognition of referral need, transfer (e.g., PLoS Medicine | www.plosmedicine.org April 2010 | Volume 7 | Issue 4 | e1000264 3 Reducing Maternal Mortality in India Reducing Maternal Mortality in India transport), and timely quality care in an appropriate EmOC facility. Management of labor and delivery depends on attendant (e.g., SBA, clean delivery) and site (e.g., expectant management in birthing center, active management in EmOC facility), as does access to specific levels of treatment (e.g., blood transfusion only available in cEmOC). doi:10.1371/journal.pmed.1000264.g001 reduce the case fatality rate through appropriate management in a referral facility (Figure 1, upper panel). tions were obtained from published data, and a plausible range for sensitivity analysis was based on review of the literature. Case fatality rates were adjusted based on complication severity (e.g., life-threatening complications requiring cEmOC) and underlying severity of anemia (Table 1; Text S1). Analytic Overview Facility levels are categorized as (1) birthing centers or health centers, which cannot provide all services necessary to qualify as a basic emergency obstetrical care (bEmOC) facility, but are staffed with SBA who provide expectant management of labor and more reliable referral when necessary than with delivery at home; (2) facilities with bEmOC capacity (e.g., first referral units); and (3) facilities with cEmOC capacity (e.g., district hospitals) [26,27]. Facilities capable of bEmOC are assumed to be capable of administering injectable antibiotics, oxytocics, and sedatives or anticonvulsants, and performing manual removal of placenta, removal of retained products, and assisted vaginal delivery. Facilities capable of cEmOC also are able to provide blood transfusion, cesarean section, and management of advanced shock. Model performance was assessed by comparing the distribution of direct causes of maternal mortality, life expectancy, propor- tionate mortality ratio, MMR, and total fertility rate (TFR) to empiric data [2,14,15,28,29,32,38,84–87] in rural and urban India. Model validation was assessed by using state-specific data from Rajasthan and Uttar Pradesh as model inputs, and comparing model-projected indicators of maternal mortality with survey-reported outcomes [14,15]. In addition, a secondary analysis assessing all strategies evaluated in the base case was conducted in Uttar Pradesh. Details of this process are included in Text S1. This model also allows us to evaluate phased approaches that involve scaling up access to services over time; the stepwise investments in infrastructure required to assure high-quality intrapartum care are designated as ‘‘upgrades.’’ In addition to reducing unmet need for family planning and unsafe abortion, four consecutively implemented strategies increased skilled atten- dants, improved antenatal/postpartum care, incrementally shifted births away from home, and improved the availability and quality of EmOC. For women delivering at home or in birthing centers, these ‘‘upgrades’’ also improved recognition of referral need, access to transport, and expedient referral to an appropriate facility (Figure 2). PLoS Medicine | www.plosmedicine.org Costs Selected costs used in the model are provided in Table 3 [18,25,80,88–94]. Details are provided in Text S1. With the exception of facility costs, salaries, and transport costs, resource requirements to deliver interventions and the costs of maternal complications were estimated from the United Nations Population Fund’s (UNFPA) Reproductive Health Costing Tools Model (RHCTM) [88]. The RHCTM uses an ingredients approach to estimate direct costs (including drugs, supplies, and personnel requirements) of 45 reproductive health interventions, as well as investments required for scale-up. We obtained personnel costs (salaries) and facility costs from public access country-specific databases [25,89], and drugs and supply costs from the UNICEF Supply Catalogue and Management Sciences for Health (MSH) International Drug Price Indicator Guide [90,91]. We leveraged public access sources and published studies to inform assumptions about the financial requirements for improving transport and scaling up facility- and human-resource capacity [7,30,80,92,93]. We assessed the face validity of model input values and established a plausible range for sensitivity analysis by comparing estimates to All models were built using TreeAge Pro 2008 (TreeAge Software Inc.) and analyzed using IBM/Lenovo Dual-Core VT Pro Desktop computers running Microsoft Windows XP, using Microsoft Excel 2007 and Visual Basic for Applications 6.5 (Microsoft Corp.). We used Monte Carlo simulation to generate the number of per woman events such as pregnancies, live births, facility-based births, and maternal complications. Model Validation Model-estimated life expectancy for a 15-y-old female was 55 y compared to the World Health Organization’s (WHO) estimate of 55.1 y [85]. The distribution of maternal deaths by cause closely approximated published regional estimates (Table 1) [29]. Model- generated estimates of TFR and MMR for India, stratified for rural and urban status, closely approximated survey-reported values [1,2,28,38], as did the state-specific models for Rajasthan and Uttar Pradesh (Table 2) [14,15]. Model predicted deaths for 2005, taking into account direct and indirect causes of maternal mortality, were 117,657, compared to 117,000 estimated by UNICEF, WHO, and UNFPA [86,87]. Increased family planning to reduce the unmet need also reduced the number of deaths attributable to unsafe abortion (Figure 3). For example, in rural India increasing contraceptive rates to 67.6% cut abortion-related deaths by more than 50%—even with no change in rates of unsafe abortion. Adding improved access to safe abortion and postabortion care for three out of four women pursuing elective termination of pregnancy prevented an additional 22% to 50% of abortion-related deaths, depending on the underlying level of unmet need; similarly, the additional cost savings ranged from 22% more, to more than double the savings expected from family planning alone. Data Alternatives evaluated in sensitivity analysis (Results and Text S1). doi:10 1371/journal pmed 1000264 g002 Figure 2. Stepwise improvements in scaling up maternal services. Four strategies that scale up access to critical maternal health services in consecutive phases are designated as upgrade 1, upgrade 2, upgrade 3, and upgrade 4. Shown are the percent increases in facility-based delivery, SBAs, recognition of referral need (by SBA at birthing/health center), transport (to appropriate referral facility), and availability/quality of EmOC (including adequate staff/supplies, appropriate clinical treatment, immediate attention), for rural and urban India. Shifts from home births assume a 70% shift to health centers/birthing centers and a 30% shift to EmOC; for routine births in EmOC, we assume 90% bEmOC and 10% cEmOC. Alternatives evaluated in sensitivity analysis (Results and Text S1). doi:10 1371/journal pmed 1000264 g002 Figure 2. Stepwise improvements in scaling up maternal services. Four strategies that scale up access to critical maternal health services in consecutive phases are designated as upgrade 1, upgrade 2, upgrade 3, and upgrade 4. Shown are the percent increases in facility-based delivery, SBAs, recognition of referral need (by SBA at birthing/health center), transport (to appropriate referral facility), and availability/quality of EmOC (including adequate staff/supplies, appropriate clinical treatment, immediate attention), for rural and urban India. Shifts from home births assume a 70% shift to health centers/birthing centers and a 30% shift to EmOC; for routine births in EmOC, we assume 90% bEmOC and 10% cEmOC. Alternatives evaluated in sensitivity analysis (Results and Text S1). doi:10.1371/journal.pmed.1000264.g002 the cost savings for a single birth cohort of 15-y-old girls (2010) that would be expected to accrue over their reproductive lifespans ranged from US$111.4 million to US$448.2 million. Reducing the unmet need, coupled with provision of safe abortion, provided synergistic benefits and saved additional costs (Table 4). Results were similar in urban India, although the amount of deaths averted and costs saved were smaller, reflecting both the lower initial TFR and the smaller population size (Table 4). those in published studies (Text S1). All costs were converted to 2006 US$. PLoS Medicine | www.plosmedicine.org Data Selected parameters and assumptions used in the model are provided in Tables 1 and 2 [7,14,15,17,22,23,28–73]. Additional details are provided in Text S1. Initial estimates of incidence and case fatality rates associated with pregnancy-related complica- April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 4 Reducing Maternal Mortality in India Figure 2. Stepwise improvements in scaling up maternal services. Four strategies that scale up access to critical maternal health services in consecutive phases are designated as upgrade 1, upgrade 2, upgrade 3, and upgrade 4. Shown are the percent increases in facility-based delivery, SBAs, recognition of referral need (by SBA at birthing/health center), transport (to appropriate referral facility), and availability/quality of EmOC (including adequate staff/supplies, appropriate clinical treatment, immediate attention), for rural and urban India. Shifts from home births assume a 70% shift to health centers/birthing centers and a 30% shift to EmOC; for routine births in EmOC, we assume 90% bEmOC and 10% cEmOC. Alternatives evaluated in sensitivity analysis (Results and Text S1). doi:10.1371/journal.pmed.1000264.g002 Figure 2. Stepwise improvements in scaling up maternal services. Four strategies that scale up access to critical maternal health services in consecutive phases are designated as upgrade 1, upgrade 2, upgrade 3, and upgrade 4. Shown are the percent increases in facility-based delivery, SBAs, recognition of referral need (by SBA at birthing/health center), transport (to appropriate referral facility), and availability/quality of EmOC (including adequate staff/supplies, appropriate clinical treatment, immediate attention), for rural and urban India. Shifts from home births assume a 70% shift to health centers/birthing centers and a 30% shift to EmOC; for routine births in EmOC, we assume 90% bEmOC and 10% cEmOC. Alternatives evaluated in sensitivity analysis (Results and Text S1). doi:10.1371/journal.pmed.1000264.g002 Figure 2. Stepwise improvements in scaling up maternal services. Four strategies that scale up access to critical maternal health services in consecutive phases are designated as upgrade 1, upgrade 2, upgrade 3, and upgrade 4. Shown are the percent increases in facility-based delivery, SBAs, recognition of referral need (by SBA at birthing/health center), transport (to appropriate referral facility), and availability/quality of EmOC (including adequate staff/supplies, appropriate clinical treatment, immediate attention), for rural and urban India. Shifts from home births assume a 70% shift to health centers/birthing centers and a 30% shift to EmOC; for routine births in EmOC, we assume 90% bEmOC and 10% cEmOC. Interventions Packaged as Integrated Services Increased family planning to reduce the unmet need (for spacing and limiting births) by amounts ranging from 25% to 100%, reduced maternal deaths by amounts ranging from 7.0% to 28.1% in rural India and 5.8% to 23.5% in urban India (Table 4). In rural India, eliminating the unmet need for family planning decreased the TFR from 2.97 to 2.14, the proportion of deaths that are pregnancy related from 16.4% to 12.3%, and the lifetime risk of maternal death from 1 in 65 to 1 in 90. In rural India alone, Our results suggest that reaching the MDG 5 goal of a 75% reduction in maternal mortality would require investments targeting the intrapartum period, in addition to family planning and safer abortion. Without these additional strategies, the model predicts a ceiling on the level of maternal mortality reduction achievable, ranging from 32% in urban India to 34% in rural India. PLoS Medicine | www.plosmedicine.org April 2010 | Volume 7 | Issue 4 | e1000264 5 Reducing Maternal Mortality in India Table 1. Selected model parameters: Incidence and mortality of pregnancy and delivery-related complications, and impact of interventions. Parameter Hemorrhage Obstructed Labor Hypertensive Disorders Sepsis Unsafe Abortion Incidence and mortality Probability of event [15,29,31–44] 0.114 0.047 0.035 0.050 0.128a Range 0.051–0.228 0.030–0.074 0.025–0.050 0.043–0.060 0.050–0.250 Probability of morbidity [28,30,45–48]b 0.008 0.022 0.001 0.400 0.120 Range 0.006–0.010 0.018–0.026 0.001–0.001 0.320–0.480 0.096–0.144 CFR [7,49–51] 0.010 0.007 0.017 0.013 0.003 Adjusted CFRc 0.023 0.019 0.021 0.028 0.009 Range 0.007–0.030 0.005–0.025 0.012–0.027 0.009–0.036 0.002–0.012 Attributable mortality [29]d 46.2% (9%–73%) 14.1% (3%–52%) 13.7% (0%–18%) 17.4% (0%–20%) 8.6% (0%–20%) Model-projected attributable mortality 40.6% 16.8% 12.3% 20.4% 9.8% Impact of interventions Decreased incidence [34,37]e 50%,75%e — — 25%, 50%f — Rangef 25%–91% — 25%–50% 0%–60% 0%–100% Decreased CFR [7,17,50,52–64]g 75% 95% 59% 90% 98%a Rangef 60%–90% 76%–100% 45%–95% 63%–93% 50%–100% See [7,14,15,17,22,23,28–64,69–73]. aIncidence of elective abortion is 0.170, and 75% are assumed to be unsafe in the base case [15,29,39–44]. Case fatality rate (CFR) of safe abortion is 0.000006, representing a 98% reduction in mortality [50,62–64]. For more details on abortion-related assumptions, see Text S1. Incidence of miscarriage (not shown) is 0.150 [69,70]. bSpecific examples of nonfatal complications include Sheehan’s Syndrome following maternal hemorrhage, fistula resulting from obstructed labor, neurological sequelae from eclampsia, pelvic inflammatory disease (PID). Interventions Packaged as Integrated Services [29], based on the entire Asia region, as well as other data to establish a range for sensitivity analysis [14,15]. Cause-specific proportions sum to 66%, reflecting approximately 33% indirect causes, although this varies from 15% to 35% in different studies. Estimates shown reflect adjustment of data from Khan et al. [29] such that a distribution is shown for the 66% of direct causes, to compare to model output. Further, anemia was reported to be responsible for 15% of deaths and was assumed to exert mortality impact on direct causes through severity of PPH, sepsis, and unsafe abortion. eIncidence of sepsis reduced by 50% with SBA and clean delivery in birthing center, bEmOC, and cEmOC; and reduced by 25% with SBA and clean delivery at home [37]. Incidence of maternal hemorrhage reduced by 50%–75% depending on expectant versus active management of labor; we assume for the status quo, all cEmOC facilities provide active management, 50% of bEmOC facilities provide active management, and birthing centers/health centers provide expectant management only [34]. Exploratory analyses that estimate the impact of community-based provision of oral misoprostol in birthing centers and at home assume a 25% to 50% reduction in PPH [22,72]. For each baseline estimate, sensitivity analysis was conducted across a plausible range based on literature review; references are documented in the Text S1. fFor each baseline estimate, sensitivity analysis was conducted across a plausible range based on literature review; references and assumptions are documented in the Text S1. gEstimates shown represent average reduction in case fatality rate provided complications necessitating surgery (e.g., cesarean section), blood transfusion, intensive hemodynamic support are treated in cEmOC. Obstructed labor is managed using assisted vaginal delivery with forceps or vacuum and, if necessary, cesarean section; severe pre-eclampsia and eclampsia treated with intravenous hydralazine and magnesium sulfate, in addition to induction of labor or emergency cesarean section when required; sepsis treated with ampicillin, gentamycin, and metronidazole or equivalent regimen followed by an 8-d course of intramuscular gentamycin and oral metronidazole (see Text S1 for details) [7,73]. doi:10.1371/journal.pmed.1000264.t001 mortality ratio by 14% to 4%, and lifetime risk of maternal mortality from one in 78 to one in 282. PLoS Medicine | www.plosmedicine.org Interventions Packaged as Integrated Services Not shown but included are the risk of infertility from PID (0.086) and the risk of severe anemia following maternal hemorrhage (0.09) [23,45,46]. cCFRs were adjusted based on complication severity (e.g., life threatening complications requiring cEmOC) and underlying severity of anemia [71]. See Text S1. dEstimates for distribution of causes of maternal mortality for India are from India overall estimates from Khan et al. [29], based on the entire Asia region, as well as other data to establish a range for sensitivity analysis [14,15]. Cause-specific proportions sum to 66%, reflecting approximately 33% indirect causes, although this varies from 15% to 35% in different studies. Estimates shown reflect adjustment of data from Khan et al. [29] such that a distribution is shown for the 66% of direct causes, to compare to model output Further anemia was reported to be responsible for 15% of deaths and was assumed to exert mortality impact on direct causes through Table 1. Selected model parameters: Incidence and mortality of pregnancy and delivery-related complications, and impact of interventions. Table 1. Selected model parameters: Incidence and mortality of pregnancy and delivery-related complications, and impact of interventions. See [7,14,15,17,22,23,28 64,69 73]. aIncidence of elective abortion is 0.170, and 75% are assumed to be unsafe in the base case [15,29,39–44]. Case fatality rate (CFR) of safe abortion is 0.000006, representing a 98% reduction in mortality [50,62–64]. For more details on abortion-related assumptions, see Text S1. Incidence of miscarriage (not shown) is 0.150 [69,70]. bSpecific examples of nonfatal complications include Sheehan’s Syndrome following maternal hemorrhage, fistula resulting from obstructed labor, neurological sequelae from eclampsia, pelvic inflammatory disease (PID). Not shown but included are the risk of infertility from PID (0.086) and the risk of severe anemia following maternal hemorrhage (0 09) [23 45 46] [ , ] bSpecific examples of nonfatal complications include Sheehan’s Syndrome following maternal hemorrhage, fistula resulting from obstructed labor, neurological sequelae from eclampsia, pelvic inflammatory disease (PID). Not shown but included are the risk of infertility from PID (0.086) and the risk of severe anemia following maternal hemorrhage (0.09) [23,45,46]. cCFRs were adjusted based on complication severity (e.g., life threatening complications requiring cEmOC) and underlying severity of anemia [71]. See Text S1. dEstimates for distribution of causes of maternal mortality for India are from India overall estimates from Khan et al. April 2010 | Volume 7 | Issue 4 | e1000264 Interventions Packaged as Integrated Services Compared to the status quo in urban India (Table 4, lower section), similar reductions in maternal deaths were predicted; although the number of absolute lives saved would be lower, the MMR, lifetime risk, and proportionate mortality ratio would be expected to decline to 113, one in 553, and 2.3%, respectively, with the most intensive strategy (upgrade 4). Table 5 shows the health and economic outcomes associated with interventions packaged as integrated services; these included phased approaches that scale up access to intrapartum services over time in rural (upper section) and urban India (lower section), coupled with incremental improvements in family planning and safe abortion. The four stepwise ‘‘upgrades’’ incorporated improvements in available SBAs for home births, recognition of referral need, transport, and availability/quality of EmOC, as well as shifts from home- to facility-based delivery. Because the stepwise improvements in each component of the integrated package (intrapartum care, family planning, and safe abortion) were assumed to occur in consecutive phases, the incremental cost-effectiveness ratio for each ‘‘upgrade’’ strategy was calculated as the difference in costs relative to the difference in effects, compared with the preceding next best strategy. While the initial strategy was cost saving in both urban and rural India, Compared to the status quo in rural India (upper section, Table 5), our model predicted that integrated strategies coupling family planning and safe abortion with four consecutive ‘‘upgrades’’ would be expected to reduce maternal deaths by 17.3% to 77.1%, the MMR to less than 200, proportionate April 2010 | Volume 7 | Issue 4 | e1000264 6 Reducing Maternal Mortality in India Table 2. Selected model parameters and assumptions: Coverage of interventions and maternal health indicators by setting. Table 2. Selected model parameters and assumptions: Coverage of interventions and maternal health indicators by setting. Interventions Packaged as Integrated Services p p g y g Parameter India India, Urban India, Rural Rajasthan Uttar Pradesh, Rural Coverage of contraception (%) [28] Family planning (any method) 56.3 64.0 53.0 47.2 39.7 Modern methods 48.5 55.8 45.3 44.4 25.2 Pill 6.4 7.0 6.2 4.5 5.2 IUD 3.7 6.1 2.4 3.6 3.2 TOL 76.9 67.7 81.9 77.0 66.7 Condom 10.9 17.9 7.3 12.8 24.6 Unmet need 13.2 10.0 14.6 14.6 23.8 Coverage of prenatal care (%) [28] Prenatal care 50.7 73.8 42.8 41.2 22.6 Treatment for anemiaa 22.3 34.5 18.1 13.1 6.7 Delivery location (%) [28] Total skilled delivery 48.3 75.2 39.1 41.0 23.8 Facility deliveryb 40.7 69.4 31.1 29.6 17.5 Home delivery with SBAb 12.8 19.0 11.6 16.2 7.6 Assumptions for available transport/interim care to appropriate facility (%) [65,66] From home to EmOC 30.4 44.4 24.4 24.4 18.1 Range 20–40 35–55 15–35 20–40 15–30 From HC or BC to EmOC 54.8 68.8 48.8 48.8 36.1 Range 40–65 60–80 40–60 30–55 25–45 From bEmOC to cEmOC 67.0 81.0 61.0 61.0 45.1 Range 55–80 70–90 50–70 50–70 35–55 Assumptions for available facility, staff/supplies, quality of care (%) [67,68] EmOC 50.0 67.5 42.5 42.5 31.5 Range 40–60 55–80 30–55 30–55 20–40 Maternal health indicators TFR [28] 2.68 2.07 2.98 3.21 4.13 Model-projected TFRc 2.70 2.07 2.97 3.24 4.13 Model-projected MMRc 440 407 520 524 633 See [14,15,28,65–68,71]. aCase fatality rates (CFRs) were adjusted based on complication severity (e.g., life threatening complications requiring cEmOC) and underlying severity of anemia [71]. See Text S1. bRoutine deliveries in EmOC facilities assume that 90% would be in bEmOC and 10% in cEmOC. Alternative assumptions explored in sensitivity analysis (Text S1). We calculated the percentage of births with skilled attendance at home by subtracting the percentage delivered in facilities (which we assume are with skilled attendance) from the total of births with skilled attendance: (total skilled delivery2facility based births)/home births; for rural India: (0.39120.311)/(120.311) = 0.116 or 11.6%. cUsing the empirically calibrated India model, we parameterized the state-level models for Rajasthan and Uttar Pradesh and adjusted for the TFR as reported in NFHS 3 [28]. To provide comparison, reported MMRs for Uttar Pradesh include an estimate of 707 from Mills [14], and prior data from SRS including the SRS 2001–2003 estimate of 517 (confidence interval [CI] 461–573), SRS 1999–2001 estimate 539 (481–596), and the SRS 1997–98 (606, CI 544–668) [15]. Interventions Packaged as Integrated Services For Rajasthan, we used the 2001–2003, special survey of deaths using RHIME, which reported 445 (371–519), and SRS 1999–2001, which reported 501 (423–580) [15]. BC, birthing center; HC, health center; IUD, intrauterine device; TOL, female sterilization. doi:10.1371/journal.pmed.1000264.t002 PLoS Medicine | www.plosmedicine.org Integrated Safe Motherhood Interventions in Rural Uttar Pradesh Costing details and methods for converting costs to 2006 US$ are id d i h T S1 provided in the Text S1. aRanges for sensitivity analyses established on the basis of assumptions and other published literature documented in the Text S1. b p aRanges for sensitivity analyses established on the basis of assumptions and other published literature documented in the Text S1. bAntenatal care includes tetanus vaccination syphilis gonorrhea chlamydia screening (and treatment) urinalysis blood tests treatment for anemia counseling (e g p aRanges for sensitivity analyses established on the basis of assumptions and other published literature documented in the Text S1. bAntenatal care includes tetanus vaccination, syphilis, gonorrhea, chlamydia screening (and treatment), urinalysis, blood tests, treatm family planning, spacing, intrapartum care). y p g, p g, p ) cPostabortion complications assumed to require manual vacuum aspiration, treatment of sepsis in 25%, surgical repair in 25% [92]. d p q p , p , g p [ ] dTotal costs reflect skill level of attendant, level of facility, and drugs and supplies. For example, delivery at birthing center (US$14.4 facility (US$4.52), and drugs and supplies (US$3.50). Other assumptions documented in the Text S1. f attendant, level of facility, and drugs and supplies. For example, delivery at birthing center (US$14.46) includes personnel (US$6.44) nd supplies (US$3.50). Other assumptions documented in the Text S1. eCommunity-based interventions evaluated in sensitivity analysis included SBA-administered misoprostol to reduce incidence of PPH in deliveries at home and in birthing centers. Costs for misoprostol (US$0.99) and training (upper bound, US$3.40) based on assumptions presented in Sutherland and Bishai [18]; these costs represent the incremental costs above routine SBA delivery. y fTransport costs include those incurred from home to a referral facility (bEmoc or cEmOC), and those incurred between facilities when necessary (e.g., bEmOC to cEmOC). Assumptions based on literature [80,93,94] and public access data described in the Text S1. gEstimates shown represent average total costs using case-specific unit costs weighted by severity. Complications requiring surgery (e.g., cesarean section), blood transfusion, intensive hemodynamic support assumed to require cEmOC. Details of unit cost assumptions for facility-specific treatment documented in Text S1. hPostpartum care includes examination, iron/folate supplementation, and counseling. doi:10.1371/journal.pmed.1000264.t003 In Figure 4 (upper panel) each cell is also color-coded to reflect the cost-effectiveness profile associated with the partic- ular strategy. PLoS Medicine | www.plosmedicine.org Integrated Safe Motherhood Interventions in Rural Uttar Pradesh incremental cost-effectiveness ratios ranged from US$150 to US$300 per YLS in rural India and from US$150 to US$350 per YLS in urban India. Cost-effectiveness ratios are also expressed as percent of the per capita GDP (US$1,068). Even the most intensive and effective strategic package was well below 50% of the per capita GDP. Figure 4 (upper panel) displays the health outcomes associated with stepwise approaches to improve maternal health in rural Uttar Pradesh. The vertical axis (from bottom to top) shows outcomes associated with increased access to family planning and safe abortion; the horizontal axis (from left to right) displays outcomes associated with investments in high-quality health-center–based intrapartum care (e.g., facility, attendance, referral, transport, EmOC). In Figure 4 (upper panel) the cell located in the far left lower corner represents current conditions in rural Uttar Pradesh. In contrast to these integrated strategies, implementing only the stepwise intrapartum care upgrades—without family planning and safe abortion—was less effective and less cost-effective. The incremental cost-effectiveness ratios ranged from US$490– US$1,060 in rural India and US$200–US$990 per YLS in urban India (Text S1). April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 7 Reducing Maternal Mortality in India Table 3. Selected model input costs. Cost Components Base Case Rangea Family planning [25,88] Oral contraceptives 10.64 6.03–15.96 Injectable contraceptives 10.20 4.92–15.30 Condoms 8.40 3.79–12.60 Intrauterine device 9.17 2.58–13.76 Female sterilization 18.98 9.49–28.47 Male sterilization 12.67 6.34–19.01 Antenatal careb [25,88] Four visits 17.82 8.54–25.61 Abortionc [25,88,94] Incomplete abortion 8.90 4.45–17.80 Elective abortion 21.87 10.94–43.74 Postabortion complications 43.40 21.70–86.80 Deliveryd [25,88] Home (TBA, SBA) 4.52, 6.44 0–9.66 Facility (birthing center, bEmOC, cEmOC) 14.46, 24.58, 32.54 7.23–48.81 Community-based interventionse Misoprostol (home, birthing center) 0.99 0.75–2.00 SBA training 3.40 0.62–5.00 Transportation costsf Home to facility 3.62–8.13 1.81–12.20 Birthing/health center/bEmOC to referral facility 4.88–7.14 2.44–10.71 Management of complicationsg [25,88] Obstructed labor 70.16 12.76–139.38 Maternal hemorrhage 67.99 18.40–212.51 Puerperal sepsis 47.92 23.15–111.02 Severe pre-eclampsia/eclampsia 65.85 33.50–153.62 Postpartum careh [25,88] One visit 4.99 1.04–7.49 See [18 25 80 88–94] Table 3. Selected model input costs. See [18,25,80,88 94]. Estimates of costs under current standard of care (2006 US$). Estimates for the base case were country-specific and from UNFPA’s Reproductive Health Costing Tools Model (RHCTM) [88] and WHO CHOICE/public databases [25,89–91], unless otherwise specified. April 2010 | Volume 7 | Issue 4 | e1000264 Integrated Safe Motherhood Interventions in Rural Uttar Pradesh Strategies that only employed family planning and safe abortion (vertical axis, from bottom to top) were generally cost saving, but reduced mortality by a maximum of 40%. Strategies that only invested in intrapartum care improvements Each of the other cells represents a unique strategy; the reduction in maternal deaths expected with each strategy, relative to current conditions, is shown. For example, a strategy that reduced the unmet need by 75%, increased safe abortion to 60%, and implemented improvements to intrapartum care consistent with upgrade 3, reduced maternal deaths by 57%. April 2010 | Volume 7 | Issue 4 | e1000264 8 Reducing Maternal Mortality in India Table 4. Health and economic outcomes of family planning to reduce the unmet need for limiting and spacing births, and safe abortion, in rural and urban India. Table 4. Health and economic outcomes of family planning to reduce the unmet need for limiting and spacing births, and safe abortion, in rural and urban India. Table 4. Health and economic outcomes of family planning to reduce the unmet need for limiting and spacing births, and safe abortion, in rural and urban India. Integrated Safe Motherhood Interventions in Rural Uttar Pradesh Strategy Lifetime Deaths per 100,000 Women Reduction in Maternal Deaths Proportionate Mortality Ratio Lifetime Risk of Death Due to Maternal Complications Model-projected Savings for a Single Birth Cohort of 15 y olds (US$)a Cost Savings for a Single Year (Current Distribution of 15–45 y Olds in India) (US$)b Rural India, current conditions (TFR, 2.97)c 1,543 — 16.4% 1 in 65 NA — Family planning Reduce unmet need 25% (56.7%) 1,435 7.0% 15.4% 1 in 70 111,357,615 60,200,655 Reduce unmet need 50% (60.3%) 1,327 14.0% 14.4% 1 in 75 223,221,615 120,611,563 Reduce unmet need 75% (64.0%) 1,218 21.1% 13.4% 1 in 82 335,439,615 181,233,496 Reduce unmet need 100% (67.6%) 1,109 28.1% 12.3% 1 in 90 448,188,615 242,067,230 Safe abortion Increase safe abortion 50% 1,517 1.7% 16.2% 1 in 66 48,080,115 42,078,125 Increase safe abortion 75% 1,473 4.5% 15.8% 1 in 68 130,739,115 114,289,234 Increase safe abortion 95% 1,433 7.1% 15.4% 1 in 70 214,460,115 167,790,590 Family planning and safe abortion Reduce unmet need (56.7%), safe abortion 75% 1,369 11.3% 14.8% 1 in 73 233,930,115 166,870,014 Reduce unmet need (60.3%), safe abortion 75% 1,265 18.0% 13.8% 1 in 79 337,386,615 219,568,526 Reduce unmet need (64.0%), safe abortion 75% 1,160 24.8% 12.8% 1 in 86 441,108,615 272,385,038 Reduce unmet need (67.6%), safe abortion 95% 1,026 33.5% 11.5% 1 in 98 580,230,615 362,579,472 Urban India, current conditions (TFR, 2.07)d 842 — 9.6% 1 in 119 NA — Family planning Reduce unmet need 25% (66.5%) 793 5.8% 9.1% 1 in 126 22,089,305 12,838,532 Reduce unmet need 50% (69.0%) 743 11.7% 8.6% 1 in 135 44,214,305 25,696,437 Reduce unmet need 75% (71.5%) 694 17.6% 8.1% 1 in 144 66,398,305 38,578,054 Reduce unmet need 100% (74.0%) 644 23.5% 7.5% 1 in 155 88,611,805 51,483,279 Safe abortion Increase safe abortion 50% 822 2.4% 9.4% 1 in 122 11,351,305 9,742,159 Increase safe abortion 75% 788 6.4% 9.1% 1 in 127 30,821,305 26,413,778 Increase safe abortion 95% 758 9.9% 8.7% 1 in 133 50,438,805 36,823,443 Family planning and safe abortion Reduce unmet need (66.5%), safe abortion 75% 741 11.9% 8.6% 1 in 135 51,235,305 37,766,193 Reduce unmet need (69%), safe abortion 75% 695 17.5% 8.1% 1 in 144 71,649,305 49,126,308 Reduce unmet need (71.5%), safe abortion 75% 648 23.0% 7.6% 1 in 154 92,122,305 60,499,204 Reduce unmet need (74%), safe abortion 95% 580 31.2% 6.8% 1 in 173 119,557,305 79,270,520 See [87]. Integrated Safe Motherhood Interventions in Rural Uttar Pradesh Reduction in direct causes of maternal mortality, including abortion-related complications, postpartum hemorrhage, hypertensive disorders, sepsis, and obstructed labor. aModel-projected cost savings reflect net costs averted over a woman’s reproductive lifespan (ages 15–45 y) applied to the current population of 15 y olds in India stratified by rural (75%) and urban (25%) settings [87]. Future costs discounted 3% annually. bCost savings for a single representative year of a successfully implemented strategy were calculated using population-level data from India [87] stratified by rural (75%) and urban (25%) settings, for the current distribution of reproductive age women (ages 15–45 y). cIn rural India model-projected TFR is 2 76 2 56 2 36 2 14 with reductions in unmet need of 25% 50% 75% 100% respectively (horizontal axis, left to right) were generally associated with the highest cost-effectiveness ratios (i.e., least attractive), reflecting the higher costs required for infrastructure improvements. An overarching strategic approach that moves along the diagonal, from the lower left corner to the upper right corner, was most effective and cost-effective; the cost savings from enhanced family planning and safe abortion offset the resources required to improve intrapartum care. PLoS Medicine | www.plosmedicine.org Sensitivity Analyses For deliveries at home and in birthing centers in rural Uttar Pradesh, removing only one ‘‘delay’’ in accessing EmOC had minimal impact (,5%) on lowering maternal mortality and was not cost-effective (e.g., US$700–US$4,900 per YLS) (Figure 4, lower panel). In contrast, an integrated strategy that made modest improvements in all components (e.g., SBA, referral, transport, and quality) reduced mortality by 22%. Cost-effectiveness of an April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 9 Reducing Maternal Mortality in India Figure 3. Averted deaths with family planning and safe abortion. Averted deaths attributable to unsafe abortion in rural India by addressing need for family planning (green shading) and providing 75% safe abortion (blue shading). Magnitude of additional averted abortion-related deaths with improved access to safe abortion depends on the amount of unmet need for contraception. doi:10.1371/journal.pmed.1000264.g003 Figure 3. Averted deaths with family planning and safe abortion. Averted deaths attributable to unsafe abortion in rural India by addressing need for family planning (green shading) and providing 75% safe abortion (blue shading). Magnitude of additional averted abortion-related deaths with improved access to safe abortion depends on the amount of unmet need for contraception. doi:10.1371/journal.pmed.1000264.g003 integrated strategy ranged from cost saving to US$170 per YLS (Text S1). uncertainty in data and assumptions used in the analysis, four critical themes emerge as robust. Universal antenatal care by itself averted fewer than 2% of maternal deaths; however, if enhanced antenatal care increased the probability of either facility-based delivery or SBA-attended birth (linked with accurate referral and transport) from 31% to 60%, health benefits increased 5-fold (Text S1). First, increasing effective family planning is the most effective individual intervention to reduce pregnancy-related mortality. If the unmet need was met in rural and urban India by 2012, our results imply that the lives of 168,000 women would be saved by the end of 2015. The cost savings over that time period would exceed US$1 billion. Because strategies to increase contraceptive options for limiting and spacing do not require the same level of infrastructure as improving intrapartum care, targeting these strategies toward rural areas with high TFRs is a promising way to initiate equitable improvements in maternal health. Sensitivity Analyses As a greater proportion of routine deliveries shifted from home to facilities, we assumed 70% would shift to birthing centers or health centers staffed by SBA and 30% to facilities with full EmOC capacity. Although the differential benefits of routine delivery in birthing/health centers versus bEmOC was dependent on expedient transfer from a center to referral EmOC if needed, provided this was assured, both approaches were cost-effective. In contrast, when we varied assumptions about the proportion of routine deliveries in cEmOC versus bEmOC, cost-effectiveness results changed drastically; as routine deliveries shifted to cEmOC, the incremental cost-effectiveness ratios became much less attractive, ranging from US$8,300 to US$27,000 per YLS. Second, two distinct—yet synergistic—approaches, family planning and safe abortion, can reduce deaths from unsafe abortion. Enhanced access to family planning by itself reduces demand for elective abortion and consequently reduces deaths attributable to unsafe abortion. In fact, reducing the unmet need for contraception can prevent one of every two abortion-related deaths. Furthermore, just a fraction of the cost savings from family planning would fully fund an intervention to provide safe abortion and postabortion care. Table 6 shows the potential incremental benefits and cost- effectiveness of adjunctive community-based SBA-administered misoprostol for births at home and birthing centers/health centers in rural India. For all four ‘‘upgrade’’ strategies, additional lives could be saved; depending on the phase of improvements in intrapartum care, an additional 7%–13% of maternal deaths were prevented. Cost-savings for a single birth cohort of 15-y-old girls (2010) expected to accrue over their reproductive lifespan (age 15– 45 y) ranged from US$128 million to US$190 million. Third, despite the substantial health and economic benefits associated with family planning and safe abortion, there is a threshold above which further reductions in mortality are impossible. MDG 5 will therefore not be achievable without involving integrated interventions that ensure reliable access to high-quality intrapartum and EmOC. These interventions could be implemented, however, in a staged, scale-up fashion. While formidable effort and financial investment would be required to scale up maternal health services over time, we identified a number of phased approaches that would ultimately prevent four out of five maternal deaths. Coupled with stepwise improvements in family planning and safe abortion, these approaches incrementally shifted home births to birthing centers or facilities with EmOC, and improved both access to SBAs as well as accurate recognition of referral need, transport, and availabil- ity/quality of EmOC. Sensitivity Analyses Successful implementation of these PLoS Medicine | www.plosmedicine.org Discussion Cost-effectiveness ratios are also expressed as percent of the per capita GDP (US$1,068), shown in the farthest right column, as interventions with cost-effectiveness ratios of less than the per capita GDP are considered very cost-effective according to criteria proposed by the Commission on Macroeconomics and Health [98]. p g gy p p p p g interventions with cost-effectiveness ratios of less than the per capita GDP are considered very cost-effective according to criteria proposed by the Commission on Macroeconomics and Health [98]. facility births; 11.6% SBA (home births); transport from home (24.4%), primary-level health center (48.8%), bEmOC (61%); recognition o primary-level health center (40%); availability and quality of EmOC (42.5%); 53% family planning. eStatus quo (rural India): 31.1% facility births; 11.6% SBA (home births); transport from home (24.4%), primary-level health center (48.8%), bEmOC (61%); recognition of referral need at home (20%), primary-level health center (40%); availability and quality of EmOC (42.5%); 53% family planning. fStatus quo (urban India): 69.4% facility births; 19% SBA (home births); transport from home (44%), primary-level health center (69%), bEmOC (81%); recognition of referral need at home (20%), primary-level health center (40%); availability and quality of EmOC (67.5%); 64% family planning. CS, cost saving; ICER, incremental cost-effectiveness ratio. eStatus quo (rural India): 31.1% facility births; 11.6% SBA (home births); transport from home (24.4%), primary-level health center (48.8%), bEmOC (61%); recognition of referral need at home (20%), primary-level health center (40%); availability and quality of EmOC (42.5%); 53% family planning. fSt t ( b I di ) 69 4% f ilit bi th 19% SBA (h bi th ) t t f h (44%) i l l h lth t (69%) bE OC (81%) iti f % facility births; 19% SBA (home births); transport from home (44%), primary-level health center (69%), bEmOC (81%); recognition of primary-level health center (40%); availability and quality of EmOC (67.5%); 64% family planning. ntal cost-effectiveness ratio. doi:10.1371/journal.pmed.1000264.t005 includes SBA-administered oral misoprostol in homes and birthing centers is likely to be cost-effective. PLoS Medicine | www.plosmedicine.org Discussion Accuracy of referral need recognition at home and in health center with SBA increase, on average, to 60%, 75%, 90%, and 95% (not shown) with upgrade 1, 2, 3, and 4 in both rural and urban India aTransport encompasses the expedient availability of means of transport (e.g., vehicle, cart), fuel (if needed), driver, and interim attendant care. Facility transport represents a weighted average of transport availability from a health center or birthing center to an EmOC facility and from a bEmOC facility to a cEmOC if indicated. Accuracy of referral need recognition at home and in health center with SBA increase, on average, to 60%, 75%, 90%, and 95% (not shown) with upgrade 1, 2, 3, and 4 in both rural and urban India. in both rural and urban India. bQuality refers to the availability and quality of services at EmOC facilities, including adequate staffing and supplies, expedient attention (e.g., without delay to collect fees or requirement for family to bring supplies), and evidence-based clinical practices. in both rural and urban India. bQuality refers to the availability and quality of services at EmOC facilities, including adequate staffing and supplies, expedient attention (e.g., without delay to collect fees or requirement for family to bring supplies), and evidence-based clinical practices. y g pp ), p traceptive use for limiting and spacing; shown are values representing the reduction in unmet need by 25%, 50%, 75%, and 100% wit ctively, for both rural and urban India. dStepwise improvements in maternal health services are assumed to occur in consecutive phases (e.g., first upgrade 1, then upgrade 2, etc.). Therefore, the incremental cost-effectiveness ratio (US$ per YLS) for each upgrade is calculated as the difference in lifetime costs relative to the difference in lifetime effects, compared with the preceding next best strategy. Cost-effectiveness ratios are also expressed as percent of the per capita GDP (US$1,068), shown in the farthest right column, as dStepwise improvements in maternal health services are assumed to occur in consecutive phases (e.g., first upgrade 1, then upgrade 2, etc.). Therefore, the incremental cost-effectiveness ratio (US$ per YLS) for each upgrade is calculated as the difference in lifetime costs relative to the difference in lifetime effects, compared with the preceding next best strategy. April 2010 | Volume 7 | Issue 4 | e1000264 Discussion We have identified several strategic options that would cost- effectively reduce maternal mortality in both rural and urban India. Our principal findings are that early intensive efforts to improve family planning and provide safe abortion, accompanied by a systematic stepwise effort to scale up intrapartum and EmOC, could reduce maternal mortality by 75%. Despite the inherent April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 10 Reducing Maternal Mortality in India Table 5. Health and economic outcomes of integrated packages of services: intrapartum care, family pla Table 5. Health and economic outcomes of integrated packages of services: intrapartum care, family planning, and safe abortion. Region Facility Birth (%) Transport- Homea (%) Transport- Facilitya (%) Quality of Careb (%) Family Planningc (%) Safe Abortion (%) Decrease in Maternal Deaths (%) MMR (Deaths per 100,000 Live Births) Maternal Deaths as Percent of Deaths Ages 15–45 y Lifetime Risk of Maternal Death Lifetime Costsd (US$) Cost- Effectivenessd ICER (US$/ YLS) ICER (% per capita GDP) Rural Indiae — — — — — — — 520 16.4 1 in 65 218.38 — — Upgrade 1 45 50 65 70 56.7 50 17.3 460 14.0 1 in 78 212.41 CS CS Upgrade 2 60 60 75 80 60.3 60 33.7 397 11.5 1 in 98 218.51 150 14 Upgrade 3 75 70 85 90 64.0 75 53.4 302 8.3 1 in 139 226.18 160 15 Upgrade 4 80 75 95 95 67.6 95 77.1 162 4.3 1 in 282 243.46 300 28 Urban Indiaf — — — — — — — 407 9.6 1 in 119 184.00 — — Upgrade 1 75 60 85 70 66.5 50 15.8 363 8.2 1 in 141 174.91 CS CS Upgrade 2 80 75 90 80 69.0 60 33.3 305 6.6 1 in 178 178.30 150 14 Upgrade 3 90 80 92.5 90 71.5 75 54.1 225 4.7 1 in 259 183.44 220 21 Upgrade 4 95 85 95 95 74.0 95 78.5 113 2.3 1 in 553 194.97 350 33 labor. aTransport encompasses the expedient availability of means of transport (e.g., vehicle, cart), fuel (if needed), driver, and interim attendant care. Facility transport represents a weighted average of transport availability from a health center or birthing center to an EmOC facility and from a bEmOC facility to a cEmOC if indicated. Discussion With the exception of anemia, we focused on direct causes of maternal mortality; a priority for future analyses is to include interventions to reduce the indirect causes of pregnancy-related mortality. The analysis would be strengthened by availability of indicators that reflect safe motherhood externalities including measures of enhanced household well-being, increased school attendance, decreased numbers of orphans, and reduced impoverishment resulting from catastrophic expenses [94,99]. Other limitations in our analysis stem from its inherent reliance on high-quality data about maternal mortality specifically. While our calibration of setting-specific models allows us to better represent within-country differences in baseline risk, coverage, and capacity than previous studies, high-quality empiric evidence for Shiffman and Smith [101] have described the importance of framing priority public health issues in a manner that resonates with both the ‘‘internal’’ community and other ‘‘external’’ decision makers. With regard to the internal community, the Table 6. Incremental benefits of community-based misoprostol in rural India. Rural India Family Planninga (%) Safe Abortion (%) Facility Birth (%) Transport- Homeb (%) Transport- Facilityb (%) Quality of Carec (%) Incremental Benefits and Cost-Effectiveness of Community-Based Misoprostold Decrease in Maternal Deaths Lives Saved with Addition of Community- based Misoprostole Cost- Effectiveness Upgrade 1 56.7 50 45 50 65 70 — — — Plus community interventiond — — — — — — 12.3% 16,992 Cost savinge Upgrade 2 60.3 60 60 60 75 80 — — — Plus community interventiond — — — — — — 13.0% 17,612 Cost savinge Upgrade 3 64.0 75 75 70 85 90 — — — Plus community interventiond — — — — — — 10.2% 13,983 Cost savinge Upgrade 4 67.6 95 80 75 95 95 — — — Plus community interventiond — — — — — — 6.9% 9,470 Cost savinge aFamily planning refers to contraceptive use for limiting and spacing; shown are values representing the reduction in unmet need by 25%, 50%, 75%, and 100% with upgrades 1, 2, 3, and 4, respectively. bTransport encompasses the expedient availability of means of transport (e.g., vehicle, cart), fuel (if needed), driver, and interim attendant care. cQuality refers to the availability and quality of services at EmOC facilities, including adequate staffing and supplies, and evidence-based clinical practices. dCommunity-based interventions assume SBA-administered misoprostol for births at home and birthing centers/health centers with a 50% (25%–60%) reduction in PPH [72]. Discussion Although alone it cannot substitute for reliable intrapartum care and EmOC, if added to a long-term plan that increases facility-based intrapartum care, it will save additional lives and will reduce costs; (4) Because settings with the highest TFRs and worst maternal health indicators also tend to be those with the greatest need for enhanced health delivery infrastructure, early consistent commitments to provide family planning and safe abortion reduce the total resources required. To place the synergistic benefits of enhanced family planning and safe abortion in context, the magnitude of cost savings from eliminating unmet need and ensuring access to safe abortion is approximately 25% of the required 10-y investment estimated by Johns et al. for scaling up maternal services in India [30]. strategies would be expected to dramatically reduce the MMR, proportionate mortality ratio, and lifetime risk of maternal death. Fourth, despite the possible variation in pace associated with scaling up maternal health services in India, systematic and consecutive phases will be cost-effective. Our results showed that—when coupled with family planning and safe abortion—both early initial strategies and late intensive strategies resulted in cost- effectiveness ratios that were just a fraction of India’s per capita GDP; these would unarguably be considered very cost-effective [95–98]. Although our general findings are consistent with earlier suggestions that interventions to reduce maternal mortality are good public health investments [7,17], our analytic approach also allowed us to identify more and less efficient ways to achieve this. In this regard, we highlight four robust insights: (1) In settings with limited infrastructure, investing in ‘‘intermediate’’ facilities (e.g., birthing centers) is very cost-effective, provided there is reliable referral capacity and transport to an appropriate EmOC facility if necessary; (2) A strategy of routine hospital-based delivery (i.e., cEmOC) is not cost-effective; (3) A community-based strategy that We may have underestimated both effectiveness and cost- effectiveness by excluding effects of certain indirect indicators and interventions. Although we purposely focused on maternal mortality, if we included neonatal health and survival, for example, most strategies would be even more cost-effective due April 2010 | Volume 7 | Issue 4 | e1000264 11 Reducing Maternal Mortality in India PLoS Medicine | www.plosmedicine.org PLoS Medicine | www.plosmedicine.org April 2010 | Volume 7 | Issue 4 | e1000264 PLoS Medicine | www.plosmedicine.org 12 Reducing Maternal Mortality in India Figure 4. Health and economic outcomes in rural Uttar Pradesh. Upper panel. Discussion Reduction in maternal deaths and cost-effectiveness with stepwise approaches to improve maternal health in rural Uttar Pradesh. The vertical axis (from bottom to top) shows outcomes associated with increased access to family planning and safe abortion. The horizontal axis (from left to right) displays outcomes associated with investments in high- quality health-center–based intrapartum care, which involved stepwise improvements in SBAs, recognition of referral need, and antenatal/ postpartum care, incrementally shifted births away from home, and improved transport, availability, and quality of EmOC. Each cell represents a unique strategy; the reduction in maternal deaths shown is relative to current conditions (far lower left corner). Shading reflects cost-effectiveness ratios, compared to status quo (pink, cost saving; blue, ,US$250/YLS; green, ,US$500/YLS; purple, ,US$750/YLS; orange, US$890/YLS). See text for details. Lower panel. Sensitivity analysis depicting the impact of improving only one component of intrapartum care services in rural Uttar Pradesh. Base case and improvements for each component are shown in numbers below the component name (e.g., quality of EmOC is 31.5% in the base case, improvements of 50%–100% were assessed). In settings where most deliveries occur at home, linkage of services in multiple domains is a critical determinant of reduction in maternal deaths. Even large improvements in skilled birth attendants for nonfacility births (yellow bar), recognition of referral need (pink bar), transport (green bar), or quality of EmOC (purple bar) fail to reduce mortality more than 2%–5% if other interdependent components are not improved as well. In contrast, even modest improvements in all four components, as shown in upgrade 1 (blue bar), reduce mortality by 22%. Cost-effectiveness of the integrated strategy ranged from cost saving to US$170 per YLS compared with US$700 to US$4,900 per YLS for single unlinked improvements. doi:10.1371/journal.pmed.1000264.g004 the effectiveness of comprehensive strategies to reduce maternal mortality and morbidity is often either lacking or inconsistent. More studies quantifying the benefits of community-level inter- ventions on preventing maternal morbidity remain a priority [100]. The additional costs that we assumed would be required to scale up interventions and build infrastructure are, at best, gross estimates. That being said, our assumption of 2- to 3-fold increases in the per woman costs to reflect the additional resources required to improve capacity is consistent with those implied by recent analyses assessing global resource needs for maternal health [30,93]. to associations between place of birth and presence of a skilled attendant, with neonatal and maternal deaths [99,100]. Supporting Information Text S1 Supplemental material accompanying the article. Part I, overview of model; part II, overview of model parameterization, calibration, performance; part III, overview of costs and estimates; part IV, supplemental results; part V, references. Finally, by placing and prioritizing safe motherhood in the context of other global health priorities [101,102], our results can also be effectively framed for policymakers who must allocate limited resources, by providing comparative and contextual information about the relative benefits and cost-effectiveness of investments in maternal health measured against other public health priorities. One of the robust findings of our analysis, for example, is that there are integrated strategies that involve improvements in family planning, safe abortion, and intrapartum care that are equally or more cost-effective or attractive than childhood immunization or treatment of malaria, tuberculosis, or HIV [95]. Found at: doi:10.1371/journal.pmed.1000264.s001 (0.53 MB PDF) Author Contributions ICMJE criteria for authorship read and met: SG SS NC UCMN DH. Agree with the manuscript’s results and conclusions: SG SS NC UCMN DH. Designed the experiments/the study: SG SS NC DH. Analyzed the data: SG SS NC UCMN DH. Collected data/did experiments for the study: SG DH. Wrote the first draft of the paper: SG. Contributed to the writing of the paper: SG SS NC UCMN DH. Wrote first draft of Text S1: DH. Acknowledgments We are appreciative of the administrative assistance from Meredith O’Shea, and constructive feedback on the model assumptions (Dan Grossman, Kelly Blanchard, Carol Levin). The Indian government has initiated several policies to improve maternal health [5], particularly in rural areas [27,103], and efforts to both implement and evaluate new strategies are ongoing [83,94,104–106]. Although our analysis is intended to catalyze actionable steps, we recognize that decisions in India about the choice of strategies and rate of stepwise investments to reduce maternal mortality will be a function not only of cost-effectiveness and affordability, but also of political will and local circumstances. Identifying approaches that can be tailored to local situations, but Reducing Maternal Mortality in India that rely on firm core principles and are cost-effective, holds considerable promise as a way to mobilize further political support and convince stakeholders that MDG 5 is within reach. strategies we identified as most effective support three crucial elements already recognized as essential to achieve MDG 5: family planning and control of fertility choices, provision of safe abortion, and assurance that all women have access to intrapartum care and EmOC. Our results reinforce this message, and extend it by quantifying the cost savings of family planning and safe abortion, and identifying efficient and cost-effective approaches to scaling-up capacity for integrated maternal health services. In particular, it is clear from our analysis that an initial focus on family planning, especially in rural poor areas, will significantly prevent pregnancy-related deaths, reduce deaths from unsafe abortion, and save resources. Providing universal access to safe abortion will further augment these benefits. The cost savings from these two strategies will partially offset the resources required to invest in the necessary infrastructure that would assure every woman access to high-quality intrapartum care and EmOC. While MDG 5 is unlikely to be met without assuring access to health- center–based intrapartum care, implementation of a phased stepwise approach, designed to reach this goal while reflecting the current realities and most feasible initial approaches in different settings, is absolutely within reach. With regard to the external community, we have tried to provide a range of outcomes that can be used to create effective ‘‘take home’’ messages for different target audiences. For example, in only 5 y, more than 150,000 lives could be saved just from increasing contraception rates by a few percentage points; nearly US$1.5 billion could be saved by adding safe abortion to family planning efforts; and finally, with stepwise investments to provide facility-based intrapartum care, the majority of maternal deaths could be prevented. In the next decade, this accomplishment would save the lives of 1 million Indian women. References 9. Rosenfield A, Maine D, Freedman L (2006) Meeting MDG-5: an impossible dream? Lancet 368: 1133–1135. 1. World Health Organization (WHO) (2007) Maternal mortality in 2005: estimates developed by WHO, UNICEF, UNFPA, and The World Bank. Available: http://www.who.int/whosis/mme_2005.pdf. Accessed 19 August 2009. 10. Government of India (2005) Reproductive and Child Health Programme Document (RCH II–Document 2): the principles and evidence base for State RCH II Programme Implementation Plans (PIPs). New Delhi, India: Government of India. Available: http://www.whoindia.org/LinkFiles/Child_ Health_in_India_PIP_Doc_Chapter01.pdf. Accessed 21 August 2009. 2. Hill K, Thomas K, AbouZahr C, Walker N, Say L, et al. (2007) Estimates of maternal mortality worldwide between 1990 and 2005: an assessment of available data. Lancet 370: 1311–1319. Health_in_India_PIP_Doc_Chapter01.pdf. Accessed 21 August 2009 3. United Nations (UN). Millennium Development Goals: Goal 5 (MDG 5). Improve maternal health. 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Available: http://www.mp. gov.in/health/Maternal_Mortality_in_India_1997-2003.pdf. Accessed 22 Au- gust 2009. 8. Discussion ePopulation-level incremental benefits (lives saved) associated with the community-based misoprostol intervention (compared to the same strategy without the community-based misoprostol intervention). These were calculated by applying model-projected outcomes to population-level data from rural India [87]. Cost-savings for a single birth cohort of 15-y-old girls (2010) expected to accrue over their reproductive lifespan (age 15–45) ranged from US$128 million to US$190 million. doi:10.1371/journal.pmed.1000264.t006 Table 6. Incremental benefits of community-based misoprostol in rural India. aFamily planning refers to contraceptive use for limiting and spacing; shown are values representing the reduction in unmet need by 25%, 50%, 75%, and 100% with upgrades 1, 2, 3, and 4, respectively. bTransport encompasses the expedient availability of means of transport (e.g., vehicle, cart), fuel (if needed), driver, and interim attendant care. cQuality refers to the availability and quality of services at EmOC facilities, including adequate staffing and supplies, and evidence-based clinical practices. dCommunity-based interventions assume SBA-administered misoprostol for births at home and birthing centers/health centers with a 50% (25%–60%) reduction in PPH [72]. ePopulation-level incremental benefits (lives saved) associated with the community-based misoprostol intervention (compared to the same strategy without the upgrades 1, 2, 3, and 4, respectively. bTransport encompasses the expedient availability of means of transport (e.g., vehicle, cart), fuel (if needed), driver, and interim attendant care. cQuality refers to the availability and quality of services at EmOC facilities, including adequate staffing and supplies, and evidence-based clinical practices. dCommunity-based interventions assume SBA-administered misoprostol for births at home and birthing centers/health centers with a 50% (25%–60%) reduction in PPH [72]. ePopulation-level incremental benefits (lives saved) associated with the community-based misoprostol intervention (compared to the same strategy without the community-based misoprostol intervention). These were calculated by applying model-projected outcomes to population-level data from rural India [87]. Cost-savings for a single birth cohort of 15-y-old girls (2010) expected to accrue over their reproductive lifespan (age 15–45) ranged from US$128 million to US$190 million. doi:10.1371/journal.pmed.1000264.t006 [72]. ePopulation-level incremental benefits (lives saved) associated with the community-based misoprostol intervention (compared to the same strategy without the community-based misoprostol intervention). These were calculated by applying model-projected outcomes to population-level data from rural India [87]. 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Laxminarayan R, Chow J, Shahid–Salles SA (2006) Intervention cost– effectiveness: overview of main messages. Disease control priorities in developing countries. 2nd edition. 35–86. New York: Oxford University Press. Available: http://www.dcp2.org/pubs/DCP/2/FullText. Accessed 4 May 2009. p 77. International Institute for Population Sciences (IIPS) (2007) Fact sheet: Uttar Pradesh. Reproductive and Child Health Project. District Level Household and Facility Survey. DLHS-3. Available: http://www.rchiips.org/pdf/rch3/state/ Uttar-Pradesh.pdf. Accessed 1 September 2009. 97. Brenzel L, Wolfson LJ, Fox-Rushby J, Miller M, Halsey NA (2006) Vaccine– preventable diseases. Disease control priorities in developing countries. 2nd edition. 389-412. New York: Oxford University Press. Available: http://www. dcp2.org/pubs/DCP/20/FullText. Accessed 4 May 2009. 78. International Institute for Population Sciences (IIPS) (2007) Fact sheet: Rajasthan. Reproductive and Child Health Project. District Level Household and Facility Survey. DLHS-3. Available: http://www.rchiips.org/pdf/rch3/ state/Rajsthan.pdf. Accessed 1 September 2009. 98. World Health Organization (WHO) (2001) Macroeconomics and health: investing in health for economic development: report of the Commission on Macroeconomics and Health. Geneva: World Health Organization. 79. Vora KS, Mavalankar DV, Ramani KV, Upadhyaya M, Sharma B, et al. (2009) Maternal health situation in India: a case study. J Health Popul Nutr 27: 184–201. 99. Ekman B, Pathmanathan I, Liljestrand J (2008) Integrating health interventions for women, newborn babies, and children: a framework for action. Lancet 372: 990–1000. 80. Iyengar SD, Iyengar K, Gupta V (2009) Maternal health: a case study of Rajasthan. J Health Popul Nutr 27: 271–292. 100. Bang RA, Bang AT, Reddy MH, Deshmukh MD, Baitule SB, et al. (2004) Maternal morbidity during labour and the puerperium in rural homes and the need for medical attention: A prospective observational study in Gadchiroli, India. BJOG 111: 231–238. 81. Iyengar SD, Iyengar K, Suhalka V, Agarwal K (2009) Comparison of domiciliary and institutional delivery-care practices in Rural Rajasthan, India. J Health Popul Nutr 27: 303–312. 82. Iyengar K, Iyengar SD, Suhalka V, Dashora K (2009) Pregnancy-related deaths in rural Rajasthan, India: exploring causes, context, and care-seeking through verbal autopsy. J Health Popul Nutr 27: 293–302. 101. Reducing Maternal Mortality in India Grimes DA (2005) Risks of mifepristone abortion in context. Contraception 71: 161. g 36. Dolea C, AbouZahr C (2003) Global burden of hypertensive disorders of pregnancy in the year 2000. Geneva, Switzerland: World Health Organization. Available: http://www.who.int/healthinfo/statistics/bod_ hypertensivedisordersofpregnancy.pdf. Accessed 23 August 2009. 65. World Bank (2002) India’s transport sector: the challenges ahead. Vol 1. Washington (D.C.): World Bank Group. Available: http://www-wds. worldbank.org/external/default/main?pagePK = 64193027&piPK=64187937 &theSitePK =523679&menuPK = 64187510&searchMenuPK = 64187283&the SitePK = 523679&entityID = 000094946_02070604022321&searchMenuPK = 64187283&theSitePK = 523679. Accessed 23 August 2009. hypertensivedisordersofpregnancy.pdf. Accessed 23 August 2009. 37. Dolea C, Stein C (2003) Global burden of maternal sepsis in the year 2000. Geneva, Switzerland: World Health Organization. Available: http://www. who.int/healthinfo/statistics/bod_maternalsepsis.pdf. Accessed 23 August 2009. 66. PMGSY (Pradhan Mantri Gram Sadak Yojana) (2006) Briefing book. Available: http://pmgsy.nic.in/. Accessed 27 July 27 2009. 38. AbouZahr C, Wardlaw T (2004) Maternal mortality in 2000: estimates developed by WHO, UNICEF and UNFPA. Geneva: World Health Organization. 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PLoS Medicine | www.plosmedicine.org PLoS Medicine | www.plosmedicine.org April 2010 | Volume 7 | Issue 4 | e1000264 April 2010 | Volume 7 | Issue 4 | e1000264 15 Editors’ Summary Background. Every year, more than half a million women— most of them living in developing countries—die from pregnancy- or childbirth-related complications. About a quarter of these ‘‘maternal’’ deaths occur in India. In 2005, a woman’s lifetime risk of maternal death in India was 1 in 70; in the UK, it was only one in 8,200. Similarly, the maternal mortality ratio (MMR; number of maternal deaths per 100,000 live births) in India was 450, whereas in the UK it was eight. Faced with the enormous maternal death toll in India and other developing countries, in September 2000, the United Nations pledged, as its fifth Millennium Development Goal (MDG 5), that the global MMR would be reduced to a quarter of its 1990 level by 2015. Currently, it seems unlikely that this target will be met. Between 1990 and 2005, global maternal deaths decreased by only 1% per annum instead of the 5% needed to reach MDG 5; in India, the decrease in maternal deaths between 1990 and 2005 was about 1.8% per annum. alone would reduce maternal deaths by 35% at most, so the researchers also used their model to test the effect of combinations of strategies on maternal death. They found that an integrated and stepwise approach (increased family planning and safe abortion combined with consecutively increased skilled birth attendants, improved care before and after birth, reduced home births, and improved emergency obstetric care) could eventually prevent nearly 80% of maternal deaths. All the steps in this strategy either saved money or involved an additional cost per year of life saved of less than US$500; given one suggested threshold for cost- effectiveness in India of the per capita GDP (US$1,068) per year of life saved, these strategies would be considered very cost-effective. What Do These Findings Mean? The accuracy of these findings depends on the assumptions used to build the model and the quality of the data fed into it. Nevertheless, these findings suggest that early intensive efforts to improve family planning and to provide safe abortion accompanied by a systematic, stepwise effort to improve integrated maternal health services could reduce maternal deaths in India by more than 75% in less than a decade. Furthermore, such a strategy would be cost-effective. Editors’ Summary Indeed, note the researchers, the cost savings from an initial focus on family planning and safe abortion provision would partly offset the resources needed to assure that every woman had access to high quality routine and emergency obstetric care. Thus, overall, these findings suggest that MDG 5 may be within reach in India, a conclusion that should help to mobilize political support for this worthy goal. Why Was This Study Done? Most maternal deaths in developing countries are caused by severe bleeding after childbirth, infections soon after delivery, blood pressure disorders during pregnancy, and obstructed (difficult) labors. Consequently, experts agree that universal access to high- quality routine care during labor (‘‘obstetric’’ care) and to emergency obstetrical care is needed to reduce maternal deaths. However, there is less agreement about how to adapt these ‘‘ideal recommendations’’ to specific situations. In developing countries with weak health systems and predominantly rural populations, it is unlikely that all women will have access to emergency obstetric care in the near future—so would beginning with improved access to family planning and to safe abortions (unsafe abortion is another major cause of maternal death) be a more achievable, more cost-effective way of reducing maternal deaths? How would family planning and safe abortion be coupled efficiently and cost-effectively with improved access to intrapartum care? In this study, the researchers investigate these questions by estimating the health and economic outcomes of various strategies to reduce maternal mortality in India. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10. 1371/journal.pmed.1000264. N UNICEF (the United Nations Children’s Fund) provides information on maternal mortality, including the WHO/ UNICEF/UNFPA/The World Bank 2005 country estimates of maternal mortality N The World Health Organization also provides information on maternal health and about MDG 5 (in several languages) What Did the Researchers Do and Find? The researchers used a computer-based model that simulates women through pregnancy and childbirth to estimate the effect of different strategies (for example, increased family planning or increased access to obstetric care) on clinical outcomes (pregnancies, live births, or deaths), costs, and cost-effectiveness (the cost of saving one year of life) in India. Increased family planning was the most effective single intervention for the reduction of pregnancy-related mortality. Reducing Maternal Mortality in India Shiffman J, Smith S (2007) Generation of political priority for global health initiatives: a framework and case study of maternal mortality. Lancet 370: 1370–1379. 83. Mavalankar DV, Vora KS, Ramani KV, Raman P, Sharma B, et al. (2009) Maternal health in Gujarat, India: a case study. J Health Popul Nutr 27: 235–248. 102. Filippi V, Ronsmans C, Campbell OM, Graham WJ, Mills A, et al. (2006) Maternal health in poor countries: the broader context and a call for action. Lancet 368: 1535–1541. 84. AMDD Working Group on Indicators (2002) Program note: using UN process indicators to assess needs in emergency obstetric services: Bhutan, Cameroon and Rajasthan, India. Int J Gynaecol Obstet 77: 277–284. 103. National Rural Health Mission (2005–2012) Mission document. Available: http://india.gov.in/outerwin.php?id = http://mohfw.nic.in/NRHM/ NRHM%28National%20Rural%20Health%20Mission%29.htm. Accessed 1 September 2009. j J y 85. World Health Organization (WHO) (2006) World Health Report: working together for health. Geneva: World Health Organization. Available: http:// www.who.int/whr/2006/en/index.html. Accessed 23 August 2009. 104. Padmanaban P, Raman PS, Mavalankar DV (2009) Innovations and challenges in reducing maternal mortality in Tamil Nadu, India. J Health Popul Nutr 27: 202–219. 86. UNICEF, WHO, UNFPA (2007) Maternal mortality declining in middle- income countries; women still die in pregnancy and childbirth in low-income countries. UNICEF, WHO, UNFPA Joint Press Release. Available: http:// www.unicef.org/media/media_41208.html. Accessed 27 July 2009. 105. Prakasamma M (2009) Maternal mortality-reduction programme in Andhra Pradesh. J Health Popul Nutr 27: 220–234. 87. United Nations (UN), Department of Economic and Social Affairs, Population Division (2007) World Population Prospects: the 2006 revision. CD-ROM 106. McPake B, Koblinsky M (2009) Improving maternal survival in South Asia- what can we learn from case studies? J Health Popul Nutr 27: 93–107. PLoS Medicine | www.plosmedicine.org April 2010 | Volume 7 | Issue 4 | e1000264 16 Reducing Maternal Mortality in India Editors’ Summary Editors’ Summary If the current unmet need for family planning in India could be fulfilled over the next 5 years, more than 150,000 maternal deaths would be prevented, more than US$1 billion saved, and at least half of abortion- related deaths averted. However, increased family planning N The United Nations Millennium Development Goals Web site provides detailed information about the Millennium Decla- ration, the MDGs, their targets and their indicators, and about MDG 5 N The Millennium Development Goals Report 2009 and its progress chart provide an up-to-date assessment of progress toward all the MDGs N Computer simulation modeling as applied to health is further discussed at the Center for Health Decision Science at Harvard University PLoS Medicine | www.plosmedicine.org April 2010 | Volume 7 | Issue 4 | e1000264 PLoS Medicine | www.plosmedicine.org 17
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Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones
Frontiers in cellular neuroscience
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OPEN ACCESS OPEN ACCESS EDITED BY Chung Yu (Keith) Chan, National Institute of Health, United States REVIEWED BY Lisi Wei, National Institutes of Health (NIH), United States Kohgaku Eguchi, Okinawa Institute of Science and Technology Graduate University, Japan *CORRESPONDENCE Hiromitsu Tanaka hiromitsu.tanaka@chiba-u.jp RECEIVED 15 August 2023 ACCEPTED 06 October 2023 PUBLISHED 19 October 2023 CITATION Tanaka H, Funahashi J and Hirano T (2023) Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones. Front. Cell. Neurosci. 17:1277729. doi: 10.3389/fncel.2023.1277729 EDITED BY Chung Yu (Keith) Chan, National Institute of Health, United States Hiromitsu Tanaka 1,2*, Junichiro Funahashi 1 and Tomoo Hirano 1 1 Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan, 2 Department of Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan Hiromitsu Tanaka 1,2*, Junichiro Funahashi 1 and Tomoo Hirano 1 Hiromitsu Tanaka 1,2*, Junichiro Funahashi 1 and Tomoo Hirano 1 Hiromitsu Tanaka 1,2*, Junichiro Funahashi 1 and Tomoo Hirano 1 1 Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan, 2 Department of Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan 1 Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan, 2 Department of Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan In presynaptic terminals 4 types of endocytosis, kiss-and-run, clathrin-mediated, bulk and ultrafast endocytosis have been reported to maintain repetitive exocytosis of neurotransmitter. However, detailed characteristics and relative contribution of each type of endocytosis still need to be  determined. Our previous live-cell imaging study demonstrated individual exocytosis events of synaptic vesicle within an active-zone-like membrane (AZLM) formed on glass using synaptophysin tagged with a pH-sensitive fluorescent protein. On the other hand, individual endocytosis events of postsynaptic receptors were recorded with a rapid extracellular pH exchange method. Combining these methods, here we  live-cell imaged endocytosed synaptophysin with total internal reflection fluorescence microscopy in rat hippocampal culture preparations. Clathrin- dependent and -independent endocytosis, which was seemingly bulk endocytosis, occurred within several seconds after electrical stimulation at multiple locations around AZLM at room temperature, with the locations varying trial to trial. The contribution of clathrin-independent endocytosis was more prominent when the number of stimulation pulses was large. The skewness of synaptophysin distribution in intracellular vesicles became smaller after addition of a clathrin inhibitor, which suggests that clathrin-dependent endocytosis concentrates synaptophysin. OPEN ACCESS Ultrafast endocytosis was evident immediately after stimulation only at near physiological temperature and was the predominant endocytosis when the number of stimulation pulses was small. COPYRIGHT © 2023 Tanaka, Funahashi and Hirano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. synaptic vesicle, endocytosis, clathrin, TIRFM, synaptophysin (SYN), cytomatrix at the active zone-associated structural protein (CAST), active zone (AZ) synaptic vesicle, endocytosis, clathrin, TIRFM, synaptophysin (SYN), cytomatrix at the active zone-associated structural protein (CAST), active zone (AZ) TYPE  Original Research PUBLISHED  19 October 2023 DOI  10.3389/fncel.2023.1277729 TYPE  Original Research PUBLISHED  19 October 2023 DOI  10.3389/fncel.2023.1277729 frontiersin.org Primary cell culture and transfection The methods for preparing the primary culture of hippocampal neurons and transfection of cDNA were described previously (Tanaka and Hirano, 2012; Tanaka et al., 2014). Briefly, hippocampi were dissected out from E18-P0 Wistar rat embryos, treated with 0.25% trypsin (Thermo Scientific, 15090-046) and dissociated by trituration with a fire-polished Pasteur pipette. Dissociated cells were seeded on poly-D-lysine-(Merck, P7280 and P6407) and NLG-coated glass (Figure  1A) in Neurobasal medium (Thermo Scientific, 21103-049) containing 1% penicillin–streptomycin (Thermo Scientific, 15140-122), 500 μM glutamine (Merck, G6392) and 2% B27 Supplement (Thermo Scientific, 17504-044). In the present study, we used total internal reflection fluorescence microscopy (TIRFM), which allows the observation of fluorescent molecules with a high signal-to-noise ratio by limiting the depth of the excitation field to approximately 100–200 nm (Axelrod, 2001; Toomre and Manstein, 2001), to improve the quality of the fluorescent images of endocytosed presynaptic proteins. In our previous studies, to efficiently visualize fluorescent molecules at active zones with TIRFM, we induced the formation of active zone-like membranes (AZLM) parallel to the glass surface by coating with neuroligin (NLG). This synaptic adhesion molecule induces presynaptic differentiation through binding with presynaptic neurexin (NRX) (Funahashi et  al., 2018). With this setup, we  recorded single exocytosis events using a synaptic vesicle protein, synaptophysin (Syp), tagged with a pH-sensitive variant of green fluorescent protein (GFP) called super-ecliptic pHluorin (SEP). SEP is non-fluorescent in the low pH intra-vesicular solution but becomes fluorescent after exposure to the neutral pH of the extracellular solution (Miesenböck et  al., 1998; Kavalali and Jorgensen, 2013). We  also visualized endocytosis events of glutamate receptors labeled with SEP in the postsynaptic membrane (Fujii et al., 2017, 2018). In those studies, the extracellular pH was changed intermittently and locally to an acidic condition using a U-tube system. At pH 6.0, cell-surface SEP signals were quenched, and only signals could be detected from recently endocytosed vesicles in which acidification had not been completed. pH-sensitive fluorophores and extracellular pH exchange have been used to study endocytic processes (Merrifield et al., 2005; Rosendale et al., 2017). Combining TIRFM, AZLM formation and rapid pH exchange using U-tube, in the present study, we  observed endocytosed Syp-SEP signals after an electrical field stimulation to trigger the exocytosis of synaptic vesicles and analyzed the distribution patterns of the endocytosed Syp-SEP signals around AZLM at several time points. DNA constructs Expression vectors for rat synaptophysin labeled with SEP (Syp- SEP), cytomatrix at the active zone-associated protein (CAST) labeled with TagRFPt (CAST-RFP), neuroligin1 with splice insertion A labeled with human immunoglobulin-Fc region (NLG-Fc) and neurexin1β without splice insertion 4 labeled with HA tag (NRX) were prepared as described previously (Funahashi et al., 2018). Animals All experimental procedures were carried out in accordance with the National Institute of Health guide for the care and use of laboratory animals and the ethical guidelines on animal experimentation of Kyoto University, and were approved by the local committee for handling experimental animals in the Graduate School of Science, Kyoto University. Each of the above methods to study presynaptic endocytosis has merits and demerits. EM captures images with ultra-high resolution but at only one-time point for each image (Watanabe et al., 2013, 2014; Jin et  al., 2019). Recording the membrane capacitance measures the change of the surface area of the presynaptic terminal with high temporal resolution (Delvendahl et al., 2016; Jin et al., 2019), but it does not provide information about the location or morphological properties of each endocytosis type. Live-cell fluorescence imaging records the movement and location of released synaptic vesicle proteins even at a single synapse, and imaging trials can be repeated in the same cell, but this approach has limited spatial resolution compared with EM and low time resolution compared with electrophysiological capacitance measurements. Overall, the distribution of each endocytosis type around individual active zones has not been demonstrated at multiple time points in living neurons.ll Primary cell culture and transfection We show the distributions of Syp-SEP during and after exo- and endocytosis around AZLM and analyze the characteristics of each type of endocytosis. Materials and methods Materials and methods activation of a presynaptic neuron, and UFE occurs immediately after the presynaptic exocytosis (Cheung and Cousin, 2013; Watanabe et al., 2013; Soykan et al., 2017). Kiss-and-run endocytosis (K&R), in which the vesicle and cell membrane fuse transiently has also been reported (Zhang et al., 2009; Ge et al., 2022), although its existence in neuronal presynaptic terminals is controversial (Granseth et al., 2006; Wu et al., 2014). Materials and methods Animals Materials and methods Introduction In presynaptic terminals, the rapid and repetitive exocytosis of synaptic vesicles occurs. Because the number of synaptic vesicles is limited, efficient synaptic vesicle retrieval processes including endocytosis are necessary. Morphological observations using electron microscopy (EM), electrophysiological capacitance measurements of the cell-surface area and live-cell imaging have been performed (Jin et al., 2019), and four types of endocytosis have been reported. After the full-fusion type of exocytosis, clathrin-mediated endocytosis (CME) and clathrin-independent one occurs in the vicinity of an active zone (Granseth et  al., 2006; Chanaday et al., 2019). The latter include activity-dependent bulk endocytosis (ADBE) and ultrafast-type endocytosis (UFE). In ADBE a large endosome is produced after repetitive Frontiers in Cellular Neuroscience Frontiers in Cellular Neuroscience 01 Tanaka et al. 10.3389/fncel.2023.1277729 Glass coating and AZLM formation FIGURE 1 Experimental scheme and system. (A) Schematic diagram of AZLM and pH exchange method. Arrows around top right U-tube indicate directions of solution flow. (B) Experimental design. An electrical field stimulation (50 pulses, 50 Hz) was applied to induce the exocytosis of synaptic vesicles containing Syp-SEP [① in (A,B)]. The endocytosed Syp-SEP was observed at each time point by repeating a pH exchange from 7.4 to 6.0 [② in (A,B)]. The green two-way arrow corresponds to the amount of endocytosed Syp-SEP. (C) The pH-exchanging speed. The right graph shows an enlargement of the region enclosed by the dotted yellow line. (D) Observation area of TIRFM in the Z-axis. The tip of a glass electrode containing CF488 dye (left) was observed under TIRFM. The middle and right TIRFM images are enlargements of the yellow-dotted square region in the left bright field + fluorescence i t 0 d 250 f th l f A i di t th l t d ti Th l th t t f th i li ti d th f TIRFM FIGURE 1 Experimental scheme and system. (A) Schematic diagram of AZLM and pH exchange method. Arrows around top right U-tube indicate directions of solution flow. (B) Experimental design. An electrical field stimulation (50 pulses, 50 Hz) was applied to induce the exocytosis of synaptic vesicles containing Syp-SEP [① in (A,B)]. The endocytosed Syp-SEP was observed at each time point by repeating a pH exchange from 7.4 to 6.0 [② in (A,B)]. The green two-way arrow corresponds to the amount of endocytosed Syp-SEP. (C) The pH-exchanging speed. The right graph shows an enlargement of the region enclosed by the dotted yellow line. (D) Observation area of TIRFM in the Z-axis. The tip of a glass electrode containing CF488 dye (left) was observed under TIRFM. The middle and right TIRFM images are enlargements of the yellow-dotted square region in the left bright field + fluorescence image at 0 and 250  nm from the glass surface. Arrows indicate the electrode tip. The length constant of the visualization depth of TIRFM was calculated to be 151 nm (right). (E) Single molecule analysis of SEP. Sequential images of the SEP signal recorded every 120 ms with TIRFM (top). The time course of the SEP fluorescence intensity, showing single-step photobleaching characteristic of a single molecule (bottom left). Distribution of single SEP fluorescence intensities at PT (bottom middle) and RT (bottom right). Glass coating and AZLM formation NLG-Fc was prepared from transfected HEK293 cells using nProtein A Sepharose (GE Healthcare, 17-5280-01). A piece of cover glass was first incubated with 43 μM biotinylated bovine serum albumin (Thermo Scientific, 29130) in buffer A (100 mM KCl, 5 mM MgCl2, 25 mM HEPES-KOH, pH7.4) at 4°C overnight (Tanaka et al., 2014). Then, the glass was incubated in buffer A containing 17 mM streptavidin (Wako, 191-12851) for 1 h, followed by incubation in buffer A containing biotin-conjugated anti-human IgG (1:100; Jackson ImmunoResearch, 109-065-098) for 1 h at RT. Then, the glass was washed and further incubated in buffer A containing 3–5 mg/mL NLG-Fc for 5 h. Expression vectors for Syp-SEP, CAST-RFPt and NRX were transfected into neurons after 10–15 days in vitro (DIV) with Lipofectamine 2000 (Thermo Scientific, 11668-019) (Funahashi et al., 2018). Half of the medium was replaced with fresh prewarmed medium 4–6 h after the transfection. AZLM was formed, and all imaging experiments were carried out 2–3 days after the transfection. Frontiers in Cellular Neuroscience 02 frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 FIGURE 1 Experimental scheme and system. (A) Schematic diagram of AZLM and pH exchange method. Arrows around top right U-tube indicate directions of solution flow. (B) Experimental design. An electrical field stimulation (50 pulses, 50 Hz) was applied to induce the exocytosis of synaptic vesicles containing Syp-SEP [① in (A,B)]. The endocytosed Syp-SEP was observed at each time point by repeating a pH exchange from 7.4 to 6.0 [② in (A,B)]. The green two-way arrow corresponds to the amount of endocytosed Syp-SEP. (C) The pH-exchanging speed. The right graph shows an enlargement of the region enclosed by the dotted yellow line. (D) Observation area of TIRFM in the Z-axis. The tip of a glass electrode containing CF488 dye (left) was observed under TIRFM. The middle and right TIRFM images are enlargements of the yellow-dotted square region in the left bright field + fluorescence image at 0 and 250  nm from the glass surface. Arrows indicate the electrode tip. The length constant of the visualization depth of TIRFM was calculated to be 151 nm (right). (E) Single molecule analysis of SEP. Sequential images of the SEP signal recorded every 120 ms with TIRFM (top). The time course of the SEP fluorescence intensity, showing single-step photobleaching characteristic of a single molecule (bottom left). Distribution of single SEP fluorescence intensities at PT (bottom middle) and RT (bottom right). Image analysis Acquired images were analyzed using MetaMorph (Molecular Devices), R (The R Project) and Excel (Microsoft). AZLM area was determined as follows. First, the CAST-RFP image was compensated for mechanical drifts of the stage. Then, the mean plus 3 times SD of the background signal intensity in each image was set as the threshold. The CAST-labeled area ranging from 0.098 to 0.38 μm2 was defined as AZLM. The active zone area in the hippocampus estimated by previous EM studies is <0.18 μm2 (mean 0.04 μm2) (Schikorski and Stevens, 1997; Holderith et al., 2012). However, we recorded a fluorescence signal from a single SEP molecule in several pixels due to diffraction and scattering (Figure  1E). Therefore, we  set the AZLM area slightly larger. Analysis was performed on AZLMs which showed significant Syp-SEP signal increase by 5 or 50 pulses stimulation.hl The Syp-SEP area reflecting exocytosis or endocytosis was determined as follows. After drift compensation (Tanaka et al., 2014), the fluorescence values of Syp-SEP were compensated for bleaching. The bleaching rate was computed by fitting the bleach curve with a double exponential function obtained from experiments without stimulation. Then, the mean plus 3 times SD of the background signal in each image was set as the threshold. Areas (> 0.098 μm2) of contiguous pixels above the threshold were defined as the Syp-SEP positive area. The centroid of the fluorescence intensity of the Syp-SEP-positive area was regarded as the center of Syp-SEP-containing vesicles. For a detailed analysis of the signal intensity, area and position of endocytosed Syp-SEP, the average of 4 consecutive high- magnification images taken from the time when the extracellular pH was changed to 6.0 was used. For example, when the pH exchange to 6.0 started 3 s after the stimulation, Syp-SEP images taken at 3.24, 3.36, 3.48 and 3.60 s were averaged and used for the quantitative analyses at 3.24 s. To exclude the Syp-SEP signal from that of organelles such as the endoplasmic reticulum, each image was subtracted from the image recorded at pH 6.0 at 3.88 s before the stimulation. If an increase in the signal was observed from the background level while the extracellular pH was 6.0, it was assumed that Syp migrated from outside the TIRFM observation area within the presynaptic terminal, and the corresponding data was excluded from the analysis. When multiple Syp-SEP containing vesicles were in close proximity, it was impossible to estimate each center. Glass coating and AZLM formation 03 Frontiers in Cellular Neuroscience frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 10.3389/fncel.2023.1277729 Image analysis However, we tried to separate a contiguous Syp-SEP positive area containing multiple intensity peaks using watershed lines (Broadhead et al., 2016; Imig et al., 2020). Watershed lines were drawn to connect local minima that existed between peaks using MetaMorph. To measure the observation depth of TIRFM, the tip of the glass electrode containing CF488 (Nacalai testque, 20 μM) was raised in the Z-axis direction at 125 nm steps from the glass surface using a micro manipulator (HEKA, MP-285). The fluorescence signal decreased as the glass tip was raised with a length constant of 151 nm (Figure 1D). The same laser intensity and illumination angle were used to record the fluorescence of single SEP molecules. SEP molecules were prepared from SEP-transfected HEK293 cells by supersonic treatment and diluted with PBS. A single SEP molecule on the glass was identified by single-step photobleaching (Figure 1E). Live-cell imaging and electrical field stimulation The intra-U-tube solution was the same as the extracellular solution, except that the pH was adjusted to 6.0 with 2-(N-morpholino) ethanesulfonic acid and KOH. The intra-U-tube solution was the same as the extracellular solution, except that the pH was adjusted to 6.0 with 2-(N-morpholino) ethanesulfonic acid and KOH. The TIRFM imaging system was composed of an inverted fluorescence microscope (Olympus, IX71) equipped with a 100× (NA 1.49) or 150× (NA 1.45) TIRFM objective lens and 1.6× intermediate lens, EM-CCD camera (Andor, iXonEM+ DU-897), 488 nm laser (Melles Griot, 85-BCD-020) and 561 nm laser (Coherent, Sapphire 561LP). Changes in Syp-SEP signals before and after the electrical stimulation were recorded with the camera using a 40 ms exposure time and the 100× objective lens. When we wanted higher resolution images, Syp-SEP and CAST-RFP images were acquired for 120 ms with the 150× objective lens and 1.6× intermediate lens using a dual-color filter (Semrock, FF01-523/610- 25). Imaging was performed in extracellular solution (120 mM NaCl, 4 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES-KOH, pH 7.3) at RT or PT. The temperature of the experimental room was controlled by air conditioning. The pH was changed intermittently, and the duration of the pH 6.0 condition was 700 ms. Within this pH 6.0 period we recorded 5 high resolution images and the later 4 were used to generate an averaged image. An electrical field stimulation (duration 1 ms, 20–26 V/cm, 5, 20, 50 or 125 pulses at 50 Hz) was applied to the cultured neurons between platinum electrodes (Tanaka et  al., 2014). To observe clathrin- independent endocytosis, pitstop  2 (pit2) was added at a final concentration of 10 μM, and imaging was performed 10 min later. Pit2 was used here to inhibit clathrin-dependent processes, because it was possible to record Syp-SEP images in a same preparation before and after its application. Previous studies also used knockdown of clathrin by RNAi or overexpression of a dominant negative form of clathrin-related protein (Granseth et al., 2006), but these manipulations take much longer time and recording endocytosis before and after the manipulation in a same cell is very difficult if not impossible. Frontiers in Cellular Neuroscience Location of endocytosed Syp-SEP signals Next, we  imaged endocytosed Syp-SEP after the rapid extracellular pH change to 6.0 at a higher magnification using 150× objective lens to examine where endocytosed Syp-SEP was localized in relation to AZLM (Figure 3A). Line scans across CAST-RFP and Syp-SEP signals from a representative case showed that the position of endocytosed Syp-SEP signal peaks at 3.24 and 7.24 s after the stimulation were about 170 nm from the CAST-RFP signal peak (Figures 3A,B). However, Syp-SEP signals at pH 6.0 were scattered, and there were multiple intensity peaks in several trials. In such cases, we attempted to divide the Syp-SEP areas by watershed lines (Figure 3C). The centroid of the Syp-SEP signal after the stimulation at pH7.4, which reflects the exocytosis of a large number of synaptic vesicles, was about 86 ± 13 nm (n = 45 clusters) inside of the edge of the CAST-RFP signal, whereas that at pH 6.0, which reflects endocytosis, was about 100 ± 28 nm (n = 69 clusters) and 73 ± 27 nm (n = 62 clusters) outside of the edge of the CAST-RFP signal at 3.24 s and 7.24 s after the stimulation (Figure 3D). The latter two values were significantly different from the former (p < 0.001, Steel-Dwass’s test). Thus, Syp-SEP signals at pH 6.0, which reflect endocytosis- related vesicles, were found in the periphery of AZLM. The centroid of the Syp-SEP signal at pH 6.0 does not always correspond to the center of an endocytosed vesicle, because the Syp-SEP signal might have come from multiple vesicles that were not separated by the watershed lines. On the other hand, we previously reported that Syp-SEP is released inside AZLM by an electric field stimulation (Funahashi et al., 2018), which is consistent with the present results. Additionally, the CAST-RFP signal was stable and moved little An electric field stimulation (50 Hz, 50 pulses) was applied to cultured neurons so that a large number of synaptic vesicles were fused to AZLM at RT, and an apparent increase in the Syp-SEP signal was detected (Figure 2A) (Funahashi et al., 2018). When the number of stimulation pulses was reduced from 50 to 20 or 5, the increase in the Syp-SEP signal decreased in proportion to the number of stimulation pulses (p < 0.05 and 0.001, Steel’s test). U-tube system The U-tube system was prepared as described previously (Bretschneider and Markwardt, 1999; Fujii et al., 2017). To improve the speed of the external fluid exchange (Figure 1C), the inner diameter of the U-tube hole was made smaller (6–8 μm), and the hole was placed 25–50 μm above the glass surface. Using the U-tube system, the pH of extracellular solution was changed from 7.4 to 6.0 in about 100 ms and from 6.0 to 7.4 in about 200 ms (Figure 1C). For the analysis of exocytosed Syp-SEP signal intensity, images recorded at 120 ms after the end of the stimulation at pH 7.4 were used. The position of exocytosis indicates the center of the entire Syp-SEP positive area, where multiple vesicles were fused to the plasma membrane, 120 ms after the onset of the stimulation. 04 frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 Frontiers in Cellular Neuroscience Visualization of Syp-SEP around AZLM after presynaptic activation To visualize Syp-SEP translocated to the plasma membrane upon the arrival of action potentials and also endocytosed Syp-SEP in presynaptic terminals, we  applied TIRFM (Figure  1A). We cultured rat hippocampal neurons on NLG-coated glass and induced AZLM formation through NLG binding to presynaptic NRX at DIV 12–18 (Funahashi et  al., 2018). Neurons were transfected with CAST labeled with tagRFPt (CAST-RFP), Syp-SEP and NRX. CAST-RFP was used as an AZLM marker (Ohtsuka et al., 2002). Then, the U-tube system was applied to this experimental system (Fujii et  al., 2017) to transiently and locally change the extracellular pH from 7.4 to 6.0 around AZLM, thus quenching SEP signals on the cell surface, and internalized Syp-SEP signals by endocytosis were detected (Figures 1A–C). The extracellular pH was changed from 7.4 to 6.0 in about 100 ms and from 6.0 to 7.4 in about 200 ms (Figure 1C). The length constant of the visualization depth of the TIRFM was 151 nm (Figure 1D). Therefore, the internalized Syp-SEP signals should have come from a region about 0–200 nm above the glass surface. We also evaluated the sensitivity of the recording system by recording fluorescence from a single SEP molecule on the glass surface (Figure 1E). A single SEP molecule was identified by one-step bleaching, and the fluorescence intensity was 2,900 ± 77 (n = 114 points) at room temperature (RT, 21–23°C) and 2,200 ± 71 (n = 102 points) at near physiological temperature (PT, 31–32°C) on an arbitrary scale of our experimental system.i Experimental design and statistical analysis exocytosis at 50 pulses, an observation consistent with a previous report (Sakamoto et al., 2018). p Endocytosis was likely to occur during the decay of the Syp-SEP signal after exocytosis. To precisely visualize the endocytosed Syp-SEP around AZLM, the pH of the extracellular solution was changed to 6.0 repeatedly so that the fluorescence from cell-surface Syp-SEP was quenched and that only the internalized Syp-SEP signals were recorded (Figures 1A,B, 2B,C). The fluorescence signal observed at pH 6.0 was more intense at 3.12, 7.12, 15.12 and 27.12 s after the stimulation onset than before the stimulation (Figures 2C,D, p < 0.001 and 0.01, Steel’s test), indicating that endocytosis primarily took place at these times after exocytosis. About 40 s after the stimulation, when the Syp-SEP fluorescence signal at pH 7.4 returned to the pre-stimulation level, the signal observed at pH 6.0 almost disappeared. The Syp-SEP signal at pH 7.4 increased and decayed similarly, irrespective of the pH exchange (Figure  2C), suggesting that the pH exchange did not significantly affect the release and uptake of vesicles containing Syp-SEP. The SEP signal intensity at pH 6.0 relative to that at pH 7.4 at 1 s after the stimulation showed a near-linear relationship (Figure  2E). The intensity of endocytosed Syp-SEP was positively correlated with the intensity of exocytosed Syp-SEP. These results confirmed that the more Syp-SEP is exocytosed, the more it is endocytosed. There was weak Syp-SEP signal at pH 7.4 before the stimulation (Figure  2C, −4.04 and −4.00 s). Thus, a small portion of the Syp-SEP signal might have been derived from Syp-SEP present on the plasma membrane before the stimulation. The sample size in each experiment was determined based on previous publications dealing with live-cell imaging (Fujii et al., 2018; Sposini et al., 2020). In each experiment, N indicates the number of Syp-SEP or CAST-RFP signal clusters and/or cells except in the single molecule fluorescence analysis. When we compared means of two groups, Mann–Whitney U test or Wilcoxon signed- rank test was used in a case the normality of data distribution and/ or equality of deviations were denied by Shapiro–Wilk test and/or F-test, otherwise we performed Student’s paired t-test. In multiple comparison of means, we used Steel’s test or Steel-Dwass’s test. All values are presented as the mean ± standard error of the mean. Statistical tests were performed using Excel and Kyplot (KyensLab, ver. 5.0.3). frontiersin.org Location of endocytosed Syp-SEP signals Conversely, the Syp-SEP signal intensity did not increase proportionally when the number of pulses was increased to 125 (Figure 2A), suggesting that the amount of release was nearly saturated at 125 and that most of the synaptic vesicles in a readily releasable pool underwent 05 Tanaka et al. 10.3389/fncel.2023.1277729 FIGURE 2 Visualization of endocytosed Syp-SEP. (A) Release of Syp-SEP induced by electrical field stimulation. In the left, averaged traces of Syp-SEP fluorescence intensity (means ± standard errors) before and after 5 (11 cells), 20 (12 cells), 50 (16 cells) or 125 (23 cells) pulses stimulation are presented with representative images of CAST-RFP (magenta) and Syp-SEP (green) before and after 50 pulses stimulation. In the right, the peak Syp-SEP signal ntensities after 5, 20, 50 or 125 pulses stimulation normalized by the intensities before the stimulation are presented. The amount of exocytosed Syp- SEP increased almost proportionally to the number of stimulation pulses (up to 50 pulses). (B) Representative images of Syp-SEP and CAST-RFP before and after the 50 pulses stimulation at pH 7.4 and 6.0. Arrows indicate Syp-SEP signal at pH 6.0 observed only after the stimulation. (C) An example of the signal intensity of Syp-SEP before and after the 50 pulses stimulation with (black) or without (yellow) pH 6.0 exchange (left graph). Kymograph showing Syp-SEP with pH exchange is presented on the top. We set the timing of pH change to 6.0 so that whole time course of endocytosis occurrence could be clarified and that later pH change to 6.0 would show the Syp-SEP endocytosed after the previous pH exchange, although we know that the Syp-SEP signal at pH 6.0 detected at 7  s include some signal endocytosed before 3 s as described in results. The light blue trace shows the Syp-SEP signal intensity without any stimulation. Right images show Syp-SEP fluorescence before (−) and after (+) the stimulation at pH 7.4 or 6.0. (D) Quantification of the Syp-SEP signal intensities at pH 6.0 at various times normalized by the intensity at pH 7.4 before the stimulation (3.12 s, n  =  43 clusters, 10 cells, 7.12  s, n  =  43 clusters, 10 cells, 15.12  s, 40 clusters, 10 cells, 27.12 s, 39 clusters, 9 cells, 39.12 s, n = 41 clusters, 10 cells). Location of endocytosed Syp-SEP signals (E) Correlation of Syp-SEP signal intensities at pH 7.4 at 1  s after the stimulation and at pH 6.0 at 3.12, 7.12 or 15.12 s after the stimulation (3.12 s, r = 0.54, 7.12  s, r  =  0.56, 15.12  s, r  =  0.58), *, **, and *** indicates p  <  0.05, 0.01, and 0.001 respectively, n.s., not significant. FIGURE 2 Visualization of endocytosed Syp-SEP. (A) Release of Syp-SEP induced by electrical field stimulation. In the left, averaged traces of Syp-SEP fluorescence intensity (means ± standard errors) before and after 5 (11 cells), 20 (12 cells), 50 (16 cells) or 125 (23 cells) pulses stimulation are presented with representative images of CAST-RFP (magenta) and Syp-SEP (green) before and after 50 pulses stimulation. In the right, the peak Syp-SEP signal intensities after 5, 20, 50 or 125 pulses stimulation normalized by the intensities before the stimulation are presented. The amount of exocytosed Syp- SEP increased almost proportionally to the number of stimulation pulses (up to 50 pulses). (B) Representative images of Syp-SEP and CAST-RFP before and after the 50 pulses stimulation at pH 7.4 and 6.0. Arrows indicate Syp-SEP signal at pH 6.0 observed only after the stimulation. (C) An example of the signal intensity of Syp-SEP before and after the 50 pulses stimulation with (black) or without (yellow) pH 6.0 exchange (left graph). Kymograph showing Syp-SEP with pH exchange is presented on the top. We set the timing of pH change to 6.0 so that whole time course of endocytosis occurrence could be clarified and that later pH change to 6.0 would show the Syp-SEP endocytosed after the previous pH exchange, although we know that the Syp-SEP signal at pH 6.0 detected at 7  s include some signal endocytosed before 3 s as described in results. The light blue trace shows the Syp-SEP signal intensity without any stimulation. Right images show Syp-SEP fluorescence before (−) and after (+) the stimulation at pH 7.4 or 6.0. (D) Quantification of the Syp-SEP signal intensities at pH 6.0 at various times normalized by the intensity at pH 7.4 before the stimulation (3.12 s, n  =  43 clusters, 10 cells, 7.12  s, n  =  43 clusters, 10 cells, 15.12  s, 40 clusters, 10 cells, 27.12 s, 39 clusters, 9 cells, 39.12 s, n = 41 clusters, 10 cells). Frontiers in Cellular Neuroscience Location of endocytosed Syp-SEP signals (E) Correlation of Syp-SEP signal intensities at pH 7.4 at 1  s after the stimulation and at pH 6.0 at 3.12, 7.12 or 15.12 s after the stimulation (3.12 s, r = 0.54, 7.12  s, r  =  0.56, 15.12  s, r  =  0.58), *, **, and *** indicates p  <  0.05, 0.01, and 0.001 respectively, n.s., not significant. 06 Frontiers in Cellular Neuroscience frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 FIGURE 3 Location of endocytosed Syp-SEP signal. (A) Example of endocytosed Syp-SEP visualized at high magnification using 150× objective lens. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. (B) Line-scan of Syp-SEP or CAST-RFP signal intensities on the yellow broken lines shown in (A). (C) Examples of binarized Syp-SEP signals and those separated by watershed segmentation. Right most images show raw data with yellow lines indicating Syp-SEP positive areas after watershed segmentation. (D) Location of centroids of the Syp-SEP signal positive areas at pH 7.4 at 0.12  s after the stimulation and at pH 6.0 at 3.24 or 7.24 s after the stimulation in relation to the edge of CAST-RFP (0.12  s, n =  45 clusters, 3.24 s, 69 clusters, 7.24 s, 62 clusters, 13 cells). (E) Stability of CAST-RFP signal. Representative images of CAST-RFP before and after the stimulation (top). Distribution of averaged CAST-RFP centroids at 10–14 s after the stimulation (black points) compared with that at 1–5  s before the stimulation (0.0, red point) (bottom left) (n = 11 clusters, 4 cells). Displacement of CAST-RFP centroids after the stimulation (bottom right). *** indicates p  <  0.001. FIGURE 3 Location of endocytosed Syp-SEP signal. (A) Example of endocytosed Syp-SEP visualized at high magnification using 150× objective lens. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. (B) Line-scan of Syp-SEP or CAST-RFP signal intensities on the yellow broken lines shown in (A). (C) Examples of binarized Syp-SEP signals and those separated by watershed segmentation. Right most images show raw data with yellow lines indicating Syp-SEP positive areas after watershed segmentation. frontiersin.org Location of endocytosed Syp-SEP signals (D) Location of centroids of the Syp-SEP signal positive areas at pH 7.4 at 0.12  s after the stimulation and at pH 6.0 at 3.24 or 7.24 s after the stimulation in relation to the edge of CAST-RFP (0.12  s, n =  45 clusters, 3.24 s, 69 clusters, 7.24 s, 62 clusters, 13 cells). (E) Stability of CAST-RFP signal. Representative images of CAST-RFP before and after the stimulation (top). Distribution of averaged CAST-RFP centroids at 10–14 s after the stimulation (black points) compared with that at 1–5  s before the stimulation (0.0, red point) (bottom left) (n = 11 clusters, 4 cells). Displacement of CAST-RFP centroids after the stimulation (bottom right). *** indicates p  <  0.001. found at different locations in different trials. The distance between the centroid of the Syp-SEP signals in the first trial and the nearest centroid in the second trial was 310 ± 42 nm (n = 21 clusters) at 3.24 s and 340 ± 50 nm (n = 19 clusters) at 7.24 s (Figure 4B), although all of the centroids were distributed in the periphery of AZLM. The average distance between the centroids at 3.24 s and 7.24 s in the same trial was 140 ± 18 nm (n = 29 clusters), which was smaller than the distance during the recording: the distance between the centroid of the CAST-RFP signal before and after the stimulation was 30.8 ± 4.2 nm (Figure 3E). It remains unclear whether synaptic vesicle proteins were endocytosed in specialized regions. To examine if there were such endocytic zones around an active zone, the same neuron was repeatedly stimulated (Figure 4A). Syp-SEP signals at pH 6.0 were 07 frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 FIGURE 4 Locations of endocytosed Syp-SEP signals in different trials. (A) Representative high magnification images of Syp-SEP at pH 6.0 after the 50 pulses stimulation in different trials using the same sample. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. Arrows indicate positions of Syp-SEP around an AZLM. The signal locations were different between 1st and 2nd trials. (B) Displacement of centroids of Syp-SEP signals at pH 6.0 in different trials (3.24 s, 7.24 s) or over a time range (3.24–7.24 s) in a trial (3.24 s, n = 21 clusters, 7.24  s, 19 clusters, 3.24–7.24  s, 29 clusters, 11 cells). Location of endocytosed Syp-SEP signals ** indicate p < 0.01. GURE 4 Locations of endocytosed Syp-SEP signals in different trials. (A) Representative high magnification images of Syp-SEP at pH 6.0 after the 50 pulses stimulation in different trials using the same sample. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. Arrows indicate positions of Syp-SEP around an AZLM. The signal locations were different between 1st and 2nd trials. (B) Displacement of centroids of Syp-SEP signals at pH 6.0 in different trials (3.24 s, 7.24 s) or over a time range (3.24–7.24 s) in a trial (3.24 s, n = 21 clusters, 7.24  s, 19 clusters, 3.24–7.24  s, 29 clusters, 11 cells). ** indicate p < 0.01. for different trials (p < 0.01, Steel-Dwass’s test). These results suggest that endocytosis locations change in different trials and that a part of the Syp-SEP signal recorded at 7.24 s might have been derived from Syp-SEP endocytosed before 3.24 s. pitstop2 (pit2) at RT (Figures 5A, 6A). When pit2 was added to the extracellular solution, the Syp-SEP signal intensity at pH 6.0 after the stimulation became smaller: at 3.24 s, the intensity was 20,700 ± 3,600 (n = 26 clusters) without pit2 and 12,200 ± 1700 with pit2 (n = 25 clusters, p < 0.05, Mann–Whitney U test); at 7.24 s, it was 16,300 ± 2,200 without pit2 (n = 26 clusters) and 10,100 ± 1,300 with pit2 (n = 26 clusters, p < 0.01, Mann–Whitney U test). The Syp-SEP signal intensity at pH 7.4 did not change significantly by pit2 application (Figure 5B, p = 0.67, Mann–Whitney U test). The pit2-sensitive component of the Syp-SEP signal at pH 6.0 presumably related to CME, whereas endocytosis that was not inhibited by pit2 was clathrin-independent. Frontiers in Cellular Neuroscience frontiersin.org Clathrin-dependent and -independent endocytosis It has been proposed that ADBE is triggered by a high load of membrane addition into the presynaptic plasma membrane through the exocytosis of a large number of synaptic vesicles and that the elevated concentration of cytoplasmic calcium in presynaptic terminals plays a role in this process. A field stimulation of 50 pulses at 50 Hz released most vesicles in a readily releasable pool. Therefore, the clathrin-independent endocytosis we  recorded here is likely to correspond to ADBE. To address whether the ADBE-dependent and CME-dependent intracellular Syp-SEP spatial distribution patterns were different, we compared the Syp-SEP signal distribution characteristics before and after pit2 application. We found that the maximum Syp-SEP signal intensity and that divided by the Syp-SEP signal-positive area at 7.24 s after the stimulation were significantly larger than those before pit2 application (Figure 6E, n = 15 clusters before application and n = 16 clusters after, signal intensity, p < 0.05 Mann–Whitney U test; signal intensity / area, p < 0.01 Student’s paired t test). The Syp-SEP signal- positive areas were not significantly different with or without pit2. These results suggest that the intracellular Syp-SEP cluster formed through CME might be more concentrated than the cluster formed through ADBE. However, it might be  also possible that some CME-mediated and ADBE-mediated Syp-SEP signals were not spatially separated and that in such cases the recorded maximum Syp-SEP signal intensity without pit2 was the sum of the CME- and ADBE-mediated signals. Next, we  took higher magnification images of Syp-SEP (Figures  6A,B). The centroids of the Syp-SEP signal at pH 6.0 detected in the presence of pit2, which presumably reflected ADBE, were located 139 ± 49 nm (n = 33 clusters) outside of the edge of the CAST-RFP signal at 3.24 s and 97 ± 44 nm (n = 31 clusters) at 7.24 s, but at pH 7.4 and 1 s after the stimulation, the centroid was 115 ± 16 nm (n = 24 clusters) inside of the edge (Figure 6C, p < 0.001 for both, Steel-Dwass’s test). Thus, ADBE are also likely to occur at the periphery of the CAST-RFP signal.i To examine whether the region where ADBE occur is fixed, the stimulation was applied repeatedly (Figure 6B). Clathrin-dependent and -independent endocytosis The distance between the centroid of the Syp-SEP signal in the first trial and the nearest centroid in the second trial was 378 ± 94 nm (n = 11 clusters) at 3.24 s and 351 ± 65 nm (n = 12 clusters) at 7.24 s (Figure 6D), which are significantly smaller than the distances recorded at 3.24 s and 7.24 s in a single trial (158 ± 23 nm, n = 25 clusters, p < 0.01 and 0.05, Steel-Dwass’s test). These Frontiers in Cellular Neuroscience Clathrin-dependent and -independent endocytosis Next, we tried to determine whether the endocytosis we studied was clathrin-dependent or -independent using the clathrin inhibitor 08 frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 FIGURE 5 Clathrin-dependent and -independent endocytosis. (A) Representative images of Syp-SEP and CAST-RFP signals at pH 7.4 or 6.0 before and after the 50 pulses stimulation in the presence of pit2. While endocytosis was inhibited in some AZLM (arrowhead, pH 6.0, 3.12 s) after the exocytosis of Syp-SEP (pH 7.4, 1.0  s), endocytosed Syp-SEP was detected in other AZLM (arrow, pH 6.0, 3.12 s). (B) Quantification of Syp-SEP at pH 7.4 at 1 s after the stimulation (blue) and at pH 6.0 at 3.24 or 7.24 s after the stimulation (red) without or with pit2 (−pit2, n = 26 clusters; +pit2, 25 clusters, 7 cells). * and ** indicates p  <  0.05 and 0.01 respectively, n.s., not significant. Clathrin-dependent and -independent endocytosis. (A) Representative images of Syp-SEP and CAST-RFP signals at pH 7.4 or 6.0 before and after the 50 pulses stimulation in the presence of pit2. While endocytosis was inhibited in some AZLM (arrowhead, pH 6.0, 3.12 s) after the exocytosis of Syp-SEP (pH 7.4, 1.0  s), endocytosed Syp-SEP was detected in other AZLM (arrow, pH 6.0, 3.12 s). (B) Quantification of Syp-SEP at pH 7.4 at 1 s after the stimulation (blue) and at pH 6.0 at 3.24 or 7.24 s after the stimulation (red) without or with pit2 (−pit2, n = 26 clusters; +pit2, 25 clusters, 7 cells). * and ** indicates p  <  0.05 and 0.01 respectively, n.s., not significant. Previous studies reported that a high-frequency stimulation triggers ADBE (Cheung and Cousin, 2013; Kokotos et al., 2018; Renard and Boucrot, 2021). It has been proposed that ADBE is triggered by a high load of membrane addition into the presynaptic plasma membrane through the exocytosis of a large number of synaptic vesicles and that the elevated concentration of cytoplasmic calcium in presynaptic terminals plays a role in this process. A field stimulation of 50 pulses at 50 Hz released most vesicles in a readily releasable pool. Therefore, the clathrin-independent endocytosis we  recorded here is likely to correspond to ADBE. results suggest that there are multiple ADBE zones around an active zone and which zone is used for ADBE varies trial to trial. Previous studies reported that a high-frequency stimulation triggers ADBE (Cheung and Cousin, 2013; Kokotos et al., 2018; Renard and Boucrot, 2021). Temperature-sensitive endocytosis occurs immediately after the stimulation Next, we examined whether endocytosis could be detected immediately after the stimulation (Figure 7). UFE, which is faster 09 frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 FIGURE 6 Pit2-resistant endocytosis. (A) Representative high magnification images of Syp-SEP signals at pH 7.4 and 6.0 before and after the 50 pulses stimulation without pit2 (upper images) and after application of pit2 (lower images) in the same sample. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. The Syp-SEP signal indicated by an arrow in the top image was clearly diminished after addition of pit2 (bottom image). (B) Representative images of Syp-SEP signals at pH 7.4 and 6.0 before and after the stimulation in the presence of pit2 in different trials using the same sample. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. Arrows indicated Syp-SEP in different locations in different trials. (C) Location of centroids of Syp-SEP signals at pH 7.4 at 0.12  s after the stimulation and at pH 6.0 at 3.24 s or 7.24  s after the stimulation in relation to the edge of the CAST-RFP signal (0.12 s, n = 24 clusters, 3.24  s, 33 clusters, 7.24  s, 31 clusters, 8 cells). (D) Displacement of centroids of Syp-SEP signal at pH 6.0 in different trials (3.24 s, 7.24 s) or over a time range (3.24–7.24  s) in a trial (3.24  s, n =  11 clusters, 7.24  s, 12 clusters, 3.24–7.24 s, 25 clusters, 8 cells). (E) Quantification of the maximum intensity, the area and the maximum intensity divided by the area of Syp-SEP signals at pH 6.0 at 7.24 s after the stimulation (Before n = 15 clusters; After, 16 clusters, 6 cells). *, **, and *** indicate p  <  0.05, 0.01 and 0.001, respectively. n.s., not significant. FIGURE 6 Pit2-resistant endocytosis. (A) Representative high magnification images of Syp-SEP signals at pH 7.4 and 6.0 before and after the 50 pulses stimulation without pit2 (upper images) and after application of pit2 (lower images) in the same sample. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. The Syp-SEP signal indicated by an arrow in the top image was clearly diminished after addition of pit2 (bottom image). Temperature-sensitive endocytosis occurs immediately after the stimulation (B) Quantification of Syp-SEP at pH 6.0 without stimulation (n = 29 clusters, 10 cells), and at 0.36 s (25 clusters, 7 cells), 0.84 s (13 clusters, 6 cells) and 3.24 s (55 clusters, 20 cells) after the 5 pulses stimulation. The intensity value (F – Fbefore) was calculated by subtracting the value 3.88 s before the stimulation (in no stimulation, the value 4.24 s before the timing of stimulation onset was subtracted). Thus, when the intensity value before the stimulation was comparably large due to the signal noise and that after was comparably small, the subtracted and presented value took small negative value. * and *** indicates p < 0.05 and 0.001 respectively; n.s., not significant. FIGURE 7 Absence of ultrafast endocytosis at RT. (A) Representative images of Syp-SEP at pH 7.4 at 4 s before and at 0.12 s (top) or 0.60 s (bottom) after the 5 pulses stimulation, and at pH 6.0 at 3.88 s before and at 0.36 s (top) or 0.84 s (bottom) after the stimulation. All Syp-SEP signals were merged with CAST-RFP signals. Endocytosed Syp-SEP signals were detected not at 0.36 s (arrowheads) but at 0.84 s (arrows) after the stimulation. (B) Quantification of Syp-SEP at pH 6.0 without stimulation (n = 29 clusters, 10 cells), and at 0.36 s (25 clusters, 7 cells), 0.84 s (13 clusters, 6 cells) and 3.24 s (55 clusters, 20 cells) after the 5 pulses stimulation. The intensity value (F – Fbefore) was calculated by subtracting the value 3.88 s before the stimulation (in no stimulation, the value 4.24 s before the timing of stimulation onset was subtracted). Thus, when the intensity value before the stimulation was comparably large due to the signal noise and that after was comparably small, the subtracted and presented value took small negative value. * and *** indicates p < 0.05 and 0.001 respectively; n.s., not significant. Lastly, the intracellular Syp-SEP signal spatial distribution after UFE was analyzed. The maximum Syp-SEP signal intensity divided by the Syp-SEP signal-positive area at 0.36 s was 8.3 ± 0.6 and at 3.24 s it was 9.6 ± 0.9 (Figure 8G, p = 0.30, Mann–Whitney U test). Temperature-sensitive endocytosis occurs immediately after the stimulation Thus, when the intensity value before the stimulation was comparably large due to the signal noise and that after was comparably small, the than other types of endocytosis, has been reported to occur around active zones at 34°C (Watanabe et al., 2013). After a single stimulus, the synaptic vesicle membrane is recovered at sites in the vicinity of the active zone within 100 ms independently of clathrin. It is not known whether UFE mediates the recovery of synaptic vesicle proteins, which should be  an important function of endocytosis at presynaptic terminals (Watanabe and Boucrot, 2017). We attempted to determine whether UFE retrieves laterally diffused Syp-SEP after exocytosis (Gimber et al., 2015; Funahashi et al., 2018) in a living neuron. At RT, we detected the Syp-SEP signal increases at pH 6.0 at 0.84 s after the stimulus but not at 0.36 s after (Figure 7B, p < 0.001 and 0.05, Steel-Dwass’s test). Thus, very fast endocytosis was not detected at RT, suggesting that UFE was unlikely to occur at RT, which is consistent with previous reports (Watanabe et al., 2013). than other types of endocytosis, has been reported to occur around active zones at 34°C (Watanabe et al., 2013). After a single stimulus, the synaptic vesicle membrane is recovered at sites in the vicinity of the active zone within 100 ms independently of clathrin. It is not known whether UFE mediates the recovery of synaptic vesicle proteins, which should be  an important function of endocytosis at presynaptic terminals (Watanabe and Boucrot, 2017). We attempted to determine whether UFE retrieves laterally diffused Syp-SEP after exocytosis (Gimber et al., 2015; Funahashi et al., 2018) in a living neuron. At RT, we detected the Syp-SEP signal increases at pH 6.0 at 0.84 s after the stimulus but not at 0.36 s after (Figure 7B, p < 0.001 and 0.05, Steel-Dwass’s test). Thus, very fast endocytosis was not detected at RT, suggesting that UFE was unlikely to occur at RT, which is consistent with previous reports (Watanabe et al., 2013). To make UFE more likely to occur, the temperature was raised to near physiological temperature (PT, 31–32°C). We also reduced the number of stimulation pulses to 5. At PT, the Syp-SEP signal was recorded when pH was changed at 120 ms after the onset of the stimulation (Figure 8A). Temperature-sensitive endocytosis occurs immediately after the stimulation (B) Representative images of Syp-SEP signals at pH 7.4 and 6.0 before and after the stimulation in the presence of pit2 in different trials using the same sample. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. Arrows indicated Syp-SEP in different locations in different trials. (C) Location of centroids of Syp-SEP signals at pH 7.4 at 0.12  s after the stimulation and at pH 6.0 at 3.24 s or 7.24  s after the stimulation in relation to the edge of the CAST-RFP signal (0.12 s, n = 24 clusters, 3.24  s, 33 clusters, 7.24  s, 31 clusters, 8 cells). (D) Displacement of centroids of Syp-SEP signal at pH 6.0 in different trials (3.24 s, 7.24 s) or over a time range (3.24–7.24  s) in a trial (3.24  s, n =  11 clusters, 7.24  s, 12 clusters, 3.24–7.24 s, 25 clusters, 8 cells). (E) Quantification of the maximum intensity, the area and the maximum intensity divided by the area of Syp-SEP signals at pH 6.0 at 7.24 s after the stimulation (Before n = 15 clusters; After, 16 clusters, 6 cells). *, **, and *** indicate p  <  0.05, 0.01 and 0.001, respectively. n.s., not significant. 10 Frontiers in Cellular Neuroscience frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 FIGURE 7 Absence of ultrafast endocytosis at RT. (A) Representative images of Syp-SEP at pH 7.4 at 4 s before and at 0.12 s (top) or 0.60 s (bottom) after the 5 pulses stimulation, and at pH 6.0 at 3.88 s before and at 0.36 s (top) or 0.84 s (bottom) after the stimulation. All Syp-SEP signals were merged with CAST-RFP signals. Endocytosed Syp-SEP signals were detected not at 0.36 s (arrowheads) but at 0.84 s (arrows) after the stimulation. (B) Quantification of Syp-SEP at pH 6.0 without stimulation (n = 29 clusters, 10 cells), and at 0.36 s (25 clusters, 7 cells), 0.84 s (13 clusters, 6 cells) and 3.24 s (55 clusters, 20 cells) after the 5 pulses stimulation. The intensity value (F – Fbefore) was calculated by subtracting the value 3.88 s before the stimulation (in no stimulation, the value 4.24 s before the timing of stimulation onset was subtracted). frontiersin.org Temperature-sensitive endocytosis occurs immediately after the stimulation Line scans across CAST-RFP and Syp-SEP images at high magnification show the position of the endocytosed Syp-SEP signal peak at 0.36 s after the stimulation was about 500 nm from the position of the CAST-RFP signal peak (Figure 8B). The Syp-SEP signal centroid at pH 6.0 was 20 ± 21 nm (n = 17 clusters) outside the edge of the CAST-RFP signal at 0.36 s, but 110 ± 25 nm (n = 17 clusters) inside the CAST-RFP signal edge at pH 7.4 at 0.12 s after the stimulation (Figure 8C, p < 0.001, Wilcoxon signed-rank test). Importantly, the Syp-SEP signal intensity at pH 6.0 was larger at 0.36 s after the stimulation than that at 3.24 s (Figure  8D, p  < 0.05, 0.01, and 0.001, Steel-Dwass’s test). These results suggest that temperature-dependent UFE occurs immediately after the stimulation, and it dominates when the number of stimulation pulses is small at PT. We did not observe a clear Syp-SEP signal at pH 6.0 inside of AZLM at 0.36 s after the onset of stimulation. Next, we addressed whether the region where UFE occurred was fixed by applying the stimulation repeatedly (Figure 8E). The distance between the centroid of the Syp-SEP signal in the first trial and the nearest centroid in the second trial at 0.36 s was 369 ± 46 nm (n = 11 clusters), which was significantly larger than the distance between the centroids of the Syp-SEP signal at 0.36 s and 3.24 s in a single trial (Figure 8F, 157 ± 27 nm, n = 14 clusters, p < 0.01, Steel- Dwass’s test). These results suggest that there are multiple UFE zones around an active zone and that the Syp-SEP signal recorded at 3.24 s included Syp-SEP endocytosed before 0.36 s. FIGURE 7 Absence of ultrafast endocytosis at RT. (A) Representative images of Syp-SEP at pH 7.4 at 4 s before and at 0.12 s (top) or 0.60 s (bottom) after the 5 pulses stimulation, and at pH 6.0 at 3.88 s before and at 0.36 s (top) or 0.84 s (bottom) after the stimulation. All Syp-SEP signals were merged with CAST-RFP signals. Endocytosed Syp-SEP signals were detected not at 0.36 s (arrowheads) but at 0.84 s (arrows) after the stimulation. Temperature-sensitive endocytosis occurs immediately after the stimulation Considering that Syp-SEP is about 32% brighter at RT than at PT, the UFE-dependent maximum Syp-SEP signal intensity divided by the signal-positive area at RT at 0.36 s was 10.9 ± 0.8, which was not significantly different from the value at 7.24 s after 50 pulses stimulation at RT with pit2 (11.5 ± 1.6, p = 0.98, Steel-Dwass’s test), but smaller than that without pit2 (21.6 ± 3.6, p  < 0.01, Steel- Dwass’s test). We could not separate the Syp-SEP signal-positive area at 0.36 s using watershed lines. 11 Frontiers in Cellular Neuroscience frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 GURE 8 ltrafast endocytosis at PT. (A) Representative high magnification images of Syp-SEP at pH 7.4 and 6.0 before and immediately after the 5 pulses timulation at PT. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. B) Line-scan of Syp-SEP and CAST-RFP signal intensities on the yellow broken line shown in (A). (C) Location of centroids of Syp-SEP signals at pH 7.4 t 0.12  s after the stimulation and at pH 6.0 immediately after the stimulation (the centroid calculated from an averaged image between 0.36 s and .72 s) in relation to the edge of CAST-RFP signal (0.12  s, n =  17 clusters, 0.36 s, 17 clusters, 7 cells). The data obtained in a trial were connected by a line. D) Quantification of Syp-SEP signal intensities at pH 6.0 without stimulation and at 0.36 s or 3.24 s after the stimulation (No stim., n = 7 clusters; 0.36 s, 6 clusters; 3.24  s, 15 clusters, 8 cells). The intensity value (F – Fbefore) was calculated by subtracting the value 3.76 s before the stimulation (in no timulation, the value 3.76  s before the timing of stimulation onset was subtracted). Thus, when the intensity value before the stimulation was omparably large due to the signal noise and that after was comparably small, the subtracted and presented value took small negative value. E) Representative images of Syp-SEP at pH 7.4 and 6.0 before and immediately after the stimulation in different trials using the same sample. The right mages are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP. Arrows indicate Syp-SEP signal. The gnal locations were different between 1st and 2nd trials. Temperature-sensitive endocytosis occurs immediately after the stimulation (F) Displacement of centroids of Syp-SEP signals at pH 6.0 in different trials (0.36 s, 3.24 s) or ver a time range (0.36–3.24  s) in a trial (0.36  s, n =  11 clusters, 3.24 s, 11 clusters, 0.36–3.24 s, 14 clusters, 4 cells). (G) Quantification of the maximum ntensity, the area and the maximum intensity divided by the area of Syp-SEP signals at pH 6.0 at 0.36 s and 3.24 s after the stimulation (0.36 s, n = 37 lusters, 3.24  s, 29 clusters, 9 cells). *, **, and *** indicate p  <  0.05, 0.01 and 0.001, respectively. n.s., not significant. FIGURE 8 Ultrafast endocytosis at PT. (A) Representative high magnification images of Syp-SEP at pH 7.4 and 6.0 before and immediately after the 5 pulses stimulation at PT. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP signals. (B) Line-scan of Syp-SEP and CAST-RFP signal intensities on the yellow broken line shown in (A). (C) Location of centroids of Syp-SEP signals at pH 7.4 at 0.12  s after the stimulation and at pH 6.0 immediately after the stimulation (the centroid calculated from an averaged image between 0.36 s and 0.72 s) in relation to the edge of CAST-RFP signal (0.12  s, n =  17 clusters, 0.36 s, 17 clusters, 7 cells). The data obtained in a trial were connected by a line. (D) Quantification of Syp-SEP signal intensities at pH 6.0 without stimulation and at 0.36 s or 3.24 s after the stimulation (No stim., n = 7 clusters; 0.36 s, 16 clusters; 3.24  s, 15 clusters, 8 cells). The intensity value (F – Fbefore) was calculated by subtracting the value 3.76 s before the stimulation (in no stimulation, the value 3.76  s before the timing of stimulation onset was subtracted). Thus, when the intensity value before the stimulation was comparably large due to the signal noise and that after was comparably small, the subtracted and presented value took small negative value. (E) Representative images of Syp-SEP at pH 7.4 and 6.0 before and immediately after the stimulation in different trials using the same sample. The right images are averages of the images enclosed by the yellow lines and were merged with an image of CAST-RFP. Arrows indicate Syp-SEP signal. The signal locations were different between 1st and 2nd trials. CME and ADBE Various types of fluorescence imaging techniques have been used to study exocytotic and/or endocytic processes in presynaptic terminals (Welzel et al., 2013; Egashira et al., 2015; Midorikawa and Sakaba, 2015; Okamoto et al., 2016; Guillaud et al., 2017; Soykan et al., 2017; Mori et al., 2021). Our method is an advancement of these studies in the following two aspects. First, the signal-to-noise ratio and z-axis spatial resolution were improved by applying TIRFM to AZLM. As described, using TIRFM, we could record signals from single SEP molecules (Figure 1E). In addition, a near two-dimensional analysis of the distribution of endocytosed synaptic proteins was performed by forming AZLM parallelly on the glass surface. Second, extracellular pH exchange using U-tube enabled fast extracellular pH switching and the recording of endocytosed synaptic vesicle proteins at different times repetitively. Previous studies have demonstrated the presence of CME and clathrin-independent endocytosis at mammalian central synapses. CME was originally thought to be  the predominant endocytic mechanism for recycling synaptic vesicles because of the sensitivity to perturbation of clathrin or clathrin-associated proteins, and its time constant was reported to be 15–30 s at RT (Granseth et al., 2006). Here, we showed that the exocytosed Syp-SEP signal after 50 pulses at 50 Hz decreased with a similar time constant of about 20 s (Figure 2A). The speed of CME is thought to be limited by the selection and gathering processes of clathrin and its associated proteins including cargo receptors (Ehrlich et al., 2004; Taylor et al., 2011; Watanabe and Boucrot, 2017). Therefore, not all endocytosis detected at 3.24 and 7.24 s after the 50 pulses stimulation was unlikely to be CME (Figures 3A, 4A). We used a 150× optical lens with a high numerical aperture (1.45) to obtain a high XY spatial resolution; nevertheless, the resolution was restricted by the diffraction limit. Super resolution techniques, such as stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), stimulated emission depletion microscopy (STED) and/or structured illumination microscopy (SIM), have been used to study exo- and endocytic processes in presynaptic terminals and endocrine cells at higher spatial resolution (Schermelleh et al., 2010; Maglione and Sigrist, 2013; Sigal et al., 2018; Schermelleh et al., 2019). Each of these methods has merits and demerits. Future studies combining our method and super- resolution imaging should provide higher quality data and more detailed information about the recycling processes of synaptic vesicles. CME and ADBE When a strong stimulation such as 50 Hz or high K+ solution is applied to neurons, a large area of the plasma membrane is internalized through the clathrin-independent mechanism ADBE (Clayton and Cousin, 2009). Synaptic vesicles are then regenerated from endosomes through a clathrin-dependent process (Kononenko et al., 2014; Watanabe et al., 2014). Recent studies have suggested that ADBE is the dominant endocytosis mechanism during and after high frequency presynaptic activation at PT. The major role of ADBE is not the immediate restoration of synaptic vesicles but it may involve the clearance of fusion sites for later exocytosis (Watanabe et al., 2014; Soykan et al., 2017; Chanaday et al., 2019; Ivanova and Cousin, 2022). On the other hand, CME might contribute to the reformation of synaptic vesicles more directly by gathering membrane proteins of synaptic vesicles through clathrin- associated adaptor proteins over a slower time course. Synaptophysin is diffused out of AZLM following exocytosis Discussion In order not to include such signals in the analysis, we excluded signals which appeared after the pH change to 6.0. It has been reported that the acidification of endocytosed vesicles takes 3–15 s (Atluri and Ryan, 2006; Granseth et  al., 2006; Egashira et  al., 2015). It has also been shown that synaptic vesicles can enter and exit the TIRFM visualization zone in about 100 ms (Midorikawa and Sakaba, 2015). Our observations indicated that the Syp-SEP signal recorded at pH 6.0 at 7.24 s includes partial signals from Syp-SEP endocytosed before 3.24 s. Methods to study endocytosis in presynaptic terminals We have established a new experimental method for the live-cell imaging of endocytosed synaptic vesicle membrane proteins at multiple time points after the exocytotic fusion of vesicles triggered by electrical stimulation. By combining the rapid extracellular pH exchange method and TIRFM observation around AZLM formed on a glass surface, we recorded clathrin-dependent and -independent endocytosis-related signals at RT and also temperature-sensitive ultrafast endocytosis- related signals. This method enabled us to directly observe the spatial pattern of the endocytosed Syp-SEP signal around AZLM repeatedly, which is in principle inaccessible with EM and electrophysiological capacitance measurements. Morphological observations by EM need fixation of the preparation and thus only provide data at one time point for a preparation (Watanabe et  al., 2013). Electrophysiological recordings provide continuous data from a live preparation with a high temporal resolution, but they cannot provide information about the localization of synaptic vesicle proteins or the morphological properties of endocytic events (Delvendahl et al., 2016). frontiersin.org Discussion within about 200 nm from the glass surface, which corresponds to the TIRFM visualization zone. Considering the thickness of the NLG coating and the extracellular space and thickness of plasma membrane, we estimate that the presynaptic intracellular space was about >50 nm above the glass surface. Most of the Syp-SEP signal observed at pH 6.0 likely came from one or multiple vesicles endocytosed just before the pH exchange, before intraluminal acidification and before the vesicles moved out of the TIRFM visualization zone. The Syp-SEP signal could have also come from endosomes having near neutral intraluminal pH and/or endoplasmic reticulum. Therefore, we  subtracted the Syp-SEP image recorded before the stimulation from that recorded after the stimulation to exclude intracellular Syp-SEP signals insensitive to the stimulation. The Syp-SEP signal may also have come from vesicles endocytosed sometime before but moved into the visualization zone from the outside. In order not to include such signals in the analysis, we excluded signals which appeared after the pH change to 6.0. It has been reported that the acidification of endocytosed vesicles takes 3–15 s (Atluri and Ryan, 2006; Granseth et  al., 2006; Egashira et  al., 2015). It has also been shown that synaptic vesicles can enter and exit the TIRFM visualization zone in about 100 ms (Midorikawa and Sakaba, 2015). Our observations indicated that the Syp-SEP signal recorded at pH 6.0 at 7.24 s includes partial signals from Syp-SEP endocytosed before 3.24 s. within about 200 nm from the glass surface, which corresponds to the TIRFM visualization zone. Considering the thickness of the NLG coating and the extracellular space and thickness of plasma membrane, we estimate that the presynaptic intracellular space was about >50 nm above the glass surface. Most of the Syp-SEP signal observed at pH 6.0 likely came from one or multiple vesicles endocytosed just before the pH exchange, before intraluminal acidification and before the vesicles moved out of the TIRFM visualization zone. The Syp-SEP signal could have also come from endosomes having near neutral intraluminal pH and/or endoplasmic reticulum. Therefore, we  subtracted the Syp-SEP image recorded before the stimulation from that recorded after the stimulation to exclude intracellular Syp-SEP signals insensitive to the stimulation. The Syp-SEP signal may also have come from vesicles endocytosed sometime before but moved into the visualization zone from the outside. Frontiers in Cellular Neuroscience Temperature-sensitive endocytosis occurs immediately after the stimulation (F) Displacement of centroids of Syp-SEP signals at pH 6.0 in different trials (0.36 s, 3.24 s) or over a time range (0.36–3.24  s) in a trial (0.36  s, n =  11 clusters, 3.24 s, 11 clusters, 0.36–3.24 s, 14 clusters, 4 cells). (G) Quantification of the maximum intensity, the area and the maximum intensity divided by the area of Syp-SEP signals at pH 6.0 at 0.36 s and 3.24 s after the stimulation (0.36 s, n = 37 clusters, 3.24  s, 29 clusters, 9 cells). *, **, and *** indicate p  <  0.05, 0.01 and 0.001, respectively. n.s., not significant. 12 Frontiers in Cellular Neuroscience frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 UFE An innovative flash-and-freeze approach using optogenetics and freezing neurons at defined time points after the stimulation revealed the existence of UFE. UFE occurs at sites lateral to fusion sites (typically <200 nm) within 100 ms after a stimulation, does not require clathrin, and is temperature dependent (Watanabe et al., 2013, 2014). The amount of membrane internalized by UFE equals the membrane exocytosed (Watanabe et al., 2013). It was suggested that any excess membrane must be removed rapidly from the plasma membrane to restore fusion sites and keep the membrane area and tension constant during a high load of membrane turnover (Rosenmund et al., 1993), something UFE seems to manage (Watanabe and Boucrot, 2017). However, it was unknown whether UFE contributes to the recovery of vesicle proteins. We found that endocytic vesicles contained Syp-SEP at 0.36 s after the onset of a stimulation at PT (Figure  8A). The maximum Syp-SEP signal intensity divided by the signal-positive area of UFE was comparable to that of ADBE after compensating for temperature effects on SEP fluorescence, suggesting that Syp-SEP densities were similar in UFE-mediated and ADBE- mediated intracellular vesicles (Figures 6E, 8G). Caveat, remaining questions and future advancement One caveat of this study is that AZLM is an artificial structure and may have characteristics that are different from normal presynaptic active zones. However, our previous study demonstrated the accumulation of various active zone proteins in AZLM and that the electrical stimulation triggered fusion of synaptic vesicles in AZLM, suggesting that AZLM shows essential properties of active zones and is a reliable model (Funahashi et al., 2018). Future study comparing the results obtained in AZLM with those in normal synapses in detail might provide useful information. It might be also possible to induce AZLM with somewhat different characteristics such as that in large or inhibitory presynaptic terminals using other synaptic adhesion molecules (Südhof, 2021). Comparison of properties among different types of AZLM could be interesting. We would like to note that Syp-SEP signal recorded at 0.36 s might have come from single vesicle in some cases because of the following reason. UFE forms a vesicle of about 100 nm diameter (Watanabe et al., 2013) and the diameter of synaptic vesicle is about 50 nm. Thus, UFE internalizes membrane area corresponding to 4 synaptic vesicles. On the other hand, according to our previous study on synaptic vesicle exocytosis in AZLM, 5 pulses stimulation at 50 Hz seems to induce fusion of around 6 synaptic vesicles (Funahashi et al., 2018). Another limitation of the present method is XY spatial resolution is restricted by the diffraction limit and is inferior to super resolution techniques such as STED, STORM, PALM and SIM (Schermelleh et al., 2010, 2019; Maglione and Sigrist, 2013; Sigal et al., 2018). Future studies combining the present method and one or some of super- resolution imaging techniques would provide more detailed information about the recycling processes of synaptic vesicle proteins. Here, we have examined only synaptophysin as a synaptic vesicle protein. Other synaptic vesicle proteins such as synaptotagmin, synaptobrevin or vglut1 might show different intracellular distribution after endocytosis, because each might be trapped on the membrane of endocytosed vesicles through different adaptor proteins (Cousin, 2017). Thus, extending the analyses to other types of synaptic vesicle protein would contribute to better understanding of retrieval processes of synaptic vesicles. Identity of Syp-SEP signal recorded at pH 6.0 The Syp-SEP signal we recorded at pH 6.0 should have come from intracellular vesicles with an intraluminal neutral pH located Frontiers in Cellular Neuroscience 13 Tanaka et al. 10.3389/fncel.2023.1277729 (Gimber et al., 2015; Funahashi et al., 2018). We showed that the maximum Syp-SEP signal intensity at pH 6.0 divided by the Syp-SEP-positive area was significantly larger before pit2 application than after, which suggests that CME-dependent intracellular vesicles concentrate Syp more than ADBE-dependent ones. This different effect by the two types of vesicles might be caused by trapping Syp through adaptor proteins in CME but not in ADBE. Thus, the density of Syp-SEP in CME-derived vesicles might be higher than that in ADBE-derived vesicles. In addition, a part of ADBE-derived large vesicle could be far from the bottom glass surface and out of TIRFM visualization zone, which might have made the Syp-SEP signal intensity weaker. It is to be noted that there is a possibility that in some cases CME-mediated and ADBE- mediated Syp-SEP signals were closely localized, such that both contributed to the signal intensity of a particular pixel. intense stimulation by high K+ solution was reported using quantum dots (Zhang et al., 2009). However, the large size of quantum dots might have affected fusion pore openings and vesicle collapse (Dittman and Ryan, 2009). K&R should take place at exocytosis sites within an active zone immediately after the exocytosis. However, we did not record events that unequivocally reflected K&R in AZLM, suggesting K&R was rare in our preparations. Frontiers in Cellular Neuroscience Endocytosis sites One merit of our experimental system is that AZLM can be  repeatedly stimulated and endocytosed SEP-tagged synaptic proteins can be imaged after each stimulation. The results show that under different conditions (5 or 50 pulses at RT or PT), the majority of endocytosed Syp-SEP signals was located <200 nm away from the edge of AZLM, but the positions varied trial to trial. Proteins involved in endocytosis might not be immobilized. However, the existence of hotspots, where a particular type of endocytosis such as UFE, ADBE and/or CME preferentially occurs, cannot be  excluded, since the number of trials in our experimental protocol was limited due to the bleaching of Syp-SEP and CAST-RFP. As for the exocytosis of synaptic vesicles, the existence of several hot spots within an active zone has been suggested (Maschi and Klyachko, 2017; Funahashi et al., 2018). References Fujii, S., Tanaka, H., and Hirano, T. (2017). Detection and characterization of individual endocytosis of AMPA-type glutamate receptor around postsynaptic membrane. Genes Cells 22, 583–590. doi: 10.1111/gtc.12493 Atluri, P. P., and Ryan, T. A. (2006). 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Data availability statement supported by grants 18H02526 to TH and 21 K06393 to HT from the Japan Society for the Promotion of Science, and grants to HT from the Takeda Science Foundation, SPIRITS 2020 of Kyoto University, and FY 2021 Kusunoki 125 of Kyoto University 125th Anniversary Fund in Japan. supported by grants 18H02526 to TH and 21 K06393 to HT from the Japan Society for the Promotion of Science, and grants to HT from the Takeda Science Foundation, SPIRITS 2020 of Kyoto University, and FY 2021 Kusunoki 125 of Kyoto University 125th Anniversary Fund in Japan. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Author contributions The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. HT: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Validation, Writing – original draft, Writing – review & editing. JF: Methodology, Writing – review & editing. TH: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. K & R An alternative fast endocytosis pathway, K&R, was reported at neuronal synapses and in neuroendocrine cells (Gandhi and Stevens, 2003; Zhang et  al., 2009), although whether K&R occurs in the mammalian central nervous system is controversial (He and Wu, 2007). The transient opening and closing of fusion pores during 14 frontiersin.org Tanaka et al. 10.3389/fncel.2023.1277729 10.3389/fncel.2023.1277729 Acknowledgments We thank S. Kawaguchi and P. Karagiannis for comments on the manuscript. The animal study was approved by Local committee for handling experimental animals in the Graduate School of Science, Kyoto University. 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https://openalex.org/W2316914652
http://old.scielo.br/pdf/rcefac/v17n6/en_1982-0216-rcefac-17-06-01929.pdf
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Tradução e adaptação brasileira do Questionnaire D'Alimentation
Revista CEFAC
2,015
cc-by
6,234
(1) Universidade Estadual de Campinas, Piracicaba, São Paulo, Brasil (2) McGill University, Montreal, Quebec, Canada (3) Universidade Cidade de São Paulo, São Paulo, São Paulo, Brasil (4) Universidade Federal de São Paulo, Diadema, São Paulo, Brasil Conflict of interest: non-existent ABSTRACT Purpose: to translate the French language instrument “Questionnaire D’Alimentation” to Brazilian Portuguese and culturally adapt it for adolescents. Methods: the translation was based on a protocol that consisted of translation to Brazilian Portuguese (by a professor with a Literary Arts degree and a Doctor of Dental Surgery, both fluent in French and Brazilian Portuguese); back-translation into French; revision by a Committee of specialists (two translators, one native and one sworn translator, and two university professors, one being a Doctor of Dental Surgery and a Speech Therapist) and cultural equivalence (pre-test). The version used in the pre-test consisted of 26 questions with five possible responses (5-Likert), distributed in five domains (Food-Mastication, Habits, Meats, Fruits and Vegetables). The pre-test and test-retest was performed with a sample of 20 adolescents (10 boys/10 girls) from public schools of Piracicaba (Brazil). At this stage, the alternative “I did not understand” was added to each question in order to identify those that were not understood. Test-retest reliability was assessed for each domain using intra-class correlation coefficients (ICCs). Results: in the pre-test, an excellent comprehension of the instrument was observed; in test-retest, ICCs ranged from 0.45 to 0.81 (moderate to excellent agreement). Conclusion: the Portuguese version of the Questionnaire D’Alimentation has shown to be easy to understand by Brazilian adolescents and useful in the evaluation of the masticatory function and feeding or swallowing disorders that may affect food intake. KEYWORDS: Translating; Mastication; Food; Adolescent KEYWORDS: Translating; Mastication; Food; Adolescent 1929 1929 KEYWORDS: Translating; Mastication; Food; Adolescent „ „ INTRODUCTION degree of malocclusion, as well as the masticatory muscles functioning, may influence the quality of an individual’s masticatory function3,4. Moreover, eating behavior may influence the amount of food taken per mouthful, then altering the rate of digestion and absorption5. Mastication is an essential part of the digestive process, because this is when food is fragmented into smaller particles and mixed with saliva, making it possible to swallow and digest1. During the masti­ cation process, contraction of various muscle groups occurs, generating pressure between the tooth cusps, which breaks down the food2. The condition of the dentition, such as the number of teeth present and in contact, the size of functional areas, and the With the aim of evaluating quality of masticatory function, the majority of studies found in the liter­ ature used objective evaluation methods, such as measurements of performance and efficiency6,7. It is believed that one type of method does not exclude the other, and an overall evaluation of the subject is desirable, particularly when it concerns the evalu­ ation of individuals who are undergoing dental and/ or orofacial treatment or who present feeding and/ or swallowing disorders, as a way of verifying the impact of certain dental conditions or orofacial motricity have on masticatory quality. Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 1930 Hilasaca-Mamani M, Barbosa TS, Feine J, Ferreira RI, Boni RC, Castelo PM Previous studies aimed to evaluate masticatory ability by means of questionnaires related to his/ her masticatory capacity, food preference and consistency8-10. Some have used the Visual Analog Scale (VAS) to make a more generalized evalu­ ation, in which the subject attributes a visual score that varies from 0 to 10 to questions such as “How well do you chew?”, or “Do you feel any discomfort when you chew?”6,8,11. A simple evaluation has also been found, such as “Are you satisfied with your chewing capacity?”, with dichotomous YES/ NO responses12. A questionnaire (Questionnaire D’Alimentation) was developed for French speaking subjects who wore partial/complete dentures living in Montreal, Quebec13,14. This questionnaire consists of 38 questions, with 29 questions being specifically related to the frequency of and difficulty chewing different types of foods during the two weeks before the evaluation. adolescents, thereby obtaining a questionnaire that can be used to evaluate the quality of masticatory function in Brazilian Portuguese language. Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 „ „ METHODS This study was approved by the Research Ethics Committee of the Piracicaba Dental School, University of Campinas (UNICAMP), Protocol Number 108/2012. Those responsible for the subjects signed the Informed Consent. It is important to point out that before the translation processes began, the authors of Questionnaire D’Alimentation were contacted (JF). The Questionnaire D’Alimentation consists of 38 questions, and the domains “Food-Mastication”, “Habits”, “Meats”, “Fruits” and “Vegetables” comprise 29 questions specifically related to the frequency of and difficulty with mastication of foods of different types of consistency, in the two weeks preceding the subject’s evaluation. The remaining nine questions relate to diet specificities (for example, appetite, allergies, reflux, nausea, and other). Each question offers five possible response indicators (5-Likert), according to the content of the question, whether it is about the intensity of the difficulty, (domain “Food-Mastication”) or about the frequency of consuming a certain food (other domains). In addition, the domains “Meats”, “Fruits” and “Vegetables” also present an alternative to be checked (not applicable - N/A) if the subject does not usually eat these foods. Difficulty with mastication is the most probable mechanism by which poor dental health conditions or feeding/swallowing disorders may affect food intake, particularly foods with greater consistency, thereby leading to inadequate intake and having a negative impact on nutritional status. A previous study has shown that the higher the number of teeth affected by caries, the greater the chance of dissat­ isfaction with mastication12. It was also observed that subjects who have masticatory problems also present a higher DMFT (number of decayed, missing and filled teeth), fewer functional teeth, and orthodontic treatment need, both in adults and children9,12,15. To measure chewing ability in relation to diet, it is important to gain an understanding of how the subject prepares and chooses their foods. Translation and cross-cultural adaptation of the Questionnaire D’Alimentation were performed in the following steps, proposed by Guillemin, et al.16: initial translation, back-translation, revision by a committee of specialists, and cultural adaptation (Figure 1). To our knowledge, there are very few instru­ ments with the aim of evaluating chewing ability specifically. Thus, the aim of the present study was to translate and cross-culturally adapt the instrument Questionnaire D’Alimentation for Brazilian Rev. CEFAC. „ „ METHODS 2015 Nov-Dez; 17(6):1929-1938 1931 Quality of masticatory function questionnaire Original Version Final Version V5 Back-Translation V4 Back-Translation V3 Pre-test (n=20) V5 in Brazilian Portuguese Revision by Committee Translation V2 Translation V1 Figure 1 – Stages of the processes of translation and cultural adaptation of the instrument Questionnaire D’Alimentation Cultural Equivalence of the Instrument Finally, to evaluate the cultural equivalence of the instrument, the version V5 in Brazilian Portuguese (Quality of Masticatory Function Questionnaire - QMFQ) was self-applied by 20 adolescents18 from public schools of Piracicaba (SP, Brazil), 13 (n=12) and 14 years of age (n=8), of both genders (10♂/10♀), selected by means of a draw from all authorizations received, under the supervision of two researchers (MHM and TSB). In version V5 the option “I didn’t understand” was added as an alternative response to all questions, as a way of identifying questions that had not been adequately understood. The percentage of “I didn’t understand” responses had to be lower than 15% so that the instrument could be considered culturally adapted19. If the established limit was exceeded, the instrument would have to be submitted to a new cultural adaptation process, until the item “I didn’t understand” had not been chosen in any question by 85% or more of the adolescents. Revision by a Committee of Specialists and Cultural Adaptation Original Version The versions V1 and V2 in Brazilian Portuguese and the Versions V3 and V4 in French, as well as the original instrument, were submitted to a Reviser Committee formed by two French language professors (one being native and the other a certified translator), a Speech Therapist – University Professor (RCB) and a Doctor of Dental Surgery - University Professor (PMC). This stage consisted of the following aspects17: • Semantic equivalence: This refers to the meaning of words; words that do not have a literal trans­ lation with a similar meaning were translated into terms in Brazilian Portuguese that had an equivalent meaning; V5 in Brazilian Portuguese • Idiomatic equivalence: The formulation of collo­ quial expressions equivalent to those in the original language; • Cultural equivalence of each question: experiences undergone within the cultural context of the society. In this stage, the cultural adaptation was performed, based on the target population of the research; that is, Brazilian adolescents who were not denture wearers. Therefore, three questions related to denture wearing were excluded from the domain “Habits” (questions 19, 23 and 24). Thus version V5 in Brazilian Portuguese was obtained. Final Version V5 Figure 1 – Stages of the processes of translation and cultural adaptation of the instrument Questionnaire D’Alimentation Initial Translation The version in French (original questionnaire) was initially translated to Brazilian Portuguese by a Professor with a degree in Literary Arts, as well as a Doctor of Dental Surgery and University Professor (RIF), both fluent in French and Brazilian Portuguese, and aware of the aim of this study, emphasizing conceptual translation rather than literal translation (versions in Brazilian Portuguese V1 and V2). Back-translation The versions in Brazilian Portuguese V1 and V2 went through back-translation into French, done by two native French teachers, who did not participate in the first stage of translation, and who had no access to the original instrument, thus obtaining the translations in French V3 and V4. The purpose of back-translation is to compare the back transla­ tioned version with the original instrument. For assessment of test-retest reliability, the same volunteers were invited to fill out a second copy of the questionnaire one week later for Intraclass Correlation Coefficient (ICC) determination using the BioEstat 5.3 (Mamirauá, Belém, PA, Brazil) statistical software package. Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 1932 Hilasaca-Mamani M, Barbosa TS, Feine J, Ferreira RI, Boni RC, Castelo PM Initial Translation and back-translation Each version in Brazilian Portuguese (V1 and V2) was independently re-translated into French (back-translation) by two native French teachers, who were not aware of the purpose of the work, thereby giving rise to the origin of versions V3 and V4 in French. Revision by a Committee of Specialists „ „ RESULTS original instrument were submitted to a Reviewing Committee. Figure 2 presents the questions in the original version and their translations, as well as a synopsis of the decision making process relative to specification of the first and second version in Portuguese (V1 and V2) made by the committee. For some questions, the translations made by both translators were identical, or practically identical; for others, one or other version was prioritized; moreover, in other questions, the option taken was to combine the two versions, generating a version of consensus, with the purpose of obtaining greater clarity of the item. Initial Translation and back-translation Figure 2 – Synopsis of the decision-making processes regards specification of the versions V1 and V2 in Portuguese for construction of the final instrument V1, choice of the first translator’s version; V2, choice of the second translator’s version; V3, choice of a modified version; V1≈V2, similarity between the two versions with specificity for the first translator; V1≈V2, similarity between the two versions with specificity for the second translator. Revision by a Committee of Specialists The versions V1 and V2 in Brazilian Portuguese and the versions V3 and V4 in French, as well as the Original Version Version Committee Question Domain Terms 10 Food-mastication La grosseur d´un dé à coudre V2 Beef/small pieces 11 Food-mastication La grosseur d´un dé à coudre V2 Small pieces 12 Food-mastication Viande hachée V1≈V2 13 Food-mastication Croquer V3 Bite 14 Food-mastication Croquer V3 Bite 15 Food-mastication Croquer V3 Bite 16 Food-mastication La pelure des fruits V1≈V2 17 Food-mastication Pain croûté V3 Bread with a hard crust 18 Food-mastication Noix et des graines V1 19 Habits L´une ou l´autre de vos prothèses V1 Your denture 20 Habits Boire en mangeant V3 Drink while eating 21 Habits La sauce a vos aliments V1 22 Habits Trempé V1 23 Habits Prothèses V1 Denture 24 Habits Prothèses V1 Denture 25 Habits Bien màchés V1 26 Meats La grosseur d´un dé à coudre V3 Small pieces 27 Meats Hacher V3 Shred 28 Meats La grosseur d´un dé à coudre V3 Small pieces 29 Meats Hacher V3 Shred 30 Meats Metre la viande em purée V3 Cook it until it falls apart 31 Fruits Croqué V1 Bit 32 Fruits D´enlever la pelure V1≈V2 Peel 33 Fruits Couper em quartiers V1≈V2 34 Fruits La grosseur d´un dé à coudre V2 Small pieces 35 Fruits Mettre em purée V1 Mash or grate 36 Vegetables Croqué V3 Bit 37 Vegetables La grosseur d´un dé à coudre V2 Small pieces 38 Vegetables Mettre em purée V2 Make puré V1 choice of the first translator’s version; V2 choice of the second translator’s version; V3 choice of a modified version; V1≈V2 Figure 2 – Synopsis of the decision-making processes regards specification of the versions V1 and V2 in Portuguese for construction of the final instrument Figure 2 – Synopsis of the decision-making processes regards specification of the versions V1 and V2 in Portuguese for construction of the final instrument Figure 2 – Synopsis of the decision-making processes regards specification of the versions V1 and V2 in Portuguese for construction of the final instrument Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 1933 Quality of masticatory function questionnaire At this stage, replacements were made of terms presented in V1 and V2 by synonyms, so that the terms would be better understood by the target population. Questions that sought to specify the size of the food (meats, fruits and vegetables) were those that generated the greatest difficulty. Revision by a Committee of Specialists For example, the literal translation of the expression “la grosseur d´un dé à coudre” would be “the thickness of a thumb”. The second translator had also suggested the term “inch”, which is commonly used by the Brazilian adult population. However, the target population (adolescents) may not know the size of an “inch” and so the Committee suggested the use of the expression “small pieces”. of the difficulty (extreme, very, moderate, hardly, no difficulty) and frequency (never, rarely, sometimes, frequently, always, not applicable). For the first item, the option was to choose the modified version (V3). For the frequency scale, the option was for the V1; however, the term “à l´occasion”, which was translated as ”occasionally”, was changed to “sometimes” by the Committee, which is the word more routinely used. After conclusion of the process of translation and revision by the Committee, three questions related to denture wearing were excluded from the domain “Habits” (questions 19, 23 and 24). In questions 13, 14, 15, 31, and 36 the term “croquer” was used, which was translated as “grind” in V1 and as “chew” in V2. The Committee therefore suggested the use of “bite”, because this would linguistically be more accept and easier to under­ stand, since “chew” would be translated into French as “mastiquer”. Cultural Equivalence of the Instrument Mash or grate hard raw fruits to eat them N/A N/A Vegetables 24. Bite into whole raw carrots X 25. Cut raw carrots into small pieces to chew them X N/A 26. Make a puré of hard vegetables to eat them X N/A Figure 3 – Pre-Test Synopsis: Description of the questions with alternative responses “not applicable” (N/A) or left blank (X) checked by six subjects Este questionário visa avaliar sua escolha por alimentos em função de sua capacidade de mastigar nas duas últimas semanas. ALIMENTAÇÃO-MASTIGAÇÃO Extrema Muita Moderada Pouca Nenhuma dificuldade 1. Você tem dificuldade para mastigar carne de vaca cortada em pedaços pequenos? □ (Assinale aqui se você não come carne de vaca) □ □ □ □ □ 2. Você tem dificuldade para mastigar frango cortado em pedaços pequenos? □ (Assinale aqui se você não come frango) □ □ □ □ □ 3. Você tem dificuldade para mastigar carne moída? □ (Assinale aqui se você não come carne moída) □ □ □ □ □ 4. Você tem dificuldade para morder legumes duros, crus, inteiros (exemplo: cenouras)? □ □ □ □ □ 5. Você tem dificuldade para morder frutas duras, cruas, inteiras (exemplo: maçãs)? □ □ □ □ □ 6. Você tem dificuldade para morder frutas duras, cruas, cortadas em quatro (exemplo: maçãs)? □ □ □ □ □ 7. Você tem dificuldade para comer a casca de frutas duras, cruas? □ □ □ □ □ 8. Você tem dificuldade de mastigar pão com casca dura? □ □ □ □ □ 9. Você tem dificuldade de mastigar nozes e grãos? □ □ □ □ □ HÁBITOS Nas duas últimas semanas: Nunca Raramente Às vezes Frequentemente Sempre Individual (gender, age) Domain Question 4 (♀,13) 7 (♀,13) 8 (♂,13) 9 (♂,13) 10 (♂,13) 16 (♂,14) Meats 15. Shred beef before eating it N/A 16. Cut chicken into small pieces N/A 17. Shred chicken before eating it N/A 18. Cook meat until it was tender before eating it N/A Fruits 19. Bite into whole raw apples N/A N/A X N/A 20. Peel apples before eating them N/A N/A N/A 21. Cut apples into quarters to chew them N/A N/A N/A 22. Cut apples into small pieces to chew them N/A N/A N/A 23. Mash or grate hard raw fruits to eat them N/A N/A Vegetables 24. Bite into whole raw carrots X 25. Cultural Equivalence of the Instrument The use of the “Quality of Masticatory Function Questionnaire” was considered easy, fast and the questions were well understood by the subjects, since the alternative “I didn’t understand” was not checked by any of the participants. On the other hand, of the 20 adolescents who participated in pre-test, six checked the alternative response N/A (not applicable) to at least one question in the domains “Meats”, “Fruits” or “Vegetables” (Figure 3). Of these six participants, only one handed in the questionnaire with the responses left blank, being one question with reference to the domain “Fruits” (19: Bite into whole raw apples) and three questions of the domain “Vegetables”. Question 17 asks whether the subject has difficult with chewing “pain croûté”. This expression was translated by both translators as “toasted bread”; whereas for the Committee this translation was not acceptable, and it was replaced by “bread with a hard crust”. The term “hacher” (questions 27 and 29) was translated as ”chop” (V1) and ”mince” (V2), and it was afterwards changed by the Committee, which proposed the term ”shred” (the meat). Question 30 also generated doubts because of the expression “metre la viande em purée”, and it was translated as “Boiling the meat till it was tender”. The test-retest reliability of the questionnaire was assessed for each domain, and the ICCs found were: 0.79 (Food-mastication), 0.45 (Habits), 0.62 (Meats), 0.74 (Fruits) and 0.81 (Vegetables), showing moderate to excellent agreement (p<0.01)20. The final version of the instrument is shown in Figure 4. As regards the alternative scales to be chosen, there were two to be translated: the scale of intensity Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 1934  Hilasaca-Mamani M, Barbosa TS, Feine J, Ferreira RI, Boni RC, Castelo PM 1934  Hilasaca Mamani M, Barbosa TS, Feine J, Ferreira RI, Boni RC, Castelo PM Individual (gender, age) Domain Question 4 (♀,13) 7 (♀,13) 8 (♂,13) 9 (♂,13) 10 (♂,13) 16 (♂,14) Meats 15. Shred beef before eating it N/A 16. Cut chicken into small pieces N/A 17. Shred chicken before eating it N/A 18. Cook meat until it was tender before eating it N/A Fruits 19. Bite into whole raw apples N/A N/A X N/A 20. Peel apples before eating them N/A N/A N/A 21. Cut apples into quarters to chew them N/A N/A N/A 22. Cut apples into small pieces to chew them N/A N/A N/A 23. FRUTAS FRUTAS Nas duas últimas semanas: Nota: Se você não comeu essa fruta, assinale a alternativa N/A (não aplicável). Nunca Raramente Às vezes Frequentemente Sempre N/A 19. Você mordeu maçãs cruas, inteiras? □ □ □ □ □ □ 20. Foi necessário descascar as maçãs antes de comê-las? □ □ □ □ □ □ 21. Foi necessário cortar as maçãs em quatro para mastigá-las? □ □ □ □ □ □ 22. Foi necessário cortar as maçãs em pedaços pequenos para mastigá-las? □ □ □ □ □ □ 23. Foi necessário amassar ou ralar as frutas duras cruas para comê-las? □ □ □ □ □ □ CARNES CARNES Nas duas últimas semanas: Nota: Se você não comeu carne, assinale a alternativa N/A (não aplicável). Nunca Raramente Às vezes Frequentemente Sempre N/A 14. Foi necessário cortar a carne de vaca em pedaços pequenos? □ □ □ □ □ □ 15. Foi necessário desfiar a carne de vaca antes de comê-la? □ □ □ □ □ □ 16. Foi necessário cortar o frango em pedaços pequenos? □ □ □ □ □ □ 17. Foi necessário desfiar o frango antes de comê-lo? □ □ □ □ □ □ 18. Foi necessário cozinhar a carne até des­ manchar antes de comê-la? □ □ □ □ □ □ Se você não comeu carne, assinale a alternativa N/A (não aplicável). 1935 Quality of masticatory function questionnaire Nunca Raramente Às vezes Frequentemente Sempre 11. Você adicionou molho aos seus alimentos para engolir melhor? □ □ □ □ □ 12. Você molhou os alimentos em líquidos para masti­ gar e engolir melhor? □ □ □ □ □ 13. Em geral, os alimentos que você engole são bem mastigados? □ □ □ □ □ Cultural Equivalence of the Instrument Cut raw carrots into small pieces to chew them X N/A 26. Make a puré of hard vegetables to eat them X N/A Figure 3 – Pre-Test Synopsis: Description of the questions with alternative responses “not applicable” (N/A) or left blank (X) checked by six subjects Este questionário visa avaliar sua escolha por alimentos em função de sua capacidade de mastigar nas duas últimas semanas. ALIMENTAÇÃO-MASTIGAÇÃO Extrema Muita Moderada Pouca Nenhuma dificuldade 1. Você tem dificuldade para mastigar carne de vaca cortada em pedaços pequenos? □ (Assinale aqui se você não come carne de vaca) □ □ □ □ □ 2. Você tem dificuldade para mastigar frango cortado em pedaços pequenos? □ (Assinale aqui se você não come frango) □ □ □ □ □ 3. Você tem dificuldade para mastigar carne moída? □ (Assinale aqui se você não come carne moída) □ □ □ □ □ 4. Você tem dificuldade para morder legumes duros, crus, inteiros (exemplo: cenouras)? □ □ □ □ □ 5. Você tem dificuldade para morder frutas duras, cruas, inteiras (exemplo: maçãs)? □ □ □ □ □ 6. Você tem dificuldade para morder frutas duras, cruas, cortadas em quatro (exemplo: maçãs)? □ □ □ □ □ 7. Você tem dificuldade para comer a casca de frutas duras, cruas? □ □ □ □ □ 8. Você tem dificuldade de mastigar pão com casca dura? □ □ □ □ □ 9. Você tem dificuldade de mastigar nozes e grãos? □ □ □ □ □ nário visa avaliar sua escolha por alimentos em função de sua capacidade de mastig mas semanas. Este questionário visa avaliar sua escolha por alimentos em função de sua capacidade de mastigar nas duas últimas semanas. 1935 „ „ DISCUSSION Originally it was proposed that this instrument should be self-applied, being necessary for the respondent to read the instructions carefully before answering the questions; therefore, future studies will be able to show whether there is equivalence. In this study, it was considered feasible for researchers to provide initial instructions to the subjects, with the aim to ensure that the rules for filling out the questionnaire were understood by the studied sample. For responses left blank or for alternative response N/A, the missing value may be replaced by the mean/median scores for each domain or for each subject28,29. An instrument may only be considered valid if it is capable of accurately capturing a certain concept21. Furthermore, in the culture for which it is being adapted, a translated instrument must be capable of obtaining the same effect as the original instrument has in the context in which it was created. The lack of cultural equivalence compromises the validity of the information collected, making it impossible to use the instrument to study a concept correctly22. This is why there are standardized instructions that are intended to minimize the loss of the original instrument characteristics that could result from the change in language16,22-24. Therefore, the methodology of the present study followed the protocol suggested by Guillemim, et al.16, by which mistakes and misinterpretations in the initial transla­ tions are revealed. During these stages, semantic equivalence was appreciated, and not the literal interpretation, between the terms, since the literal interpretation is not always shown to be more advantageous to express concepts or situations of the new population one wishes to study16,24-26. The process of translation and cultural adaptation of health-related questionnaires must keep a conceptual basis and follow standardized methodologies, since differences in subject’s habits and culture may lead to misunderstandings, thereby altering the psychometric properties of the instrument. Therefore, in order to fully achieve cultural adaptation, it is also necessary to conduct a study of equivalence measurement, evaluating the reliability and validity of this new version in different populations and conditions (e.g. subjects with dental caries and/or periodontal disease, patients presenting feeding and/or swallowing disorders, denture users, patients undergoing orthognathic or bariatric surgery, and others). In this process, using and comparing more than one version is relevant. LEGUMES Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 Nas duas últimas semanas: Nota: Se você não comeu esse legume, assinale a alternativa N/A (não aplicável). Nunca Raramente Às vezes Frequentemente Sempre N/A 24. Você mordeu cenouras cruas inteiras? □ □ □ □ □ □ 25. Foi necessário cortar as cenouras cruas em pedaços pequenos para mastigá-las? □ □ □ □ □ □ 26. Foi necessário fazer purê com os legumes duros para comê-los? □ □ □ □ □ □ OBRIGADO POR SUA COLABORAÇÃO! The English-version of this questionnaire is available at: Muller K, Morais J, Feine J. Nutritional and anthropometric analysis of eden­ tulous patients wearing implant overdentures or conventional dentures. Braz Dent J. 2008;19:145-50. Figure 4 – QUESTIONÁRIO DE AVALIAÇÃO DA QUALIDADE DA MASTIGAÇÃO (QAQM) Nas duas últimas semanas: Nota: Se você não comeu esse legume, assinale a alternativa N/A (não aplicável). Nunca Raramente Às vezes Frequentemente Sempre N/A 24. Você mordeu cenouras cruas inteiras? □ □ □ □ □ □ 25. Foi necessário cortar as cenouras cruas em pedaços pequenos para mastigá-las? □ □ □ □ □ □ 26. Foi necessário fazer purê com os legumes duros para comê-los? □ □ □ □ □ □ OBRIGADO POR SUA COLABORAÇÃO! Se você não comeu esse legume, assinale a alternativa N/A (não aplicável). OBRIGADO POR SUA COLABORAÇÃO! The English-version of this questionnaire is available at: Muller K, Morais J, Feine J. Nutritional and anthropometric analysis of eden­ tulous patients wearing implant overdentures or conventional dentures. Braz Dent J. 2008;19:145-50. Figure 4 – QUESTIONÁRIO DE AVALIAÇÃO DA QUALIDADE DA MASTIGAÇÃO (QAQ Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 1936 Hilasaca-Mamani M, Barbosa TS, Feine J, Ferreira RI, Boni RC, Castelo PM „ „ DISCUSSION In addition to making it possible to choose items to incorporate, or to allow the junction of items arising from different versions, this strategy allows to carefully examine the sequence of the procedures, including the translations themselves, their re-translation and the appreciation that follows. The importance of a general criticism made by the committee of specialists must also be pointed out; among the members of the committee there must be individuals who are specialists in the disease investigated, in the measure used and in the concept explored, and they should preferably be bilingual16. „ „ CONCLUSION The Portuguese version of the Questionnaire D’Alimentation has shown to be easy to understand by Brazilian adolescents and useful in the evalu­ ation of the masticatory function and feeding or swallowing disorders that may affect food intake. The differences between definitions, beliefs and behaviors demand that the use of an instrument drawn up in other cultural contexts should, in addition to reliable translation, be preceded by cross-cultural adaptation to the country in which it will be applied, in order to keep the same concepts as those of the original27. At the stage of cross-cultural adaptation, the number and characteristics of volunteers involved in pre-test were chosen in agreement with methodological criteria used for cultural adaptation of questionnaires16,18. RESUMO Objetivo: traduzir o instrumento em língua francesa “Questionnaire D’Alimentation” para o português brasileiro e realizar a adaptação transcultural em adolescentes brasileiros. Métodos: a tradução foi realizada com base em protocolo que consistiu na tradução para o português brasileiro (por um professor formado em Letras e uma Cirurgiã Dentista fluentes em francês), tradução reversa para o francês (realizada por dois professores de francês), revisão por Comitê de especialistas (duas tradutoras, uma nativa e uma juramentada, e duas professoras universitárias, sendo uma Cirurgiã Dentista e uma Fonoaudióloga) e equivalência cultural (pré-teste). A versão utilizada no pré- teste consistiu de 26 questões com cinco respostas possíveis (5-Likert), distribuídas em cinco domínios (Alimentação- mastigação, Hábitos, Carnes, Frutas e Legumes). O pré-teste e teste-reteste foram realizados com uma amostra de 20 adolescentes (10 meninos/10 meninas) de escolas públicas de Piracicaba (Brasil). Nesta fase, a alternativa “não entendi” foi incluída a cada questão, a fim de iden­ tificar aquelas não compreendidas. A confiabilidade teste-reteste foi avaliada para cada domínio utili­ zando os coeficientes de correlação intra-classe (CCI). Resultados: no pré-teste, foi observada uma excelente compreensão do instrumento; no teste-reteste, os CCIs variaram entre 0,45-0,81 (mode­ rada a excelente concordância). Conclusão: a versão em Português do Questionnaire D’Alimentation mostrou ser de fácil compreensão por parte dos adolescentes brasileiros e útil na avaliação da função mastigatória e dos distúrbios de alimentação e deglutição que podem alterar a ingestão de alimentos. DESCRITORES: Tradução; Mastigação; Alimentos; Adolescente „ „ ACKNOWLEDGEMENTS The authors would like to thank Prof. Fábio Nunes da Silva for his help in the translation process. This study was supported by the State of São Paulo Research Foundation (FAPESP, SP, Brazil, n. 2012/04492-2). Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 1937 Quality of masticatory function questionnaire RESUMO Objetivo: traduzir o instrumento em língua francesa “Questionnaire D’Alimentation” brasileiro e realizar a adaptação transcultural em adolescentes brasileiros. Método realizada com base em protocolo que consistiu na tradução para o português b professor formado em Letras e uma Cirurgiã Dentista fluentes em francês), tradu o francês (realizada por dois professores de francês), revisão por Comitê de es tradutoras, uma nativa e uma juramentada, e duas professoras universitárias, sen Dentista e uma Fonoaudióloga) e equivalência cultural (pré-teste). A versão utiliza consistiu de 26 questões com cinco respostas possíveis (5-Likert), distribuídas em (Alimentação- mastigação, Hábitos, Carnes, Frutas e Legumes). O pré-teste e te realizados com uma amostra de 20 adolescentes (10 meninos/10 meninas) de esc Piracicaba (Brasil). Nesta fase, a alternativa “não entendi” foi incluída a cada questã tificar aquelas não compreendidas. A confiabilidade teste-reteste foi avaliada para c zando os coeficientes de correlação intra-classe (CCI). Resultados: no pré-teste, fo excelente compreensão do instrumento; no teste-reteste, os CCIs variaram entre 0 rada a excelente concordância). Conclusão: a versão em Português do Questionnai mostrou ser de fácil compreensão por parte dos adolescentes brasileiros e útil na ava mastigatória e dos distúrbios de alimentação e deglutição que podem alterar a inges DESCRITORES: Tradução; Mastigação; Alimentos; Adolescente RESUMO Objetivo: traduzir o instrumento em língua francesa “Qu brasileiro e realizar a adaptação transcultural em adoles realizada com base em protocolo que consistiu na tra professor formado em Letras e uma Cirurgiã Dentista f o francês (realizada por dois professores de francês), tradutoras, uma nativa e uma juramentada, e duas pro Dentista e uma Fonoaudióloga) e equivalência cultural consistiu de 26 questões com cinco respostas possíve (Alimentação- mastigação, Hábitos, Carnes, Frutas e L realizados com uma amostra de 20 adolescentes (10 m Piracicaba (Brasil). Nesta fase, a alternativa “não entend tificar aquelas não compreendidas. A confiabilidade teste zando os coeficientes de correlação intra-classe (CCI). R excelente compreensão do instrumento; no teste-reteste rada a excelente concordância). Conclusão: a versão em mostrou ser de fácil compreensão por parte dos adolesce mastigatória e dos distúrbios de alimentação e deglutição DESCRITORES: Tradução; Mastigação; Alimentos; Ado DESCRITORES: Tradução; Mastigação; Alimentos; Adolescente „ „ REFERENCES comparisons of implant–supported mandibular prostheses: psychometric evaluation. J Dent Res. 1994;73:1096-104. 1. Pereira LJ, Gavião MB, Engelen L, Van der Bilt A. Mastication and swallowing: influence of fluid addition to foods. J Appl Oral Sci. 2007;15:55-60. 9. Hassan AH, Amin Hel-S. Association of orthodontic treatment needs and oral health-related quality of life in young adults. Am J Orthod Dentofacial Orthop. 2010;137:42-7. 2. Felicio CM, Melchior MO, Silva MAMR, Celeghini RMS. 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Nutritional and anthropometric analysis of edentulous patients wearing implant overdentures or conventional dentures. Braz Dent J. 2008;19:145-50. 8. de Grandmont P, Feine JS, Taché R, Boudrias P, Donohue WB, Tanguay R et al. Within–subject Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 1938 Hilasaca-Mamani M, Barbosa TS, Feine J, Ferreira RI, Boni RC, Castelo PM screening of violence against pregnant women. Rev Saude Publ. 2000;34:610-6. 15. Cushing AM, Sheiham A, Maizels J. Developing socio-dental indicators the social impact of dental disease. Community Dent Health. 1986;3:3-17. 23. „ „ REFERENCES da Mota Falcão D, Ciconelli RM, Ferraz MB.Translation and cultural adaptation of quality of life questionnaires:an evaluation of methodology. J Rheumatol. 2003;30:379-85. 16. Guillemin F, Bombardier C, Beaton D. Cross- cultural adaptation of health-related quality of life measures: literature review and proposed guidelines. J Clin Epidemiol. 1993;46:1417-32. 24. Herdman M, Fox-Rushby J, Badia X. “Equivalence” and the translation and adaptation of Health-Related Quality of Life Questionnaires. Qual Life Res. 1997;6:237-47. 17. Beaton DE, Bombardier C, Guillemin F, Ferraz MB.Guidelines for the process of cross- cultural adaptation of self-report measures. Spine. 2000;25:3186-91. 25. Reichenheim ME, Moraes CL.Pillars for assessing validity in epidemiological studies. Rev Bras Epidemiol. 1998;1:131-48. 18. Yusuf H, Gherunpong S, Sheiham A, Tsakos G. Validation of an English version of the Child-OIDP index, an oral health-related quality of life measure for children. Health Qual Life Outcomes. 2006;4:38. 26. Tesch FC, Oliveira BH, Leão A. Semantic equivalence of the Brazilian version of the Early Childhood Oral Health Impact Scale. Cad Saude Publica. 2008;24:1897-909. 19. Ciconelli RM, Ferraz MB, Santos W, Meinão I, Quaresma MR. Brazilian-Portuguese version of the SF-36. A reliable and valid quality of life outcome measure. Rev Bras Reumatol. 1999;39:143-50. 27. Barbosa TS, Gavião MB.Quality of life and oral health in children - Part II: Brazilian version of the Child Perceptions Questionnaire 11-14. Cien Saude Colet. 2011;16:3267-76. 20. Fleiss JL. Reliability of Measurement. In: Fleiss JL. The design and analysis of clinical experiments. New York: Wiley; 1986. pp. 1-32. 28. Shrive FM, Stuart H, Quan H, Ghali WA.Dealing with missing data in a multi-question depression scale:a comparison of imputation methods. BMC Med Res Methodol. 2006;6,57. 21. Barbosa TS, Serra MD, Gavião MBD.Quality of life and oral health in children - Part I: Brazilian version of the Child Perceptions Questionnaire 8-10. Cien Saude Colet. 2011;16:4077-85. 21. Barbosa TS, Serra MD, Gavião MBD.Quality of life and oral health in children - Part I: Brazilian version of the Child Perceptions Questionnaire 8-10. Cien Saude Colet. 2011;16:4077-85. 22. Reichenheim ME, Moraes CL, Hasselmann MH. Semantic equivalence of the Portuguese version of the Abuse Assessment Screen tool used for the 29. Eekhout I, de Boer RM, Twisk JW, de Vet HC, Heymans MW. Missing data: a systematic review of how they are reported and handled. Epidemiology. 2012;23:729-32. 22. Reichenheim ME, Moraes CL, Hasselmann MH. Semantic equivalence of the Portuguese version of the Abuse Assessment Screen tool used for the http://dx.doi.org/10.1590/1982-021620151764715 Received on: April 13, 2015 Accepted on: September 11, 2015 Mailing address: Paula Midori Castelo Depto. Ciências Biológicas - Universidade Federal de São Paulo (UNIFESP) R. São Nicolau, 210 – 1. Andar Diadema – SP – Brasil CEP: 09913-030 E-mail: pcastelo@yahoo.com Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938 Rev. CEFAC. 2015 Nov-Dez; 17(6):1929-1938
W3023666651.txt
https://zenodo.org/records/1669673/files/article.pdf
en
Optical Activity and Chemical Composition
Science
1,900
public-domain
660
SCIENCE. [N.S. VOL.XI. NO. 282. taken up isomeric compounds, including stereoisomers, homologous series, influence of the mode of linkage of the carbon atoms, summation of the rotatory actions of several asymmetric groups, optical superposition and the dependence of the rotatory power of ao active atomic grouping on the masses of the four radicles united to the asymmetric carbon atom, the hypothesis of Guye. The translation is well done and the subject is brought up to date by notes and additions by Optical Activity and Chemical Composition. By the translator. The subject is presented in a DR. H . LANDOLT, Professor of Chemistry in very attractive and readable form and the book the University of Berlin. Translated, with can be heartily recommended to anyone, who MCCRAE, desires to know the present state of our knowlthe author's permission, by JOHN Ph.D. Whittaker and Co., London, and the edge regarding the relation existing between Macmillan Co., 66 Fifth Ave., New York. optical activity and chemical composition ; 1899. Small8vo. Pp. 158. Price, $1.00. though for more detailed information Landolt's This little book forms a translation of the Das optische Drehungsvermiigen organischer eighth chapter of the first volume of Graham- Substanzen und dessen praktische AnwendungOtto's ' Lehrbuch der Chemie ' and is a smaller en ' must be used. W. R. ORNDORFF. and condensed edition of the author's wellknown ' Das optische Drehungsvermiigen organischer Substanzen und desseu praktische AnSCIENTIFIC JOURNALS A N D ARTICLES. wendungen,' published in 1898. The subject THE Osprey for April, a little belated, opens is treated under three heads: I. General with the fourth part of 'Birds of the Road,' by Principles of Optical Activity ; 11. Connection Paul Bartsch. Wm. L. Wells describes the between the Rotatory Power and the Chemical 'Nesting of some Rare Birds,' including the Composition of Carbon Compounds, and 111. yellow rail and solitary sandpiper, and TheoConnection between Degree of Rotation and dore Gill presents the second part of ' William Chemical Constitution. Under the first head Swainson and his Times ' which carries Swainare discussed such subjects as crystal rotation, son through his journey to Brazil. I n editorial liquid rotation, molecular rotation, measure- comments under ' Birds and Women ' the situment of rotation, specific rotation, variations ation is summed up in a few words ' L If the deof specific rotation with concentration and mand exists for anything, that demand will be change of rotatory power of dissolved sub- supplied if it can be done with aprofit." Under stances with time, multirotation. Under the Notes is to be found a n extraordinary account second head are treated optical modifications, of ' How Two Lions stopped an African Railthe investigations of Pasteur, the van't Hoff road,' and other matters of interest. and Le Be1 theory, calculation of the number A Bulletin of Mathematics and of the Physical of optically active isomers of a compound from and Natural Sciences, to be published semithe number of asymmetric carbon atoms which monthly in the interest of teachers in Italian it contains, the formation and properties of schools, has been established by Professor racemic compounds, resolution of racemic Alberto Conti, of Bologne. substances into the antipodes, formation and properties of the active modifications, transSOCIETIES AND A CADEJIIES. formation of one antipode into the other, the GEOLOGICAL SOCIETY O F WASHINGTON. configurationally inactive non-decomposable THE lOlst meeting of the Society was held a t modifications and their differences from racemic the Cosmos Club April 11, 1900. inactive isomers. Under the third head are beginners. A student with some knowledge of organic chemistry could use it as a text-book if i t were possible for him to resolutely confine his attention to the coarse print.' But it is as a reference book for the student who wishes to refresh his memory not merely of one compound, but of the complete chemistry of a group of compounds, that the work is of peculiar value, and may be cordially recommended. EDWARDRENOUF.
https://openalex.org/W2010653101
https://europepmc.org/articles/pmc3027619?pdf=render
English
null
Prioritizing Conservation of Ungulate Calving Resources in Multiple-Use Landscapes
PloS one
2,011
cc-by
15,183
Received June 23, 2010; Accepted December 15, 2010; Published January 26, 2011 Copyright:  2011 Dzialak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Pioneer Natural Resources Company funded this work. Hayden-Wing Associates, LLC provided in-kind contributions including travel costs associated with dissemination of this work, and materials associated with data collection and analysis including radio-telemetry equipment, GPS devices, and statistical analysis software. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This work was funded by a commercial source, Pioneer Natural Resources Company. Hayden-Wing Associates, LLC provided in-kind contributions including travel costs associated with dissemination of this work, and materials associated with data collection and analysis including radio- telemetry equipment, GPS devices, and statistical analysis software. Having received funding from this source, the consultancy could reasonably be perceived as a financial competing interest. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. ¤ Current address: New Mexico Game and Fish Department, Santa Fe, New Mexico, United States of America Matthew R. Dzialak*, Seth M. Harju, Robert G. Osborn¤, John J. Wondzell, Larry D. Hayden-Wing, Jeffrey B. Winstead, Stephen L. Webb Hayden-Wing Associates, LLC, Natural Resource Consultants, Laramie, Wyoming, United States of America Hayden-Wing Associates, LLC, Natural Resource Consultants, Laramie, Wyoming, United States of America Hayden-Wing Associates, LLC, Natural Resource Consultants, Laramie, Wyoming, United States of America PLoS ONE | www.plosone.org Abstract Background: Conserving animal populations in places where human activity is increasing is an ongoing challenge in many parts of the world. We investigated how human activity interacted with maternal status and individual variation in behavior to affect reliability of spatially-explicit models intended to guide conservation of critical ungulate calving resources. We studied Rocky Mountain elk (Cervus elaphus) that occupy a region where 2900 natural gas wells have been drilled. Methodology/Principal Findings: We present novel applications of generalized additive modeling to predict maternal status based on movement, and of random-effects resource selection models to provide population and individual-based inference on the effects of maternal status and human activity. We used a 262 factorial design (treatment vs. control) that included elk that were either parturient or non-parturient and in areas either with or without industrial development. Generalized additive models predicted maternal status (parturiency) correctly 93% of the time based on movement. Human activity played a larger role than maternal status in shaping resource use; elk showed strong spatiotemporal patterns of selection or avoidance and marked individual variation in developed areas, but no such pattern in undeveloped areas. This difference had direct consequences for landscape-level conservation planning. When relative probability of use was calculated across the study area, there was disparity throughout 72–88% of the landscape in terms of where conservation intervention should be prioritized depending on whether models were based on behavior in developed areas or undeveloped areas. Model validation showed that models based on behavior in developed areas had poor predictive accuracy, whereas the model based on behavior in undeveloped areas had high predictive accuracy. Conclusions/Significance: By directly testing for differences between developed and undeveloped areas, and by modeling resource selection in a random-effects framework that provided individual-based inference, we conclude that: 1) amplified selection or avoidance behavior and individual variation, as responses to increasing human activity, complicate conservation planning in multiple-use landscapes, and 2) resource selection behavior in places where human activity is predictable or less dynamic may provide a more reliable basis from which to prioritize conservation action. Citation: Dzialak MR, Harju SM, Osborn RG, Wondzell JJ, Hayden-Wing LD, et al. (2011) Prioritizing Conservation of Ungulate Calving Resources in Multiple-Use Landscapes. PLoS ONE 6(1): e14597. doi:10.1371/journal.pone.0014597 Editor: Michael Somers, University of Pretoria, South Africa Received June 23, 2010; Accepted December 15, 2010; Published January 26, 2011 Prioritizing Conservation of Ungulate Calving Resources in Multiple-Use Landscapes Matthew R. Dzialak*, Seth M. Harju, Robert G. Osborn¤, John J. Wondzell, Larry D. Hayden-Wing, Jeffrey B. Winstead, Stephen L. Webb January 2011 | Volume 6 | Issue 1 | e14597 Capturing elk p g In February and March 2006–2009, helicopter net-gunning was used to capture yearling (1.5 years) and adult ($2.5 years) female elk throughout and adjacent to the gas field. Elk were fitted with Global Positioning Systems (GPS) collars (TGW-3590, Telonics, Inc., Mesa, AZ 85204) configured with store-on-board and Very High Frequency (VHF) beacon options. Twenty-five female elk were fitted with GPS collars in 2006, 40 in 2007, 50 in 2008, and 50 in 2009. GPS collars attempted to record location information every 3 h resulting in a maximum of 8 locations/elk/day. Age of elk was estimated based on dental eruption and wear patterns [26]. Blood samples were collected from captured elk in 2008 and 2009 to determine pregnancy, but not from elk captured in previous years. Animal capture and handling protocols were approved by the Colorado Division of Wildlife (Permit #s 06TR1083, 07TR1083, 08TR1083 and 09TR1083A001). Areas associated with parturition are important because female ungulates make resource-related choices that affect offspring development during gestation and provisioning of recently born calves that are susceptible to malnutrition and predation [12], [13]. While there is little evidence of consistent parturition site fidelity in many ungulates, strong fidelity among females to seasonal ranges, particularly around calving time, has been demonstrated [2], [14]. New light has been shed on the adaptive significance of resource selection during the period that encompasses reproductive activity in many vertebrates through the study of maternal effects – developmental mechanisms by which parents translate their environmental experience into adaptive variation in their offspring [15], [16], [17]. The adaptive significance of resource selection during reproductive periods suggests that conservation strategies designed around ungulate parturition areas might be most reliable when based on parturient females rather than samples including both parturient and non-parturient individuals. Study area The 1845 km2 study area encompassed northern portions of the Raton basin in south-central Colorado, USA. Topography is rugged with steep slopes, rocky outcrops, ridges, and valleys ranging in elevation from 2000–3000 m. Mean annual precipita- tion is about 40–53 cm depending on elevation [23]. Vegetation includes conifer forest, montane shrub, and grassland. Dominant species include ponderosa pine (Pinus ponderosa), one-seed juniper (Juniperus monosperma), two-needle pinyon (Pinus edulis), Gambel oak (Quercus gambelii) which commonly forms shrub-thickets on southern aspects, antelope bitterbrush (Purshia tridentata), skunkbush sumac (Rhus trilobata), and willow (Salix spp.) in riparian areas. Predators of elk (including neonates) include black bear (Ursus americanus), mountain lion (Felis concolor), and coyote (Canis latrans); no wolf (Canis lupus) pack occurred in the study area. The study area encompassed historic and ongoing energy development. Bituminous coal mining was a dominant land use during 1873– 1970. Coal-bed methane development was initiated in Raton basin in 1982 and accelerated in the late 1990s [24], [25]. In 2009, there were about 2900 wells associated with methane development in the Basin (Figure 1). A key approach in studying wildlife-human interaction is resource selection modeling [4]. Resource selection is a funda- mental ecological process that structures animal movement and distribution [5]. The choices animals make as they move throughout the landscape reflect trade-offs between selecting resources that meet their needs for survival and reproduction, and minimizing perceived risk of harm – such risk often is a function of interaction with predators or humans [6], [7], [8]. A set of analytical methods used with increasing frequency for investigating resource selection in animals is the estimation of RSFs [9]. RSFs describe the relative probability of occurrence of animals as a function of behavioral responses to features of the environment. The probability of occurrence is described as relative because RSFs are estimated in a use-versus-availability framework in which selection is quantified relative to available but presumed non-used features. Environmental features can include a wide range of variables such as vegetation, terrain, group/herd size, risk of predation, or human-modifications of the landscape [10], [11]. RSFs have strong application in conservation planning where wildlife-human interaction is a concern; specifically, in establishing a spatially explicit basis from which to prioritize conservation action such as reclamation, mitigation, or minimizing human activity in particular habitats. Introduction Model-based methods to designate maternal status were necessary because data bearing directly on maternal status were unavailable for most elk. Second, we estimated group- dependent random-effects resource selection functions (RSFs) to identify how resource selection patterns differed relative to maternal status and human activity, and how behavior varied among individuals. We applied results by developing and validating group-dependent predictive maps of critical calving resources, and quantifying discrepancies among maps relative to predictive accuracy. Introduction wildlife and their habitats [3]. Understanding how human activity such as resource extraction interacts with wildlife, and developing tools to guide conservation planning in areas where human activity is widespread or increasing are ongoing challenges in conservation science. In this paper, we investigated interactions between human activity associated with energy development and resource selection by female Rocky Mountain elk (Cervus elaphus) during calving season with the larger goal of informing conservation planning for ungulates in places where human activity is widespread or increasing. We asked: 1) what are the relative influences of maternal status (parturiency) and human activity on resource selection, 2) to what extent does behavior vary among individuals, Identifying resources associated with critical life-history phases in ungulates is a conservation priority. Winter range, parturition areas, and migration routes are important seasonal habitats in North America that provide resources necessary for survival and reproduction such as high-quality forage, reduced exposure to inclement conditions, and reduced risk of predation [1], [2]. In the Intermountain West USA, these habitats have become increas- ingly fragmented. Here, energy development is of broad conservation interest because its prominence has increased in recent decades along with concern about its potential impact on PLoS ONE | www.plosone.org January 2011 | Volume 6 | Issue 1 | e14597 January 2011 | Volume 6 | Issue 1 | e14597 1 Ungulate Conservation situation-specific basis [22] making it difficult to generalize behavior across the population and thus effectively guide conservation planning. 3) how does individual variation interact with human activity, and 4) how can our findings be applied in conservation planning and decision making? First, we describe a novel application of generalized additive models for designating maternal status using movement data. Model-based methods to designate maternal status were necessary because data bearing directly on maternal status were unavailable for most elk. Second, we estimated group- dependent random-effects resource selection functions (RSFs) to identify how resource selection patterns differed relative to maternal status and human activity, and how behavior varied among individuals. We applied results by developing and validating group-dependent predictive maps of critical calving resources, and quantifying discrepancies among maps relative to predictive accuracy. 3) how does individual variation interact with human activity, and 4) how can our findings be applied in conservation planning and decision making? First, we describe a novel application of generalized additive models for designating maternal status using movement data. PLoS ONE | www.plosone.org Grouping Elk Relative to Maternal Status and Human Activity We predicted maternal status (parturient versus non-parturient) by using generalized additive models (GAMs) to parameterize response curves depicting daily movement of elk during calving season. GAMs are semi-parametric extensions of generalized linear models [27]. The central concept is that the function of a covariate is estimated nonparametrically from the data by means of scatterplot smoothers. The functional form of the relationship between the response and covariate(s) is therefore determined by the data rather than being restricted to a parametric form [28]. Formally, the linear regression model Another key feature of animal ecology that warrants further attention as part of conservation planning is variation in behavior among individuals. Individual variation is widespread and well- known in many animal species [18] and can reflect long-term selection for a given trait or learned behavior [19], [20]. Fitness is influenced by the choices individuals make in terms of resource selection because each resource type has particular costs and benefits to the individual [21]. In risky or rapidly changing environments such as those in which human activity is increasing, optimal behavioral strategies may vary among individuals on a y~az X I i~1 bi(Xi)ze is generalized by modeling y as being related to covariates additively by is generalized by modeling y as being related to covariates additively by January 2011 | Volume 6 | Issue 1 | e14597 2 Ungulate Conservation Figure 1. The Raton Basin gas field and adjacent areas in south-central Colorado, USA. Each natural gas well is encircled by a 1-km buffer (shaded region). We designated elk locations occurring within the buffered region to be ‘‘inside of the gas field’’ whereas locations adjacent but external to the buffered region were designated ‘‘outside of the gas field’’. doi:10.1371/journal.pone.0014597.g001 Figure 1. The Raton Basin gas field and adjacent areas in south-central Colorado, USA. Each natural gas well is encircled by a 1-km buffer (shaded region). We designated elk locations occurring within the buffered region to be ‘‘inside of the gas field’’ whereas locations adjacent but external to the buffered region were designated ‘‘outside of the gas field’’. doi:10.1371/journal.pone.0014597.g001 the effective number of parameters of a smoother) is described by the quantity equivalent degrees of freedom (df). Span is related inversely to df so as span increases df decreases [27]. Data that are best described by a straight line (a single parameter comprises the smoother) correspond to a span of 100% and thus 1 df. PLoS ONE | www.plosone.org Grouping Elk Relative to Maternal Status and Human Activity This data summary (i.e., PLoS ONE | www.plosone.org January 2011 | Volume 6 | Issue 1 | e14597 January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org 3 Ungulate Conservation [35] [36]) W h th i d th t d i l i ti t d i ti d il h i th di t d b Figure 2. Movement in female ungulates during calving time. Generalized from the literature [34], [35], [36], parturient females often will make long-distance movements associated with pre-parturient ‘‘restlessness’’ within days of parturition and then exhibit reduced movement associated with provisioning the neonate (a). This pattern may provide a quantifiable distinction between parturient and non-parturient females. Also shown (b) is parameterization (equivalent degrees of freedom) as a function of span based on generalized additive models of distance moved between consecutive locations in 103 female Rocky Mountain elk. Loess smoothing and automated calculation of degrees of freedom using the generalized cross validation method were specified. doi:10.1371/journal.pone.0014597.g002 Ungulate Conservation [35], [36]). We hypothesized that during calving time, movement i d i h l d li d ki i i lk response curves depicting dai lk b i l i Figure 2. Movement in female ungulates during calving time. Generalized from the literature [34], make long-distance movements associated with pre-parturient ‘‘restlessness’’ within days of parturition associated with provisioning the neonate (a). This pattern may provide a quantifiable distinction between par shown (b) is parameterization (equivalent degrees of freedom) as a function of span based on generaliz between consecutive locations in 103 female Rocky Mountain elk. Loess smoothing and automated calcu generalized cross validation method were specified. doi:10.1371/journal.pone.0014597.g002 Figure 2. Movement in female ungulates during calving time. Generalized from the literature [34], [35], [36], parturient females often will make long-distance movements associated with pre-parturient ‘‘restlessness’’ within days of parturition and then exhibit reduced movement associated with provisioning the neonate (a). This pattern may provide a quantifiable distinction between parturient and non-parturient females. Also shown (b) is parameterization (equivalent degrees of freedom) as a function of span based on generalized additive models of distance moved between consecutive locations in 103 female Rocky Mountain elk. Loess smoothing and automated calculation of degrees of freedom using the generalized cross validation method were specified. doi:10.1371/journal.pone.0014597.g002 [35], [36]). Grouping Elk Relative to Maternal Status and Human Activity Conversely, data that are best described by gradients or turning points (several or many parameters comprise the smoother) correspond to a smaller span and thus to a larger number of df [27], [30]. The analyst may set the span by specifying df based on visual examination of the data, or implement generalized cross validation methods in which appropriate df are identified automatically given the data. From a GAM perspective, movement of female ungulates during the calving season likely contains information on maternal status. Restlessness and the seeking of solitude characterize imminent parturition [31], [32], [33] and establish a general pattern of increased daily movement pre-partum and decreased movement post-partum relative to barren females (Figure 2a; [34], g(y)~az X I i~1 fi(Xi)ze where g is the link function and e is a random error term. Functions fi may be linear or nonparametric functions defined by smoothers such as smoothing splines or locally estimated scatterplot smoothers (loess; also referred to as locally estimated, or weighted, polynomial regression). Smoothers provide a series of data summaries of the response that are specific to regions of the covariates; a well known smoother is the moving average [29]. The amount of smoothing is calibrated by the size of the neighborhood, or percentage of the data points, over which averaging is done; a quantity known as span. A larger span yields a smoother data summary (less curvature) whereas a smaller span yields a less smooth data summary (more curvature). This data summary (i.e., where g is the link function and e is a random error term. Functions fi may be linear or nonparametric functions defined by smoothers such as smoothing splines or locally estimated scatterplot smoothers (loess; also referred to as locally estimated, or weighted, polynomial regression). Smoothers provide a series of data summaries of the response that are specific to regions of the covariates; a well known smoother is the moving average [29]. The amount of smoothing is calibrated by the size of the neighborhood, or percentage of the data points, over which averaging is done; a quantity known as span. A larger span yields a smoother data summary (less curvature) whereas a smaller span yields a less smooth data summary (more curvature). PLoS ONE | www.plosone.org Grouping Elk Relative to Maternal Status and Human Activity We hypothesized that during calving time, movement associated with restlessness and solitude seeking in parturient elk provides a general pattern of complexity that is not observed in non-parturient females, and we predicted that smoothers associ- ated with movement data on parturient elk consistently would be comprised of more parameters and thus more df than smoothers associated with non-parturient elk. response curves depicting daily changes in the distance moved by elk between successive locations. We used PROC GAM in SASH (SAS Institute, Inc., Cary, North Carolina, USA), specifying automated calculation of df using the generalized cross validation method and loess smoothing, to assign df to response curves depicting calving season movement patterns in each elk. We established the following prediction: movement described by ,3 df in GAMs would depict non-parturiency whereas movement described by $3 df would depict parturiency. This prediction was based on two observations. First, the shape of the response curve depicting relatively simple movement, as in non-parturient individuals, appears to correspond with #2 regions that differ in We used GAMs to regress distance traveled within a 24 h period (using locations recorded at 1200 h on consecutive days) against date. Date encompassed the 15 May – 1 July calving season [33] in each of the 4 years comprising the study period. We used the generalized cross validation option to assign df to PLoS ONE | www.plosone.org January 2011 | Volume 6 | Issue 1 | e14597 4 Ungulate Conservation slope, each of which being calibrated over a span of $50% of the data (Figure 2a; [34], [35]). Second, by plotting df as a function of span, our data show that a span of 50% corresponds to ,3 df (Figure 2b). Random intercepts can account for unbalanced data, correlation among observations, and provide improved model fit and parameter estimation [39] (but see [11]). Random-effects (or mixed-effects) RSF models provide information on individual behavior, how individuals contribute to population-level observa- tions, and how their responses to a resource may change as a function of its availability – a process known as a functional response [40], [41]. Analytical approaches to model functional responses in resource selection are particularly important when there is a trade-off in selection for a particular resource [4], [40], which would be the case if human activity is perceived as a risk of harm [7]. Grouping Elk Relative to Maternal Status and Human Activity We tested the GAM approach using blood samples obtained from elk during capture in 2008 and 2009, and field observation of females and calves in those same years. Blood sera were tested for presence of pregnancy-specific protein-B (PSPB; BioTracking, LLC, Moscow, Idaho, USA; [37]). We conducted field observation from dawn to 0900 and from 1800 until dusk using binoculars and spotting scopes to watch for behavior that suggested a maternal bond between a female and calf. This behavior included nursing and licking bouts, traveling as a female/calf unit, and heightened attentiveness between a female and calf [13], [38]. Females for which PSPB testing indicated pregnancy and for which field observation suggested a strong female-calf bond were designated as parturient. Determining non-parturiency is never definitive; however, we designated females as non-parturient with negative PSPB results and for which field observation was unable to associate the female with a calf [13]. We thus established two groups of elk relative to maternal status by which we analyzed resource selection. We incorporated random effects into the use versus availability design [9] in which covariates representing important resources are compared at used and available (but presumed non-used) locations using ^w(x)~exp(^b0z^b1x1z^b2x2z:::z^bnxn) where ^w(x) is the relative probability of use as a function of covariates xn with coefficients ^bn estimated from logistic regression. Availability was defined for each elk by including random locations within 100% minimum convex polygon seasonal use area estimates; the number of random locations generated was 3 times the number of used locations for each elk. We examined resource selection within seasonal use areas (i.e., 3rd order selection). We modeled resource selection separately during day and night because we expected behavior of elk to differ between day and night. We assigned time of day at random to available locations for day versus night comparisons; times assigned to available locations corresponded to times associated with used locations (e.g., every 3 hours on the sampled hour). Day models included the times 0900, 1200, 1500, and 1800 h whereas night models included 0000 and 0300 h. Using a Geographic Informa- tion System (GIS; ArcGIS 9.2), we calculated 7 covariates at used and random locations (Table 1). Four of these covariates including cover type, slope, elevation, and habitat edge density were calculated at locations both within and outside of the gas field. Grouping Elk Relative to Maternal Status and Human Activity Three covariates including road density, distance to a human-built structure, and industrial development footprint (Table 1) were calculated only within the gas field because human activity in areas adjacent but outside of the gas field was less intense and not associated with industrial development. Raster data for cover type were developed from annual aerial photography of the study area, terrain covariates were calculated from a 30-m resolution digital elevation model, and human activity covariates including roads, structures, and industrial development footprint were heads-up- digitized from aerial photography and analyzed as year-specific covariates (Table 1). We used Spatial Analyst in ArcGIS to extract values from raster data for all covariates. where ^w(x) is the relative probability of use as a function of covariates xn with coefficients ^bn estimated from logistic regression. Similarly, we established two groups of elk relative to human activity: elk occupying developed areas versus elk occupying undeveloped areas. Elk locations occurring within 1 km of a gas well were considered to be in the developed area (Figure 1). Human activity was apparent in the areas we called undeveloped including some ranching and residences; however, no industrial development occurred in undeveloped areas and human activity was limited relative to developed areas. Given the short temporal window within which we conducted these analyses, calving season use areas (100% minimum convex polygons) generally occurred wholly within developed areas or wholly within undeveloped areas. Only a small number of elk occupied both areas during the calving season – we discuss these elk separately. We estimated minimum convex polygons because the temporal window of the study was relatively short, calving season use areas comprised a portion of the annual use areas, and we wanted to err on the side of inclusiveness rather than potentially omitting a portion of critical calving range from the analysis. In comparing resource selection between elk occupying developed versus undeveloped areas, a key assumption is that human activity associated with energy development is the primary difference between areas and that other factors to which elk respond were similar between areas. We compared landscape and habitat covariate values between areas to inform this assumption. January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org Random-effects Resource Selection Modeling Modeling variables as random effects can improve our understanding of resource selection, which will enhance the practical application of RSFs in management decisions [4], [39]. Random effects models assume that sample units are drawn at random from a larger population and that the data are structured hierarchically (i.e., within subject responses are more similar than between-subject responses). Mechanistically, assumptions of ran- dom-effects models are: (1) the random effects are distributed normally with mean equal to 0 and unknown variance, (2) within- group correlation is constant through time, and (3) the analyst has correctly specified the variance-covariance structure (see [39] for a review of the application of random-effects models in resource selection analysis). In RSF models, it is appropriate to model intercepts and/or covariates as random effects when variance among sample units is of interest, animal response to gradients in a resource is suspected, or population-level inference is of interest. We estimated a three-level random-effects model in which locations i = 1…I occurred within strata representing individual elk j = 1…J [39]. Considering a random intercept and random coefficients, the RSF is estimated by g(x)~b0zb1x1ijz:::zbkxkijzckjxkjzc0j g(x)~b0zb1x1ijz:::zbkxkijzckjxkjzc0j where covariates k (k = 1…K) have values x, c0j is the random intercept and ckj is the random coefficient of xk for elk j, which is the difference in the intercept and coefficient for elk j from the mean population-level intercept b0 and coefficient bkj, respectively. We estimated models using the GLIMMIX procedure in SASH. We specified the conditional probability distribution of the data as where covariates k (k = 1…K) have values x, c0j is the random intercept and ckj is the random coefficient of xk for elk j, which is where covariates k (k = 1…K) have values x, c0j is the random intercept and ckj is the random coefficient of xk for elk j, which is the difference in the intercept and coefficient for elk j from the mean population-level intercept b0 and coefficient bkj, respectively. We estimated models using the GLIMMIX procedure in SASH. We specified the conditional probability distribution of the data as PLoS ONE | www.plosone.or January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org 5 Ungulate Conservation Table 1. Covariates used in random-effects resource selection models and their descriptions. Covariate Description Slope Digital elevation model (DEM) provided at a resolution of 1.52 m and re-sampled for covariate calculation to a resolution of 30 m and measured in degrees. Values for slope calculated at point locations in our study ranged from 0.02 (flat ground) to 41.8 degrees (very steep). Elevation DEM re-sampled for covariate calculation to a resolution of 30 m and measured in m. Elevation at sample points in our study ranged from 1918.7–2970.0 m. Cover type Raster dataset compiled from 1-foot resolution aerial imagery of the study area using Image AnalysisTM and re-sampled to 30 m resolution for covariate calculation. From an elk-centric perspective, habitat in the Raton basin functioned in one of two ways: as security cover or as forage resources. Raster cells were assigned one of two values (binary covariate) representing habitat that functioned to provide cover versus habitat that did not function to provide cover. All tree or oak-thicket dominated habitats were considered security cover, whereas all shrub and grassland dominated habitats were considered non-cover. Edge density Density of line features depicting the interface of cover and non-cover habitat calculated for the central grid cell within a 990 m2 moving window. Values in our study were 22.2–136.0 km/km2. Mapping Responses We applied the results from random-effects models to map relative probability of use across the study area which encom- passed developed and undeveloped areas. We used marginal coefficients from logistic regression to derive an RSF at a resolution of 30 m using w(x)~exp X K k~1 b kxk ! w(x)~exp X K k~1 b kxk ! where covariates k (k = 1…K) have values x [9]. We were interested in differences in resource selection patterns depending on maternal status and between elk that occupied developed versus undevel- oped areas. It is important to note that, although we analyzed an independent group of field-observed parturient elk to facilitate comparison of marginal estimates, these elk were withheld from final RSF development as a validation sample. Thus, we examined differences in how the relative probability of use was assigned throughout the landscape depending on whether this probability was based on models of: 1) all sample elk inside the gas field, 2) elk predicted to be parturient based on GAMs inside the gas field, and 3) all sample elk outside of the gas field (groups 1,2, and 5 above). We conducted this examination using the following methods separately for day and night. We developed annual RSF maps for each of the 3 groups identified above. We estimated quantiles in SASH (PROC RANK, PROC MEANS) by which pixels comprising the raster surface were partitioned into 5 equal-sized subsets based on pixel value. In GIS we reclassified RSF values based on quantiles establishing 5 ranks of the relative probability of use (1 = low probability, 5 = high probability). We summed within-year maps across all years and ranked relative probability of use as described above yielding 6 multi-year predictive maps (day map and night map for each group) with relative probability of use ranging from 1 (low) to 5 (high). We validated predictive maps using locations from 24 elk that were observed in the field to be ^bbk~ 1 J X J j~1 ^bkj where ^bkj was the estimate of coefficient k for elk j [43], [44], and we estimated variance using where ^bkj was the estimate of coefficient k for elk j [43], [44], and we estimated variance using var ^bk   ~ 1 J{1 X J j~1 (^bkj{^bbk)2 Population-level coefficient estimates (^bbk) are similar to the average of the conditional estimates because the conditional coefficient estimates are constrained to have means ^bk [44]. g(x)~b0zb1x1ijz:::zbkxkijzckjxkjzc0j Density of line features depicting the interface of cover and non-cover habitat calculated for the central grid cell within a 990 m2 moving window. Values in our study were 22.2–136.0 km/km2. Density of line features depicting roads calculated for grid cells at the center of a 990 m2 moving window. Road density was calculated as a year-specific variable – that is, new road features were added to the data set as annual aerial imagery became available. Values in our study, calculated within the gas field only, were 0.0–7.6 km/km2. Linear distance from a sample location to a human-built structure including houses, agricultural facilities, and industrial facilities. Distance to structure was a year-specific variable and was analyzed as natural log transformed distance +0.1 to allow its magnitude to decrease with increasing distance. Values in our study, calculated within the gas field only, were 0.0–5854.9 m. Area (density) of physically modified ground calculated from aerial photography as a year-specific variable. Physically modified ground primarily reflected industrial development including well pads, pipe yards, pipelines, construction areas, or clearing for facilities development. Industrial development footprint was calculated for the central grid cell within a 990 m2 moving window. This covariate excludes physical disturbance associated with roads. Values in our study, calculated only within the gas field, were 0.0–0.62 km2/km2. Industrial development footprint Area (density) of physically modified ground calculated from aerial photography as a year-specific variable. Physically modified ground primarily reflected industrial development including well pads, pipe yards, pipelines, construction areas, or clearing for facilities development. Industrial development footprint was calculated for the central grid cell within a 990 m2 moving window. This covariate excludes physical disturbance associated with roads. Values in our study, calculated only within the gas field, were 0.0–0.62 km2/km2. doi:10.1371/journal.pone.0014597.t001 binomial and used a logit link function. We included ordinal date as a class variable and specified a variance components covariance structure in which random intercepts for ordinal date were nested within each individual to address within-day autocorrelation among locations. R-side, or marginal, random-effects models estimated using GLIMMIX provide conditional coefficient estimates for each individual, that is, estimates for individual animals that are conditional on the distribution of coefficient estimates across all individuals in the population. g(x)~b0zb1x1ijz:::zbkxkijzckjxkjzc0j To estimate marginal (i.e., population-level) coefficients we assumed that conditional coefficients for each elk represented a random sample from a normal distribution with the mean of that distribution representing the population-level effects of covariates on the probability of use [42]. We estimated marginal coefficients using g(x)~b0zb1x1ijz:::zbkxkijzckjxkjzc0j Road density Density of line features depicting roads calculated for grid cells at the center of a 990 m2 moving window. Road density was calculated as a year-specific variable – that is, new road features were added to the data set as annual aerial imagery became available. Values in our study, calculated within the gas field only, were 0.0–7.6 km/km2. Distance to structure Linear distance from a sample location to a human-built structure including houses, agricultural facilities, and industrial facilities. Distance to structure was a year-specific variable and was analyzed as natural log transformed distance +0.1 to allow its magnitude to decrease with increasing distance. Values in our study, calculated within the gas field only, were 0.0–5854.9 m. Industrial development footprint Area (density) of physically modified ground calculated from aerial photography as a year-specific variable. Physically modified ground primarily reflected industrial development including well pads, pipe yards, pipelines, construction areas, or clearing for facilities development. Industrial development footprint was calculated for the central grid cell within a 990 m2 moving window. This covariate excludes physical disturbance associated with roads. Values in our study, calculated only within the gas field, were 0.0–0.62 km2/km2. doi:10.1371/journal.pone.0014597.t001 Table 1. Covariates used in random-effects resource selection models and their descriptions. Table 1. Covariates used in random-effects resource selection models and their descriptions. Digital elevation model (DEM) provided at a resolution of 1.52 m and re-sampled for covariate calculation to a resolution of 30 m and measured in degrees. Values for slope calculated at point locations in our study ranged from 0.02 (flat ground) to 41.8 degrees (very steep). DEM re-sampled for covariate calculation to a resolution of 30 m and measured in m. Elevation at sample points in our study ranged from 1918.7–2970.0 m. Raster dataset compiled from 1-foot resolution aerial imagery of the study area using Image AnalysisTM and re-sampled to 30 m resolution for covariate calculation. From an elk-centric perspective, habitat in the Raton basin functioned in one of two ways: as security cover or as forage resources. Raster cells were assigned one of two values (binary covariate) representing habitat that functioned to provide cover versus habitat that did not function to provide cover. All tree or oak-thicket dominated habitats were considered security cover, whereas all shrub and grassland dominated habitats were considered non-cover. Status #Elk #Locations Year Groupa Day Night Day Night 2006 Inside – all sample elk 11 10 1,759 879 Inside – GAM-predicted parturient 5 5 917 458 Outside – all sample elk 7 7 122 61 2007 Inside – all sample elk 26 24 3,281 1,641 Inside – GAM-predicted parturient 9 9 1,174 587 Outside – all sample elk 16 14 900 451 2008 Inside – all sample elk 19 19 2,056 1,455 Inside – GAM-predicted parturient 7 7 776 438 Inside – field-observed parturient 12 12 1,598 802 Inside – field-observed non-parturient 5 5 425 227 Outside – all sample elk 19 16 417 230 2009 Inside – all sample elk 15 15 2,735 1,482 Inside – GAM-predicted parturient 8 8 1,381 757 Inside – field-observed parturient 12 12 1,976 1,062 Inside – field-observed non-parturient 5 5 685 496 Outside – all sample elk 16 14 411 215 Random-effects Resource Selection Modeling – Marginal Inference Mapping Responses In all, five groups of elk were available for comparison: 1) all sample elk inside of the gas field, 2) elk predicted to be parturient based on GAMs inside the gas field, 3) field-observed parturient elk inside of the gas field, 4) field-observed non-parturient elk inside of the gas field, and 5) all sample elk outside of the gas field (sample size was too low to develop maternal groups; see Results). January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org 6 Ungulate Conservation parturient (see below); these elk were withheld from final RSF development so they represent an independent validation sample. Locations from these elk were plotted on multi-year predictive maps. We tested whether the number of locations that occurred within each predicted probability of use rank (1–5) differed from expectation using a chi-square test for specified proportions (PROC FREQ, SASH). To provide a measure of the amount to which a map that validated well differed from a map that validated poorly, we calculated the number of pixels comprising the raster surface that differed between maps in terms of relative probability rank. were minimal. Based on 25,290 GIS-generated random sample points (12,645 inside and 12,645 outside of the gas field, respectively), x 6 SD values inside versus outside of the gas field respectively were 2,347.7 6 146.4 m and 2,382.6 6 243.4 m for elevation, 10.6 6 6.2 degrees and 9.4 6 7.0 degrees for slope, and 82.0 6 10.2 km/km2 and 77.5 6 12.7 km/km2 for edge density. The proportion of sample points that occurred within security habitat was 0.84 and 0.80 inside versus outside of the gas field, respectively. Topographic covariates (elevation and slope) were not ground truthed; however, vegetation attributes were ground- truthed based on field-established polygons (n = 1177) of known vegetation type. Status #Elk #Locations Year Groupa Day Night Day Night 2006 Inside – all sample elk 11 10 1,759 879 Inside – GAM-predicted parturient 5 5 917 458 Outside – all sample elk 7 7 122 61 2007 Inside – all sample elk 26 24 3,281 1,641 Inside – GAM-predicted parturient 9 9 1,174 587 Outside – all sample elk 16 14 900 451 2008 Inside – all sample elk 19 19 2,056 1,455 Inside – GAM-predicted parturient 7 7 776 438 Inside – field-observed parturient 12 12 1,598 802 Inside – field-observed non-parturient 5 5 425 227 Outside – all sample elk 19 16 417 230 2009 Inside – all sample elk 15 15 2,735 1,482 Inside – GAM-predicted parturient 8 8 1,381 757 Inside – field-observed parturient 12 12 1,976 1,062 Inside – field-observed non-parturient 5 5 685 496 Outside – all sample elk 16 14 411 215 aInside refers to areas within the gas field and outside refers to areas adjacent but external to the gas field. GAM-predicted parturient refers to female elk that we predicted to be parturient based on results of generalized additive modeling (GAM) of movement data. Field-observed refers to elk for which we observed behavior in the field to assess parturition status. Total sample fitted with Global Positioning Systems collars was 25, 40, 50, and 50 in 2006, 2007, 2008, and 2009, respectively. Within-year deviation from the available sample arises from 3 sources: 1) some collared elk moved out of the study area during calving, 2) the GAM-predicted sample was nested within the total sample, and 3) some individuals occupied areas both inside and outside of the gas field. doi:10.1371/journal.pone.0014597.t002 PLoS ONE | www.plosone.org Table 2. Resource selection by Rocky Mountain elk during calving time; sample size and number of locations used in analyses. Table 2. Resource selection by Rocky Mountain elk during calving time; sample size and number of locations used in analyses. Results Generalized Additive Modeling to Designate Maternal Status aInside refers to areas within the gas field and outside refers to areas adjacent but external to the gas field. GAM-predicted parturient refers to female elk that we predicted to be parturient based on results of generalized additive modeling (GAM) of movement data. Field-observed refers to elk for which we observed behavior in the field to assess parturition status. Total sample fitted with Global Positioning Systems collars was 25, 40, 50, and 50 in 2006, 2007, 2008, and 2009, respectively. Within-year deviation from the available sample arises from 3 sources: 1) some collared elk moved out of the study area during calving, 2) the GAM-predicted sample was nested within the total sample, and 3) some individuals occupied areas both inside and outside of the gas field. Random-effects Resource Selection Modeling – Marginal Inference We found little evidence of lack of fit or overdispersion in RSF models; generalized chi-square/degrees of freedom was 0.79–0.92 for night time models and 0.87–0.97 for day time models. The most notable response by elk inside of the gas field was strong avoidance of the industrial development footprint during the day among all groups (at the population-level; Figure 3a). Other notable behavior in field-observed parturient elk included strong selection for cover and avoidance of high road density during the day relative to other groups (Figure 3b,c). While field-observed parturient elk showed some day-time preference for flatter areas and lower elevation relative to the larger sample, differences among maternal groups in selection for these and other resources were small, inconsistent, or not apparent. Table 3. Parameterizing movement using generalized additive models. Year/Group df,2a 2,df,3 df$3 2008 Field-observed parturient 2 1 9 2009 Field-observed parturient 5 3 4 2008 Field-observed non-parturient 2 2 1 2009 Field-observed non-parturient 3 2 0 aAs part of generalized additive modeling we specified the generalized cross validation option to assign parameters (df) to polynomials depicting daily changes in the distance moved by elk between successive locations. Maternal status was assigned to 34 female Rocky Mountain elk (10 non- parturient and 24 parturient) using information from blood samples (pregnancy-specific protein-B) and field observation (788 h). We assessed how generalized additive models of location data parameterized movement, and how parameterization corresponded with calf status as determined in the field. Results show that among 14 elk for which movement was parameterized by $3 df, 13 were observed in the field to be with calf. doi:10.1371/journal.pone.0014597.t003 Table 3. Parameterizing movement using generalized additive models. aAs part of generalized additive modeling we specified the generalized cross validation option to assign parameters (df) to polynomials depicting daily changes in the distance moved by elk between successive locations. Maternal status was assigned to 34 female Rocky Mountain elk (10 non- parturient and 24 parturient) using information from blood samples (pregnancy-specific protein-B) and field observation (788 h). We assessed how generalized additive models of location data parameterized movement, and how parameterization corresponded with calf status as determined in the field. Results show that among 14 elk for which movement was parameterized by $3 df, 13 were observed in the field to be with calf. January 2011 | Volume 6 | Issue 1 | e14597 Status We applied results from PSPB and field observation of female elk in 2008 and 2009 to test the GAM approach, in which movement patterns parameterized by ,3df were predicted to depict non-parturiency whereas movement patterns parameterized by $3 df were predicted to depict parturiency. Based on PSPB results and 788 hours of field observation, we assigned maternal status to 34 elk; 24 were identified as parturient and 10 as non- parturient. Movement during calving time was highly variable, especially among parturient elk, with parameterization ranging from 1.24–16.9 df. However, among elk observed to be parturient in the field, 93% (13/14) were parameterized by $3 df (Table 3). We fitted 25 female elk with GPS collars in 2006, 40 in 2007, 50 in 2008, and 50 in 2009. The entire within-year sample was unavailable for these analyses because the analyses spanned a relatively short period during calving (Table 2), and in each year several elk moved from the study area to alpine habitat for parturition. Inadequate sample size (i.e., #2 elk comprising maternal groups) in undeveloped areas restricted a more comprehensive assessment. Differences in elevation, slope, edge density, and cover type between developed and undeveloped areas parturition. Inadequate sample size (i.e., #2 elk comprising maternal groups) in undeveloped areas restricted a more comprehensive assessment. Differences in elevation, slope, edge density, and cover type between developed and undeveloped areas Table 2. Resource selection by Rocky Mountain elk during calving time; sample size and number of locations used in analyses. Random-effects Resource Selection Modeling – Marginal Inference doi:10.1371/journal.pone.0014597.t003 Total sample fitted with Global Positioning Systems collars was 25, 40, 50, and 50 in 2006, 2007, 2008, and 2009, respectively. Within-year deviation from the available sample arises from 3 sources: 1) some collared elk moved out of the study area during calving, 2) the GAM-predicted sample was nested within the total sample, and 3) some individuals occupied areas both inside and outside of the gas field. g doi:10.1371/journal.pone.0014597.t002 January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org 7 Ungulate Conservation Figure 3. Group-dependent population-level responses: inside of the gas field. Marginal coefficient estimates 6 95% CL of selection for human disturbance (a), security cover (b), and road density (c) by female elk during 2006-2009 in Raton Basin, Colorado, USA. Day time results are displayed among maternal statuses for elk inside of the gas field. doi:10.1371/journal.pone.0014597.g003 Figure 3. Group-dependent population-level responses: inside of the gas field. Marginal coefficient estimates 6 95% CL of s human disturbance (a), security cover (b), and road density (c) by female elk during 2006-2009 in Raton Basin, Colorado, USA. Day time displayed among maternal statuses for elk inside of the gas field. doi:10.1371/journal.pone.0014597.g003 Figure 3. Group-dependent population-level responses: inside of the gas field. Marginal coefficient estimates 6 95% CL of selection for human disturbance (a), security cover (b), and road density (c) by female elk during 2006-2009 in Raton Basin, Colorado, USA. Day time results are displayed among maternal statuses for elk inside of the gas field. doi:10.1371/journal.pone.0014597.g003 We estimated selection for 4 landscape/habitat covariates in both developed and undeveloped areas including security cover, slope, elevation, and habitat edge density. Considering population- level coefficient estimates, selection for resources that functioned to provide security cover differed markedly between elk that occupied developed areas versus undeveloped areas (Figure 4). Elk inside of the gas field consistently showed strong selection for security cover during the day (Figure 4a) and strong selection for non-cover or forage habitats during the night (Figure 4b) throughout the study period whereas elk occupying areas outside of the gas field generally selected randomly for security cover with coefficient estimates near zero. Another difference between elk inside versus outside of the gas field involved selection for elevation. Elk outside of the gas field generally selected randomly for elevation with coefficient estimates always near zero. Random-effects Resource Selection Modeling – Marginal Inference Elk inside of the gas field generally selected for higher elevation during the day (Figure 4c; however, 95% CL overlapped zero), and for lower elevation at night (Figure 4d). Diurnal patterns in selection for slope and edge density were apparent but differences in selection for these features between elk inside versus outside of the gas field generally were small (Table 4). PLoS ONE | www.plosone.org Random-effects Resource Selection Modeling – Conditional Inference Conditional estimates revealed a complex association between human activity and individual variation in response to environmental and anthropogenic features (Figure 5). Elk inside the gas field showed greater heterogeneity among individuals relative to elk outside of the January 2011 | Volume 6 | Issue 1 | e14597 8 Figure 4. Population-level responses: inside versus outside of the gas field. Marginal coefficient estimates 695% CL of day-time (a) and night-time (b) selection for security cover, and day-time (c) and night-time (d) selection for elevation by female elk during 2006-2009 in Raton Basin, Colorado, USA. Inside refers to elk occupying an active gas field whereas outside refers to elk occupying adjacent undeveloped areas; maternal status is not considered (i.e., all sample elk are grouped depending on whether they occurred inside versus outside of the gas field). doi:10.1371/journal.pone.0014597.g004 Ungulate Conservation Ungulate Conservation gas field in their responses to edge density and elevation. Selection for l (Fi 5 b) d d d it (Fi 5 d) ti t d Seasonal use areas of 15 e d l d I di id l Figure 4. Population-level responses: inside versus outside of the gas field. Marginal coefficient e night-time (b) selection for security cover, and day-time (c) and night-time (d) selection for elevation by fema Colorado, USA. Inside refers to elk occupying an active gas field whereas outside refers to elk occupying adjace is not considered (i.e., all sample elk are grouped depending on whether they occurred inside versus outsid doi:10.1371/journal.pone.0014597.g004 Figure 4. Population-level responses: inside versus outside of the gas field. Marginal coefficient estimates 695% CL of day-time (a) and night-time (b) selection for security cover, and day-time (c) and night-time (d) selection for elevation by female elk during 2006-2009 in Raton Basin, Colorado, USA. Inside refers to elk occupying an active gas field whereas outside refers to elk occupying adjacent undeveloped areas; maternal status is not considered (i.e., all sample elk are grouped depending on whether they occurred inside versus outside of the gas field). doi:10.1371/journal.pone.0014597.g004 Seasonal use areas of 15 elk overlapped both developed and undeveloped areas. Individuals showed notable behavioral differ- ences, including diurnal variation, relative to selection for security cover and elevation depending on whether they were in developed versus undeveloped areas (Figure 6). January 2011 | Volume 6 | Issue 1 | e14597 Random-effects Resource Selection Modeling – Conditional Inference During the day, 13 of 15 elk showed stronger selection for security cover when inside the gas field, but at night this pattern broke down with no apparent consistent behavioral response. Selection for elevation was variable inside the gas field during day and night with some elk showing relatively strong selection for higher or lower elevations. When elk were outside of the gas field they generally used elevation at random (Figure 6). gas field in their responses to edge density and elevation. Selection for slope (Figure 5a,b) and edge density (Figure 5c, d) was estimated more precisely among elk inside of the gas field; however, elk outside the gas field selected (randomly) for elevation with high precision (Figure 5e, f). Also apparent inside the gas field but not outside was a temporal trend of increasing avoidance of high edge density (Figure 5c). Within-year marginal estimates of day time selection for road density were positive (i.e., elk selected for higher road density) in 3 of 4 years. However, conditional estimates revealed that, across all years, only 37% of sampled elk selected for relatively high road density; 32% selected neither for nor against high road density, and 31% avoided high road density (Figure 5g). gas field in their responses to edge density and elevation. Selection for slope (Figure 5a,b) and edge density (Figure 5c, d) was estimated more precisely among elk inside of the gas field; however, elk outside the gas field selected (randomly) for elevation with high precision (Figure 5e, f). Also apparent inside the gas field but not outside was a temporal trend of increasing avoidance of high edge density (Figure 5c). Within-year marginal estimates of day time selection for road density were positive (i.e., elk selected for higher road density) in 3 of 4 years. However, conditional estimates revealed that, across all years, only 37% of sampled elk selected for relatively high road density; 32% selected neither for nor against high road density, and 31% avoided high road density (Figure 5g). PLoS ONE | www.plosone.org January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org 9 Ungulate Conservation Table 4. Marginal coefficient estimates (^bbn[SE]) for two-level random-effects models of resource selection by Rocky Mountain elk during calving time (15 May - 1 July), 2006–2009. Random-effects Resource Selection Modeling – Conditional Inference Day Night Effect/Year Inside Outside Inside Outside Slope 2006 20.001 (0.002) 20.003 (0.034) 20.064 (0.013) - Slope 2007 0.011 (0.006) 0.005 (0.013) 20.030 (0.010) 20.040 (0.016) Slope 2008 20.003 (0.007) 0.003 (0.003) 20.035 (0.007) 20.045 (0.008) Slope 2009 0.012 (0.005) 0.024 (0.014) 20.023 (0.011) 20.027 (0.013) Edge Density 2006 0.015 (0.016) 20.022 (0.004) 20.007 (0.021) - Edge Density 2007 0.001 (0.013) 20.003 (0.012) 0.006 (0.011) 20.013 (0.004) Edge Density 2008 20.026 (0.009) 20.008 (0.003) 20.026 (0.008) 20.010 (0.002) Edge Density 2009 20.027 (0.009) 20.006 (0.010) 20.036 (0.012) 20.044 (0.021) - Model failed to converge. doi:10.1371/journal.pone.0014597.t004 Table 4. Marginal coefficient estimates (^bbn[SE]) for two-level random-effects models of resource selection by Rocky Mountain elk during calving time (15 May - 1 July), 2006–2009. Table 4. Marginal coefficient estimates (^bbn[SE]) for two-level random-effects models of resource selection by Rocky Mountain elk during calving time (15 May - 1 July), 2006–2009. Table 4. Marginal coefficient estimates (^bbn[SE]) for two-level random-effects models of resource selection by Rocky Mountain elk during calving time (15 May - 1 July), 2006–2009. Figure 5. Population-level and individual responses. Marginal (dash) and conditional (circles) coefficient estimates (SE) of day-time selection for slope (a, b) edge density (c, d), elevation (e, f), and road density (g) by female elk during 2006-2009 in Raton basin, Colorado, USA. Results are displayed by year for elk that occupied developed (panels a, c, e, and g) and undeveloped (panels b, d, and f) areas. Orange depicts elk that were predicted to be parturient using generalized additive modeling; open circles (#) indicate conditional estimates for field-observed parturient elk in 2008 and 2009. doi:10.1371/journal.pone.0014597.g005 Figure 5. Population-level and individual responses. Marginal (dash) and conditional (circles) coefficient estimates (SE) of day-time selection for slope (a, b) edge density (c, d), elevation (e, f), and road density (g) by female elk during 2006-2009 in Raton basin, Colorado, USA. Results are displayed by year for elk that occupied developed (panels a, c, e, and g) and undeveloped (panels b, d, and f) areas. Orange depicts elk that were predicted to be parturient using generalized additive modeling; open circles (#) indicate conditional estimates for field-observed parturient elk in 2008 and 2009. doi:10 1371/journal pone 0014597 g005 Figure 5. Population-level and individual responses. January 2011 | Volume 6 | Issue 1 | e14597 Random-effects Resource Selection Modeling – Conditional Inference Symbols represent conditional coefficient estimates (y-axes) for each elk in 2007 (squares; n = 6), 2008 (circles; n = 4), and 2009 (triangles; n = 5); lines show how selection changed within each elk depending on whether it was inside versus outside of the gas field. Solid lines depict larger selection coefficients inside the gas field whereas dashed lines depict larger coefficients outside of the gas field. Coefficients are informed by 36-293 location per elk inside the gas field, and 20-248 locations per elk outside the gas field. doi:10.1371/journal.pone.0014597.g006 Functional Responses resources near physical disturbance and, during the day, showed a stronger functional response to the proportion of cover than did elk in undeveloped areas (Figure 7b, c). Consistent with a functional response in resource selection, elk selected randomly for disturbed areas when disturbance was minimal. As human activity increased (i.e., across individual elk calving season areas, or through time), elk showed stronger avoidance of the industrial development footprint during the day but not at night; this spatiotemporal pattern of avoidance revealed that elk continued to use physically disturbed areas but modified their behavior to avoid human activity (Figure 7a). One way elk avoided human activity in the day was by modifying selection for security cover. In developed areas elk were constrained to select Random-effects Resource Selection Modeling – Conditional Inference Marginal (dash) and conditional (circles) coefficient estimates (SE) of day-time selection for slope (a, b) edge density (c, d), elevation (e, f), and road density (g) by female elk during 2006-2009 in Raton basin, Colorado, USA. Results are displayed by year for elk that occupied developed (panels a, c, e, and g) and undeveloped (panels b, d, and f) areas. Orange depicts elk that were predicted to be parturient using generalized additive modeling; open circles (#) indicate conditional estimates for field-observed parturient elk in 2008 and 2009. doi:10 1371/journal pone 0014597 g005 doi:10.1371/journal.pone.0014597.g005 doi:10.1371/journal.pone.0014597.g005 January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org 10 Ungulate Conservation Figure 6. Movement from within the gas field to areas adjacent to the gas field. A subsample of female elk (n = 15) occupied areas within the gas field as well as areas outside but adjacent to the gas field. This figure shows differences in day-time (a) and night-time (b) selection for habitat that provides security cover, and day-time (c) and night-time (d) selection for elevation among these elk depending on whether they were inside versus outside of the gas field. Symbols represent conditional coefficient estimates (y-axes) for each elk in 2007 (squares; n = 6), 2008 (circles; n = 4), and 2009 (triangles; n = 5); lines show how selection changed within each elk depending on whether it was inside versus outside of the gas field. Solid lines depict larger selection coefficients inside the gas field whereas dashed lines depict larger coefficients outside of the gas field. Coefficients are informed by 36-293 location per elk inside the gas field, and 20-248 locations per elk outside the gas field. doi:10.1371/journal.pone.0014597.g006 Figure 6. Movement from within the gas field to areas adjacent to the gas field. A subsample of female elk (n = 15) occupied areas within the gas field as well as areas outside but adjacent to the gas field. This figure shows differences in day-time (a) and night-time (b) selection for habitat that provides security cover, and day-time (c) and night-time (d) selection for elevation among these elk depending on whether they were inside versus outside of the gas field. PLoS ONE | www.plosone.org Mapping Responses Dashes (-) and dashed lines symbolize night-time resource selection, and circles (N) and solid lines symbolize day-time resource selection. Dashes and circles outlined by open circles (#) or boxes (%) indicate conditional estimates for field-observed parturient elk in 2008 and 2009. doi:10.1371/journal.pone.0014597.g007 Figure 7. Functional responses in resource selection by female elk. Selection for the human disturbance footprint (a), security cover inside of the gas field (b), and security cover outside of the gas field (c) change as a function of availability. Availability was calculated at the seasonal use area level and conditional coefficients were estimated from generalized linear random-effects resource selection models. Dashes (-) and dashed lines symbolize night-time resource selection, and circles (N) and solid lines symbolize day-time resource selection. Dashes and circles outlined by open circles (#) or boxes (%) indicate conditional estimates for field-observed parturient elk in 2008 and 2009. doi:10 1371/journal pone 0014597 g007 and 3505 day locations) and then summed day and night values within the three groups of interest to estimate overall within-group model performance. For both models based on elk behavior inside of the gas field (Figure 8a), habitat with low predicted probability of use was modeled accurately; however, validation elk used habitat predicted to have high probability of use only as much as would be expected if elk used all predictive classes equally (Figure 8c). The model based on elk behavior outside of the gas field (Figure 8b) validated well with few locations occurring in lowest ranked areas and many locations occurring in highest ranked areas (Figure 8d). The proportion of pixels ranked differently between the model that validated most poorly and the model that validated best was 0.72–0.88 depending on year. Mapping Responses We developed six models (day and night separately for each of three groups): the first was based on all sample elk that occurred inside of the gas field; the second was a subset of the first group for which GAMs predicted females to be parturient; and the third was based on all sample elk that occurred outside of the gas field. We validated day and night models separately (1830 night locations January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org 11 Figure 7. Functional responses in resource selection by female elk. Selection for the human disturbance footprint (a), security cover inside of the gas field (b), and security cover outside of the gas field (c) change as a function of availability. Availability was calculated at the seasonal use area level and conditional coefficients were estimated from generalized linear random-effects resource selection models. Dashes (-) and dashed lines symbolize night-time resource selection, and circles (N) and solid lines symbolize day-time resource selection. Dashes and circles outlined by open circles (#) or boxes (%) indicate conditional estimates for field-observed parturient elk in 2008 and 2009. doi:10.1371/journal.pone.0014597.g007 Ungulate Conservation Ungulate Conservation Figure 7. Functional responses in resource selection by female elk. Selection for the human disturbance the gas field (b), and security cover outside of the gas field (c) change as a function of availability. Availability wa level and conditional coefficients were estimated from generalized linear random-effects resource selection m symbolize night-time resource selection, and circles (N) and solid lines symbolize day-time resource selection. circles (#) or boxes (%) indicate conditional estimates for field-observed parturient elk in 2008 and 2009. doi:10.1371/journal.pone.0014597.g007 Figure 7. Functional responses in resource selection by female elk. Selection for the human disturbance footprint (a), security cover inside of the gas field (b), and security cover outside of the gas field (c) change as a function of availability. Availability was calculated at the seasonal use area Figure 7. Functional responses in resource selection by female elk. Selection for the human disturbance footprint (a), security cover inside of the gas field (b), and security cover outside of the gas field (c) change as a function of availability. Availability was calculated at the seasonal use area level and conditional coefficients were estimated from generalized linear random-effects resource selection models. Discussion and 3505 day locations) and then summed day and night values within the three groups of interest to estimate overall within-group model performance. For both models based on elk behavior inside of the gas field (Figure 8a), habitat with low predicted probability of use was modeled accurately; however, validation elk used habitat predicted to have high probability of use only as much as would be expected if elk used all predictive classes equally (Figure 8c). The model based on elk behavior outside of the gas field (Figure 8b) validated well with few locations occurring in lowest ranked areas and many locations occurring in highest ranked areas (Figure 8d). The proportion of pixels ranked differently between the model that validated most poorly and the model that validated best was 0.72–0.88 depending on year. We successfully integrated GAMs, field observation, and random-effects RSF modeling to designate parturiency, describe individual and population-level resource selection, and determine the relative influence of maternal status and human activity on the reliability of spatially explicit models intended to guide conserva- tion of critical ungulate calving resources. We assumed that planning tools such as spatially explicit models would be more reliable if they account for adaptive behavior which, among parturient elk, should be reflected in movement and resource selection [45]. This was the basis for using GAMs to designate parturiency based on movement; our thinking was that ungulate PLoS ONE | www.plosone.org PL January 2011 | Volume 6 | Issue 1 | e14597 PLoS ONE | www.plosone.org January 2011 | Volume 6 | Issue 1 | e14597 12 Ungulate Conservation Figure 8. Assigning the relative probability of use throughout the landscape. Comparison of how the relative predicted probability of use was assigned throughout the landscape depending on whether RSF mapping was based on resource selection behavior among female elk that occupied a natural gas field (a) or occupied areas adjacent to but outside of a natural gas field (b). Maps depict day-time probability of use. Wells are depicted as black dots. Borders of wildlife areas managed by the state of Colorado are depicted in red. Probability of use is scaled from low to high with each of 5 ordinal bins representing quantiles of the total number of pixels (30-m resolution) comprising the area. Discussion doi:10.1371/journal.pone.0014597.g008 behavior contains information on reproductive status and GAMs would reveal a difference in the shape and relative complexity of the response curve between parturient and non-parturient females. Maternal status was evident in elk movement with our application of GAMs correctly predicting parturiency 93% of the time. We note, however, the GAM approach as presented would require further development if improved sensitivity (reduced false negative rate) is desirable. GAMs have been a standard tool in epidemiologic analyses and have found broad application in ecology for modeling tolerance thresholds and spatial distributions [46], [47], [48], [49]. Using movement data to designate functional groups, seasonality, or behavior in animals has increased in prevalence as GPS-based research has become more frequent [50], [51]. Although we found some potentially important differences in resource selection patterns between parturient and non-parturient elk (see below), behavior in these groups was more behavior contains information on reproductive status and GAMs would reveal a difference in the shape and relative complexity of the response curve between parturient and non-parturient females. Maternal status was evident in elk movement with our application of GAMs correctly predicting parturiency 93% of the time. We note, however, the GAM approach as presented would require further development if improved sensitivity (reduced false negative rate) is desirable. GAMs have been a standard tool in epidemiologic analyses and have found broad application in ecology for modeling tolerance thresholds and spatial distributions [46], [47], [48], [49]. Using movement data to designate functional groups, seasonality, or behavior in animals has increased in prevalence as GPS-based research has become more frequent [50], [51]. Although we found some potentially important differences in resource selection patterns between parturient and non-parturient elk (see below), behavior in these groups was more similar than expected. Had maternal status been more apparent in shaping resource selection, methods to designate status might have found better application in landscape-level mapping of the relative predicted probability of use. Here, both parturient and non- parturient elk were examined inside the gas field where any behavior distinctly associated with parturiency was obscured by apparent risk-aversive behavior related to human activity during the day time. At the population-level parturient elk, while avoiding roads and selecting for security cover more strongly than other elk, conformed to a general pattern of avoiding human activity during the day by occupying upland forest or Q. Discussion Charts (c, d) display map validation with columns depicting the number of locations from an independent sample of elk that, when plotted on RSF maps (a, b), occurred within each ordinal bin. The black horizontal line depicts expectation if resources were selected at random. The map based on behavior inside of the gas field validated poorly (c; x2 = 577.46, df = 4, p,0.001) relative to the map based on behavior outside of the gas field (d; x2 = 849.18, df = 4, p,0.001) with the distribution of validation locations differing significantly from random in both cases. RSF maps (a, b) are based on all sample elk inside and outside of the gas field, respectively; not shown is the RSF map based on elk predicted to be parturient using generalized additive models. doi:10.1371/journal.pone.0014597.g008 Figure 8. Assigning the relative probability of use throughout the landscape. Comparison of how the relative predicted probability of use was assigned throughout the landscape depending on whether RSF mapping was based on resource selection behavior among female elk that occupied a natural gas field (a) or occupied areas adjacent to but outside of a natural gas field (b). Maps depict day-time probability of use. Wells are depicted as black dots. Borders of wildlife areas managed by the state of Colorado are depicted in red. Probability of use is scaled from low to high with each of 5 ordinal bins representing quantiles of the total number of pixels (30-m resolution) comprising the area. Charts (c, d) display map validation with columns depicting the number of locations from an independent sample of elk that, when plotted on RSF maps (a, b), occurred within each ordinal bin. The black horizontal line depicts expectation if resources were selected at random. The map based on behavior inside of the gas field validated poorly (c; x2 = 577.46, df = 4, p,0.001) relative to the map based on behavior outside of the gas field (d; x2 = 849.18, df = 4, p,0.001) with the distribution of validation locations differing significantly from random in both cases. RSF maps (a, b) are based on all sample elk inside and outside of the gas field, respectively; not shown is the RSF map based on elk predicted to be parturient using generalized additive models. Discussion gambelii thickets, and selecting forage resources in valley bottoms at night regardless of proximity to infrastructure. Trade-offs that structure resource selection in many ungulates, most notably between forage requirements and risk avoidance [6], [52], often are amplified in PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org January 2011 | Volume 6 | Issue 1 | e14597 13 January 2011 | Volume 6 | Issue 1 | e14597 Ungulate Conservation parturient females through nutritional demands to support body condition, lactation, and neonatal defense [53]. Here, responses to human activity were more apparent than differences in resource selection as a function of parturiency with elk exhibiting a clear spatiotemporal avoidance of human activity at the population- level and modified patterns of selection for environmental features, notably security cover and elevation, in developed areas relative to undeveloped areas. Research has shown that ungulates exhibit avoidance behavior relative to human development, recreation, hunting, and other activities [54]. Modeling resource selection separately during day and night offered insight into whether the physical presence of infrastructure versus the operation and maintenance of such infrastructure (only occurring during the day) was more important in avoidance behavior. Elk inside the gas field used cover and elevation to modulate avoidance of human activity during the day [55] whereas cover and elevation were less influential outside of the gas field suggesting that human activity during the day was the factor to which elk were responding [3]. At night during calving time female elk showed no aversion to infrastructure and selected for areas characterized by valley- bottoms and foraging resources that in many instances were in close proximity to, or directly associated with, disturbances such as well pads and roads [56]. Day time refugia characterized by minimal human disturbance, security cover, and upper slope positions will be critical for maintenance of calving and perhaps other seasonal habitats in multiple-use landscapes. drome, individuals have a behavioral type such as risk-aversion (i.e., more risk-averse versus less risk-averse types; [58]). The notion that individuals can be more or less risk aversive implies a limit to their range of behavioral plasticity. From a conservation perspective, animals exhibiting limited plasticity in environments undergoing rapid change, such as those affected by industrial development, may be less able to adapt. Elk clearly show some ability to adapt to human activity [59]. Discussion Nonetheless, if the effect of human activity is a threshold phenomenon [60], we might expect there to exist a limit of physical disturbance corresponding to a limit in the range of behavioral plasticity in ungulates, beyond which redistribution, social, or demographic effects may be observed [61], [62]. Examining only average responses across populations obscures variability among individuals that may have important implica- tions for management or provide new ecological insight. For example, management strategies designed to conserve a resource that is important, on average, to the population may overlook resources that are critical to individuals that comprise a smaller demographic segment that functions disproportionately in popu- lation persistence. It has been stated ‘‘information on individual resource use is necessary if we are to make the transition from phenomenological models of population dynamics to mechanistic models in which the dynamics of a population are predicted from the properties of its components’’ [18]. Conditional estimates also provide information on why marginal estimates may be counter- intuitive, or how individuals assemble to comprise the marginal estimate. For example, the marginal estimate of selection for road density in our study indicated that elk selected for higher road density in 3 of the 4 years comprising the study period (Figure 5). This observation is counter to most research on roads and ungulates [63], [64]. Conditional estimates revealed that ,1/3 of elk showed a positive association with higher road density but just as many avoided high road density; this observation sets the stage for examining potential links between a particular behavioral strategy such as road avoidance and demographic responses. The increase in the within-year proportion of elk occupying areas with higher road density from 2007 to 2009 (Figure 5d), concurrent with an increase in new road development, suggests that elk maintained relatively consistent calving season use areas during the study period, but modified their behavior as they became increasingly constrained to select resources in proximity to development. Such modification was consistent with a functional response in resource selection; elk response to human activity changed as a function of availability and with time of day. During the day when humans are active elk showed avoidance behavior that strengthened with increasing disturbance. This response spilled over to elk selection for security cover which appeared to have high importance to elk inside of the gas field as a day time refugium. Discussion Outside of the gas field elk showed relatively weak day time selection for cover and, as might be expected, selection weakened as the proportion of cover within the seasonal use area increased (sensu [40], Figure 7). These observations are consistent with the hypothesis that ungulates face a trade-off that is mediated by human activity in multiple-use landscapes – that is, the strength of the trade-off varies in direct relation to resource availability which is driven by human activity [41]. To our knowledge, fully random-effects RSF models do not appear in the published literature. The random-effects framework provided insight into individual behavior and how elk modified patterns of calving season resource selection relative to develop- ment. Individuals that moved between developed and undevel- oped areas (Figure 6) were spatially aware and showed marked changes in their behavior that were consistent with the hypothesis that human activity during the day was perceived as a source of risk [6]. An observation that warrants further attention in wildlife- human interaction studies is that heterogeneity among individuals in response to their environment was apparent, and this variation was amplified in developed areas relative to undeveloped areas. Associated with this observation was generally more precise estimation of selection within individuals in developed areas (except selection for elevation). Among-individual variation can comprise the majority of a population’s niche width, and only when the within-individual component of total niche width is constrained does between-individual variation become prominent. Of particular relevance here is that trade-offs remain among the most plausible mechanisms for the observation of limited within- individual variation [18]. If ungulates in developed areas must make trade-offs associated with avoiding human activity, partic- ularly during daylight hours, we might expect constrained within- individual variation and thus more heterogeneity among individ- uals. Human activity functioning to constrain decision-making in ungulates is consistent with the notion that risky environments impose pressures that disallow animals to respond to other features as they otherwise would [57]. This could make it difficult to establish or predict general patterns of resource selection during periods in which ungulates show fidelity to historic ranges, yet human activity rapidly modifies the landscape. In fact, we showed that RSF maps based on day-time resource selection behavior in developed areas had poor predictive accuracy. Spatially explicit models of relative predicted probability of use validated poorly when they were based on resource selection behavior inside of the gas field. PLoS ONE | www.plosone.org Discussion The model based on behavior outside of the gas field validated well. To our knowledge, this treatment/control concept is a novel approach in RSF-based conservation planning. Typically, quantifying relative predicted probability of use in human-modified areas is based on animal The study of behavioral syndromes offers a relevant framework within which to discuss the conservation implications of individual variation in human-wildlife interaction studies [58]. A behavioral syndrome is a suite of correlated behaviors reflecting among- individual consistency across multiple contexts. Within a syn- January 2011 | Volume 6 | Issue 1 | e14597 January 2011 | Volume 6 | Issue 1 | e14597 14 Ungulate Conservation extensive portions of the landscape, potentially resulting in large errors in where conservation action would have its greatest impact. In population response research it is always desirable to include spatial and temporal controls (before-after, control-impact design), and demographic responses. Availability of such components is uncommon in large-scale wildlife-human interaction studies. In such situations the approach we describe, including efforts to account for possible adaptive behavior among reproductive groups, attention to the treatment/control concept, and a random-effects modeling framework, should have general appli- cation in human-wildlife interaction research particularly among species that inhabit places where human activity is intense, or among special status species for which little information on resource needs exists. behavior in those areas. This approach has been used effectively; it has been shown that predictive maps based on mule deer (Odocoileus hemionus) behavior in human-modified areas validated well [3]. In their study [3], variation among individuals was present but limited relative to our findings. We suggest that ungulates in developed areas often respond to human activity in situation-specific ways. In our study, the local attributes of human activity varied in space and time within the seasonal use area of each elk making it difficult to generalize across the population. Situation-specific responses, including individual variation in the strength of selection or avoidance response, induced heterogeneity which complicated the application of models based on such behavior for conservation planning. 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Rice, of the Colorado Division of Wildlife for providing insightful comments on an earlier version of this paper. We thank J.J. Cox and B. Augustine of the University of Kentucky Department of Forestry for providing comments on earlier versions. We thank landowners in Raton Basin who participated in this effort for their patience and ongoing enthusiasm for this work. Discussion If conservation objec- tives include establishing zones within such landscapes intended to function as refugia from human activity and promote long-term population persistence, information on resource selection patterns in existing refugia would be expected to provide valuable guidance in prioritizing the creation of new zones in modified landscapes. Establishing refugia based on resource selection patterns that reflect responses to human activity risks uncertainty in the performance of such refugia once human disturbance pressure is released allowing animals to respond to features as they otherwise would have [57]. In our study, models based on resource selection patterns that reflected responses to human activity classified the relative probability of use differently from the models based on behavior in the absence of industrial development throughout Author Contributions Conceived and designed the experiments: MRD SMH RGO LDHW JBW. Performed the experiments: MRD SMH. Analyzed the data: MRD SMH SLW. Wrote the paper: MRD SMH SLW. Field observation: MRD SMH RGO JJW. Data collection: SMH RGO JJW. Conceived and designed the experiments: MRD SMH RGO LDHW JBW. Performed the experiments: MRD SMH. Analyzed the data: MRD SMH SLW. Wrote the paper: MRD SMH SLW. Field observation: MRD SMH RGO JJW. Data collection: SMH RGO JJW. 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Friar JL, Merrill EH, Beyer HL, Morales JM (2008) Thresholds in landscape connectivity and mortality risks in response to growing road networks. Journal of Applied Ecology 45: 1504–1513. 47. Yuan LL (2004) Assigning macroinvertebrate tolerance classifications using generalized additive models. Freshwater Biology 49: 662–677. 48. Potvin MJ, Drummer TD, Vucetich JA, Beyer Jr. DE, Peterson RO, et al. (2005) Monitoring and habitat analysis for wolves in upper Michigan. Journal of Wildlife Management 69: 1660–1669. 65. Moilanen A, Franco AMA, Early RI, Fox R, Wintle B, Thomas CD (2005) Prioritizing multiple-use landscapes for conservation: methods for large multi- species planning problems. Proceedings of The Royal Society B 272: 1885–1891. PLoS ONE | www.plosone.org January 2011 | Volume 6 | Issue 1 | e14597 16
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Identifying priority areas for European resource policies: a MRIO-based material footprint assessment
Journal of economic structures
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*Correspondence: stefan.giljum@wu.ac.at 1 Institute for Ecological Economics, Vienna University of Economics and Business (WU), Vienna, Austria Full list of author information is available at the end of the article Identifying priority areas for European resource policies: a MRIO‑based material footprint assessment Stefan Giljum1*, Hanspeter Wieland1, Stephan Lutter1, Martin Bruckner1, Richard Wood2, Arnold Tukker3,4 and Konstantin Stadler2 Stefan Giljum1*, Hanspeter Wieland1, Stephan Lutter1, Martin Bruckner1, Richard Wood2, Arnold Tukker3,4 and Konstantin Stadler2 Giljum et al. Economic Structures (2016) 5:17 DOI 10.1186/s40008-016-0048-5 Open Access Abstract In the context of the transformation toward a “green economy,” issues related to natural resource use have rapidly increased in importance in European and international policy debates. The large number of studies applying economy-wide material flow analysis so far mostly produced aggregated national indicators, making the results difficult to connect to policies, which are often designed for single sectors or consumption areas. This paper provides a detailed assessment of the composition of EU’s material foot- print in its global context, aiming at identifying the main product groups contributing to overall material consumption and specifying the geographical sources for the raw materials required to satisfy EU’s final demand. Based on multi-regional input–output (MRIO) modeling, we apply production layer decomposition to assess supply chains and their structural changes from 1995 to 2011. The global MRIO database used in this study is EXIOBASE 3, which disaggregates 200 products and 163 industries, of which 33 represent material extraction sectors. By that means, we increase the level of detail to a degree where policies can more easily connect to. We find that the generally grow- ing material footprint of the EU was characterized by a dramatic shift regarding the origin of raw materials, with the share of materials extracted within the EU territory falling from 68 % in 1995 to 35 % in 2011. In 2011, raw materials extracted in China to produce exports to the EU already contributed an equal share to EU’s material footprint as material extraction within the EU itself. Import dependency is most critical for the material group of metal ores, with only 13 % of all metals required as inputs to EU final demand stemming from within the EU. Regarding product composition, construction was confirmed as the most important sector contributing to the material footprint, fol- lowed by the group of manufacturing products based on biomass. Materials embodied in service sector activities together contributed a quarter to the total material footprint in 2011, making services an important, but currently disregarded area for European resource policies. We also find that supply chain structures became more complex over time, with a growing part located outside the EU territory. Keywords:  Material footprint, Multi-regional input–output analysis, Production layer decomposition, Supply chains, Resource policies © 2016 The Author(s). © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 1.1  Policy background Economic development in the past decades was characterized by steadily increasing levels of global resource use and rising human pressures on global ecosystems (Giljum et al. 2014; Krausmann et al. 2009; UNEP 2011a). Driven by rapidly growing levels of material consumption in emerging economies together with continued high levels of per capita resource use in the industrialized world, the global economy is also characterized by increasing competition between world regions regarding the access to raw materials. Against this background issues related to material consumption and resource productiv- ity have rapidly increased in importance in European and international policy debates in the past few years. In response to these environmental and economic challenges, numerous policy ini- tiatives have been introduced aiming at significantly increasing resource efficiency to reduce negative environmental impacts, while addressing issues related to material sup- ply security and import dependency. Examples for these strategies on the international level with a strong focus on resource efficiency include the “Green Economy” concept promoted by UNEP (2011b) or the “Green Growth Strategy” of the OECD (2011). The most important policy strategy at the European level promoting resource efficiency is the Commission’s Flagship Initiative “A resource-efficient Europe” (European Commis- sion 2011b) as well as the subsequent “Roadmap to a resource-efficient Europe” (Euro- pean Commission 2011c), both part of the implementation of the “Europe 2020” strategy. The overall objective of the Roadmap is to ensure that “by 2050 the EU’s economy has grown in a way that respects resource constraints and planetary boundaries” and to identify priority areas and actions “to put us on a path to resource efficient and sustain- able growth” (p. 3). Three consumption areas receive particular attention in the Road- map: food, housing and mobility. The food value chains are identified as causing major global impacts, in particular related to the production of animal-based products. Regard- ing the housing and building sector, the Commission recognizes the significant material flows related to construction and maintenance activities of buildings. It is argued that the aggregated impacts of housing and infrastructure account for around 15–30 % of all environmental pressures caused by European consumption. The third high-impact area regarding resource use identified in the Roadmap is mobility with its high demand for material-intensive transport infrastructure and the high dependency on fossil fuels. In some consumption areas, notably food consumption, the Commission acknowl- edges that European consumption causes negative global impacts. Abstract This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Page 2 of 24 Giljum et al. Economic Structures (2016) 5:17 1.1  Policy background In general, however, the Commission is rather concerned about the negative impacts on the EU economy caused by international developments such as restrictions in raw material availability or increasing raw material and food prices (European Commission 2011a). The most prom- inent related policy strategy focusing on aspects of import dependency, resource supply and access to natural resources is the Raw Materials Initiative (European Commission 2008, 2011d). In these documents, the Commission focuses on critical materials, in par- ticular metal ores with high import dependency rates and high relevance for European industries, and describes measures to secure and improve access to these raw materials. In the initiative, certain materials with specifically high dependency rates are identified, such as indium, titanium and niobium, where the EU faces an import dependency rate Giljum et al. Economic Structures (2016) 5:17 Page 3 of 24 of 100 % (European Commission 2008). However, the supply and dependency aspects are solely tackled from a perspective of direct imports and inputs into the European economy, not from a supply chain perspective. Issues such as supply chain security and dependency on indirect supplies via imported manufactured goods are not mentioned. Recently, the EU has also committed itself to the vision of a circular economy and has presented a package of legislative and voluntary measures. This initiative aims at closing the loops of materials and thus maintaining the value of resources and products as long as possible within the economy, while minimizing the amount of generated waste (Euro- pean Commission 2015). Given the increased demand for robust measures from the policy side, discussions on the most suitable indicators to measure resource use, productivity and criticality of raw materials are intensively ongoing. In recent years, awareness of policy makers generally increased regarding the significance not only of materials and products directly used by a national economy, or a regional economy such as the EU, but also of indirect resource use required along supply chains and embodied in internationally traded products. Consideration of all indirect effects leads to a consumption—or footprint—perspective, allowing illustrating the global impacts related to final demand. As a consequence, there is increasing research on the differences between production-based versus consump- tion-based trends of material use and its relation to GDP, in order to evaluate whether decoupling is being achieved (UNEP 2011a). 1.1  Policy background Recent results suggest that assessments of decoupling can lead to very different results when measured with different indica- tors. Industrialized countries and regions, such as the USA or the EU, have in general achieved a relative decoupling when measured with indicators of direct material use, i.e., production-oriented indicators. Some countries, such as Japan, were even able to reduce direct material use in absolute terms (Giljum et al. 2014; Steinberger et al. 2013). How- ever, when consumption-based—or footprint-type—indicators are applied, the decou- pling performance is much weaker or even reversed, with material footprints growing faster than GDP (Wiedmann et al. 2015). 1.2  Methodological background A rapidly growing body of literature assessing material flows considers indirect mate- rial flows embodied in internationally traded products and applies consumption-based or footprint-type indicators with the objective of providing complementary information for the design of resource policies. Studies include national assessments (for example, Kovanda and Weinzettel 2013; Schaffartzik et  al. 2014), EU-wide studies (Arto et  al. 2012; Schoer et al. 2012) as well as international assessments (for example, Bruckner et al. 2012; Giljum et al. 2015; Wiedmann et al. 2015). Existing studies so far mostly pro- duced aggregated national indicators on material consumption and resource productiv- ity, making it difficult to use MFA results for policy making, which is often focused on specific sectors or consumption areas. The drivers underlying these aggregated indica- tors as well as the hotspot products and supply chains mostly contributing to the overall results have rarely been investigated (the study by Acosta-Fernández (2007) on identify- ing sectoral hotspots of material use in the German economy and related priority areas of policy action is one of the very few exceptions). A more detailed understanding of developments on the level of economic sectors and supply chains is essential to identify Giljum et al. Economic Structures (2016) 5:17 Page 4 of 24 areas in the production–consumption system, which needs to be transformed, if abso- lute levels of material consumption should be reduced. Several methods are available to calculate consumption-based MFA indicators, each having its advantages and drawbacks (Lutter et  al. 2016; Schoer et  al. 2013). Three methodological approaches have been developed and applied so far: coefficient-based approaches building on process analysis, environmental input–output analysis and hybrid approaches combining elements from both approaches. Coefficient-based approaches allow calculating material footprint-type indicators at a very high level of detail, as coefficients can be calculated for a large number of single products without any restrictions of pre-defined sectors or product groups. However, material input coef- ficients are difficult to calculate for higher manufactured products and services with complex supply chains. Coefficients are often not specific regarding geographical origin of raw materials—in many cases, the geographical origin of a raw material is different to the geographical origin of an imported manufactured good. Furthermore, based on coef- ficient approaches, it is likely that the calculated total of global environmental pressures related to consumption does not equal the pressures of worldwide production. 1.2  Methodological background Input–output analysis, in particular in the context of multi-regional input–output (MRIO) models, has the key advantage of depicting the whole global economy and thus allowing calculating material footprints for all products and sectors independently of the complexity of the underlying supply chains. MRIO models also avoid double counting as supply chains are clearly distinguished from each other. Furthermore, these mod- els allow determining the geographical origin of each raw material related to the final demand of a country or region. For these reasons, in this paper, we apply a MRIO-based approach, based on the database EXIOBASE, which was specifically developed for envi- ronmental-related assessments and distinguishes a large number of different raw materi- als (see Sect. 2 for more details). 2.1  MRIO database For the material footprint assessments in this paper, we apply the MRIO database EXIOBASE (Tukker et al. 2013, 2014; Wood et al. 2015), a very detailed global MRIO database, developed in particular for environmental assessments of the EU. With 200 commodities and 163 industries, of which 33 represent the primary sector of the econ- omy, EXIOBASE provides the highest consistent level of product and sector detail by country across all currently available MRIO models (see Additional file  1: Full list of EXIOBASE products and product groups). In EXIOBASE, agricultural activities are rep- resented by 15 product groups and thus in a similar detail as in the GTAP model (Andrew and Peters 2013). In contrast to most available MRIO tables, which aggregate mining and quarrying of fossil fuels and mineral resources into one or two sectors (see, Giljum et al. 2015), EXIOBASE contains 12 extraction sectors for fossil fuels, 8 metal extraction sec- tors and three sectors representing the extraction of industrial and construction minerals. It thus represents a major advantage in terms of detail for assessing global material flows. l On the downside, EXIOBASE only details 44 countries (28 EU member states plus 16 major trade partners of the EU, together adding up to around 95 % of global GDP) and covers all other countries in the form of five rest-of-the-world regions (see Addi- tional file 2: Full list of EXIOBASE countries and regions). In contrast, the Eora database (Lenzen et al. 2013) distinguishes 187 countries/regions, and the latest version of GTAP (Narayanan et  al. 2015) separately models 140 countries and regions. However, for assessments of EU’s global environmental impacts, EXIOBASE is a very suitable data- base. In this paper, we aggregated all results of the EU-28 countries into one region, in order to provide an overall assessment of the EU as a whole. Future research may focus on specific patterns of single EU countries. i Furthermore, a high level of regional detail as provided by Eora or GTAP is required to avoid errors caused by product and price inhomogeneity between countries in a cer- tain region and becomes relevant when there are large trade flows from those regions. 1.3  Objectivesh This paper provides a detailed assessment of the composition of the EU’s material con- sumption in a global context, with the aim to identify primary product groups con- tributing to the EU’s global material footprint. We further aim at assessing the supply chain structure of various product groups, thus identifying in which parts of the supply chain major material extractions take place. The temporal and geographical coverage of EXIOBASE further allows for the assessment of changes over time, for example regard- ing the importance of raw material inputs stemming from non-European countries. This analysis elucidates whether outsourcing of environmental pressures through inter- national trade occurred and illustrates which world regions serve as major suppliers of raw materials directly and indirectly required to satisfy EU’s final demand. Based on the empirical assessment, we aim at reflecting current priority areas of EU resource policy making and at deriving recommendations for the priority areas and topics the EU should focus in its future policy efforts. f The remainder of the paper is structured as follows: Section 2 starts with a descrip- tion of the MRIO and material flow data as well as of the applied analytical methods. Section  3 presents the various results from the MRIO assessment both on the level of overall EU material consumption and on the level of different product groups and Page 5 of 24 Giljum et al. Economic Structures (2016) 5:17 their underlying supply chains. It also discusses the results in the context of current EU resource policy priorities. The final Sect. 4 concludes. 2.2  Material extraction data and indicatorsh The material extensions in EXIOBASE were compiled using the WU Global Material Flow database (WU 2015, available at www.materialflows.net). This database contains material extraction data for all countries worldwide, for all raw material categories and in annual time series from 1980 to 2013. The database is compiled following the stand- ards of economy-wide material flow accounting (EW-MFA) provided by Eurostat and the OECD (EUROSTAT 2013; OECD 2007). Four main data sources are used for the compilation of the database—the British Geo- logical Survey (BGS) and the US Geological Survey (USGS) for metal and mineral data; the International Energy Agency (IEA) for the data on fossil fuels; and the FAO for the data on biomass extraction. The quality of the material extension data varies across the different material categories, depending on the data source and necessary estimation or manipulation steps. In general, the primary data from BGS, USGS, IEA and the FAO are of good quality. However, for some types of material extraction, estimation techniques need to be applied due to a lack of official statistics. This holds especially true for the cases of the extraction of construction minerals, biomass uptake by rooming animals (grazing) and the generation of by-products from agricultural harvest. For more infor- mation on the compilation of the underlying material flow database, for example, regard- ing the treatment of coupled production in metal mining, see Additional file 3: Technical details on the compilation of the global material flow database and WU (2015). l In order to generate the physical satellite to the MRIO system, the around 380 catego- ries of primary raw material extraction data are aggregated into the 33 extraction sectors of EXIOBASE (see Additional file 4: Correspondence table between raw materials and EXIOBASE sectors). All MFA data are fed into the MRIO system in the unit of 1000 t. The main indicator generated with the MRIO system is the economy-wide indicator raw material consumption (RMC), also known as material footprint (Giljum et al. 2015; Wiedmann et al. 2015). The RMC or material footprint indicator comprises all domes- tically and foreign raw materials that were directly and indirectly required to produce goods and services of the EU final demand. The material footprint thus expresses the amounts of materials embodied in final demand and aims at understanding the socio- economic drivers, such as private or public consumption, of raw material extraction. 2.1  MRIO database We argue that for the analysis of the flows of raw materials, limited trade flows to the EU come directly from the aggregate regions, and because those materials traded have prices being mainly determined on global markets, price differences between countries within an aggregate region will be smaller compared to price differences between raw materials. Consequently, for this study a high level of commodity detail is regarded as more relevant. In this paper, we apply EXIOBASE in its version 3.1, covering the time period of 1995 to 2011. In order to adequately represent the global economic structure over time, EXIOBASE 3.1 combines several data sources to capture economic changes. Principle data include macroeconomic accounts (GDP, total trade, final demand from UN SNA database) and other detailed data sources (IEA, FAOSTAT, Comtrade, various mining databases, BACI and others) to estimate product and industry output over time as well as product-specific bilateral trade. The structural change of domestic economies was estimated by inter/extrapolate observed changes of national IO tables over time (for 38 Giljum et al. Economic Structures (2016) 5:17 Page 6 of 24 of the 44 EXIOBASE countries, i.e., excluding Mexico, Luxembourg, Croatia, Indone- sia, Turkey and Canada). Inconsistencies within the database caused by conflicting data sets were resolved by balancing the systems using a flexible mathematical programming approach where information gain is minimized (Lenzen et al. 2009). The estimation of the uncertainty associated with such techniques has not received a great deal of atten- tion in research, albeit Lenzen et al. (2010) have attempted to capture some of the impact of these techniques for the case of greenhouse gas emissions. 2.3  Analytical methods The data analysis starts on the aggregated level illustrating the overall material footprint time series of the EU followed by decompositions into the four main material categories, into sectors and product groups as well as into source countries for raw materials. The latter are identified for the total material footprint and separately for each aggregated material group. This is carried out for the whole time period of 1995–2011 in order to determine whether and to which regions the EU has outsourced material extraction via international trade. We also analyze the sector composition, identifying the sec- tor groups contributing most to the EU’s material footprint, and how this composition changed over time, thus analyzing structure and temporal dynamic of the EU material footprint. Apart from these standard analyses, MRIO models representing the entire global economy and all underlying supply chains allow for the application of additional ana- lytical tools. These tools assess supply chain structures underlying the total material footprint of a product or product group and thus visualize the type and complexity of intermediate deliveries. In this paper, we apply production layer decomposition, PLD (compare Lenzen and Crawford 2009; Llop and Ponce-Alifonso 2015; Wiedmann et al. 2009), which is an aggregated form of structural path analysis (SPA). SPA allows open- ing up the black box of national and international supply chains uncovering the larg- est inter-sector and inter-country pathways of flows of goods and services through the global economy (Lenzen 2007; Peters and Hertwich 2006). The benefit of the very high level of detail through identifying all paths in the global economy related to final demand in a certain country or region entails the disadvantage of producing very large data sets, in particular when an SPA is performed for various types of raw materials and with time- series data. Therefore, in order to derive more general conclusions from the MRIO model in terms of the biophysical dimension of supply chain structures and temporal dynamics, in con- trast to SPA, production layer decomposition does not calculate the “weight” of single paths but aggregates all material extractions occurring at discrete supply chain layers. In its core, PLD can be expressed as: (1) ek,m = ˆsmAk ˆy ek,m = ˆsmAk ˆy (1) where ek,m equals the material extraction of raw material group m on layer k along the path of a product or product group, which directly or indirectly serves final demand. 2.2  Material extraction data and indicatorsh It is important to note that this approach differs from other types of material flow analysis, which aim at physically tracking actual flows of certain raw materials or substances (for example, Graedel et al. 2015; Laner et al. 2015). Furthermore, it needs to be emphasized that the approach applied in this paper focuses on the footprint of primary materials. Integrating secondary raw materials from recycling in this assessment would require additional material flow data (see Haas et al. 2015). Page 7 of 24 Giljum et al. Economic Structures (2016) 5:17 In MFA, indicators are generally decomposed into four major material categories, i.e., biomass, fossil fuels, minerals and metal ores. In this paper, we present indicators both on the aggregated level across all materials and on the level of the four main material groups. 2.3  Analytical methods ˆs represents the diagonalized direct intensities of material group m, which results from dividing the domestic extraction vector of m by the sectors gross output vector x, and where ˆx = Lˆy, with L being the Leontief inverse matrix and ˆy the diagonalized final demand vector. Subsequently, ˆse = ˆmˆx−1. A is the technical coefficients matrix. For the case of layer 0, i.e., direct deliveries material extraction sectors to final demand: ek=0,m = ˆsmIˆy, where I represents the identity matrix. where ek,m equals the material extraction of raw material group m on layer k along the path of a product or product group, which directly or indirectly serves final demand. ˆs represents the diagonalized direct intensities of material group m, which results from dividing the domestic extraction vector of m by the sectors gross output vector x, and where ˆx = Lˆy, with L being the Leontief inverse matrix and ˆy the diagonalized final demand vector. Subsequently, ˆse = ˆmˆx−1. A is the technical coefficients matrix. For the case of layer 0, i.e., direct deliveries material extraction sectors to final demand: ek=0,m = ˆsmIˆy, where I represents the identity matrix. Page 8 of 24 Giljum et al. Economic Structures (2016) 5:17 Due to the fact that there are an infinite number of layers, we calculate the residual er+ via via (2) Lr+ = L −I − r  k=1 Ak and (2) Lr+ = L −I − r  k=1 Ak (3) e ˆs L ˆy Lr+ = L −I − r  k=1 Ak (2) and (3) er+,m = ˆsmLr+ˆy er+,m = ˆsmLr+ˆy (3) where r is the selected threshold regarding the number of layers separately analyzed. where r is the selected threshold regarding the number of layers separately analyzed. We can then relate the single-layer results ek to the overall material footprint MF through their summation: (4) MF = r−1  k=0 ek  + er+ (4) Moreover, we can use production layer decomposition to disaggregate the individual layer results into the amount of raw materials extracted within and outside the EU. 3.1  Resultsh The results section of the paper is structured by starting with aggregated results for the EU material footprint and subsequently decomposing the overall trends in various dimensions. The first perspective is the origin of the various raw materials related to EU final demand, followed by an analysis of the main products and product groups contrib- uting to the EU material footprint. Based on the PLD, we then illustrate the structure of supply chains behind each of the aggregated product groups and assess how the layer structure evolved over time. For selected product groups, we also discuss how the distri- bution between materials extracted in the EU versus abroad changed on each layer from 1995 to 2011. Finally, we provide a disaggregation of the material footprint layers by the main material categories, thereby analyzing the structural differences in the use of cer- tain material groups in the economic system. Figure 1 illustrates the overall development of the material footprint of the EU, disag- gregated by the four main material groups (top) and by the countries and world regions, where the raw materials were extracted to directly and indirectly serve final demand in the EU (bottom). The overall material footprint of the EU rose from 8.4 billion tonnes in 1995 to 12.2 billion tonnes in 2011. After a 5-year period of relatively strong increases between 2002 and 2007, the impact of the economic crisis in 2008 and in particular in 2009 is clearly visible in the overall trend. In these two years, the EU material footprint dropped by around 13 % compared to the maximum level of 2007. In 2011, the EU material footprint regained an absolute level that was equal to the level in 2005/2006. When analyzed by main material groups, it is clearly visible that the category of industrial and construction minerals contributes the largest share to the material foot- print (54 % in 2011, up from 43 % in 1995). Minerals were also the group that most Page 9 of 24 Giljum et al. Economic Structures (2016) 5:17 Fig. 1  Overall material footprint of the EU-28, by main material category (top) and main countries of origin of raw materials (bottom), 1995–2011, in billion tonnes Fig. 3.1  Resultsh 1  Overall material footprint of the EU-28, by main material category (top) and main countries of origin of raw materials (bottom), 1995–2011, in billion tonnes significantly declined in the period of the economic crisis, contributing around half of the overall reduction between 2007 and 2009. This indicates the strong impact of the crisis on the construction sector, in particular in countries such as Spain, Italy or the UK, but also in some Eastern European countries such as Romania. Direct and indirect extraction of biomass grew by 35 % over the whole time period, and the required metal ores to satisfy EU final demand was increased by 116 %, but from a lower absolute level. Fossil fuels were the only category where the EU consumed less in 2011 compared to 1995 (−14 %). From the perspective of the origin of raw materials, dramatic shifts can be observed over the analyzed time period. The share of raw materials that were extracted within the territory of the EU fell significantly from 68 % in 1995 to 35 % in 2011. This illustrates that today around two-thirds of the raw materials that are directly and indirectly needed to satisfy European final demand is extracted in other world regions. As illustrated in Fig. 1 (bottom), the most significant shift occurred between the EU and China, which in 2011 already contributed an equal amount of raw materials related to EU’s final demand Page 10 of 24 Giljum et al. Economic Structures (2016) 5:17 for goods and services. This particular pattern is closely related to the current develop- ment stage of China, which—as a fast-growing emerging market—is in the process of building up its housing, transport and energy infrastructure. On the one hand, this leads to significant amounts of raw materials required for investments and capital formation, which dominate China’s own material footprint (see Giljum et al. 2015). On the other hand, construction materials also serve as indirect inputs to manufacturing industries, which produce goods exported to the EU (see also Wang et al. 2014; Wiedmann et al. 2015). This indirect flow of construction materials grew from around 230 million tonnes in 1995 to more than 3 billion tonnes in 2011 and was thus far above flows of other key raw materials extracted in China and serving EU final demand, such as coal (394 million tonnes) or iron ores (157 million tonnes). 3.1  Resultsh As the material footprint calculations can be performed separately for various raw materials and then be aggregated into the four major material groups, Fig. 2 depicts the regional origin of raw materials serving the EU final demand for biomass, fossil fuels, minerals and metal ores. The overall development as well as the regional patterns of origin differs signifi- cantly across the four main material categories. However, two general trends can be observed for all four raw material groups. First, extraction of raw materials within the EU decreased, while the material footprint increased for all four categories except fossil fuel consumption, which in 2011 was on an equal level as in the mid-1990s. This implies that the EU is to an increasing extent directly as well as indirectly dependent on imports from other world regions. Second, the Asia and Pacific region serves as the main source Fig. 2  Biomass, fossil fuels, minerals and metal footprint of the EU-28, by main world region of origin of raw materials, 1995–2011, in million tonnes Fig. 2  Biomass, fossil fuels, minerals and metal footprint of the EU-28, by main world region of origin of raw materials, 1995–2011, in million tonnes Giljum et al. Economic Structures (2016) 5:17 Page 11 of 24 of raw materials embodied in European imports across all four major categories, with China playing an outstanding role. Biomass is the material category, where the highest share of raw materials still stems from within the EU territory. The share was 76 % in 1995 and decreased to 47 % in 2011. With 35 % in 2011, biomass originated to a significant extent from Asia, up from 15 % in 1995. China played an important role contributing 16 % to biomass extraction in 2011, in particular related to the production of cattle and other meat. Three percentage of bio- mass in the EU material footprint was extracted in India with a similar product focus as in the Chinese case. With 1 %, Indonesia was the third largest Asian contributor, with a clear focus on the sector of “oil seeds,” i.e., palm oil. Smaller amounts of biomass were embodied in imports from America and Africa. Fossil fuels extraction within the EU decreased significantly, from around 900 million tonnes in 1995 to around 570 million tonnes in 2011, in particular due to a decrease in coal mining. 3.1  Resultsh With 41 %, the Asia and Pacific region made up the largest fraction of fos- sil fuels embodied in EU final demand in 2011. Fossil fuels embodied in imports from China were rapidly increasing in the 2000s and made up 23 % of the total fossil fuel foot- print of the EU-28 in 2011. These fossil fuels included, for example, coal (394 million tonnes in 2011) that was extracted in China and used in electricity production, which is then embodied in Chinese exports. Russia also plays an important role as a direct exporter of particularly natural gas to the EU. The Middle East contributed 15 % to the fossil fuel footprint in 2011. Minerals extraction within the EU also showed a decreasing trend, with a particu- larly sharp decrease during the years of the economic crisis, underpinning the argument above that the construction sector in many EU countries was one of the most effected parts of the economies. At the same time, the minerals embodied in imports from the Asia and Pacific region grew almost by a factor of 5. With more than 3 billion tonnes in 2011, China alone contributed 48 % to the total EU mineral footprint, almost exclusively in the categories of construction materials, such as sand, gravel and stone. From a European policy perspective, the situation regarding metal ores can be regarded as most critical, as Europe has only a minor production of metals within its territory and is thus heavily dependent on imports from other world regions. In 2011, only 13 % of all metals required as direct and indirect inputs for products and services finally consumed in the EU stemmed from within the European territory. Again, the Asia and Pacific region plays the most important role as a raw material provider (50 %). Most important extrac- tion countries are China, which accounts for 27 % of total EU metal footprint, mostly in the categories of iron ore and copper. Australia delivered 6  % of metals serving as indirect inputs to EU final demand, with iron ore, copper and precious metals being the most important commodities. Three percent of the metal footprint originated in Indone- sia, dominated by lead and zinc. Thirty-two percentage of metals embodied in EU’s final demand were extracted in the Americas, with Latin American countries providing almost three quarters, mostly iron ore, copper, lead, zinc and precious metals. In Fig. 3.1  Resultsh 3, we turn to another dimension, which the MRIO model allows to put into focus, i.e., the product groups contributing to the material footprint of the EU. In order to facilitate visualization, we aggregated the 200 EXIOBASE products into 11 broad product groups (see Additional file 1: Full list of EXIOBASE products and product groups). Page 12 of 24 Giljum et al. Economic Structures (2016) 5:17 Fig. 3  Sectoral composition of the EU-28 material footprint, 1995–2011 Fig. 3  Sectoral composition of the EU-28 material footprint, 1995–2011 Figure  3 illustrates that the composition of the EU material footprint in terms of aggregated product groups remained relatively stable across the observed time period, despite the overall growing material footprint (compare Fig. 1 above). The main aggre- gated sector contributing to the material footprint was construction, which held a share of 25 % in 1995, falling slightly to 23 % in 2011. Construction was followed by the group of manufacturing products based on biomass, most importantly from food-processing sectors. Its share was 18 % in 2011, similar to the contribution in 1995. Manufactur- ing of products mainly based on fossil fuels ranked third in 2011, with a share of 14 %. Most important products were motor gasoline with a material footprint of 189 million tonnes, followed by diesel oil (92 million tonnes). The fourth biggest contributing sec- tor was the large group of manufacturing products based on metals and minerals (8 %). With a material footprint of 393 million tonnes, motor vehicles were the most important product group within this aggregated sector, followed by machinery and equipment (232 million tonnes) and radio, television and communication products (129 million tonnes). The contribution of agriculture and forestry products to the EU material footprint in 2011 was 5 %, than that of mining products 3 %. Although raw materials are extracted only in these two aggregated sectors (see Fig. 4; Table 2), the overwhelming part of the extracted raw materials are not directly delivered to final demand, but purchased by other sectors of the economy for further processing. Materials embodied in service sec- tor activities together contributed 25 % to the total material footprint, as the group of “other products” mainly contains service sectors, including the large service sectors of health, education and public administration and defense (see Table 1 for more details). 3.1  Resultsh As indicated in the introduction, one key strength of EXIOBASE is the very high prod- uct detail. In Table 1, we further decompose the product groups contributing to the EU material footprint and show the top 30 EXIOBASE products (the full list with the foot- prints all EXIOBASE products can be found in the Additional file 5: Detailed material footprint (MF) results). These 30 products made up around 80 % of the total EU material footprint in 2011. Table 1 indicates that among the top 30 contributing products, almost all of the 11 aggre- gated product groups are represented. The product “construction work,” which is iden- tical to the aggregated group “construction” as illustrated above, constitutes by far the Page 13 of 24 Giljum et al. Economic Structures (2016) 5:17 Fig. 4  Production layer decomposition of material footprint of EU-28 aggregated product groups, 2011 largest product group in the overall EU material footprint (23.2 % in 2011). Construction had both a high share in total EU final demand (9.9 %) as well as a high material intensity or almost 2.3 kg of direct and indirect material extraction required to produce one Euro of final demand. Given this huge contribution of just one product, it would be desirable to further split up this product group. It is interesting to note that on the more detailed sector level, service sectors are also found among the top contributors. For example, health and social work services ranked second in the product list, with a contribution of 6.6 % to the total material footprint in 2011. This sector contributed 12.8 % to EU’s monetary final demand in that year; however, material intensity was significantly lower (half a kg of material input per €). The reason, why this sector has a significant material footprint is that the provision of services in the health sector requires a large number of material-intensive products serving as indirect inputs, including, for example, chemical and pharmaceutical products. Also, food provi- sion in hospitals and other social institutions induces significant flows of biomass, with meat and animal products contributing most to the biomass footprint. Apart from health and social services, also public administration and defense (4.5 %) as well as real-estate services (3 %) has significant shares in the overall material footprint of the EU-28. 3.1  Resultsh i Following aggregated product groups such as “food products nec” and “chemicals nec,” some specific manufacturing product groups are also located among the top contribu- tors. They include motor vehicles (with a share of 3.2 %) and machinery (1.9 %). Ani- mal products, which are well known for their high material footprint (Lettenmeier et al. 2014), are not found among the top 10 EXIOBASE products. This fact is a consequence of the construction principles of EXIOBASE, which further split up environmentally sensitive products. In fact, EXIOBASE contains 11 products related to production of meat and dairy products. If the material footprint of these sectors is again aggregated, animal products together contribute almost 900 million tonnes, or 7.1 %, to the total material footprint of the EU-28 and rank second after construction. Page 14 of 24 Giljum et al. Economic Structures (2016) 5:17 Table 1  Top 30 products contributing to the EU-28 material footprint (MF), values of final demand (FD) and material intensities (kg/€), 1995 and 2011 Product Product group MF 1995 MF 2011 % change FD 2011 Intensity 2011 1000 t 1000 t 1995–2011 million € kg per € Construction work Construction 2,135,125 2,834,192 33 1,239,222 2.29 Health and social work services Other products 371,974 799,155 115 1,607,816 0.50 Food products nec Manuf. products (biomass) 542,018 654,621 21 280,307 2.34 Public administration and defense services Other products 357,479 543,163 52 1,036,643 0.52 Chemicals nec Manuf. products (fos- sil fuels) 234,641 509,761 117 157,823 3.23 Motor vehicles, trailers and semi-trailers Manuf. products (metals/min) 217,156 393,023 81 348,919 1.13 Real-estate services Financial services 311,943 362,505 16 1,113,134 0.33 Furniture and other manufactured products nec Manuf. products (metals/min) 149,487 277,609 86 240,462 1.15 Membership organization services nec Other products 128,809 236,885 84 455,624 0.52 Machinery and equip- ment nec Manuf. products (metals/min) 194,722 232,110 19 206,048 1.13 Education services Other products 145,041 222,463 53 893,586 0.25 Recreational, cultural and sporting services Other products 117,288 218,279 86 496,697 0.44 Vegetables, fruit, nuts Agri. and for. prod- ucts 170,744 193,087 13 79,544 2.43 Motor gasoline Manuf. products (fos- sil fuels) 229,335 188,913 −18 129,213 1.46 Products of meat cattle Manuf. products (biomass) 176,350 185,881 5 25,177 7.38 Hotel and restaurant services Sales and retail services 108,763 175,816 62 605,336 0.29 Wearing apparel; furs Manuf. products (biomass) 109,155 170,524 56 160,629 1.06 Meat products nec Manuf. products (biomass) 93,166 155,681 67 75,501 2.06 Fish products Manuf. 3.1  Resultsh products (biomass) 77,426 155,457 101 89,126 1.74 Products of meat poultry Manuf. products (biomass) 79,197 136,148 72 72,534 1.88 Radio, television and communication equip- ment Manuf. products (metals/min) 54,644 129,304 137 128,149 1.01 Dairy products Manuf. products (biomass) 80,227 122,533 53 78,554 1.56 Electricity by coal Electricity and water supply 107,615 115,171 7 14,537 7.92 Distribution services of gaseous fuels Electricity and water supply 125,924 114,488 −9 67,903 1.69 Fabricated metal prod- ucts, except machinery Manuf. products (metals/min) 66,117 104,590 58 79,618 1.31 Transport services equip- ment Manuf. products (metals/min) 38,244 99,858 161 88,686 1.13 Basic plastics Manuf. products (fos- sil fuels) 51,881 99,469 92 89,099 1.12 Table 1  Top 30 products contributing to the EU-28 material footprint (MF), values of final demand (FD) and material intensities (kg/€), 1995 and 2011 Page 15 of 24 Giljum et al. Economic Structures (2016) 5:17 Table 1  continued Product Product group MF 1995 MF 2011 % change FD 2011 Intensity 2011 1000 t 1000 t 1995–2011 million € kg per € Sand and clay Mining products 110,445 97,418 −12 1354 71.93 Ceramic goods Manuf. products (metals/min) 54,608 96,508 77 8511 11.34 Leather and leather products Manuf. products (biomass) 44,832 95,763 114 59,771 1.60 Other product groups 1,741,897 2,472,081 42 2,603,563 Total footprint 8,426,255 12,192,456 45 12,533,084 Table 1  continued The material intensities differ significantly across the various products. More than 7 kg of material extraction served as direct and indirect material input to produce one € of final demand of cattle meat. For vegetables and fruits, it was almost 2.5 kilograms. To produce one € of final demand of motor vehicles, around 1.1 kg of raw materials were required. The highest material intensities can be observed for mining activities, such as the extraction of sand and clay (almost 72 kilograms of extraction per € of final demand in 2011). Production layer decomposition allows investigating the material dimension of supply chains and illustrating the structure and complexity of intermediate deliveries behind the final use of a certain product or service. The numbering of the layers reflects the distance to the extraction of the various raw materials required to produce the final product, i.e., the number of intermediate deliveries before a product or service ends up in final demand. Figure 4 depicts the composition of all layers of the respective supply chains to the absolute material footprints of the 11 aggregated product groups. 3.1  Resultsh All layers together constitute the total footprint of a product group. In order to keep complexity on a manageable level, we illustrate Layers 0 to 4 separately and add up all higher levels in the category Layer 5+. Table 2 complements the information in Fig. 4 by detailing the percentage contributions of each layer to the product group total, both in the year 1995 and in 2011. As explained in Methods section above, Layer 0 only contains values for those product groups, which are directly related to material extraction activities, i.e., biomass extrac- tion from agriculture and forestry as well as mining of fossil fuels, minerals and metal ores. These two aggregated product groups are thus the only ones that have direct deliv- eries from material extraction to final demand. For agriculture and forestry products, the share of Layer 0 was 56 % in 2011, for mining products 72 %. Material extractions taking place on Layer 1 are located one step away from the prod- uct group in focus, i.e., they represent the final intermediate deliveries before a product is being brought to its final purpose. Taking the example of the group of manufacturing products from biomass, we observe that Layer 1 has the largest contribution (31 % in 2011). These are mainly deliveries of biotic raw materials from the agricultural sectors to food-processing sectors as well as of harvested timber to sectors producing wood-based products. Page 16 of 24 Giljum et al. Economic Structures (2016) 5:17 Table 2  Share of layers in material footprint of product groups, 1995 and 2011 Layer 0 (%) Layer 1 (%) Layer 2 (%) Layer 3 (%) Layer 4 (%) Layer 5  + (%) Material footprint (1000 t) Agri. and for. products 1995 69 12 8 5 3 4 543,263 2011 56 13 11 7 5 8 638,561 Mining products 1995 78 15 3 2 1 1 295,577 2011 72 17 4 2 2 3 334,663 Manuf. products (biomass) 1995 0 44 21 14 8 12 1,521,136 2011 0 31 22 16 11 20 2,169,646 Manuf. products (metals/min) 1995 0 37 25 15 9 14 713,056 2011 0 31 26 16 10 18 1,004,042 Manuf. 3.1  Resultsh products (fossil fuels) 1995 0 17 24 21 14 24 996,043 2011 0 13 20 19 15 33 1,742,524 Electricity and water supply 1995 0 60 20 9 5 7 353,418 2011 0 51 18 11 7 13 370,134 Construction 1995 0 38 36 14 6 7 2,135,125 2011 0 31 33 16 8 12 2,834,192 Sales and retail services 1995 0 9 40 21 12 18 126,164 2011 0 5 33 22 14 26 194,515 Transport services 1995 0 10 28 22 15 24 95,321 2011 0 6 22 20 16 35 178,545 Financial services 1995 0 11 33 25 13 18 458,940 2011 0 7 23 23 16 31 608,436 Other products 1995 0 12 31 22 14 21 1,188,212 2011 0 6 24 23 16 31 2,117,199 Table 2  Share of layers in material footprint of product groups, 1995 and 2011 Layer 2 contains extractions of raw materials, which are two intermediate steps away from the final product. An example from the construction sector, which is characterized by the largest share of inputs from Layer 2 across all aggregated product groups (33 % in 2011), is the sequence of limestone being extracted in mining, delivered as an input to cement production, with cement then purchased by construction activities. With regard to the composition and temporal development of the structure of sup- ply chains across the various layers, two key observations can be drawn from Table 2. First, the distribution across the 5 analyzed layers changes across sectors, as supply chains increase in complexity. While products from the primary sectors contain by far the largest parts of inputs on Layer 0, the manufacturing sectors typically peak on Layers 2 or 3. Services have an even higher share of high-layer extractions in their total material Page 17 of 24 Giljum et al. Economic Structures (2016) 5:17 footprint. The second important conclusion concerns the changes of layer structures over time. Table 2 illustrates that in the past 20 years, supply chains across all product groups have become more complex, i.e., percentage contributions of layers with lower numbers were declining in almost all cases, whereas Layers 3–5 + gained in importance. This illustrates that supply chains are increasingly complex and differentiated in their organization, and an ever-increasing number of production steps is interlinked for the production of goods and services delivered to final demand. 3.1  Resultsh i Figure 5 takes the analysis one level further by illustrating to what extent the changes in the supply chain structures were linked to the overall trends of outsourcing mate- rial extraction away from the EU territory to other world regions discussed for the over- all material footprint in Figs. 1 and 2. For illustrative purposes, we select two product groups with a major importance in the overall EU material footprint, i.e., construction and manufactured products based on biomass. Figure  5 illustrates that significant geographical shifts can be observed on all lay- ers when comparing the years 1995 and 2011, i.e., the contribution of material extrac- tions within the EU was shrinking, while extractions in the non-EU countries gained in importance (compare the aggregated results in Figs. 1 and 2 above). Construction is a pronounced example: While 83 % of extractions as inputs to construction activities— in particular on Layers 1 and 2—were taking place within the EU borders in 1995, this value decreased to only 38 % in 2011. In the latter year, significant material extractions took place in the non-EU countries on all layers. For example, on Layer 1, large material extractions outside the EU took place regarding construction minerals, such as sand and clays, which are then further processed into construction materials imported into the EU. On Layer 2, metal ores used in construction, notably iron ore, as well as fossil fuels (coal and oil) serving as energy inputs into manufacturing of construction materials play major roles. Fig. 5  Material extraction per layers in the EU versus the non-EU countries for construction and biomass- based manufactured products, 1995 and 2011 Fig. 5  Material extraction per layers in the EU versus the non-EU countries for construction and biomass- based manufactured products, 1995 and 2011 Page 18 of 24 Giljum et al. Economic Structures (2016) 5:17 A similar, but less accentuated development could be observed for the group of manu- factured products from biomass. Also here the ratio of domestic to foreign extractions changed from 73:27 % in 1995 to 40:60 % in 2011. However, there are still significant shares of extraction within the EU on Layer 1, indicating that the EU food- and wood- processing industries are still to an important extent supplied by agricultural and for- estry products harvested within the EU. 3.2.1  Priorities for European resource policies The results generated with the global MRIO model with detailed material extensions as applied in this paper confirm the priorities of EU resource policy initiatives with regard to product groups, i.e., we find that construction as well as the group of manufactured biomass-based products are the top 2 product groups constituting EU’s material foot- print. Transport services as such play only a minor role as a final demand category; however, construction activities also cover the resources used in developing transport infrastructure. Our analysis illustrated that there are important product groups in addi- tion to the commonly known high-impact activities of construction, food and mobil- ity. Most notably, we found that, with around a quarter of the total, the service sectors contribute a significant share to the overall material footprint of the EU. Examples for service sectors with high absolute importance are health and education services, public administration and defense as well as real-estate services. These activities are character- ized by very complex supply chains, i.e., the provision of services typically requires a large variety of highly manufactured products as inputs. As the layer analysis illustrated, these products are characterized by a significant number of processing stages. The tran- sition toward a “knowledge-based service economy” is often assumed to be an environ- mentally positive development. While services do have a lower material intensity per € of generated added value compared to other parts of the economy (see Table 1 above and the detailed table in the Additional file 5), the growing absolute importance of the service sectors in generating European GDP, however, results in high absolute material consumption of services. This is in line with similar finding regarding the carbon foot- print of countries (Hertwich and Peters 2009; Piaggio et al. 2015). A service-oriented economy thus is not automatically dematerialized or decarbonized, when taking a full supply chain perspective. This fact is currently being overlooked in the European resource policy context. Production layer decomposition, which revealed the underlying structure of supply chains for different product groups as well as for aggregated raw material groups, also provides important insights for EU resource policies. On the one hand, the disaggre- gation by material groups suggests that resource policies should take into account the different levels of complexities of supply chains. 3.1  Resultsh However, raw materials for higher processed inputs to the production processes of biomass-based manufactured products are origi- nating to a growing extent outside the EU. This trend can also be observed for other product groups. In the final analytical step, we disaggregate the information from the layer analysis by the four main material groups of MFA. Figure 6 illustrates on which of the layers each of the four raw material groups was mainly extracted to serve the overall EU final demand for products and services in the year 2011. We can observe different patterns across the four aggregated material categories. The use of minerals shows the steepest distribution across all layers, peaking at Layer 2. This implies that products being produced from extracted mineral resources have relatively short supply chains (compare also the layer structure of the construction sector in Fig. 4 and Table 2). This is not surprising, as the major products from minerals are construc- tion and industrial minerals, which are either directly delivered from the mining sector (e.g., sand and stones) or delivered from the sectors producing further processed con- struction materials, such as cement. Biomass also has a curve, which peaks at Layer 1, indicating that there are only one or two steps of processing before the largest share of extracted biomass is being delivered to final demand (compare the structure of the agri- cultural/forestry product group and the biomass-based manufactured products in Fig. 5 above). The distribution of the other two main material categories, i.e., fossil fuels and metal ores, shows a different pattern. Their slope is much lower, indicating that a larger fraction of the overall fossil and metal footprint is generated on higher layers, i.e., further away from the final product. Fossil fuels and metal ores thus mainly serve as inputs to goods and services with more complex supply chains, indicating their fundamental char- acter for the production processes of various sectors across the whole economy. Fig. 6  Distribution of main material groups across production layers, EU-28 material footprint, 2011 Page 19 of 24 Giljum et al. Economic Structures (2016) 5:17 3.2.1  Priorities for European resource policies Products based on minerals and bio- mass are characterized by less complex intermediate delivery structures compared to fossil fuels and metal ores, which serve as inputs for a large number of different products across numerous economic sectors. Given the very high levels of import dependency in these two product groups (compare Fig. 2 above), this puts the EU in a vulnerable position. On the other hand, the layer analysis illustrated that an increasing share of materials is extracted outside the EU on each of the single layers, thus pointing to the importance of considering the overall supply chain, if an absolute reduction in global material extraction related to EU consumption should be achieved. Secondary materials from recycling activities were not explicitly considered in this study, which focused on the material footprint of primary extraction. Recent estimates illustrate that there exist huge potentials to increase the degree of circularity in the European economy. The aggregated recycling rate, calculated as the amounts of recy- cled materials as a share of all processed materials, was only 12.6 % in 2005 (Haas et al. Giljum et al. Economic Structures (2016) 5:17 Page 20 of 24 2015). An ambitious implementation of the circular economy package would reduce the European demand for primary raw materials and thus would also decrease the mate- rial footprint as presented in this paper. Moving to a circular economy is therefore also closely connected to economic issues, such as import dependence and stable supply of raw materials for European industries (European Commission 2015). The global impacts related to material consumption in Europe are mentioned in some of the EU policy documents, most notably with regard to biomass consumption. How- ever, the results of the MRIO-based assessments in this paper suggest that a supply chain perspective is of key importance for the EU economy, which has a higher material import dependence than all other world regions (see Giljum et al. 2015). The consump- tion of goods and services in the EU is to a large and rapidly growing extent dependent on indirect material extractions taking place outside the EU’s territory. This has environ- mental implications, as raw materials originate to an increasing extent from develop- ing and emerging economies, which often have lower levels of environmental standards compared to industrialized countries (for example, Hilson 2012). 3.2.1  Priorities for European resource policies The expansion of raw material extraction activities also often leads to an increasing number of environmental conflicts with the local population, which faces, e.g., dislocations due to expansions of mining areas or accumulations of air and water pollution with severe negative effects on their daily lives (Martinez-Alier 2014). Thus, if considering pollution and other negative environmental consequences related to material extraction in impact-oriented material footprint indicators (van der Voet et al. 2009), the geographical location of raw mate- rial extraction could play an increasingly important role in determining Europe’s envi- ronmental performance. Also, labor conditions are often very low in developing country activities related to agricultural production or mining, and material extraction often fos- ters enclave types rather than inclusive forms of development (for example, Bird 2014). As a final consumer of products to which these raw materials serve as direct and indirect inputs, Europe should acknowledge its share in the responsibility for the environmen- tal and social conditions in other world regions. Europe could more actively engage in financial, technological and knowledge support in extraction countries to raise environ- mental and social standards, as underlying, for example, the idea of a Global Marshall Plan (Yunker 2014). This responsibility is currently not sufficiently assumed in EU poli- cies related to raw materials. For example, the raw material initiative is strongly focused on securing access to resources in particular in developing countries, which sometimes contradicts the objectives of EU development policies, which aim at helping develop- ing countries combat poverty through investing in the sustainable management of their natural resources (Heinrich Böll Foundation 2011). 3.2.2  Methodological aspects The MRIO analysis performed in this paper allocates material extraction due to eco- nomic activities through the global supply chains to the final consumers. As such, MRIOs foremost rely on reliable data on domestic material extraction, as any errors or uncertainty in the extraction data are passed through the full analysis and directly affect the footprint results. Although extraction data are similar across global databases with regard to categories such as agricultural production or extraction of fossil fuels (Fis- cher-Kowalski et al. 2011), estimations need to be applied for quantitatively important Giljum et al. Economic Structures (2016) 5:17 Page 21 of 24 categories, such as construction materials (see above and Additional file 3 for details). This calls for further international efforts to align existing estimation approaches, including the consideration of recycled construction materials (Hashimoto et al. 2009; Wiedenhofer et al. 2015). Allocating material extraction to those industries or users extracting the material requires detailed knowledge about the composition of each industry—data that are not readily available. For example, rock aggregate is a high mass but low-value commodity that is sometimes traded and sometimes extracted directly. Allocating rock aggregate extraction to the quarrying industry, as is done in EXIOBASE, assumes that all rock aggregate is traded, whereas in some countries, quarrying may be limited to higher- value commodities, and rock aggregate may be extracted directly by the construction industry (Schaffartzik et al. 2014; Schoer et al. 2013). f High sector detail can help reducing this uncertainty (de Koning et al. 2015; Lenzen 2011). EXIOBASE provides the highest harmonized sector detail of MRIO models, but because data are not available at such detail for all countries, a combination of LCA data, proxy country IO data (from some specific, highly detailed national IO tables) and sector-specific information (e.g., energy balances from the IEA for the energy sector) was used for disaggregation (Wood et al. 2015) where regional differences can have an impact, particularly in assessments of material flows (Stadler et al. 2014). l A general caveat to current MRIO analyses is that the production of capital goods including infrastructure is not allocated to the users of the infrastructure (by sector, or over time), such that results show the annual resource requirement of various activi- ties. 3.2.2  Methodological aspects This approach provides consistency with national accounting, but could be relaxed by endogenizing capital goods and infrastructure in the coefficient matrix (see Lenzen 2001) to allocate infrastructure to goods and services consumed by households and government. 4  Conclusions In this paper, we provided a detailed assessment of the composition of the material foot- print of the EU and its underlying supply chain structures. We argued that recent devel- opments in the compilation of multi-regional input–output databases open up a range of new analytical options for the assessment of global material flows related to final consumption of a country or world region. A particular strength of the MRIO approach to analyze material flows is the possibility to identify the most important products and product groups contributing to the material footprint as well as to investigate the inter- national supply chains underlying the final goods and services delivered to final demand through applying production layer decomposition. This type of analysis goes far beyond the calculation of headline indicators on material consumption and material productiv- ity, which so far has dominated the MFA literature, and allows connecting much better to issues related to the development of resource policies, which are currently emerging in the context of European policy making. The objective of this paper was to provide an illustration of the analytical potentials starting from the macro-level of the overall EU material footprint and disaggregating it in several dimensions. However, there is still a lot of room to expand this type of research further and to derive more specific results. One option is to focus the analysis on single Giljum et al. Economic Structures (2016) 5:17 Page 22 of 24 raw materials instead of aggregated material groups. Full structural path analysis (SPA) could also be applied for selected products of particular policy relevance, in order to identify hotspots of material extraction on each layer of the supply chain, for example, with regard to service sectors with important contributions to the overall material foot- print. Finally, it is important to continue work on uncertainty analyses in connection with material footprint models based on various MRIO databases. Author details 1 1 Institute for Ecological Economics, Vienna University of Economics and Business (WU), Vienna, Austria. 2 Industrial Ecol- ogy Programme, Norwegian University of Science and Technology (NTNU), Trondheim, Norway. 3 Institute of Environ- mental Sciences (CML), Leiden University, Leiden, The Netherlands. 4 Netherlands Organisation for Applied Scientific Research (TNO), Delft, The Netherlands. Additional files Additional file 1: Full list of EXIOBASE products and product groups. Additional file 2: Full list of EXIOBASE countries and regions. Additional file 3: Technical details on the compilation of the global material flow database. Additional file 4: Correspondence table between raw materials and EXIOBASE sectors. Additional file 5: Detailed material footprint (MF) results. Acknowledgementsi We acknowledge financial support for this research from the European Commission through the FP7 project DESIRE “Developing a system of indicators for a resource efficient Europe” (Project Number: 308552). References Acosta-Fernández J (2007) Identifikation prioritärer Handlungsfelder für die Erhöhung der gesamtwirtschaftlichen Res- sourcenproduktivität in Deutschland [Identification of priority areas of action for increasing economy-wide resource productivity in Germany]. Wuppertal Institut, Wuppertal y y Andrew RM, Peters GP (2013) A multi-region input–output table based on the global trade analysis project database (GTAP-MRIO). Econ Syst Res 25:99–121 y y Andrew RM, Peters GP (2013) A multi-region input–output table based on the global trade analysis project database (GTAP-MRIO). Econ Syst Res 25:99–121 Arto I, Genty A, Rueda-Cantuche JM, Villanueva A, Andreoni V (2012) Global resources use and pollution: vol. 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Synthesis and evaluation of delayed anti-high temperature gel plugging agent
Frontiers in energy research
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cc-by
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OPEN ACCESS OPEN ACCESS EDITED BY Xianbin Huang, China University of Petroleum (East China), China REVIEWED BY Hanyi Zhong, China University of Petroleum, Huadong, China Hui Mao, Chengdu University of Technology, China Yb He, China University of Petroleum, China *CORRESPONDENCE Yang Bai, baiyanghyq@foxmail.com SPECIALTY SECTION This article was submitted to Advanced Clean Fuel Technologies, a section of the journal Frontiers in Energy Research RECEIVED 26 July 2022 ACCEPTED 31 October 2022 PUBLISHED 14 November 2022 CITATION Bai Y, Wu L, Luo P and Li D (2022), Synthesis and evaluation of delayed anti-high temperature gel plugging agent. Front. Energy Res. 10:1003473. doi: 10.3389/fenrg.2022.1003473 Yang Bai*, Lingfeng Wu, Pingya Luo and Daoxiong Li State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation, School of Oil and Natural Gas Engineering, Southwest Petroleum University, Chengdu, China State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation, School of Oil and Natural Gas Engineering, Southwest Petroleum University, Chengdu, China The malignant leakage of complex formations seriously restricts the drilling time and has become a major drilling technical problem that needs to be solved urgently at this stage. In particular, the malignant leakage of large fractures and fractured formations is difficultto construct,andthesuccessrateofpluggingis low.In thispaper,accordingto the above formation conditions, byanalyzing the leakage mechanism of the malignant leakage formation, the method of plugging the large fractures and broken fractured formations is summarized. The adiabatic copolymerization co-hydrolysis method was used as the gel synthesis method, 2-methacrylic acid amide and acrylamide (AM) were used as the main raw materials, and the redox initiation system composed of sodium sulfate and sodium bisulfite was used as the initiator. Using its redox initiation characteristics, high-strength bonding between organic polymer covalent bonds was achieved by a self-made cross-linking agent; using the thermoplasticity of hot-melt adhesive, the initiator is wrapped in hot-melt adhesive particles to achieve high-temperature slow-release initiation, and finally, a gel plugging agent with good compatibility with drilling fluid, controllable gel formation time, stable gel formation, and good gel breaking performance was synthesized, which is suitable for severe lost circulation under high-temperature and high-pressure conditions. A compatibility study of gel and common drilling fluid systems, the effect of temperature on gel formation, gel rheological properties under high temperature and high pressure, gel start-up pressure, and gel breakability tests were evaluated under laboratory conditions. gel synthesis, delay, anti-high temperature gel, malignant leakage plugging, drilling engineering TYPE Original Research PUBLISHED 14 November 2022 DOI 10.3389/fenrg.2022.1003473 TYPE Original Research PUBLISHED 14 November 2022 DOI 10.3389/fenrg.2022.1003473 TYPE Original Research PUBLISHED 14 November 2022 DOI 10.3389/fenrg.2022.1003473 Introduction In view of the deficiency of the above gel plugging materials that need the cooperation of a mud system and are not resistant to high temperatures, a new gel plugging material, ZND-6, is developed. The gel is compatible with the general water-based drilling fluid, and it can be mixed with the drilling fluid on the ground to maintain the relatively low viscosity. It may enter the target formation with the drill pipe pump and utilize the temperature of the formation within a specified time to form a gel polymer with certain strength so as to block the formation. Its performance is superior to that of the general gel, and it has features such as a controlled gelling time, high temperature resistance of 150°C, good pressure bearing capacity, etc. It solves the problem of the compatibility of the general loss- stopping agent with the leakage fracture size and can effectively block the malignant leakage caused by large fractures and broken formations. Loss of circulation is a phenomenon where various working fluids (including drilling fluid, cement slurry, completion fluid, and other fluids) leak into the formation under the action of pressure differences during various downhole operations such as drilling, cementing, and testing (Fan et al., 2018). The “vicious leakage” mainly refers to the phenomenon where the mud pumped into the wellbore has no circulation and no return during the drilling process. It has the characteristics of high leakage speed, huge leakage, difficult leakage processing, and easy recurrence of leakage. Malignant leakage mainly occurs in karst formations and fractured formations with large widths (Zhou et al., 2012). Half of the widths of wide fractures are more than millimeters, and even some fractures can reach about 10 mm in width; the underground voids of karst strata are much larger than wide fractures malignant leakage will seriously restrict the drilling speed during the drilling process. In particular, the malignant leakage that occurs in complex formations often requires long-term, multiple times, multiple methods, and multiple leakage plugging construction, which is inconsistent with the drilling goal of “safety, high quality, high efficiency, and economy” (Li and Xiang, 2020). The malignant leakage in complex formations has become a major drilling technology problem that has not been fully solved in the current stage. OPEN ACCESS The experimental results show that the gel has good compatibility with common drilling fluid systems; with the increase of temperature, the gel time of the gel plugging agent gradually decreases, and the gel time can be adjusted from 1 to 3 h at a high temperature of 150°C; when the concentration of the gel in the experimental slurry exceeds 2%, its rheological properties change abruptly; under certain conditions, the starting pressure of the gel can reach up to 6.5 MPa; and under the condition of 150°C, the gel breaking rate was over 98% after 24 h. Various experiments show that the gel’s performance meets the requirements for plugging malignant leakage formations, and the effect is good. Compared with traditional gel materials, the gel plugging agent synthesized in this paper has the characteristics of a simple configuration process and superior performance with drilling fluid, which provides a new idea for malignant leakage plugging. COPYRIGHT © 2022 Bai, Wu, Luo and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Energy Research 01 frontiersin.org 10.3389/fenrg.2022.1003473 Bai et al. Reagent and main instruments Acrylamide (AM), analytically pure (Elsharafiand Bai, 2017); initiator (self-made); 2-methacrylic acid amide, analytically pure; sodium hydroxide (NaOH), analytically pure; deionized water; sodium persulfate (Na2S2O8), analytically pure (Zhao et al., 2018); sodium bisulfite (NaHSO3), analytically pure; sodium sulfate (Na2SO4), analytically pure; sodium formate, analytically pure; surfactant OP-10, analytically pure. FFD-50 adiabatic reactor; 85-1 magnetic stirrer; HHS-11- 6 electric water bath; GJD-B12K variable-frequency and high- speed mixer; Granulator. With the continuous emergence of new materials and new technologies in the petroleum industry, the gel plugging materials have been improved and upgraded for the malignant leakage of the complex formation. Jianshan (2019) takes the rigid aromatic ring as the main body of the structural unit and develops the SMSHIELD intelligent gel plugging agent with the carbon–nitrogen bond, carbon–carbon bond, and other chemical bonds with high stability and outstanding rigidity as the main chain. Such gel materials are characterized by strong deformation and easy formation, and they could effectively bond the components in the mud cake and fully fill all spatial positions of the mud cake to make it denser. Such intelligent gel can be combined with other treating agents, but they were not compatible with the drilling fluid of other systems. Suming et al. (2007) have developed a pressure- bearing delay plugging agent, OC L-GYDL. Such a plugging agent has a strong pressure bearing capacity, and the gel time is adjustable. However, it could not be consistent with high temperature; therefore, it could not be applied to high- temperature formations. Introduction The study believes that for micro and narrow fractures, the use of irregular rigid and flexible plugging materials has a better effect; for large fractures and fractured formations, inorganic plugging materials are difficult to construct, the plugging success rate is low, it is difficult to significantly improve the pressure bearing capacity of the formation, the cost of plugging is high, and it is difficult to deal with the plugging of malignant leakage in complex formations (Du et al., 2019). Synthesis The synthesis method of gel is the adiabatic copolymerization co-hydrolysis method. This method does not require a separate hydrolysis operation, which not only reduces the production cost but also saves the production cycle. The steps of preparing gel by adiabatic copolymerization co-hydrolysis are as follows. First, 2-methacrylic acid amide and acrylamide were added to an adiabatic reactor according to a certain feed ratio. According to the requirements of gel fluidity, the acrylate monomer was added, deionized water and instant additives were added, and an appropriate amount of alkali was added to adjust the pH value of the system. After stirring, a monomer solution with a concentration of 35% was formed. Then the solution was controlled to the polymerization temperature (40°C) (Kumar et al., 2014), and an initiator was added after removing oxygen by nitrogen. After adiabatic polymerization for 8 h (Barreira and Negrão, 2017), the colloid was taken out for granulation, drying, crushing, and packaging to obtain the gel sample. Frontiers in Energy Research 02 frontiersin.org Bai et al. 10.3389/fenrg.2022.1003473 FIGURE 1 Cementation state of the sample before and after the high-temperature aging. FIGURE 1 Cementation state of the sample before and after the high-temperature aging. other and have a strong structural force. We used a scanning electron microscope to observe the microstructure of the samples after aging, as shown in Figure 2. It was found that the gel particles were completely gelled under the conditions of 10 h and high temperature, and a dense three-dimensional network structure is formed between the gel molecules. The reticular formation of the gel molecular closely combines with other gel molecules that are independently distributed to form the large gel mass so that the sample has strong structural strength (Wang et al., 2020). The initiator is a redox initiation system composed of sodium persulfate and sodium bisulfite, and its addition amount is 2% of the total weight of the monomer solution (Quiroz et al., 2022). The instant additive is composed of sodium sulfate, sodium formate, and surfactant OP-10, and the dosage is 3% of the total weight of the monomer solution (Liu et al., 2018). Compatibility study of gel and common drilling fluid systems KPS, analytical reagent (Gregolin et al., 2010); HV-CMC, analytical reagent; SPNH, analytical reagent; the formula for the experimental pulp is as follows: H2O + bentonite + barytes, +1%HV-CMC+3%SPNH, the experimental mud density is 1.5. In order to explore the effect of gel ZND-6 mixed into drilling fluid on the performance of drilling fluid, the compatibility experiment of gel with NaCl, polysulfonic acid, and polymer drilling fluid systems was carried out in the laboratory. The drilling fluid before and after aging after adding gel was mainly investigated for changes in rheology and dehydration wall-building. The experimental results are shown in Tables 1, 2, 3. M7500 high-temperature and high-pressure rheometer; QD- 2 test device on plugging materials; XGRL-4 aging furnace. Frontiers in Energy Research Frontiers in Energy Research Microstructure characterization According to the experimental pulp formula, we add 3% gelatin ZND-6 to the configured experimental pulp, stir for 10 min at low speed, and then put it into the aging tank. After aging for 10 h at 150°C, gel particles are gelatinized. The sample status before and after the experiment is shown in Figure 1. It could be found that the sample has a strong thixotropy after adding the gel particles. Before aging, the gel particles in the sample are distributed independently in the experimental slurry, and there is no cementation between them. After aging, the gel particles agglomerate with each It can be seen from Table 1 that adding the gel ZND-6 to the NaCl drilling fluid can enhance the static shear force of the system, reduce the filtration loss, and enhance its water loss and wall-building ability without affecting the fluidity of the system. Gel ZND-6 has good compatibility with NaCl drilling fluid. It can be seen from Table 2 that the effect of gel ZND-6 on the system is generally favorable after adding the polysulfonic drilling fluid, which can improve the rheological properties of the system and improve the ability of water loss and wall-building. Frontiers in Energy Research 03 frontiersin.org Bai et al. 10.3389/fenrg.2022.1003473 FIGURE 2 Micromorphology of the gel after aging. FIGURE 2 Micromorphology of the gel after aging. Evaluation of the gel’s rheological property To determine the rheological data of the gel ZND-6 under the downhole high-temperature and high-pressure conditions, the rheological property of the gel ZND-6 is tested for 10H in a high- temperature and high-pressure environment with the M7500 high-temperature and high-pressure rheometer produced by Grace Company. The time length includes the time before and after the gelation of gel ZND-6 and the period after gelation so as to determine the viscosity change before gelation and the stability after gelation. can improve the water loss and wall-building properties as well as the viscosity and static shear force of the drilling fluid. The compatibility between the gel and the commonly used drilling fluid system is sufficient to meet the requirements of solving the malignant loss of large fractures and broken formations. Add the gel ZND-6 with a concentration of 1.5% to the experimental pulp and increase the temperature to 150°C. Then, test the sample viscosity every 20 min under the environment at a pressure of 20 MPa, with the experimental results shown in Figure 3. The samples are gelatinized at 60 min, and the highest viscosity reaches 19000 mPa s. Besides, the sample viscosity basically remains stable in the remaining experiment time. resulting in a faster release of the initiator from the hot-melt adhesive and a shortening of the gel formation time (Li, X et al., 2022). resulting in a faster release of the initiator from the hot-melt adhesive and a shortening of the gel formation time (Li, X et al., 2022). TABLE 4 Impact of temperature in clear water on gelling time. Temperature/°C 80 100 120 150 tgelation/h 4.2 3.1 1.5 1.1 TABLE 5 Impact of temperature in experimental pulp on gelling time. Temperature/°C 80 100 120 150 tgelation/h 6.8 5.6 4.1 3.3 Frontiers in Energy Research TABLE 1 Compatibility of ZND and NaCl drilling fluids (150°C for 16 h aging). ZND-6/% Experimental conditions AV/mPa·s PV/mPa·s YP/Pa Gel 10 min/Pa Fl/ml Filter cake thickness/mm 0 Before ageing 30.0 25 5 5.1 8.5 0.5 1.0 Before ageing 82.5 87 6 12.7 6.1 0.5 2.0 Before ageing 142.2 156 4 75.3 5.3 0.5 3.0 Before ageing 204.8 265 6 164.4 3.7 0.5 0 After ageing 15.0 15 0 2.0 19.0 0.5 1.0 After ageing 44.1 51 4 6.4 14.6 0.5 2.0 After ageing 86.2 97 0 34.8 8.2 0.5 3.0 After ageing 102.2 124 12 99.2 7.7 0.5 ZND-6/% Experimental conditions AV/mPa·s PV/mPa·s YP/Pa Gel 10 min/Pa Fl/ml Filter c nditions AV/mPa·s PV/mPa·s YP/Pa Gel 10 min/Pa Fl/ml Filter cake thickness/mm TABLE 2 Compatibility of ZND and Polysulfonic Drilling Fluid (150°C for 16 h of aging). TABLE 2 Compatibility of ZND and Polysulfonic Drilling Fluid (150 C for 16 h of aging). ZND-6/% Experimental conditions AV/mPa·s PV/mPa·s YP/Pa Gel10 min/Pa FL/mL Filter cake thickness/mm 0 Before ageing 34.0 31 3 6.1 10.0 1.0 1.0 Before ageing 66.2 66 6 15.3 8.2 1.0 2.0 Before ageing 131.9 124 4 41.3 6.5 0.8 3.0 Before ageing 184.7 156 0 115.8 4.8 0.5 0 After ageing 31.2 21 3 3.4 12.1 0.5 1.0 After ageing 37.1 24 4 7.6 7.5 0.5 2.0 After ageing 64.1 62 4 22.9 8.8 0.5 3.0 After ageing 122.7 98 3 59.7 6.8 0.5 Gel ZND-6 has good compatibility with polysulfonic drilling fluid. fluid loss of the drilling fluid is low. The performance after aging also meets the general requirements for drilling fluids and has good compatibility with polymer drilling fluids. It can be seen from Table 3 that ZND-6 gel can improve the fluidity of the drilling fluid after adding the polymer drilling fluid, while maintaining the static shear force of the system, and the To sum up, the gel ZND-6 has good compatibility with drilling fluids such as NaCl, polysulfonic acid, and polymers and Frontiers in Energy Research Frontiers in Energy Research 04 frontiersin.org 10.3389/fenrg.2022.1003473 Bai et al. TABLE 3 Compatibility of ZND and Polymer Drilling Fluid (150°C for 16 h aging). nditions AV/mPa·s PV/mPa·s YP/Pa Gel10 min/Pa FL/mL Filter cake thickness/mm TABLE 4 Impact of temperature in clear water on gelling time. Influence of temperature on the gelling time The effect of temperature on the gel formation time is obvious: the half-life of the gel polymer initiator decreases exponentially with the increase in temperature. The gelling times of gelatin in water and the experimental slurry at 80°C–150°C are studied, respectively, to determine the relevant time. The experimental results are shown in Tables 4, 5. According to Figures 4, 5, the rheological properties of the samples only increase linearly with the gel concentration when the gel concentration is 0.5%, 1.0%, or 1.5%. After the gel concentration reaches 2%, all rheological data of the sample show sudden changes. When the gel concentration is relatively low, the gel particles in the sample are far away; therefore, it is not easy for them to contact each other and form a three- dimensional network structure between the particles. Actually, they just change the rheological property of the samples through the swelling and gelation of each individual gel particle. After the gel concentration reaches or exceeds 2%, not only the individual gel particle has an influence on the rheological property of the samples. Besides, with the increase It can be seen from the experimental results that the gel- forming time of the gel ZND-6 gradually decreases with an increase in the experimental temperature. The reasons and results may be that ① the half-life of the initiator decreases with the increase of temperature, which shortens the gel formation time (Li et al., 2015) and ②with the increase of the experimental temperature, the melting rate of the hot-melt adhesive capsule in the gel increases, Frontiers in Energy Research 05 frontiersin.org Bai et al. 10.3389/fenrg.2022.1003473 FIGURE 3 Viscosity property change of the samples within 10 h. FIGURE 4 Comparison of shearing stress at different rotating speeds under different concentrations of gel. FIGURE 3 Viscosity property change of the samples within 10 h. FIGURE 3 Viscosity property change of the samples within 10 h. FIGURE 4 Comparison of shearing stress at different rotating speeds under different concentrations of gel. FIGURE 4 Comparison of shearing stress at different rotating speeds under different concentrations of gel. property of the slurry will change suddenly when the concentration of the gel ZND-6 exceeds 2%. Hence, the optimal dosage concentration of gel ZND-6 should not be less than 2%. Influence of temperature on the gelling time of gel particles per unit volume, the distances between the gel particles are reduced so as to form a network structure among the particles and create a larger gel mass. Such large gel masses could obviously affect the rheological properties of samples more than single gel particles. It could be judged from the above experiments that the rheological property of gel could basically maintain stability under the downhole high-temperature and high- pressure conditions, and it could block the broken formation stably for a long time, which could prolong the safe operation time effectively and strengthen the safety of well control (Li, Z et al., 2022). Besides, the rheological Frontiers in Energy Research Starting pressure test of gel After reaching the position of lost circulation and penetrating into large fractures and fractured formations, the gel swells into glue in the well slurry to block large pores and fractures. After the gel plugging, there is a Frontiers in Energy Research 06 frontiersin.org frontiersin.org Bai et al. 10.3389/fenrg.2022.1003473 FIGURE 5 Comparison of viscosity at different rotating speeds under different concentrations of gel. TABLE 6 ZND-6 start-up experiment. No. Sample dosage/% Measuring temperature/°C Holding time/h Start-up pressure/MPa 1 3 80 5 1.5 2 3 100 5 2.0 3 3 120 5 2.0 4 3 150 5 4.0 5 5 150 5 6.5 6 5 170 5 4.0 6 5 180 5 3.5 FIGURE 5 Comparison of viscosity at different rotating speeds under different concentrations of gel. Comparison of viscosity at different rotating speeds under different concentrations of gel. TABLE 6 ZND-6 start-up experiment. No. Sample dosage/% Measuring temperature/°C Holding time/h Start-up pressure/MPa 1 3 80 5 1.5 2 3 100 5 2.0 3 3 120 5 2.0 4 3 150 5 4.0 5 5 150 5 6.5 6 5 170 5 4.0 6 5 180 5 3.5 TABLE 6 ZND-6 start-up experiment. No. Sample dosage/% Measuring temperature/°C Holding time/h Start-up pressure/MPa 1 3 80 5 1.5 2 3 100 5 2.0 3 3 120 5 2.0 4 3 150 5 4.0 5 5 150 5 6.5 6 5 170 5 4.0 6 5 180 5 3.5 pressure with 0.5 MPa as the gradient to observe whether there is a sample extrusion. After the samples are extruded, stop the experiment and record the pressure, which is the start-up pressure of the gel. The start-up pressures vary at different temperatures, gel concentrations, and high-preservation standing times. The experimental results are as shown in Table 6. pressure difference inside and outside the wellbore. After the gelatinization, the pressure difference that the plugging material can withstand is the start-up pressure of the gel, and it is a key metric to evaluate its plugging performance (Yokoi et al., 2022). The QD-2 test device on plugging materials is adopted to simulate leakage layer cracks with different seam widths (1, 2, 3, 4, and 5 mm). We add the gel to the experimental pulp and investigate the plugging pressure-bearing capacity of the gel plugging materials on the large pores and fractures causing the malignant leakage under the high-temperature and high- pressure conditions. Frontiers in Energy Research Well Shuangmiao 1 Well Shuangmiao 1 in Dazhou is a key vertical exploration well in southwest China. There are six leakage points in the 2083–3,573 m well section. A high-pressure gas layer was drilled at a depth of 3,446–3,448 m, and the bottom hole overflowed. In order to seal the leakage layer and protect the gas layer, the on-site technicians have successively adopted bridge plug plugging, nine times of cement plugging, and bridge plug and cement composite plugging methods, all of which have not been effective, taking up to 2 months, and the phenomenon of simultaneous leakage and leakage has not been controlled. Wellhead two single ram blowout preventers in the upper ram damage failure, four anti-nozzle lines in three damaged, throttling blowout, and casing pressure are extremely difficult to reduce. The leakage layer of 3,446–3,448 m is the main factor to induce complex downhole conditions. air blockage but also cements with the tank externally. With the rise in temperature, the strength of the network structure will increase. Once it exceeds the limiting temperature of gel, the strength of the structure will decrease. Therefore, the start-up pressure of gel will increase with the rise in temperature in the range from 80°C to 150°C. After it exceeds 150°C, the start-up pressure will decrease with the rise in temperature. Similarly, the strength of the gel’s network structure will be enhanced with the concentration of gel in the experimental pulp. Starting pressure test of gel Data in Table 6 indicates that under the same concentration, the start-up pressure increases with the rise in temperature, and the maximum start-up pressure is 6.5 MPa in the temperature range of 80°C–150°C; when the temperature exceeds 150°C, the sample’s start-up pressure decreases with the rise in temperature. Under the same temperature, the ZND-6 start-up pressure increases as the gel concentration increases. At 150°C and a concentration of 5%, the gel will form a tight and elastic air blockage, as shown in Figure 6. The specific experimental method is as follows: add the gel ZND-6 to the experimental pulp and put it into the plugging material test device after mixing evenly, pressurize the upper valve rod by 0.5 MPa, close the lower valve rod, and let it stand for several hours at a certain temperature. After the gel in the samples seals the joint board, open the foot valve and increase the This phenomenon occurs mainly because the hydrophobic group in gel ZND-6 will form a network structure in the slurry. This network structure not only makes the gel combine into an Frontiers in Energy Research 07 frontiersin.org 10.3389/fenrg.2022.1003473 Bai et al. 10.3389/fenrg.2022.1003473 D is the gel breaking rate; m is the gel quality before gel breaking, g; and m0 is the remaining gel quality after gel breaking, g. FIGURE 6 Air Blockage formed by examples. Add 3% gel to the experimental pulp, and then add 1% KPS. After mixing evenly, pour it in the aging tank and take out the sample after rolling it in the roller furnace for a certain time at 150°C. Then, screen out the remaining gel with the screen and weigh it to calculate the gel breaking rate. The experimental results are as shown in Figure 7. It can be seen from Figure 7 that the gel breaking rate could reach about 50% after 18 h at 150°C; after 24 h, the gel breaking rate could reach 98%. At this time, the gel breaking of gel is basically complete, meeting the requirements of the reverse drainage of the general reservoir. Author contributions 2. The gel plugging materials ZND-6 could penetrate the broken formation to form the air blockage. When the gel concentration is 1.5% at 150°C, the sample viscosity reaches 19,000 mPa s and the stable time is greater than 10 h. YB: ideas and suggestions; LW: data collection and drafting of papers; PL: approval of the final version of the paper for publication; and DL: made important revisions to the paper. 3. It could be found from the experiment that after the concentration of gel reaches 2% in the slurry, it is easier to form the three- dimensional network structure and become the larger gel mass, and the sample performance may change suddenly. In view of the actual construction and economic benefits, it is suggested to control the gel concentration at 3%–5%. Gel breaking test In order to effectively plug the three leakage layers at a depth of 3,483, 3,446, and 3,436 m, the special gel plugging technology is tried. A total of 70 m3 of gel was injected into the three layers, and then 25 m3 of cement was added. The construction was successful. After pressure relief, the casing pressure drops to 12 MPa and then shuts down. The standpipe pressure is zero, and the casing pressure remains unchanged. Shut-in observation riser pressure is still zero, the casing pressure is still zero, to achieve the purpose of blocking the annulus, cut off the bottom hole high-pressure gas layer. The logging results show that the cement sheath is in the well depth of 2,250–3,200 m, the cement return height is in the well depth of 2,355 m, the ZND-6 gel column in the annulus of the leakage layer is nearly 100 m, and the plugging is successful. The core material of the oxidation breaker is potassium persulfate or ammonium persulfate. The principle is that the breaker can be dissolved into a highly active, solid strong oxidant in contact with water (Guo et al., 2016). The advantage of oxidative gel breakers is that they reduce the effect of gel breakers on the rheological properties of fracturing fluid. After the reservoir leakage plugging, it is necessary to reverse the gel breaking in the lost circulation plugging slurry to ensure the downhole production capacity. Hence, the gel-breaking test shall be carried out. The calculation formula of the gel breaking rate is: D  m −m0 m × 100%. m Frontiers in Energy Research 08 frontiersin.org Bai et al. 10.3389/fenrg.2022.1003473 FIGURE 7 Change of gel breaking rate of samples with time. FIGURE 7 Change of gel breaking rate of samples with time. Well Liu 67–72 5. Under the condition of 150°C, the gel breaking rate is over 98% after 24 h, which meets the requirement of general reservoir reverse flow. 5. Under the condition of 150°C, the gel breaking rate is over 98% after 24 h, which meets the requirement of general reservoir reverse flow. 5. Under the condition of 150°C, the gel breaking rate is over 98% after 24 h, which meets the requirement of general reservoir reverse flow. Well Liu 67–72 in Changqing leakage occurred at a depth of 1,629 m. The bridge slurry (the particle size of the bridging material is 3 ~ 5 mm) and the cement plug failed nine times. In order to plug the leakage point, ZND-6 gel plugging technology is adopted. Drill down the drill pipe a depth of 1,500 m, pump 34 m3 concentration of 1.2% gel ZND-6, and then inject 12 m3 water; next, pull out the drill, and allow it to stand for 6 h. Down to the bottom of the well, open the pump return, normal displacement. After circulating the drilling fluid for 15 min, drilling 8 m at 150 kN and 65 r/min, the pump pressure displacement is normal and the plugging is successful. 6. According to field application, the innovation of gel plugging agent ZND-6 is that it can be used alone with drilling fluid and has a good effect on plugging malignant leakage layers. It can well solve the leakage problems that cannot be solved by methods such as fractured, porous, and broken formations; bridge plugging; and plugging while drilling. It is unique in dealing with the leakage problems of aquifers and the same layer of leakage. 6. According to field application, the innovation of gel plugging agent ZND-6 is that it can be used alone with drilling fluid and has a good effect on plugging malignant leakage layers. It can well solve the leakage problems that cannot be solved by methods such as fractured, porous, and broken formations; bridge plugging; and plugging while drilling. It is unique in dealing with the leakage problems of aquifers and the same layer of leakage. Conclusion The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author. 1. The high-temperature gel plugging agent ZND-6 is synthesized with a controlled gelling time, and the free radical is aggregated to form the high-strength gel with dense cross-linking. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 4. The temperature resistance of the gel ZND-6 could reach 150°C. When the temperature exceeds 150°C, the gel strength would decrease. Frontiers in Energy Research 09 frontiersin.org frontiersin.org 10.3389/fenrg.2022.1003473 10.3389/fenrg.2022.1003473 Bai et al. References Li, Z., and Xiang, C. (2020). Experimental investigation of a new weak-gel- type clay-free and water-based drilling fluid with high-temperature and high- salt resistance for determining its optimized formulation. Energy Sources Part A Recovery Util. Environ. Eff. 107, 1–18. doi:10.1080/15567036.2020. 1763516 Barreira, E. M., and Negrão, C. O. R. (2017). Performance of pressure relief devices on flow start-up of gelled waxy crude oils in pipelines. J. Petroleum Sci. 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Spleen Spleen Spleen A E H N O F I J K G P B C Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 25 Total Splenocyte Viable Cells (x 10*7) 0 2 4 6 8 0 5 10 15 20 25 Tumor weight(g) r = 0.9108 p = 0.0002 Total Splenocyte Viable Cells (x 10*7) CD3+ CD4+ CD8+ Treg CD19+ CD11b+ NK Macro Neut DC 0 20 40 60 80 ns ns ns ns Percent of Live CD45+(%) Veh-D9 Veh-D21 Anlo-D9 Anlo-D21 Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 100 GzmB+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 IFN- +CD4+/CD4+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 IFN- +CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 TNF- +CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 TNF- +CD8+/CD8+(%) ns 0 2 4 6 8 0 10 20 30 40 50 r = 0.9690 p<0.0001 CD11b+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 r = -0.6849 p = 0.0289 CD8+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 20 25 r = -0.7522 p = 0.0121 CD4+CD45+/CD45+(%) Spleen Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 PD1+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 PD1+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 Ki67+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 Ki67+CD8+/CD8+(%) ns CD3+ CD4+ CD8+ Treg CD19+ CD11b+ NK Macro Neut DC 0 10 20 30 40 50 ns ns Percent of Live CD45+(%) ns ns D L M ns ns ns ns ns ns ns Blood Spleen A B C Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 25 0 2 4 6 8 0 5 10 15 20 25 Tumor weight(g) r = 0.9108 p = 0.0002 Total Splenocyte Viable Cells (x 10*7) CD3+ CD4+ CD8+ Treg CD19+ CD11b+ NK Macro Neut DC 0 20 40 60 80 ns ns ns ns Percent of Live CD45+(%) Veh-D9 Veh-D21 Anlo-D9 Anlo-D21 ns Spleen B 0 2 4 6 8 0 5 10 15 20 25 Tumor weight(g) r = 0.9108 p = 0.0002 Total Splenocyte Viable Cells (x 10*7) A Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 25 Total Splenocyte Viable Cells (x 10*7) ns C A B Total Splenocyte Viable Cells (x 10*7) E H N Q O R F I J K G P S Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 Total Splenocyt 0 2 4 6 8 0 5 Tumor weight(g) p 0 000 Total Splenocyt CD3+ CD4+ CD8+ Treg CD19+ CD11b+ NK Macro Neut DC 0 20 ns ns ns Percent of Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 100 GzmB+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 IFN- +CD4+/CD4+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 IFN- +CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 TNF- +CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 TNF- +CD8+/CD8+(%) ns 0 2 4 6 8 0 10 20 30 40 50 Tumor weight(g) r = 0.9690 p<0.0001 CD11b+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 Tumor weight(g) r = -0.6849 p = 0.0289 CD8+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 20 25 Tumor weight(g) r = -0.7522 p = 0.0121 CD4+CD45+/CD45+(%) 0 2 4 6 8 0 10 20 30 40 50 Tumor weight(g) CD11b+CD45+/CD45+(%) r = 0.8857 p = 0.0006 0 2 4 6 8 0 2 4 6 8 10 Tumor weight(g) r = -0.7536 p = 0.0118 CD8+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 Tumor weight(g) r = -0.8712 p = 0.0010 CD4+CD45+/CD45(%) Tumor Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 PD1+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 PD1+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 Ki67+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 Ki67+CD8+/CD8+(%) ns CD3+ CD4+ CD8+ Treg CD19+ CD11b+ NK Macro Neut DC 0 10 20 30 40 50 ns ns Percent of Live CD45+(%) ns ns D L M ns ns ns ns ns ns Blood E F G Tumor weight(g) C C C C CD M Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 100 GzmB+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 IFN- +CD4+/CD4+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 TNF- +CD4+/CD4+(%) ns CD3+ CD4+ CD8+ Treg CD19+ D11b+ NK Macro Neut DC 0 10 20 30 40 50 ns ns Percent of Live CD45+(%) ns ns D ns ns Blood G M Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 100 GzmB+CD8+/CD8+(%) ns 5 D G 100 G 100 Percent of Live CD45+(%) D H N Q O R I J K P S D9 D21 D9 D21 D9 D21 D9 D21 Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 IFN- +CD8+/CD8+(%) D9 D21 D9 D21 Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 TNF- +CD8+/CD8+(%) ns 0 2 4 6 8 0 10 20 30 40 50 Tumor weight(g) r = 0.9690 p<0.0001 CD11b+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 Tumor weight(g) r = -0.6849 p = 0.0289 CD8+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 20 25 Tumor weight(g) r = -0.7522 p = 0.0121 CD4+CD45+/CD45+(%) 0 2 4 6 8 0 10 20 30 40 50 Tumor weight(g) CD11b+CD45+/CD45+(%) r = 0.8857 p = 0.0006 0 2 4 6 8 0 2 4 6 8 10 Tumor weight(g) r = -0.7536 p = 0.0118 CD8+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 Tumor weight(g) r = -0.8712 p = 0.0010 CD4+CD45+/CD45(%) Tumor Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 PD1+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 PD1+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 Ki67+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 Ki67+CD8+/CD8+(%) ns CD CD CD Tr CD1 CD11 Mac Ne L M ns ns ns ns H I J K Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 IFN- +CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 20 40 60 80 TNF- +CD8+/CD8+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 PD1+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 PD1+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 5 10 15 20 Ki67+CD4+/CD4+(%) ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 Ki67+CD8+/CD8+(%) ns C C C CD CD M L M ns ns ns ns Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 PD1+CD8+/CD8+(%) Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 Ki67+CD8+/CD8+(%) ns L M ns ns H N Veh D9 Veh D21 Anlo D9 Anlo D21 0 10 20 30 40 50 IFN- +CD8+/CD8+(%) C C C ns I 8 H 50 J 20 Ki67+CD8+/CD8+(%) N O P Veh D9 Veh D21 Anlo D9 Anlo D21 Veh D9 Veh D21 Anlo D9 Anlo D21 0 2 4 6 8 0 10 20 30 40 50 Tumor weight(g) r = 0.9690 p<0.0001 CD11b+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 Tumor weight(g) r = -0.6849 p = 0.0289 CD8+CD45+/CD45+(%) 0 2 4 6 8 0 5 10 15 20 25 Tumor weight(g) r = -0.7522 p = 0.0121 CD4+CD45+/CD45+(%) Tumor Veh D9 Veh D21 Anlo D9 Anlo D21 Veh D9 Veh D21 Anlo D9 Anlo D21 Veh D9 Veh D21 Anlo D9 Anlo D21 Veh D9 Veh D21 Anlo D9 Anlo D21 N D9 D21 D9 D21 D9 D21 D9 D21 0 2 4 6 8 0 5 10 15 20 25 Tumor weight(g) r = -0.7522 p = 0.0121 CD4+CD45+/CD45+(%) N O O 0 2 4 6 8 0 5 10 15 Tumor weight(g) r = -0.6849 p = 0.0289 CD8+CD45+/CD45+(%) D9 D21 D9 D21 9 9 P 0 2 4 6 8 0 10 20 30 40 50 Tumor weight(g) r = 0.9690 p<0.0001 CD11b+CD45+/CD45+(%) P CD11b+CD45+/CD45+(%) CD4+CD45+/CD45+(%) CD8+CD45+/CD45+(%) Tumor weight(g) Q 0 2 4 6 8 0 5 10 15 Tumor weight(g) r = -0.8712 p = 0.0010 CD4+CD45+/CD45(%) R 0 2 4 6 8 0 2 4 6 8 10 Tumor weight(g) r = -0.7536 p = 0.0118 CD8+CD45+/CD45+(%) S 0 2 4 6 8 0 10 20 30 40 50 Tumor weight(g) CD11b+CD45+/CD45+(%) r = 0.8857 p = 0.0006 Q 1 R S CD11b+CD45+/CD45+(%) R CD8+CD45+/CD45+(%) CD4+CD45+/CD45(%)
https://openalex.org/W2025131735
http://medpet.journal.ipb.ac.id/index.php/mediapeternakan/article/download/8795/6870
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Impacts of Domestic Maize Price Changes on the Performance of Small-scale Broiler Farming in Indonesia
Media Peternakan
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cc-by-sa
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ISSN 0126-0472 EISSN 2087-4634 Accredited by DGHE No: 66b/DIKTI/Kep/2011 ISSN 0126-0472 EISSN 2087-4634 Accredited by DGHE No: 66b/DIKTI/Kep/2011 ISSN 0126-0472 EISSN 2087-4634 Accredited by DGHE No: 66b/DIKTI/Kep/2011 Media Peternakan, December 2014, 37(3):198-205 DOI: 10.5398/medpet.2014.37.3.198 Available online at http://medpet.journal.ipb.ac.id/ Media Peternakan, December 2014, 37(3):198-205 DOI: 10.5398/medpet.2014.37.3.198 Available online at http://medpet.journal.ipb.ac.id/ Impacts of Domestic Maize Price Changes on the Performance of Small-scale Broiler Farming in Indonesia S. J. K. Umboha,b,*, D. B. Hakimc, B. M. Sinagac, & I K. Kariyasad aStudy Program of Agricultural Economics, Faculty of Economics and Management, Graduate School, Bogor Agricultural University bFaculty of Animal Husbandry, Sam Ratulangi University Jln. Kampus Unsrat, Bahu Manado-North Sulawesi 95115, Indonesia cFaculty of Economics and Management, Bogor Agricultural University Jln. Kamper, Kampus IPB Darmaga Bogor-West Java 16680, Indonesia dIndonesian Center for Food Crops Research and Development Jln. Merdeka No 147 Bogor-West Java 16111, Indonesia (Received 09-06-2014; Reviewed 01-09-2014; Accepted 03-11-2014) S. J. K. Umboha,b,*, D. B. Hakimc, B. M. Sinagac, & I K. Kariyasad aStudy Program of Agricultural Economics, Faculty of Economics and Management, Graduate School, Bogor Agricultural University bFaculty of Animal Husbandry, Sam Ratulangi University Jln. Kampus Unsrat, Bahu Manado-North Sulawesi 95115, Indonesia cFaculty of Economics and Management, Bogor Agricultural University Jln. Kamper, Kampus IPB Darmaga Bogor-West Java 16680, Indonesia dIndonesian Center for Food Crops Research and Development Jln. Merdeka No 147 Bogor-West Java 16111, Indonesia (Received 09-06-2014; Reviewed 01-09-2014; Accepted 03-11-2014) ABSTRACT This research aimed at analyzing the impact of maize price changes on the performance of small-scale broiler farming in Indonesia using a multimarket model analysis. The multimarket model analysis is partial equilibrium analysis that contains six blocks of equations: prices, supply, input demand, consumption, income and equilibrium. This model analysis was originally designed in General Algebric Modelling System (GAMS) using the Path NLP solver. Employed data in this study were classified into 3 types, namely: (1) production and input, consumption, and household income; (2) inputs and outputs, and (3) elasticities. Decreased domestic maize price was responded by farmer through reducing maize planted area and fertilizers uses. It further had undesired impact on the maize production and maize farmer’s income. Whereas, this policy had positive impact on meat production and small-scale broiler farming income. The opposite impact will happen on those variables, if government increases domestic maize price. This policy caused the maize demand for feed industry decreased. As a result of this condition, it decreased the chicken meat production and small-scale broiler farming income. Key words: maize, broiler, price, multimarket *Corresponding author: E-mail: sintyajkumboh@yahoo.co.id Kata kunci: jagung, ayam ras pedaging, price, multimarket Key words: maize, broiler, price, multimarket INTRODUCTION This research excluded feed market into model, therefore feed demand was approximated by a proxy from maize demand. This proxy was reliable as maize proportion in feed ingridient was constant about 66%. During 2000-2010, the feed price of broiler and maize price tended to increase (Table 1). The fluctuation of maize price was being concern of feed manufacturer to decide production capacity. This finding was in line with Kariyasa & Sinaga (2007) that concluded that feed manufacturer concerned more to maize price than g p p y g The main components of chicken feed are maize, soybean meal, and fish flour. The composition of maize in the feed ranges from 55%-65%. In 2010, maize demand for broiler feed was 1.11 million tons and 1.65 million tons for layer. The average increase in maize demand for broiler and layer feeds during the period 2000-2010 increased by 6.85% and 8.31%, respectively (Swastika et al., 2011; Directorate General of Livestock and Animal Health, 2012, processed). Some factors caused the use of maize for feed is high, among others are its prices is relatively cheap, easy to produce, as well as palatable to poultry. In addition, maize contains high calorie and protein with complete amino acids are also as consideration or reasons why the use of it is still higher compared to others (Swastika et al., 2011). Attempts have been done to replace maize with other grains are not succesfully. Therefore maize remains as the main raw material for livestock feed. INTRODUCTION therefore could affect the sustainability of small-scale broiler farming. Broiler farming in Indonesia has become an indus­ try with complete components from upstream to down­ stream. The contribution of Gross Domestic Product (GDP) of livestock sub-sector to the agriculture sector GDP based on market prices reached 12%. Of this fig­ ure, poultry meat contribution was 64%, and of the total poultry meat contribution, it is about 70.24 percent com­ ing from chicken meat (Saptana & Sumaryanto, 2009; Directorate General of Livestock and Animal Health, 2012). This figure has shown that the chicken meat has an important role in providing meat for Indonesian (Ilham, 2009). It also explained that chicken meat has been the main driver on the development of the poultry business in Indonesia. Related to feed, maize, and chicken meat price data, the increasing of average feed price have the same trend with increasing of average maize price of by 6.78% and 7.96%, respectively, while chicken meat price incereased around 5.90% (Pusdatin, 2012).fi Viewed from efficiency as a result of business integration, breeding farms and feed mills which belong to foreign investors sell DOC and feed to independent farmers in high prices, making their business inefficient. Indarsih et al. (2010) revealed that there was a disparity in feed prices to be paid by large-scale and small-scale broiler farming, which was around 30%-50%. No wonder the production costs of small-scale broiler farming are much higher that those of broiler farming large-scale. If all chicken meat production goes to traditional markets, independent farmers will lose money because they can not keep up with the production cost of large-scale. In cost structure side, feed placed the biggest por­ tion in broiler and egg production cost. It was account­ ing to 70% of the total production cost, while other costs such as DOC (Day Old Chick) was only about 13 percent (Yusdja & Pasandaran, 1998). Feed demand for broilers and layers during the period 2000-2010 increased by 6.85% and 8.31%, respectively (Swastika et al., 2011). In 2010, its demand for broiler reached 3.51 million tons and 2.06 million tons for layers (Directorate General of Livestock and Animal Health, 2012). This indicated that feed available plays a significant role in broilers and eggs production. Therefore, if there is shock happening in feed prices, it brings a great influence on broiler farm­ ing performance, especially small-scale broiler farming. ABSTRAK Penelitian ini bertujuan untuk menganalisis dampak perubahan harga jagung domestik terhadap kinerja usaha peternakan rakyat ayam ras pedaging di Indonesia dengan menggunakan analisis model multimarket. Analisis model multimarket merupakan model keseimbangan parsial yang terdiri atas enam blok persamaan: harga, penawaran, permintaan input, konsumsi, pendapatan, dan kondisi keseimbangan. Model ini secara original dibangun dalam software General Algebric Modelling System (GAMS) dengan metode solusi Path NLP. Data terdiri atas: (1) data produksi dan input, konsumsi, dan pendapatan rumahtangga, (2) harga input dan output, dan (3) elastisitas. Untuk data produksi, penggunaan input, konsumsi, pendapatan dan harga menggunakan data dari Badan Pusat Statistik dan Kementerian Pertanian, sedangkan untuk elastisitas menggunakan data hasil penelitian sebelumnya. Penurunan harga jagung domestik direspon petani dengan mengurangi luas pertanaman jagung dan penggunaan input pupuk sehingga menyebabkan turunnya produksi dan pendapatan dari usahatani jagung. Sebaliknya kebijakan ini berdampak pada meningkatnya permintaan jagung untuk pakan sehingga meningkatkan produksi daging ayam ras dan pendapatan peternak rakyat ayam ras pedaging. Kondisi berbeda jika terjadi peningkatan harga jagung domestik. Kebijakan ini menyebabkan permintaan jagung untuk pakan menurun, akibatnya produksi daging ayam ras dan pendapatan peternak rakyat ayam ras pedaging menurun. Kata kunci: jagung, ayam ras pedaging, price, multimarket 198 December 2014 UMBOH ET AL. / Media Peternakan 37(3):198-205 Source: BPS (2012), Pusdatin Ministry of Agriculture (2012), processed INTRODUCTION 231 232 233 Price block. The producer prices (PP) are lower than the consumer prices (PC) due to the presence of the domestic marketing margin (MARG), hence: ) ( g y, ) PCc,h = PMc * (1+RMARG) Multimarket model is a partial equilibrium model was used to analyze the impact of changes in price and quantity in a particular market on household income and expenditures. Multimarket model analysis was used to measure the impact of policy changes on the poor households (Dorosh et al., 1995; Minot & Goletti, 1998), and it was focused on analyzing the impact of food im­ ports on the poverty level of poverty in Mozambique. In Indonesia, Hutabarat et al. (2012) investigate the impact of climate changes on the production, imports, and con­ sumption of horticulture. 234 235 236 237 238 PCc,h= PPc,h * (1 + MARG) ...................................(1) arge scale(PRSHN) ofbroiler consumer price diff .(1) e diff where the subscripts c and h refer to commodity and household type, respectively. The border prices (PM) for tradable products are linked to the world price by the exchange rate (er), import tariffs (tm) and the international marketing margin (RMARG). y an internal marketing margin (INTMARG) (Sugema & 010). PCc,pr = PCc,p * (1 + INTMARG) where the subscripts c and h refer to commodity and household type, respectively. The border prices (PM) for tradable products are linked to the world price by the exchange rate (er), import tariffs (tm) and the international marketing margin (RMARG). y an internal marketing margin (INTMARG) (Sugema 010). PCc,pr = PCc,p * (1 + INTMARG) PMc= PWc * er *(1 + tmc) ....................................(2) PCc,h = PMc * (1+RMARG)................................(3) PCc,rt = PCc,pr l d d i i d f h h h ld Based on the description above, this research ana­ lyzed the impact of domestic maize price changes on the performance of broiler industry in Indonesia with the problem statement of how the domestic maize price changes has impacted the performance of maize farm­ ing, broiler farming, and income of small-scale broiler farming household. This study therefore was conducted with the aims at analyzing the impact of domestic maize price changes on: (1) the performance of maize farming, (2) the performance of broiler farming, and (3) small- scale broiler farming household income. INTRODUCTION Year Domestic price of maize (IDR) Impor price of maize (IDR) Feed price of broiler (IDR) 2000 1 440 1 029 2 345 2001 1 450 1 227 2 470 2002 1 493 1 069 2 577 2003 1 547 1 084 2 687 2004 1 605 1 497 2 796 2005 1 362 1 577 2 905 2006 1 500 1 425 3 194 2007 2 269 1 963 3 250 2008 3 301 3 160 3 693 2009 2 585 2 378 4 461 2010 2 547 2 340 4 450 Trend (%/year) Domestic price of maize (%) Impor price of maize (%) Feed price of broiler (%) 2000-2001 0.69 19.24 5.33 2001-2002 2.96 -12.87 4.33 2002-2003 3.61 1.40 4.27 2003-2004 3.74 38.09 4.06 2004-2005 -15.14 5.34 3.89 2005-2006 10.13 -9.60 9.95 2006-2007 51.27 37.75 1.75 2007-2008 45.48 60.97 13.63 2008-2009 -21.69 -24.74 20.80 2009-2010 -1.47 -1.60 -0.25 7.96 11.40 6.78 Source: BPS (2012), Pusdatin Ministry of Agriculture (2012), processed Table 1. Maize and feed prices in Indonesia (Period 2000–2010) Table 1. Maize and feed prices in Indonesia (Period 2000–2010) The sustainability of broiler farming was deter­ mined by price changes of inputs and outputs. The change of input and output prices affected to input allo­ cation (feed) and the production decision of small-scale broiler farming. Saptana & Sumaryanto (2011) revealed that large-scale broiler farming dominated market struc­ ture of chicken meat. In addition, the large-scale broiler farming was dominant in the input market of poultry industry by making business agreements with feed companies and such association of integrated firms that operated from upstream to downstream activities, e.g. PT. Charoen Phokphan Indonesia, PT. Japfa Comfeed, and PT. Sierad Produce. Feed price was determined by feed industry that collaborated with poultry companies, December 2014 199 PMc = PWc 230 UMBOH ET AL. / Media Peternakan 37(3):198-205 (9) model definition, (10) fixing variables, (11) model closure, (12) display of parameters and variables, (13) solution statement, (14) optional solution, and (15) solu­ tion reports. he border price (PM) of the importable productsare link change rate (er), import tariffs (tm), and the inte feed price when decided the quantity of feed produc­ tion. Moreover, the quantity demanded of maize was determined by maize price itself rather than feed price. The problem of this research was how the change of domestik maize prices impact on the performance of small-scale broiler farming. INTRODUCTION 239 240 241 242 243 In addition, the large-scale (PRSHN) of broiler consumer prices differ from the small-scale (PTRYT) by an internal marketing margin (INTMARG). The consumer prices can be defined as: e included a price index for each household group eighted by their shares of consumption: ∗ PC 1h i PCc,pr= PCc,p * (1 + INTMARG)..............................(4) PCc,rt = PCc,pr.............................................................(5) PINDEX PCWTh,i PC0h,i i w is the budget share for each commodity. The sup We included a price index for each household group to reflect changes in prices weighted by their shares of consumption: periods 0 denote starting prices and 1 end of simulation p Product Categories PINDEX= Si PCWTh,i * (PC1 h,i/PC0 h,i) .................(6 ly Block PINDEX= Si PCWTh,i * (PC1 h,i/PC0 h,i) .................(6) where w is the budget share for each commodity. The superscript on the PC terms refers to periods 0 denote starting prices and 1 end of simulation prices. pp y ousehold’s supply of rice and maize are determined by ailable to each household; b) the share of that land alloca There are 6 (six) commodities in the model, namely: rice (rc), maize (m), broiler (br), eggs (lr), urea (ur), and triple super phosphate (TSP). The model assumes that commodities (rice, maize, chicken meat, and eggs) are used as final consumption by household and maize as input in feed industry. 246 247 248 Supply block. Household’s supply of food products is determined by total amount of land available, share of that land allocated to the specific crops, and yield of the crops. The share of land (SH) is a function of: the associated yield for the crops. The share of land ( op by household group h is a function of all crop prices: Data log(SHh,f) = αh,f s + βh,f,ff s ff log(PPh,ff) ........(7) rs to crop commodities (rice and maize). rs to crop commodities (rice and maize). D) f f b h h ld h f ti f This research used data of 2011 consisted of: (1) production, input utilization, consumption, and income, (2) price of inputs and outputs, and (3) elasticity. The data source of production, input utilization, consump­ tion, and income were Bureau of Statistics Central, Directorate General of Livestock and Animal Health, Bureau of Food Security of Ministry of Agriculture. 250 wh 251 Yie 252 as w 253 254 whe 251 Yie 252 as w 253 254 where f refers to crop commodities (rice and maize). (YLD) for crops f by household groups h are a function of out (YLD) for crops f by household groups h are a function of Yields (YLD) of household groups are determined by output producer price and input consumer prices. ( ) p y g p p l as land. l as land. log YLDh,f = αh,f y + βh,f y log PPh,f + γh,f,iin y iin log(PCh,iin) (8) log YLDh,f = αh,f + βh,f log PPh,f + γh,f,iin iin log(PCh,iin) 15) RESULTS AND DISCUSSION h,f,in h,in βh,f,in f g h,f γh,in g ( h,in) and for urea and TSP are given by: and for urea and TSP are given by: Total demand for urea and TSP are given by: g HDFEh f = αl + βl l log PPh l + γl ff l DINin= Sh HDINh,in ..............................................(14) i = HDINh i h ( , , , , , efer to maize for livestock feed Supply and Demand of Maize log HCh,i = αh,i h + βh,i,j h j log PCh,j + γh,i h log⁡(YHh) (17) log HCh,i = αh,i h + βh,i,j h j log PCh,j + γh,i h log⁡(YHh) consumption is given by : log (YHh)(17) where YH is household income, PC are consumer prices. hold income, PC are consumer prices. log HCh,i = αh,i h + βh,i,j h j log PCh,j + γh,i h log⁡(YHh) (17) log HCh,i = αh,i h + βh,i,j h j log PCh,j + γh,i h log⁡(YHh) consumption is given by : log (YHh)(17) where YH is household income, PC are consumer prices. mption is given by : S = HC NSi = HCh,i h Total consumption is given by : i h,i h ck ock CONSi= Sh HCh,i ..............................................(18) .(18) Agricultural income (YHAG) for rural households is the sum of crop an ock revenue minus input costs: Agricultural income (YHAG) for rural households is th Income block. Agricultural income (YHAG) is the sum of crop and poultry revenue minus input costs: YHAGh=∑f(PPf*SCRh,f)+∑1(PP1*SLVh,1)–(PCin*DINh,in)-(PCfe*DFEh,fe) (1 tal household income (YH) is the sum of agricultural income and the exogenous ck revenue minus input costs: YHAGh=∑f(PPf*SCRh f)+∑1(PP1*SLVh 1)–(PCin*DINh in)-(P YHAGh=∑f(PPf*SCRh,f)+∑1(PP1*SLVh,1)–(PCin*DINh,in)- (PCfe*DFEh,fe) ........................(9) mined non-agricultural income (YHNAG). The latter component is adjusted by ndexand the price index is as defined in equation (6). YHh = YHAGh + YHNAG h ∗PINDEX (20) tal household income (YH) is the sum of agricultural income mined non-agricultural income (YHNAG). The latter compon ndexand the price index is as defined in equation (6) and total household income (YH) is the sum of agricultural income and the exogenously determined non-agricultural income (YHNAG). The latter component is adjusted by a price index and the price index is as defined in equation (6). d the exogenously t is adjusted by a (20) To meet domestic demand for maize, some at­ tempts have been done such as increasing productivity, expanding planted maize areas, improving technical assistance, empowering agricultural institution, and providing financial support. As a result, there was a significant production increase of 7.86% per year during the period 2005-2011 (BPS, 2012). It is hoped that the domestically produced maize will be able to close the domestic demand. h = YHAGh + YHNAG h ∗PINDEX YHh= YHAGh + YHNAGh * PINDEX ................(20) brium Conditions Equilibrium conditions. In this block, it is assumed that there is “market clearing condition” for all commodity brium Conditions Equilibrium conditions. Structure of the Model Those are mathematically defined as SCRf= Sh SCRh,f .......................................(10) livestock supply is modelled as a function of livestock p livestock feed products, where the subscript ffe refer SCRf= Sh SCRh,f .......................................(10) livestock supply is modelled as a function of livestock ivestock feed products, where the subscript ffe ref The poultry production by households is a function of poultry prices and poultry input prices of feed. of livestock feed products, where the subscript ffe refers to livestoc cts. cts. log SLVh l = αh l 1 + βh l ll 1 ll log PPh l + γh l ffe 1 ffe log⁡(PCh ffe 1) SCRf + SLV1 + MC = CONSi + DFEC .....................(21) log SLVh,l = αh,l 1 + βh,l.ll 1 ll log PPh,l + γh,l,ffe 1 ffe log⁡(PCh,ffe) log (PCh,ffe) (11) livestock supply is given by: 1) where M equals imports and CONS and DFE denote human and livestock consumption respectively. stock supply is given by: Total poultry supply is given by: LVl = SLVh,l h Model Simulation = SLVh,l h t Bl k SLV1= Sh SLVh,1 .....................................................(12) nput Block and for (PP) and mand for (13) (14) Two simulations were conducted to answer the ob­ jectives of this study, i.e: (1) Decreased domestic price of maize by 10%, and (2) Increased domestic price of maize by 10%. These simulations concerned to the increasing trend of maize price during 2000-2010 that reached about 7.96%. In 2011, maize price increased remarkably from IDR 2547 to IDR 2933 or increased around 15%. nd Input Block Household demand for input (HDIN) is a function of output prices (P prices (PC), where the subscript in refers to urea andTSP. Household dem Demand input block. Household demand for input (HDIN) is a function of output prices (PP) and input pric­ es (PC), where the subscript in refers to Urea and TSP. Household demand for urea and TSP: Household demand for input (HDIN) is a function of output price prices (PC), where the subscript in refers to urea andTSP. Household nd TSP: DINin = HDINh in h d TSP: og HDINh,f,in = αh,in f + βh,f,in f f log PPh,f + γh,in f log⁡(PCh,in) mand for urea and TSP are given by: log HDINh,f,in = αh,in f + βh,f,in f f log PPh,f + γh,in f log⁡(PCh,in) emand for urea and TSP are given by: log (PCh,in)(13) Total demand for urea and TSP are given by: old demand maize for livestock feed: log HDFEh f = αh f l + βh l f l l log PPh l + γh f ff l ff lo nd TSP: log HDINh,f,in = αh,in f + βh,f,in f f log PPh,f + γh,in f log⁡(PCh,in) demand for urea and TSP are given by: log HDINh,f,in = αh,in f + βh,f,in f f log PPh,f + γh,in f log⁡(PCh,in) demand for urea and TSP are given by: log (PCh,in)(13) Total demand for urea and TSP are given by: ehold demand maize for livestock feed: log HDFEh f = αh f l + βh l f l l log PPh l + γh f ff l ff lo nd TSP: log HDINh,f,in = αh,in f + βh,f,in f f log PPh,f + γh,in f log⁡(PCh,in) log HDINh,f,in = αh,in f + βh,f,in f f log PPh,f + γh,in f log⁡(PCh,in) log (PCh,in)(13) hold demand maize for livestock feed: Supply and Demand of Maize Household demand maize for poultry feed: , demand maize for livestock feed: nd for maize is given by: (16) During the period 2005-2011, the development trend of harvested area, production and productivity of maize nationally showed an increase by 2.62%, 7.86% and 5.23%, respectively. In 2011, the national maize harvested area reached 3.86 million hectares with pro­ duction and productivity levels of 17.63 million tons and 4.57 ton/ha, respectively (BPS, 2012). log HDFEh,fe = αh,fe l + βh,l,fe l l log PPh,l + γh,fe,ffe l ffe log PCh,ffe (15 (15) g y DFEfe = HDFEh fe h e fe refer to maize for livestock feed. demand for maize is given by: where fe refer to maize for poultry feed. Total demand for maize is given by: umption Block fe = HDFEh,fe h DFEfe= Sh HDFEh,fe ..............................................(16) mand for the consumption items (HC) by the househ oups, where the i and eggs). YH is (17) (18) um of crop and *DFEh,fe) (19) p y ( ) Although increased, the national maize productiv­ ity is still low due to most of farmers still have used seeds local varieties. Some other farmers eventhough they grow maize hybrids and composites, but they managed it less intensively. This conditions have caused maize yield is still far from its potency (Swastika et al., 2005). Indeed, if farmers are willing to manage their maize farm intensively or use input according to recom­ mended technology, the productivity can reach 6-10 tons per hectare for maize hybrid and 5-8 tons/ha for maize composite. This means that farmers have not utilized the production potential optimally and the possibility in getting more income through using new superior variet­ ies of maize. This condition is caused by several things, among others are: (1) farmers do not have enough money to buy superior seeds, fertilizers and pesticides needed and (2) the seed available is frequent delay, so that planting activity cannot be done on time. As a result, the maize productivity level is not as expected (Yusdja & Agustian, 2003). umption Block Demand for the consumption items (HC) by the household groups, where th o commodities households purchase (rice, maize, chicken meat, and eggs). YH Consumption block. The demand for each commodity is modeled as the consumption function of household groups as follow: o commodities households purchase (rice, maize, chicken me hold income, PC are consumer prices. hold income, PC are consumer prices. Structure of the Model the coefficients (α, β, γ)represent the price elasticities. where the coefficients (a, b, g) represent the price elasticities. the coefficients (α, β, γ)represent the price elasticities. tal household supply to the market is then determined as the pr This research employed multimarket model from Umboh et al. (2014) and it was divided into 6 blocks of equations, namely: (1) price, (2) supply, (3) input de­ mand, (4) consumption, (5) income, and (6) equilibrium condition. The model was originally designed in General Algebric Modelling System (GAMS) using the Path NLP solver consist of fifteen steps, namely: (1) set declaration, (2) database, (3) parameter declaration, (4) parameter definition, (5) variable declaration, (6) variable defini­ tion, (7) equation declaration, (8) equation definition, The 256 are 257 Ad 258 con 259 260 The 261 The 256 area 257 Adj 258 con 259 260 The 261 tal household supply to the market is then determined as the produ under cultivation, the share of land devoted to the crop, an tmentsare made for losses and use of the output for seed (loss), and f rsionfactors (conv). The production of food crops by each household is determined by total land devoted to the crop, the share of land devoted to the crop, and the yield. After that, total production available at the market is adjusted by losses, use of the food crops for seed (loss), and conversion factors (conv), hence: tal household supply to the market is then determined as the pr under cultivation, the share of land devoted to the crop mentsare made for losses and use of the output for seed (loss), a sionfactors (conv). SCRh,f = AREA ∗SHh,f ∗YLDh,f ∗(1 −lossf) ∗convf SCRh,f = AREA ∗SHh,f ∗YLDh,f ∗(1 −lossf) ∗convf (9) (9) 200 December 2014 onvf (9) f h h ld l (10) UMBOH ET AL. / Media Peternakan 37(3):198-205 The total supply of each of the food crops are: Rf = SCRh,f h livestock supply is modelled as a function of livestock The total supply of each of the food crops are: Rf = SCRh,f h livestock supply is modelled as a function of livestock 0) nput feed nd input ock feed markets. In this regard, the sum of quantity supplied (do­ mestic production plus net imports) is equal to the quan­ tity demanded for both human consumption and feed demand. Supply and Demand of Maize and Chicken Meat in Table 2. For maize farmers, the reduction in domestic maize price was responded by reducing the land share for planted maize land by 3.120% for small-scale broiler farming households and by 3.133% for other house­ holds, respectively. At the same time both two types of households have decided to reduce in Urea and TSP fer­ tilizers uses of by 5.668% and 2.611%, respectively. The changes of farmer decision for maize planted area size and fertilizers uses, then had impact on maize produc­ tion decrease by 2.615%. But, opposite impact accoured when the maize price goes down, in which farmers will be more interesting to increase rice planted area size. In this condition, planting rice gives more interesting ben­ efit than that of planting maize. This was showed by the increased share of rice planted land for small-scale broil­ er farming households and other households by 1.1436% and 1.140%, respectively. Therefore, when there was a decline in the maize price, farmers would divert their planted maize area to rice farming. In this case, the farm­ ers also are willing to use more input production for rice that had positive impact on rice productivity. Therefore, at level of productivity and land share increase would lead to the increase in rice production by 1.7481%. Comparing with the national maize production data, in 2005 national maize production reached 12.52 million ton, then decreased to 11.61 million ton in 2006 and increased again to 17.63 million ton in 2011. Based on that, in 2006 there was a deficit so for answered the demand, the government did an import around 1.84 million ton. In 2007-2011, national maize production reached over the demand. However, maize still im­ ported around 414 thousand ton in 2007 and increased to 3.1 million ton in 2011 (BPS, 2012). Maize import was done by the feed industries with some reasons such as, the difficulties to find maize from the farmer, national maize production does not available in a whole year, and maize import buying mechanism is more easier than local corn buying, because they do not need to get in touch with the farmers or producers di­ rectly (Malian, 2004; Hakim, 2005; Swastika et al., 2011). In 2005, the total maize requirement reached around 11.86 million ton, then increased to 13.71 million ton in 2008, and became 16.50 million ton in 2011 (BPS, 2012). Impact of Domestic Maize Price Changes on the Performance of Broiler Farming Domestic maize price decrease and the existing margin between the large-scale and small-scale broiler farming by 20%, where by IDR 2933 on the broiler farm­ ing of large-scale and IDR 3519.60 on the small-scale, respectively (Table 2), had an impact on the performance of broiler farming as shown in Table 3. For small-scale broiler farming, when the price of maize to be paid by the small-scale was 20% higher than the price paid by the broiler large-scale, increasing the maize demand for feed by 0.5268%, this in turn would increase chicken meat production by 0.5279%. The same phenomenon also occured in large-scale production, where the increased maize demand for broiler feed triggered the increase in production large-scale of by 0.5285%. Furthermore, the impact of domestic maize price decrease by 10% leading to the increase in maize demand for feed by the large and small-scale broiler farming, increasing the national The most broiler population are located in 14 provinces, namely West Java, East Java, Central Java, Banten, North Sumatra, Riau, East Kalimantan, South Kalimantan, West Kalimantan, South Sumatra, South Sulawesi, Lampung, Riau, and Jambi. Meanwhile, for layer are located in 12 provinces: East Java, Central Java, West Java, North Sumatra, West Sumatra, Banten, South Sulawesi, South Sumatra, Bali, Lampung, and West Kalimantan (Directorate General of Livestock and Animal Health, 2012.) Supply and Demand of Maize and Chicken Meat Related to production and price chicken meat, al­ though there was broiler feed price increasing because of maize price increasing, there was still broiler production increasing. This was because of the input price increas­ ing (feed) compensated with the output price increasing (chicken meat), so that this gave an incentive to the farmer to increase their production. During 2000-2010, chicken price and production increased around 5.86% and% 14.453, respectively (BPS, 2012). p y Domestic maize price increased was responded by farmers by increasing maize planted area by 2.84% and 2.876% on the households of small-scale broiler farming and other households, respectively. In addition, the im­ provement of maize price led to Urea and TSP fertilizers uses for maize farming increased by 5.371% and 5.383%, respectively and then improved maize productivity by 2.39971 and 2.39910 on the households of small-scale and other households. Increased land share for and pro­ ductivity of maize would cause the increase in national maize production by 5.34606%. On the other side, this condition has a negative impact on rice production by 1.41942%. This indicated that when the price of maize increases, farmers would use part of their rice land for planting maize. These results were in line with other research results, such as Sayaka et al. (2007); Huang et al. (2009); and Dorosh et al. (2009). Theoritically, the cause of chicken meat price increasing, besides caused by production cost increas­ ing, also caused by the increasing of demand because of population, nutritional consumption awareness, and income increasing (Ilham, 2009; Ilham & Yusdja, 2010). By the time, chicken meat contribution to the national meat consumption has reached around 70.24% (Saptana & Sumaryanto, 2009). During 2000-2009, chicken meat consumption was around 2.33 kg per capita per year. The demand for chicken meat continuously increased to 3.10 kg per capita in 2010 and reached around 4.53 kg per capita per year in 2011 (Bureau of Food Security of Ministry of Agriculture, 2012). Supply and Demand of Maize In this block, it is assumed that there is “market clearing condition” for all commodity December 2014 201 UMBOH ET AL. / Media Peternakan 37(3):198-205 202 December 2014 Note: *) increasing domestic maize price by 10%; **) decreasing domestic maize price by 10%. Impact of Domestic Maize Price Changes on the Performance of Maize Farming The reseach result showed that domestic maize price decrease by 10% had a negative impact on the performance of maize farming in Indonesia as shown 202 December 2014 UMBOH ET AL. / Media Peternakan 37(3):198-205 Note: *) increasing domestic maize price by 10%; **) decreasing domestic maize price by 10%. Variables Base value (Unit) Alternative simulation Simulation 1* Simulation 2** (Unit) (%) (Unit) (%) PMm (Imported Maize Price) 2346.40 0.00 0.00 0.00 0.00 PCm,p (Maize Price of PRSHN) 2933.00 293.30 10.00 -293.30 -10.00 PCm,pr (Maize Price of PTRYT) 3519.60 351.96 10.00 -351.96 -10.00 PMrc (Imported Rice Price) 6150.40 0.00 0.00 0.00 0.00 PCrc (Domestic Rice Price) 7688.00 0.00 0.00 0.00 0.00 SH1,2 (Rice Land Share of PTRYT) 0.078 -0.001 -0.872 0.001 1.144 SH1,2 (Rice Land Share of RTOTH) 0.526 -0.005 -0.880 0.006 1.14 YLD1,2 (Rice Productivity of PTRYT) 4.207 -0.018 -0.419 0.025 0.594 YLD1,2 (Rice Productivity of RTOTH) 5.58 -0.023 -0.419 0.033 0.591 HSCR1 (Rice Production of PTRYT) 2598.32 -36.877 -1.419 45.425 1.748 HSCR2 (Rice Production of RTOTH) 23240.54 -329.885 -1.419 406.30 1.748 SCR1,2(Rice Production of Indonesia) 25838.86 -366.762 -1.419 451.69 1.748 SH3,4 (Maize Land Share of PTRYT) 0.025 0.001 2.84 -0.001 -3.12 SH3,4 (Maize Land Share of RTOTH) 0.160 0.005 2.876 -0.005 -3.133 YLD3,4 (Maize Productivity of PTRYT) 3.50 0.084 2.400 -0.091 -2.611 YLD3,4 (Maize Productivity of RTOTH) 4.56 0.109 2.399 -0.119 -2.610 HSCR3 (Maize Production of PTRYT) 1107.53 59.209 5.346 -62.80 -5.67 HSCR4 (Maize Production of RTOTH) 9211.81 492.47 5.346 -522.24 -5.669 SCR3,4 (Maize Production of Indonesia) 10319.34 551.68 5.346 -585.03 -5.669 HDIN1 (Demand for Urea PTRYT) 1.679 0.09 5.36 -0.095 -5.658 HDIN2 (Demand for Urea RTOTH) 1049.36 56.48 5.382 -59.50 -5.67 DIN1,2 (Demand for Urea Indonesia) 1051.04 56.57 5.371 -59.58 -5.668 HDIN3 (Demand forTSP PTRYT) 0.705 0.038 5.39 -0.04 -5.673 HDIN4 (Demand for TSP RTOTH) 440.73 23.72 5.381 -11.53 -2.616 DIN3,4 (Demand for TSP Indonesia) 441.43 23.763 5.383 -11.53 -2.611 CONS1,2 (Consumption for Rice) 24789.08 42.60 0.172 -90.65 -0.365 CONS3,4 (Consumption for Maize) 7941.02 -52.46 -0.66 124.32 1.565 IMrc (Net Import of Rice) 4499.991 409.36 9.096 -542.339 -12.052 IMm (Net Import of Maize) 3182.356 -607.83 -19.10 709.364 22.29 Table 2. The results of simulation of domestic maize price changes on the performance of maize farming in Indonesia Table 2. The results of simulation of domestic maize price changes on the performance of maize farming in Indonesia ble 3. Impact of Domestic Maize Price Changes on the Performance of Maize Farming The results of simulation of domestic maize price changes on the performance of broiler farming in Indone esults of simulation of domestic maize price changes on the performance of broiler farming in Indonesia Table 3. The results of simulation of domestic maize price changes on the performance of broiler farming in Indonesia Variables Base value (Unit) Alternative Simulation Simulation 1* Simulation 2** (Unit) (%) (Unit) (%) PCbr (Domestic Chicken Meat Price) 27500.00 0.000 0.000 0.000 0.000 PClr (Domestic Egg Price) 18058.00 0.000 0.000 0.000 0.000 HSLV1(Chicken Meat Production of PRSHN) 1137.12 -5.40 -0.474 6.010 0.529 HSLV2(Chicken Meat Production of PTRYT) 181.83 -0.867 -0.477 0.960 0.528 SLV1,2 (Chicken Meat Production of Indonesia ) 1318.95 -6.207 -0.470 6.970 0.528 HSLV3( Egg Production of PTRYT) 0.10 -0.000 -0.460 0.001 1.300 HSLV4( Egg Production of RTOTH) 1025.91 -4.879 -0.476 5.420 0.528 SLV3,4 ( Egg Production of Indonesia) 1026.01 -4.880 -0.477 5.427 0.529 HDFE1 (Demand for Maize PRSHN) 1066.37 -5.070 -0.475 5.640 0.529 HDFE2 (Demand Maize for Broiler Feeds PTRYT) 227.00 -1.080 -0.476 1.196 0.527 HDFE3 (Demand Maize for Layer Feeds PTRYT) 0.127 -0.001 -0.472 0.001 0.787 HDFE4 (Demand for Maize RTOTH) 1292.25 -6.140 -0.475 6.834 0.529 DFE1,2,3,4 (Demand for Maize Indonesia) 2585.747 -12.290 -0.475 13.671 0.528 CONSP1,2 (Consumption for Chicken Meat Indonesia) 1137.21 -0.630 -0.055 1.032 0.090 CONSP1,2 (Consumption for Eggs Indonesia) 1027.80 -3.980 -0.387 7.717 0.750 Note: *) increasing domestic maize price by 10%; **) decreasing domestic maize price by 10%. December 2014 203 UMBOH ET AL. / Media Peternakan 37(3):198-205 Table 4. The results of simulation of domestic maize price changes on households income of small-scale broiler farming (Million Rupiahs) Table 4. The results of simulation of domestic maize price changes on households income of small-scale broiler farming (Million Rupiahs) Variables Base value (Unit) Alternative Simulation Simulation 1* Simulation 2** (Unit) (%) (Unit) (%) a. YHAG1 (Income of PRSHN) 21888976 -415240.45 -1.897 430092.870 1.965 b. YHAG2 (Income of PTRYT) 22286608 -153717.82 -0.690 210125.455 0.943 1. Rice 15968858.530 -226640.48 -1.419 279174.770 1.748 2. Maize 3115440.070 167759.37 5.385 -176713.795 -5.672 3. Broiler 3201210.80 -94787 -2.961 97182.470 3.036 4. Laying hens 997.65 -49.08 -4.920 58.510 5.865 c. YHAG3 (Income of RTOTH) 1049290416 -7219118.062 -0.688 9621993.115 0.917 Total (a+b+c) 1093466000 -10597552.96 -0.969 10262211.120 0.938 d. YHNAG1(Income of PRSHN) 0.00 0.00 0.000 0.000 0.000 e. YHNAG2(Income of PTRYT) 3039080 0.00 0.000 0.000 0.000 f. Impact of Domestic Maize Price Changes on the Performance of Maize Farming YHNAG3(Income of RTOTH) 6445640124 0.00 0.000 0.000 0.000 Total (d+e+f) 6448679204 0.00 0.000 -3025496.54 -1.878 g. YH1 (Income of. PRSHN) 21888976 -415240.45 -1.897 430092870 1.965 h. YH2 (Income of PTRYT) 25325688 -153718.77 -0.607 210124.51 0.829 i. YH3 (Income of RTOTH) 7494930540 -9768894 -0.130 9621993 1.284 Total (g+h+i) 7542145204 -10337853 -0.137 10262211.17 0.136 Note: *) increasing domestic maize price by 10%; **) decreasing domestic maize price by 10%. Impact of Domestic Maize Price Changes on the Household Income of Small-scale Broiler Farming Impact of Domestic Maize Price Changes on the Household Income of Small-scale Broiler Farming The decrease of domestic maize price by 10% and if the price margin to be paid by small-scale broiler farming was 20% higher than the price large-scale had to pay, the household income of small-scale broiler farming would decrease by 5.572% from maize farm­ ing, but it increased their income from rice farming, broiler business and layer business by 1.748%, 3.036%, and 5.865%, respectively (Table 4). This policy caused the increase in agricultural income (YHAG) as well as national income (YH) of by 0.938% and 0.136%, respec­ tively. In contrast, when government issued the policy of increasing domestic maize price of by 10%, it caused the household income of small-scale broiler farming from maize increased by 5.385%, but decrease of the income of small-scale broiler farming from rice farming, broiler, and layer business by 1.419%, 2.961%, and 4.920%, respectively. Furthermore, this policy caused the agri­ cultural sector income (YHAG) and the national income (YH) go down by 0.969% and 0.137%, respectively. CONCLUSION production of by 0.5284%. This condition also had a positive impact on the improvement of layers produc­ tion of by 0.5289%. The price policy to increase maize price had positive impacts on performance of maize farming and farmer income by increasing maize cultivated area and input utilization to increase domestic maize production and to reduce maize import. On the other hand, the lowering of domestic maize price was responded by farmers through reducing maize planted land and fertilizer input uses, causing maize production and farmer income from maize farming decline; in addition, price policy to increase maize price showed a negative impact on the performance and income of small-scale broiler farming as it caused the increase of production cost due to the increasing feed price. In contrast, reduction maize price would have an impact on increasing maize demand for feed industry. Aside from that, other impacts from this policy were increasing broilers production and small- scale broiler farming income. In general, this policy could improve the level of agricultural income and national income. y In contrast, when the domestic maize price in­ creased by 10%, in which maize demand by both large and small-scale broiler farming went down by 0.4754% and 0.4757%, respectively. Consequently, the produc­ tion for meat also decreased by 0.474% and 0.477%, respectively, both in large and small-scale broiler farm­ ing. These results were in accordance with Kariyasa & Sinaga (2007). REFERENCES Bureau of Food Security of Ministry of Agriculture. 2012. De­ velopment of Food Consumption Directory. Bureau of Food Security of Ministry of Agriculture, Jakarta. BPS. 2012. Statistic Indonesia. Central Bureau of Statistic, Ja­ karta. Directorate General of Livestock and Animal Health. 2012. Livestock and Animal Health Statistics. Directorate Gen­ eral of Livestock and Animal Health, Jakarta. Dorosh, P. A., C. del Ninno, & D. E. Sahn. 1995. Poverty allevi­ ation in Mozambique: a multimarket analysis of the role of food aid. Agricultural Economics 13:89-99. http://dx.doi. org/10.1016/0169-5150(95)01156-0 204 December 2014 UMBOH ET AL. / Media Peternakan 37(3):198-205 Malian, A. H. 2004. International trade policy of agricultural commodities. 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K. S., M. Manikmas, B. Sayaka, & I. K. Kariyasa. 2005. The behavior of maize supply and demand in Indo­ nesia. Jurnal Penelitian Pertanian Tanaman Pangan 24:67- 75. g j p Hutabarat, B., A. Setiyanto, R. Kustiari, & T. B. Sulser. 2012. Conjecturing production, imports and consumption of horticulture in Indonesia in 2050: A GAMS simulation through changes in yields induced by climate change. Jur­ nal Agro Ekonomi 30:1-23. Swastika, D. K. S, A. Agustian, & T. Sudaryanto. 2011. Gap analysis of supply and demand of corn forage production approach sync center, feed plant, animal and population in Indonesia. Informatika Pertanian 2:65-75. g Ilham, N. 2009. Shortage of meat production: policy indication and implication. Analisis Kebijakan Pertanian 7:43-63.l p Ilham, N. & Y. Yusdja. 2010. Impact of Avian Influenza on pro­ duction and contribution of poultry to small-scale farmers income in Indonesia. Jurnal Agro Ekonomi 28:39-68. Umboh, S. J. K, D. B. Hakim, B. M. Sinaga, & I. K. Kariyasa. 2014. REFERENCES Impact of maize import tariff policy changes on pro­ duction and consumption in Indonesia: A multimarket model analysis. IJFAE 2: 113-126. g Indarsih, B., M. H. Tamsil, & M. P. Nugroho. 2010. A study of contract broiler production in Lombok, NTB: An opportu­ nity of introducing syariah partnership. Med. Pet. 33:124- 130. http://dx.doi.org/10.5398/medpet.2010.33.2.124 y Yusdja, Y. & E. Pasandaran. 1998. Restructurization direction of Indonesia poultry agribusiness. Jurnal Agro Ekonomi 16:21-32. f Yusdja, Y. & A. Agustian. 2003. Maize tariff policy analysis. Analisis Kebijakan Pertanian 1:36-54. t p g p Kariyasa, I. & B. M. Sinaga. 2007. Feed and chicken meat mar­ kets behaviour analysis in Indonesia: simultaneous econo­ metric model approach. Journal on Socio-Economics of Agriculture and Agribusiness (SOCA) 7:158-166. December 2014 205
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Rumination and Default Mode Network Subsystems Connectivity in First-episode, Drug-Naive Young Patients with Major Depressive Disorder
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Rumination and Default Mode Network Subsystems Connectivity in First-episode, Drug-Naive Young Patients with Major Depressive Disorder received: 19 August 2016 accepted: 18 January 2017 Published: 22 February 2017 Xueling Zhu1,2,3, Qiuling Zhu4, Huaizhen Shen2, Weihua Liao5 & Fulai Yuan1 Neuroimaging evidence implicates the association between rumination and default mode network (DMN) in major depressive disorder (MDD). However, the relationship between rumination and DMN subsystems remains incompletely understood, especially in patients with MDD. Thirty-three first- episode drug-naive patients with MDD and thirty-three healthy controls (HCs) were enrolled and underwent resting-sate fMRI scanning. Functional connectivity analysis was performed based on 11 pre-defined regions of interest (ROIs) for three DMN subsystems: the midline core, dorsal medial prefrontal cortex (dMPFC) and medial temporal lobe (MTL). Compared with HCs group, patients with MDD exhibited increased within-system connectivity in the dMPFC subsystem and inter-system connectivity between the dMPFC and MTL subsystems. Decreased inter-system connectivity was identified between the midline core and dMPFC subsystem in MDD patients. Depressive rumination was positively correlated with within-system connectivity in the dMPFC subsystem (dMPFC-TempP) and with inter-system connectivity between the dMPFC and MTL subsystems (LTC-PHC). Our results suggest MDD may be characterized by abnormal DMN subsystems connectivity, which may contribute to the pathophysiology of the maladaptive self-focus in MDD patients. Rumination is defined as a mode of responding to distress that involves repetitively and passively focusing on symptoms of distress and on the possible causes and consequences of these symptoms1,2. It is strongly and con- sistently related to depressive symptoms3. Evidence from prospective longitudinal and experimental studies have emphasized the role of rumination in the onset and maintenance of symptoms and the diagnosis of major depres- sive disorder (MDD)2,4,5, onset of depressive symptomatology in non-depressed people2, and risk of depressive relapse in remitted patients6. p p Although the pathophysiological mechanisms underlying MDD remain unclear, neuroimaging studies have shown that MDD has been conceptualized as a neural network-level disease7–10. Among the neural networks iden- tified in MDD, the default mode network (DMN) has received growing attention. It encompasses a specific set of brain regions, including the medial prefrontal cortex (MPFC), precuneus/posterior cingulate cortex (PCC), and medial, lateral, and inferior parietal cortex11,12. Researchers have consistently reported that the DMN is involved in internal mentation, including self-referential processing, the memory retrieval process and autobiographical memory retrieval13–15, which is closely associated with depressive symptomatology15,16. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 19 August 2016 accepted: 18 January 2017 Published: 22 February 2017 Rumination and Default Mode Network Subsystems Connectivity in First-episode, Drug-Naive Young Patients with Major Depressive Disorder MDD: major depressive disorder; HC: healthy control; Data value are Mean ± SD; F/M: female/male; SD. CES-D: Center for Epidemiologic Studies Depression; RSQ: Response to Stress Questionnaire. aChi-square test; bTwo sample t-test. Table 1. Demographic and clinical characteristics for subjects. MDD: major depressive disorder; HC: healthy control; Data value are Mean ± SD; F/M: female/male; SD. CES-D: Center for Epidemiologic Studies Depression; RSQ: Response to Stress Questionnaire. aChi-square test; bTwo sample t-test. Recent imaging studies have suggested the intuitive association between the self-focused rumination in MDD and the self-referential operations performed by DMN25,26, which has prompted further interest in examining the role of DMN in MDD. It was revealed that people with MDD showed stronger functional connectivity with DMN in the subgenual prefrontal cortex (sgPFC), the degree of activation being modulated by the level of maladaptive rumination27,28. Similarly, our previous study also reported increased functional connectivity with DMN in ante- rior medial cortex including the sgPFC in MDD patients29. Furthermore, effective connectivity analysis revealed mutually propagating activation between the sgPFC and ventral MPFC in people with MDD, which predicted higher levels of depressive rumination30. Convergent findings from neuroimaging studies have identified the role of the sgPFC in the DMN and its relationship with rumination in MDD, which suggest that the sgPFC might be unique to depression and a neural substrate of depressive rumination15,26. However, the association between the rumination and DMN in MDD still remains unclear and requires further investigation in this field. q gi Although the DMN originally was regarded as a large-scale brain system, current studies have noted the DMN is not as homogenous as previously assumed and suggested that its architecture can be further subdivided into smaller anatomical–functional subsystems31. Furthermore, the subsystems and the interaction among them have been shown to provide interesting insights for exploring mental diseases32–35. Recent evidence both from monkeys and humans have suggested that the DMN comprises a set of interacting subsystems, including a mid- line core constituted by hub regions in the anterior medial prefrontal and posterior cingulate cortices as well as two functionally dissociable subsystems: the dorsal medial prefrontal cortex (dMPFC) subsystem involved in self-referential processes and the medial temporal lobe (MTL) subsystem involved in episodic memory31. Furthermore, altered interaction among the three subsystems within the DMN might be crucial in the psychopa- thology of some neuropsychiatric disorders, including Alzheimer’s disease32, obsessive-compulsive disorder33 and schizophrenia34. Rumination and Default Mode Network Subsystems Connectivity in First-episode, Drug-Naive Young Patients with Major Depressive Disorder Striking differences in the activity and functional connectivity of the DMN between individuals with MDD and healthy controls (HCs) have been identified in a large number of studies using either task-based17–19 or resting-state fMRI20–23. Despite inconsistent findings, the DMN has been considered to play a central role in the physiopathology of MDD24. 1Health Management Center, Xiangya Hospital, Central South University, Changsha, 410008, China. 2School of Humanities and Social Sciences, National University of Defense Technology, Changsha, 410074, China. 3Medical Psychological Institute, Second Xiangya Hospital, Central South University, Changsha, 410011, China. 4Obstetrics Department, Jinan Maternity and Child Care Hospital, Jinan, 250001, China. 5Department of Radiology, Xiangya Hospital, Central South University, Changsha, 410008, China. Correspondence and requests for materials should be addressed to F.Y. (email: fulaiyuan2010@163.com) Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 1 www.nature.com/scientificreports/ Characteristic MDD (n = 31) HC (n = 32) P value Gender (F/M) 17/14 17/15 0.657a Age (years) 20.53 ± 1.78 20.96 ± 1.28 0.434b Education (years) 13.62 ± 0.76 13.75 ± 0.82 0.611b Illness duration (years) 0.29 ± 0.20 NA CES-D 36.90 ± 6.79 17.97 ± 7.93 <0.001b RSQ-rumination 7.74 ± 1.18 4.65 ± 1.68 <0.001b Table 1. Demographic and clinical characteristics for subjects. MDD: major depressive disorder; HC: healthy control; Data value are Mean ± SD; F/M: female/male; SD. CES-D: Center for Epidemiologic Studies Depression; RSQ: Response to Stress Questionnaire. aChi-square test; bTwo sample t-test. Characteristic MDD (n = 31) HC (n = 32) P value Gender (F/M) 17/14 17/15 0.657a Age (years) 20.53 ± 1.78 20.96 ± 1.28 0.434b Education (years) 13.62 ± 0.76 13.75 ± 0.82 0.611b Illness duration (years) 0.29 ± 0.20 NA CES-D 36.90 ± 6.79 17.97 ± 7.93 <0.001b RSQ-rumination 7.74 ± 1.18 4.65 ± 1.68 <0.001b Table 1. Demographic and clinical characteristics for subjects. MDD: major depressive disorder; HC: healthy control; Data value are Mean ± SD; F/M: female/male; SD. CES-D: Center for Epidemiologic Studies Depression; RSQ: Response to Stress Questionnaire. aChi-square test; bTwo sample t-test. Characteristic MDD (n = 31) HC (n = 32) P value Gender (F/M) 17/14 17/15 0.657a Age (years) 20.53 ± 1.78 20.96 ± 1.28 0.434b Education (years) 13.62 ± 0.76 13.75 ± 0.82 0.611b Illness duration (years) 0.29 ± 0.20 NA CES-D 36.90 ± 6.79 17.97 ± 7.93 <0.001b RSQ-rumination 7.74 ± 1.18 4.65 ± 1.68 <0.001b Table 1. Demographic and clinical characteristics for subjects. Rumination and Default Mode Network Subsystems Connectivity in First-episode, Drug-Naive Young Patients with Major Depressive Disorder Previous studies have reported that patients with MDD exhibited abnormal connectivity in pos- terior, ventral and core DMN subsystems along with reduced interactions from the anterior to the ventral DMN subsystems35, which suggested the role of these subsystems and the interplay among them in the pathophysiology of MDD. To date, however, the extent to which altered function and interaction of three DMN subsystems (the midline core, dMPFC and MTL subsystems) can contribute to the pathophysiology of MDD is not known. y p p y gy To the best of our knowledge, limited research has investigated neural correlates of rumination and three DMN subsystems in MDD. In the current study, our first aim was to investigate DMN subsystems connectivity in first-episode treatment-naïve young adults with MDD. The second goal was to examine the association between depressive rumination and DMN subsystems connectivity. We hypothesized that MDD patients would exhibit altered activity in DMN subsystems, which would correlate with depressive rumination. Figure 1. The left panels of (A) and (B) shows half of the averaged connectivity matrices among 11 ROIs of the DMN for MDD patients (A) and HCs group (B). The color bar indicates the value of correlation strength. The right panels of (A) and (B) showed visualized brain network for the averaged correlation coefficient matrix threshold by 0.35, where the thickness of each line reflects correlation strength. The left panel of (C) shows the two-sample t-tests of functional connectivity matrix in MDD compared with HCs group, where the white “*” denotes significantly altered connectivity (p < 0.05 with false discovery rate (FDR) correction). The color bar indicates t value. The right panel of (C) shows visualized brain network for the two-sample t-tests, where the red and blue lines respectively show increased and decreased functional connectivity, and the thickness of each line reflects t value. Figure 1. The left panels of (A) and (B) shows half of the averaged connectivity matrices among 11 ROIs of the DMN for MDD patients (A) and HCs group (B). The color bar indicates the value of correlation strength. The right panels of (A) and (B) showed visualized brain network for the averaged correlation coefficient matrix threshold by 0.35, where the thickness of each line reflects correlation strength. The left panel of (C) shows the two-sample t-tests of functional connectivity matrix in MDD compared with HCs group, where the white “*” denotes significantly altered connectivity (p < 0.05 with false discovery rate (FDR) correction). The color bar indicates t value. The right panel of (C) shows visualized brain network for the two-sample t-tests, where the red and blue lines respectively show increased and decreased functional connectivity, and the thickness of each line reflects t value. Figure 1. The left panels of (A) and (B) shows half of the averaged connectivity matrices among 11 ROIs of the DMN for MDD patients (A) and HCs group (B). The color bar indicates the value of correlation strength. The right panels of (A) and (B) showed visualized brain network for the averaged correlation coefficient matrix threshold by 0.35, where the thickness of each line reflects correlation strength. The left panel of (C) shows the two-sample t-tests of functional connectivity matrix in MDD compared with HCs group, where the white “*” denotes significantly altered connectivity (p < 0.05 with false discovery rate (FDR) correction). The color bar indicates t value. Results Demographic and clinical data comparisons. The demographic and clinical data were summarized in Table 1. There was no significant difference between the MDD and HCs groups in gender, age and years of education (Table 1). Compared with HCs group, MDD patients showed higher levels of Center for Epidemiologic Studies Depression (CES-D) (t = 10.030, p < 0.001) and rumination of Response to Stress Questionnaire (RSQ- rumination) (t = 6.980, p < 0.001). Differences in functional connectivity in 11 regions of interest (ROIs). The average half of 11 × 11 functional connectivity matrices is displayed for both MDD (Fig. 1A) and HCs (Fig. 1B) groups with a threshold of 0.3534. The MDD patients showed similar connectivity patterns among 11 ROIs as HCs group. As illustrated in Fig. 1C, compared with HCs, MDD patients showed increased connectivity in dMPFC-TempP, TPJ-LTC, TPJ-PHC, LTC- PHC, TempP-vMPFC, TempP-pIPL, TempP-Rsp and TempP-PHC (p < 0.05 with false discov- ery rate (FDR) correction). Of all, the dMPFC-TempP and TPJ-LTC were within-system connectivity in the dMPFC subsystem, whereas the others were inter-system connectivity between the dMPFC and MTL subsystems. Meanwhile, the decreased inter-system interactions were reported in MDD patients between the midline core and dMPFC subsystems, including aMPFC-dMPFC and PCC-dMPFC (p < 0.05 with FDR correction). Differences in functional connectivity of DMN subsystems. The average within-system and inter-system connectivity of DMN subsystems were respectively exhibited in Figs 2 and 3 in MDD and HCs groups. Relative to HCs group, the patients with MDD exhibited significantly increased within-system con- nectivity in dMPFC subsystem (t = 5.155, p < 0.001), but not in the midline core (t = 0.408, p = 0.685) or MTL subsystem (t = 1.835, p = 0.072). Additionally, the significantly increased inter-system connectivity of DMN Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The right panel of (C) shows visualized brain network for the two-sample t-tests, where the red and blue lines respectively show increased and decreased functional connectivity, and the thickness of each line reflects t value. Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 3 www.nature.com/scientificreports/ Figure 2. Bar diagrams of average within-system functional connectivity of DMN subsystems in MDD and HCs group. Figure 2. Bar diagrams of average within-system functional connectivity of DMN subsystems in MDD and HCs group. Figure 3. Bar diagrams of average inter-system functional connectivity of DMN subsystems in MDD and HCs group. Figure 2. Bar diagrams of average within-system functional connectivity of DMN subsystems in MDD and HCs group. Figure 3. Bar diagrams of average inter-system functional connectivity of DMN subsystems in MDD and HCs group. Figure 3. Bar diagrams of average inter-system functional connectivity of DMN subsystems in MDD and HCs group Figure 3. Bar diagrams of average inter-system functional connectivity of DMN subsystems in MDD and HCs group. Figure 4. Significantly positive correlations between RSQ-rumination score and DMN subsystem connectivity in patients with MDD (p < 0.05). Figure 4. Significantly positive correlations between RSQ-rumination score and DMN subsystem connectivity in patients with MDD (p < 0.05). subsystems was reported in dMPFC-MTL interaction in MDD patients (t = 2.039, p = 0.048), but not in the m line core-dMPFC (t = 1.126, p = 0.267) or midline core-MTL interaction (t = 0.195, p = 0.846). Correlation analysis. After controlling for CES-D score, age, education and illness duration as covariates RSQ-rumination scores were positively correlated with functional connectivity in patients with MDD, including MPFC-TempP (r = 0.382, p = 0.038) and LTC-PHC (r = 0.416, p = 0.020) (Fig. 4). Correlation analysis. After controlling for CES-D score, age, education and illness duration as covariates, RSQ-rumination scores were positively correlated with functional connectivity in patients with MDD, including dMPFC-TempP (r = 0.382, p = 0.038) and LTC-PHC (r = 0.416, p = 0.020) (Fig. 4). Discussion To date, few studies have focused on the neural activity of DMN subsystems in MDD patients, although previous work has investigated different aspects of the DMN. The present study aimed to investigate three DMN subsys- tems resting-state functional connectivity and the association with rumination in patients with MDD. Four prin- cipal findings emerged from our study. Firstly, we found significantly increased within-system connectivity in the Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 4 www.nature.com/scientificreports/ dMPFC subsystem in patients with MDD (dMPFC-TempP and TPJ-LTC). Secondly, the increased inter-system connectivity was revealed between the dMPFC and MTL subsystems in MDD patients (TPJ-PHC, LTC-PHC, TempP-vMPFC, TempP-pIPL, TempP-Rsp and TempP-PHC). Thirdly, the decreased connectivity was observed between the midline core and dMPFC subsystem in individuals with MDD (aMPFC-dMPFC and PCC-dMPFC). Finally, correlation analysis revealed that the connectivity of dMPFC-TempP and LTC-PHC were positively cor- related with depressive rumination in the MDD group.i p g p In present study, we found significantly greater connectivity in the dMPFC subsystem in MDD group, includ- ing dMPFC-TempP and TPJ-LTC. These observations might reflect resting-state hyperconnectivity in the dMPFC subsystem in MDD patients. The dMPFC subsystem is thought to play more of a social-reflective role, allowing individuals to infer the mental states of other people and reflect on their own mental states14,31,36. In particular, as the key brain region in Mayberg’s model of MDD37,38, the dMPFC reportedly is involved in the process of evaluating whether stimuli are self-referential39–41 and is linked to a variety of cognitive functions, appraisal and the expression of negative emotion42. The neuropathological and neurochemical studies in major depression con- sistently reported abnormalities in the dMPFC subsystem43–45. Our findings of positive correlations between the rumination score and connectivity of dMPFC-TempP and LTC-PHC further support the view that the dMPFC subsystem is involved in self-abnormalities in MDD. Our study provides novel evidence of the notion that the dMPFC subsystem abnormalities in MDD might reflect the ruminative nature of MDD patients and further suggests that the resting-state signal in the dMPFC subsystem may be a marker for a ruminative response style in depression. p Furthermore, increased inter-system connectivity was observed between the dMPFC and MTL subsystems in MDD patients relative to HCs. It has been suggested that these two subsystems interact when individuals are left to think alone and undisturbed46. Discussion In such cases, people tend to engage in self-relevant internal cognitive pro- cesses, predominantly about significant past and future events31. The increased inter-system connectivity between the dMPFC and MTL subsystems in our study showed stronger self-relevant neural activity in MDD patients, which might be related to self-abnormalities in MDD. Consistent with this hypothesis, the following correlation results indicated that a positive correlation existed between LTC-PHC connectivity with rumination score. As far as we know, no study has investigated the relationship between rumination score and interaction within the dMPFC and MTL subsystems in subjects with MDD. Our findings provide new pathophysiological evidence that the interaction between the dMPFC and MTL subsystems may contribute to the ruminative response style in MDD, which is consistent with the previous studies mentioned above46. Our results support the notion that abnormal interaction between DMN subsystems might be involved in the pathophysiology of self-relevant symp- toms in MDD. In MDD patients, we detected decreased connectivity between the midline core and dMPFC subsystem, including aMPFC-dMPFC and PCC-dMPFC. Evidence from anatomical and imaging studies has revealed that both aMPFC and PCC have extensive connectivity with the dMPFC subsystem47,48. It has been suggested that midline core is critical to making self-relevant, affective decisions, which is strongly correlated with the dMPFC subsystem, especially in affective self-referential cognition31. The current finding of decreased inter-system con- nectivity might indicate impaired interaction between the midline core and dMPFC subsystem. It is worthy of mention that the result relating to the diminished connectivity of PCC-dMPFC is consistent with our previous study on first-episode treatment-naïve young adults with MDD49. The results were corroborated by other studies on resting-state connectivity in MDD50, suggesting decreased interaction between the anterior and posterior DMN in young people with depression. Nevertheless, increased PCC-dMPFC connectivity has been reported in late-life depression51–53, which implies that there is a relationship between PCC-dMPFC connectivity and patient age and medication status. Taken together, all the findings suggest that the pathophysiology of MDD could also be related to reduced central role of the midline core subsystem in the DMN.hi y The results of this study must be interpreted in light of its limitation. Discussion Firstly, as we selected first-episode, medication-naïve young patients with MDD to minimize the confounding influences of chronicity, treatment or comorbidity, the relatively small sample size may limit the generalizability of our results as well as our ability to detect more relationships between clinical variables and neuroimaging findings. Secondly, consistent with previous work31, seed-based analysis with the predefined 11 ROIs was used to calculate DMN subsystems con- nectivity. However, defining DMN based on data-driven approaches is also worth studying54. Finally, the acqui- sition parameters with large slices (4 + 1 mm), relatively short scanning time and high TR may have effect on the consistency of resting-state findings. More optimized acquisition parameters deserve investigation in future work. Conclusions h In summary, the present study examined the relationship between DMN subsystems connectivity and rumi- nation in patients with MDD. Relative to HCs MDD patients demonstrated disrupted communication within DMN subsystems, which supported and extended the widely acknowledged large-scale network dysfunction in MDD55,56. The MDD patients showed increased within-system connectivity in the dMPFC subsystem, decreased inter-system connectivity between the midline core and dMPFC subsystem, and increased inter-system connec- tivity between the dMPFC and MTL subsystems. Importantly, abnormal connectivity in DMN subsystems had a significant positive correlation with rumination scores in MDD patients. Our findings suggest that disrupted integration of DMN subsystems may be closely associated with the pathophysiology of MDD patients, which highlights the importance of DMN subsystems connectivity. Methods Participants. Patients with MDD were recruited from the psychiatric clinic at Second Xiangya Hospital of Central South University in Changsha, China. Patients with MDD were diagnosed according to the Structured Clinical Interview for DSM-IV57 by independent assessments of two psychiatrists. All of the patients were Participants. l h The following parameters were applied: repetition time = 2000 ms, echo time = 30 ms, flip angle = 80°, field of view (FOV) = 240 mm × 240 mm, matrix = 64 × 64, slice thickness = 4 mm, slice gap = 1 mm, number of slices = 32. For each participant, 216 vol- umes were obtained, and the scan lasted 432 s. High-resolution 3-dimensional (3D) structural images were acquired using a T1-weighted, magnetization- prepared rapid gradient-echo sequence. The following parameters were applied: repetition time = 1900 ms, echo time = 2.01 ms, flip angle = 9°, FOV = 256 mm × 256 mm, matrix = 256 × 256, slice thickness = 1 mm, slice gap = 0 mm, and number of slices = 176. Data preprocessing. Image preprocessing was performed using the Data Processing Assistant for Resting-State fMRI (DPARSF) professional software62 (http://www.restfmri.net). For each individual participant, the first 10 functional images were excluded from analysis. Subsequent images were corrected by slice timing and realigned for head motion. Two MDD patients and one healthy subject were excluded because their translation or rotation exceeded ±1.5 mm ±1.5°. The individual T1-weighted structural images were coregistered to functional images. The transformed structural images were then segmented into gray matter, white matter, and cerebro- spinal fluid and normalized to Montreal Neurologic Institute (MNI) space. These transformation parameters were also applied to the functional images. The normalized functional images were resampled at a resolution of 3 × 3 × 3 mm3 and spatially smoothed with a 6-mm full width at half maximum Gaussian kernel. The sources of spurious variance were regressed out including 6 parameters from head-motion correction (Friston 24-parameter model)63, white matter and cerebrospinal fluid signal. Finally, functional images with linear trend were removed by temporal bandpass filtering (0.01–0.08 Hz).l y p pi g In view of the influence of head motion on functional connectivity results64–66, the data was further performed with the scrubbing method to remove time points affected by head motions. As recommended by Yan et al.66,67, the framewise displacement (FD) was calculated by translation and rotation parameters of head motion based on the formula from previous study64. The image frames (FD > 0.5 mm) were identified as bad time points. Along with the bad time points, 1 preceding and 2 following points were deleted to assure exclusion of motion-related confounds. Participants. l h Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 5 www.nature.com/scientificreports/ experiencing their first episode of depression and had never received medication. Closely matched healthy sub- jects were recruited through advertisements from several colleges in Changsha. All subjects were right-handed. The shared exclusion criteria for patients and control subjects included any major medical illnesses; clinical diag- nosis of neurologic trauma; any history of psychiatric disorder in the control subjects or any history of psychiatric disorder, except major depression, in the MDD patients; any history of substance abuse or alcohol in the past 6 months; and any contraindications to imaging scanning. Finally, 33 patients with MDD and 33 matched HCs were recruited. Research ethics. The study protocol was approved by Second Xiangya Hospital of Central South University. Written informed consent was obtained from all participants prior to the study, which was approved by the Institutional Review Board of Second Xiangya Hospital of Central South University for Brain Research. The methods were conducted in accordance with relevant approved guidelines and regulations. Measures. Depression. Depressive severity was measured using the CES-D scale58, a 20-item self-report instrument to assess depressive symptoms in the general population. The Chinese version of the CES-D has been found to have high degrees of reliability and validity59. In this study, the internal consistency of the CES-D was good (Cronbach’s alpha = 0.93). Rumination. Level of rumination was assessed using the rumination subscale of RSQ in Chinese version60,61, which focuses on the feelings and thoughts that are associated with negative events. The rumination subscale of the RSQ exhibited high degrees of reliability and validity in both English and Chinese versions. Data acquisition. Images were obtained using a Siemens Skyra 3T scanner with a standard head coil. Participants wore a standard head coil fitted with foam padding to minimize head movement and diminish scan- ner noise. During scanning, all participants were required to remain motionless, keep their eyes closed and sys- tematically try not to think of anything. After scanning, the participants were asked about their statement during scanning. Resting-state fMRI images were acquired with a single-shot, gradient-recalled echo-planar imaging sequence oriented parallel to the line of the anterior-posterior commissure. Participants. l h No difference was observed for the mean FD between MDD and HCs groups (Mann Whitney U-test, P = 0.27). For all subjects, the remaining imaging data contained more than 128 volumes. Definition of 11 ROIs. According to previous work31–34, 11 ROIs of the DMN were defined using 8-mm radius spheres (see Fig. 5 and Table 2), including the anterior MPFC (aMPFC), PCC, dorsal MPFC (dMPFC), temporo-parietal junction (TPJ), lateral temporal cortex (LTC), temporal pole (TempP), ventral MPFC (vMPFC), retrosplenial cortex (Rsp), posterior inferior parietal lobule (pIPL), parahippocampal cortex (PHC) and hip- pocampal formation (HF). The aMPFC and PCC constitute the midline core. The dMPFC subsystem includes the dMPFC, TPJ, LTC and TempP. The MTL subsystem comprises the vMPFC, pIPL, Rsp, PHC and HF. Functional connectivity analysis. Functional connectivity analysis was performed on the left-lateralized 11 ROIs mentioned above (see Fig. 5 and Table 2) with the Resting-State fMRI Data Analysis Toolkit63. The mean time series from the voxels within each ROI were extracted. Pearson’s correlations were calculated between any two nodes of 11 ROIs. The correlation coefficients referred to the functional connectivity strength correspond- ing to the paired ROIs. The 11 × 11 functional connectivity matrix was obtained for each subject68,69. Fisher’s r-to-z transformation was applied to normalize the correlation coefficients. Apart from the individual edge Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 6 www.nature.com/scientificreports/ Figure 5. 11 regions of interest (ROIs) in the default mode network (DMN) are depicted with BrainNet Viewer70, including the midline core denoted by red color, dMPFC subsystem denoted by green color, and MTL subsystem denoted by blue color. The midline core includes posterior cingulate cortex (PCC) and anterior medial prefrontal cortex (aMPFC). The dMPFC subsystem includes dorsal medial prefrontal cortex (dMPFC), temporo-parietal junction (TPJ), lateral temporal cortex (LTC) and temporal pole (TempP). The MTL subsystem includes ventral medial prefrontal cortex (vMPFC), posterior inferior parietal lobule (pIPL), retrosplenial cortex (Rsp), parahippocampal cortex (PHC) and hippocampal formation (HF). Figure 5. 11 regions of interest (ROIs) in the default mode network (DMN) are depicted with BrainNet Viewer70, including the midline core denoted by red color, dMPFC subsystem denoted by green color, and MTL subsystem denoted by blue color. The midline core includes posterior cingulate cortex (PCC) and anterior medial prefrontal cortex (aMPFC). The dMPFC subsystem includes dorsal medial prefrontal cortex (dMPFC), temporo-parietal junction (TPJ), lateral temporal cortex (LTC) and temporal pole (TempP). Participants. l h group differences in average connectivity of three DMN subsystems, the two-sample t-tests were applied to explore the average within-system and inter-system connectivity between patients with MDD and HCs. Correlation analysis. To examine the association of DMN subsystems connectivity with the level of depressive rumination, linear correlations were calculated in MDD patients between the z-value of all edges and RSQ-rumination scores with SPSS 18.0 software (IBM SPSS Inc., USA). Given that these analyses were explora- tory, we used an uncorrected statistical significance level of p < 0.05. Correlation analysis. To examine the association of DMN subsystems connectivity with the level of depressive rumination, linear correlations were calculated in MDD patients between the z-value of all edges and RSQ-rumination scores with SPSS 18.0 software (IBM SPSS Inc., USA). Given that these analyses were explora- tory, we used an uncorrected statistical significance level of p < 0.05. Participants. l h The MTL subsystem includes ventral medial prefrontal cortex (vMPFC), posterior inferior parietal lobule (pIPL), retrosplenial cortex (Rsp), parahippocampal cortex (PHC) and hippocampal formation (HF). DMN Subsystem Regions Abbreviation BA MNIxyz midline core   Anterior medial prefrontal cortex aMPFC 10, 32 −6, 52, −2   Posterior cingulate cortex PCC 23,31 −8, −56, 26 dMPFC self system   Dorsal medial prefrontal cortex dMPFC 9, 32 0, 52, 26   Temporal parietal junction TPJ 40, 39 −54, −54, 28   Lateral temporal cortex LTC 21, 22 −60, −24, −18   Temporal pole TempP 21,38 −50, 14, −40 MTL memory system   Ventral medial prefrontal cortex vMPFC 11, 24, 25, 32 0, 26, −18   Posterior inferior parietal lobule pIPL 39 −44, −74, 32   Retrosplenial cortex Rsp 29, 30, 19 −14, −52, 8   Parahippocampal cortex PHC 20, 36, 19 −28, −40, −12   Hippocampal formation HF 20, 36 −22, −20, −26 Table 2. Coordinates of 11 regions of interest (ROIs) in the default mode network (DMN). BA: Brodmann area; MNI: Montreal Neurological Institute; MTL: medial temporal lobe Table 2. Coordinates of 11 regions of interest (ROIs) in the default mode network (DMN). BA: Brodmann area; MNI: Montreal Neurological Institute; MTL: medial temporal lobe. corresponding to one paired ROIs, the average connectivity of all edges is another indicator to represent the global functional connectivity strength. Then, the average within-system and inter-system connectivity of three DMN subsystems for each subject were calculated48. The average within-system connectivity is defined as the mean of the sum of all edges in one subsystem, while the average inter-system functional connectivity is referred to the mean of the sum of functional connectivity strength of all edges between two subsystems. Statistical analysis. To examine group differences in functional connectivity of 11 ROIs, the two-sample t-tests were performed on half of 11 × 11 correlation coefficients matrices of MDD and HCs groups due to sym- metry. The statistical significance level was set at p < 0.05 (FDR correction). 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This research was supported by the National Natural Science Foundation of China (grant number 81301211 to X.Z., Q.Z. and F.Y.) and Foundation for the Author of National Excellent Doctoral Dissertation of PR China (grant number 201411 to X.Z.). Author Contributions X.Z., Q.Z. and F.Y. conceived and designed the experiments. H.S. and W.L. conducted the experiments and collected data. Q.Z. and H.S. analyzed the results. X.Z. and Q.Z. wrote the main manuscript text. All authors reviewed the manuscript. X.Z., Q.Z. and F.Y. conceived and designed the experiments. H.S. and W.L. conducted the experiments and collected data. Q.Z. and H.S. analyzed the results. X.Z. and Q.Z. wrote the main manuscript text. All authors reviewed the manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 39. Gusnard, D. A., Akbudak, E., Shulman, G. L. & Raichle, M. E. Medial prefrontal cortex and self-referential mental activity: relation to a default mode of brain function. Proc. Natl. 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Limbic-cortical dysregulation: a proposed mo Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 8 Scientific RePorTS | 7:43105 | DOI: 10.1038/srep43105 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Zhu, X. et al. Rumination and Default Mode Network Subsystems Connectivity in First-episode, Drug-Naive Young Patients with Major Depressive Disorder. Sci. Rep. 7, 43105; doi: 10.1038/ srep43105 (2017). How to cite this article: Zhu, X. et al. Rumination and Default Mode Network Subsystems Connectivity in First-episode, Drug-Naive Young Patients with Major Depressive Disorder. Sci. Rep. 7, 43105; doi: 10.1038/ srep43105 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium
Journal of biological chemistry/˜The œJournal of biological chemistry
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Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium* y y Received for publication,December 9, 2013, and in revised form, January 24, 2014 Published, JBC Papers in Press,February 7, 2014 Namthip Witayavanitkul‡, Younss Ait Mou‡, Diederik W. D. Kuster‡, Ramzi J. Khairallah‡, Jason Sarkey‡, Suresh Govindan‡, Xin Chen§, Ying Ge§, Sudarsan Rajan¶, David F. Wieczorek¶, Thomas Irving, Margaret V. Westfall**, Pieter P. de Tombe‡1, and Sakthivel Sadayappan‡1,2 From the ‡Department of Cell and Molecular Physiology, Health Sciences Division, Loyola University Chicago, Maywood, Illinois 60153, the §Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706, the ¶Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, the Department of Biological and Chemical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616, and the **Department of Cardiac Surgery, University of Michigan, Ann Arbor, Michigan 48109 From the ‡Department of Cell and Molecular Physiology, Health Sciences Division, Loyola University Chicago, Maywood, Illinois 60153, the §Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706, the ¶Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, the Department of Biological and Chemical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616, and the **Department of Cardiac Surgery, University of Michigan, Ann Arbor, Michigan 48109 Background: Myocardial infarction (MI) leads to proteolytic cleavage of cMyBP-C (hC0C1f) and decreased contractility. Results: hC0C1f can incorporate into the human cardiac sarcomere, depressing force generation and increasing tension cost. Conclusion: Interaction between hC0C1f and both actin and -tropomyosin causes disruption of intact cMyBP-C function. Significance: Proteolytic cleavage of cMyBP-C is sufficient to cause contractile dysfunction following MI. Myocardial infarction (MI) is associated with depressed car- diac contractile function and progression to heart failure. Car- diac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cul- tured cardiomyocytes results in decreased Ca2 transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mech- anisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recom- binant human C0-C1f (hC0C1f) and incorporated it into per- meabilized human left ventricular myocardium. Mechanical properties were studied at short (2 m) and long (2.3 m) sar- comere length (SL). * Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth Grants R01HL105826 and K02HL114749 (to S. S.); HL007692 (to J. S.); HL101297, HL75494, and HL62426 (to P. P. d. T.); 2P41RR008630-17 and 9P41GM103622–17 (to T. I.); and HL096971 (to Y. G.). This work was also supported by American Heart Association Midwest Fellowships 11POST5260038 (to Y. A.), 13POST14720024 (to S. G.), and 13POST17220009 (to D. W. D. K.). Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Argonne National Laboratory, was supported by the U.S. Department of Energy under Contract DE-AC02-06CH11357. The Bio- CAT facility is supported by grants from the National Institute of General Medical Sciences of the National Institutes of Health (9 P41 GM103622-18). Author’s Choice—Final version full access. 1 1 The abbreviations used are: MI, myocardial infarction; cMyBP-C, cardiac myosin-binding protein-C; I/R, ischemia/reperfusion; -TM, -tropomyo- sin; SL, sarcomere length; IAF, 5-iodoacetamidofluorescein. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 13, pp. 8818–8827, March 28, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 13, pp. 8818–8827, March 28, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 13, pp. 8818–8827, March 28, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Author’s Choice 2 To whom correspondence should be addressed: Dept. of Cell and Molecular Physiology, Health Sciences Division, Loyola University of Chicago, 2160 S. First Ave., Maywood, IL 60153. Tel.: 708-216-7994; Fax: 708-216-6308; E-mail: ssadayappan@lumc.edu. EXPERIMENTAL PROCEDURES Human Samples—Non-age/sex-matched, deidentified hearts were procured via the National Disease Research Inter- change. Tissue procurement and processing was approved by the University of Michigan Institutional Review Board, and the Institutional Review Board at Loyola University Chicago approved the protocol for the use of deidentified human donor hearts. Prior to explant, hearts were flushed with ice-cold car- dioplegia solution and arrived on ice 12 h postexplant. Sec- tions of left ventricular tissue were then immediately frozen in liquid N2 and stored at 80 °C until use. Top-Down Analysis by Mass Spectrometry—His-tagged recombinant hC0C1f proteins were expressed in Escherichia coli and purified as described previously (4, 15). Recombinant hC0C1f (20–100 g) was desalted by offline reverse phase pro- tein trap (Michrom Bioresources). The samples were intro- duced to the mass spectrometer via an automated chip-based nano-electrospray ionization source (Triversa NanoMate; Advion BioSciences, Ithaca, NY) with a spray voltage of 1.2–1.6 kV versus the inlet of the mass spectrometer, resulting in a flow of 50–200 nl/min. Intact protein molecular ions were analyzed using 7T linear ion trap/FTICR (LTQ FT Ultra) hybrid MS (Thermo Scientific Inc., Bremen, Germany) as described previ- ously (17). Up to 1,500 transients were averaged per spectrum to ensure high quality spectra. All FTICR spectra were pro- cessed with in-house developed MASH Suite software (version 1.0), using the THRASH algorithm with a signal to noise threshold of 3 and fit factor of 60% and validated manually. Single Permeabilized Cardiac Myocyte Preparation—Single skinned cells were prepared as previously described (18, 19) with adaptations to allow the use of human tissue. Briefly, frozen human left ventricle samples were thawed in ice-cold relaxing solution containing 10 mM EGTA, 10 mM creatine phosphate, 10 units/mlcreatinekinase,100mMN,N-bis(2-hydroxyethyl)taurine; N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid, 6.3 mM ATP, 6.48 mM magnesium chloride, 49.76 mM potassium propio- nate, and protease inhibitors mixture (Sigma-Aldrich) at pH 7.0. Single cells were obtained by mechanically homogenizing left ven- tricular tissues for 3 s at 10,000 rpm using a homogenizer (Power- Gen 700D; Fisher Scientific). The homogenate was centrifuged at 120  g for 1 min, and the resulting pellet was filtered to remove any unhomogenized tissue. The run-through was washed with fresh ice-cold relaxing solution and resuspended in relaxing solu- tion containing 1% Triton X-100 for 30 min at room temperature under agitation to permeabilize the cells. N-terminal Fragment of cMyBP-C Impairs Myofilament Function myosin function by the presence of C0-C1f fragments in the sarcomere, including its interaction with thick and thin fila- ment proteins and its effects on ATPase activity, myofilament Ca2 sensitivity, and contractility. myosin function by the presence of C0-C1f fragments in the sarcomere, including its interaction with thick and thin fila- ment proteins and its effects on ATPase activity, myofilament Ca2 sensitivity, and contractility. cross-bridge cycling kinetics and tension cost, effectively break- ing the interaction between cMyBP-C and actomyosin. Further, we establish that the effects of hC0C1f occur through direct interaction with the thin filament proteins actin and -TM. y y The C0-C1f fragment contains the cardiac-specific C0 domain, the proline-alanine (Pro-Ala)-rich region, the C1 domain, and the first 17 residues of the M domain (1–271 res- idues; C0-C1f) of cMyBP-C (2). The N-terminal region of cMyBP-C interacts with both actin and myosin and is believed to function as a critical regulator of contraction (5–7). The car- diac isoform of cMyBP-C differs from the two skeletal isoforms by having an extra C0 domain at the N-end (8). Thus, the C0 domain is exclusively unique and specific to cMyBP-C. The C0 domain is able to directly bind with both actin (9, 10) and reg- ulatory light chain of myosin (11), thereby placing the N-termi- nal region of cMyBP-C in close proximity to the motor domain of myosin (11). However, the relative binding affinity of the C0 domain to actin was found to be less than that of C1-C2 (9, 10). The C1-M-C2 region binds to the S2 fragment of myosin and directly influences myofilament Ca2 sensitivity, myofibrillar tension development, cross-bridge cycling kinetics, and sar- comere length (SL)-tension relationships (5–7). The C1-M-C2 region also binds to actin, supporting the idea that cMyBP-C may play a role in the regulation of cardiac contraction by mod- ulating actin-myosin interaction (9, 10, 12, 13). Furthermore, the interaction of the C1-M-C2 region of cMyBP-C with actin and myosin is dynamically regulated in an on-off fashion by phosphorylation within the M domain (6, 9, 13, 14). Phosphor- ylation by PKA prevents the cMyBP-C N-region from binding to actin (9) and myosin S2 (15) or altering myofilament Ca2 sensitivity (16). Interestingly, the cleaved C0-C1f fragment gen- erated by the proteolytic degradation of cMyBP-C following ischemia/reperfusion (I/R) injury does not contain these regu- latory phosphorylation sites (2). N-terminal Fragment of cMyBP-C Impairs Myofilament Function Because the C0-C1f region retains strong interaction with actin but lacks the phosphory- lation sites necessary for phosphorylation-dependent on-off interaction with myosin and actin, we hypothesized that the presence of the C0-C1f fragments in the sarcomere would alter actin-myosin interaction by constitutively interacting with thin filament proteins, such as actin and -tropomyosin (-TM), in turn having direct consequences on force generation of sar- comere function. Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium* Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca2 sen- sitivity. Moreover, hC0C1f led to increased cooperative activa- tion, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and -tro- pomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2 sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and -tropomyo- sin, suggesting that cleaved C0-C1f could act as a poison poly- peptide and disrupt the interaction of native cardiac myosin- binding protein C with the thin filament. Myocardial infarction (MI)3 leads to depressed cardiac con- tractile function and often progresses to heart failure. This pro- gression is preceded by impaired Ca2 handling, altered myo- filament phosphorylation, and reduced cross-bridge cycling rates. Surprisingly, cardiomyocytes in remote noninfarcted regions, unaffected by ischemia, also display decreased contrac- tility, despite restoration of blood flow. The mechanisms lead- ing to this dysfunction are still incompletely understood (1). We have previously shown that MI is associated with catalytic cleavage of cardiac myosin binding protein C (cMyBP-C), an important regulator of cardiac contractility (2). Following MI, this proteolytic fragment can be detected in the ischemic region but is also present in the noninfarcted adja- cent and remote regions, as well as in nonischemic heart failure (2–4), presumably after in situ cleavage. Furthermore, cleaved cMyBP-C is released into the blood following MI, such that its N-terminal fragment (C0-C1f) can be used as a marker of MI (2). The C0-C1f fragment has been shown to disrupt myosin- regulated contraction (3) and potentially lead to global dysfunc- tion in the heart, not just in the ischemic area. Therefore, the study aimed to identify the mechanisms underlying altered 8818 8818 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 13•MARCH 28, 2014 N-terminal Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function The aluminum clips holding the fiber were mounted on hooks, slightly larger than the holes in the clips, so that they were held securely between a force transducer (KG7; World Precision Instruments, Sarasota, FL) and a servomotor (Cam- bridge Technologies model 308B; Aurora Scientific, Aurora, Canada) mounted on micromanipulators. During the experi- ment, relaxing solution was continuously perfused through the chamber using a peristaltic pump. All experiments were done at room temperature. A highly sensitive CCD detector (Aviex PCCD 16080) with a 160-mm  80-mm active area and 39-m pixels was used to collect x-ray patterns. Prior to analysis, x-ray diffraction patterns were corrected for dark current flat field and spatial distortions. Spacings on detector images were meas- ured using the FIT2D program on a UNIX work station. The accuracy of these measurements reached to 1 pixel of 200– 600 (i.e., 0.5%) or better. Measured spacings of the 1,0 and 1,1 equatorial reflections from the diffraction pattern were con- verted to d1,0 lattice spacing (thin filament spacing) using Bragg’s Law, which can then be converted to the interfilament spacing. Localization of C0-C1f after Exchange in Permeabilized Cardiomyocytes—Permeabilized human cardiac myocytes were attached to two needles controlled by a custom microme- chanics system using optical glue (NOA 63; Norland Products Inc.) and mounted on a laser scanning confocal microscope (LSM 410; LSM Tech Inc.). After attachment, nonspecific bind- ing was blocked by incubating in blocking solution containing 1% (w/v) BSA, 0.1% (w/v) gelatin, and 0.1% (v/v) Tween 20 for 30 min at room temperature. Mouse monoclonal cardiac -ac- tinin (Sigma; #A7811) was used to stain the Z-disc at a 1:200 dilution in blocking buffer overnight at room temperature. Unbound antibody was removed by washing three times with PBS. The secondary antibody, goat anti-mouse IgG (Invitrogen; A11001; 1:100 dilution in blocking buffer) conjugated to a flu- orescein 488, was then added in blocking solution for 1 h at room temperature. Unbound antibody was washed away with PBS. The cell was then attached and imaged at 488/525 excita- tion/emission. Following the first image, the cell was then incu- bated with excess of hC0C1f-IAF (30 M) for 20 min at room temperature. The unbound hC0C1f-IAF was washed three times with relaxing solution, and the cell was imaged again at 488/525. Both -actinin and hC0C1f-IAF fluorescence signals were measured simultaneously. N-terminal Fragment of cMyBP-C Impairs Myofilament Function col provided by Molecular Probes. Briefly, labeled hC0C1f-IAF was prepared by dissolving lyophilized hC0C1f in dissolving buffer containing 6 M urea, 0.02 M Tris-HCl, and 0.001 M DTT at pH 8.0 to an initial concentration of 2 mg/ml and incubated at room temperature until completely dissolved (3 h). The DTT was removed by dialyzing the solution against a DTT-free buffer containing 6 M urea, 0.1 M NaCl, 0.02 M Tris-HCl, and 0.001 M EDTA, at pH 7.5, overnight at 4 °C. To ensure high labeling efficiency, hC0C1f was incubated with a 4 molar excess of IAF for 8 h at room temperature. IAF covalently binds via disulfide bonds with cysteine residues. After 8 h, the reac- tion was stopped by adding 1 mM DTT. To remove unbound IAF, the solution was dialyzed against a postlabeling buffer con- taining 6 M urea, 1 M NaCl, 0.02 M Tris-HCl, 0.005 M MgCl2, and 0.001 M DTT at pH 8.0, overnight at 4 °C. Finally, the solution was dialyzed against relaxing solution overnight at 4 °C. The stock solution was aliquoted (10 l) and stored at 80 °C. Labeling efficiency was based on the relative molar concentra- tions of dye/protein, where the dye concentration was deter- mined spectroscopically by absorbance at 492 nm using a molar extinction coefficient of 80,000–85,000 M1 cm1, and the protein concentration was determined by the Bradford method. Å wavelength). All flight paths were evacuated, except for a small gap around the sample chamber itself (1 mm down- stream, 2 mm upstream of the sample). The beam size at the sample position was collimated to about 0.4 by 0.8 mm and about 0.065 by 0.15 mm at the detector and contained a maxi- mum incident flux of 3  1012 photons/s. Permeabilized human cardiac tissue was clipped using aluminum T-clips and mounted in a small trough with dimensions of 0.8 mm wide  40 mm long  5 mm deep. The bottom of the trough was a glass coverslip allowing the muscle to be viewed by a long working distance (4 mm) 4 objective and also allowed for sarcomere length measurement via laser diffraction (21). One end of the trough expanded into a larger reservoir for mounting the fiber. The slides of the trough were hollowed out, allowing the x-ray beam to pass through the fiber via KaptonTM windows 0.0005 inch thick. N-terminal Fragment of cMyBP-C Impairs Myofilament Function The resulting image was then subtracted from the -actinin staining to reveal the localization of the hC0C1f-IAF. The experiment was also repeated, starting with hC0C1f-IAF incubation and followed by -actinin staining. p g Simultaneous Measurement of Isometric Tension and ATPase Activity—Permeabilized human cardiac tissue was obtained by adapting previously described methods (19). Our novel approach consisted of cutting the frozen tissue into 2–4-mm pieces (0.04 g) in ice-cold relaxing solution, fol- lowed by homogenizing at low speed (1,000 rpm, 3 s) three times (Power Gen 700D; Fisher Scientific) in relaxing solution. The preparation was allowed to settle, and the supernatant was discarded, after which the tissue was resuspended in relaxing solution. The tissue was permeabilized overnight in 1% Triton X-100 (Amresco) at 4 °C, which served to remove cell mem- branes and intracellular membrane-bound structures. The tis- sues were then extensively washed in fresh ice-cold relaxing solution, stored on ice, and used within 8 h. The strips of fibers were selected for appearance under a dissecting microscope. They were then attached to a force transducer (KG4A; World Precision Instruments, Sarasota, FL) and high speed length controller (model 315C; Aurora Scientific) using aluminum T-Clips. Muscle dimensions were determined using an ocular micrometer mounted in the dissection microscope (resolution, 10 m). Sarcomere length was measured in the passive relaxed condition by laser diffraction as previously described (21), adjusted to 2 m and then 2.3 m. The skinned fibers were Simultaneous Measurement of Isometric Tension and ATPase Activity—Permeabilized human cardiac tissue was obtained by adapting previously described methods (19). Our novel approach consisted of cutting the frozen tissue into 2–4-mm pieces (0.04 g) in ice-cold relaxing solution, fol- lowed by homogenizing at low speed (1,000 rpm, 3 s) three times (Power Gen 700D; Fisher Scientific) in relaxing solution. The preparation was allowed to settle, and the supernatant was discarded, after which the tissue was resuspended in relaxing solution. The tissue was permeabilized overnight in 1% Triton X-100 (Amresco) at 4 °C, which served to remove cell mem- branes and intracellular membrane-bound structures. The tis- sues were then extensively washed in fresh ice-cold relaxing solution, stored on ice, and used within 8 h. The strips of fibers were selected for appearance under a dissecting microscope. They were then attached to a force transducer (KG4A; World Precision Instruments, Sarasota, FL) and high speed length controller (model 315C; Aurora Scientific) using aluminum T-Clips. EXPERIMENTAL PROCEDURES The resulting skinned cells were washed three times with ice-cold relaxing solution and suspended in 1.5 ml of fresh ice-cold relaxing solution. The cells were kept on ice and used the same day. We have previously shown that expression of C0-C1f protein in neonatal and adult cardiomyocytes induces contractile dys- function (4). Furthermore, transgenic mice expressing this fragment display sarcomere dysgenesis, increased fibrosis, and impaired contractility, leading to the development of heart fail- ure (3), abnormalities suggesting that cleaved C0-C1f could act as a poison polypeptide. We hypothesize that C0-C1f interferes with the binding of native cMyBP-C to thick and thin filaments, thereby altering its regulation of actomyosin interaction. This would then result in the depressed myocardial function observed in post-MI and in heart failure. Here, to determine the impact of C0-C1f on sarcomere function, we applied human C0-C1f (hC0C1f) recombinant protein fragment to permeabi- lized donor human myocardium and assayed force-Ca2/ force-ATPase relationships, length-dependent activation, and cross-bridge cycling kinetics. We show that hC0C1f increases 5-Iodoacetamidofluorescein Labeling of hC0C1f—To deter- mine whether the hC0C1f correctly localized to the sarcomere after incubation, hC0C1f was labeled with a thiol-sensitive maleimide fluorophore (5-iodoacetamidofluorescein (IAF)). The labeling procedure was conducted according to the proto- MARCH 28, 2014•VOLUME 289•NUMBER 13 8819 JOURNAL OF BIOLOGICAL CHEMISTRY MARCH 28, 2014•VOLUME 289•NUMBER 13 N-terminal Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function Isometric tension and ATPase activity were measured at various levels of Ca2 activation as previously described (22, 23). Briefly, the isolated muscle was exposed to a range of calcium solutions obtained by proportional mixing of activating and relaxing solutions, and the force generated and ATP consumed were measured simultaneously during the con- traction. Activating solution contained 20 mM Ca2-EGTA, 1.55 mM potassium propionate, 6.59 mM MgCl2, 100 mM N,N- bis(2-hydroxyethyl)taurine; N,N-Bis-(2-hydroxyethyl)-2-ami- noethanesulfonic acid, 5 mM sodium azide, 1 mM DTT, 10 mM phosphoenolpyruvate, 0.01 mM oligomycin, 0.1 mM PMSF, and 0.02 mM A2P5, as well as protease inhibitor mixture. Relaxing solution was identical, except it contained 20 mM EGTA, 21.2 mM potassium propionate, and 7.11 mM MgCl2. The preactivat- ing solution contained 0.5 mM EGTA, 19.5 mM 1,6-diamino- hexane-N,N,N,N-tetraacetic acid, and 21.8 mM potassium pro- pionate. All solutions contained 0.5 mg/ml pyruvate kinase and 0.05 mg/ml lactate dehydrogenase (Sigma) and had an ionic strength of 180 mM, 5 mM free ATP, and 1 mM free magnesium, as determined by the Fabiato program (24). Statistical Analysis—Tension- and ATPase-pCa relation- ships were fit with a modified Hill equation (23, 26, 27), and stiffness and tension costs were determined by linear fit to the tension-stiffness and tension-ATPase data, respectively. The data were analyzed using 1) one-way repeated measures analy- sis of variance for the dose response of control and hC0C1f on maximum force development, 2) an unpaired t test for compar- ing control and hC0C1f, 3) two-way analysis of variance, and 4) a Holm-Sidak post hoc test with a level of statistical significance set at p  0.05. The data are presented as means  S.E. y g ATPase activity was measured by a UV-coupled optical absorbance enzyme assay (25). Briefly, ATP hydrolysis into ADP and inorganic phosphate (Pi) inside the fiber was coupled to the oxidation of NADH to NAD catalyzed by pyruvate kinase and lactate dehydrogenase. Because NADH absorbs light at 340 nm and NAD does not, the oxidation of NADH to NAD, and thus ATP consumption, was determined by meas- uring the absorbance of UV light at 340 nm at steady state within the measurement chamber. This absorbance signal was calibrated by repeated injections of 50 nl of 10 mM ADP into the measuring chamber. N-terminal Fragment of cMyBP-C Impairs Myofilament Function The ADP injection induced a rapid step reduction in fluorescence, and the rate of ATP consumption was calculated by the magnitude of this step from the fluores- cent decay rate at 340 nm. In addition, the ADP injection served to confirm that the chemical response time and the bath stir- ring were adequate. Furthermore, the rate of force redevelop- ment following a release-restretch maneuver, ktr, was measured during a final contraction at maximum Ca2 as previously described (23). Only muscles that maintained greater than 80% maximal tension were included for analysis. N-terminal Fragment of cMyBP-C Impairs Myofilament Function Briefly, the isolated muscle was exposed to a range of calcium solutions obtained by proportional mixing of activating and relaxing solutions, and the force generated and ATP consumed were measured simultaneously during the con- traction. Activating solution contained 20 mM Ca2-EGTA, 1.55 mM potassium propionate, 6.59 mM MgCl2, 100 mM N,N- bis(2-hydroxyethyl)taurine; N,N-Bis-(2-hydroxyethyl)-2-ami- noethanesulfonic acid, 5 mM sodium azide, 1 mM DTT, 10 mM phosphoenolpyruvate, 0.01 mM oligomycin, 0.1 mM PMSF, and 0.02 mM A2P5, as well as protease inhibitor mixture. Relaxing solution was identical, except it contained 20 mM EGTA, 21.2 mM potassium propionate, and 7.11 mM MgCl2. The preactivat- ing solution contained 0.5 mM EGTA, 19.5 mM 1,6-diamino- hexane-N,N,N,N-tetraacetic acid, and 21.8 mM potassium pro- pionate. All solutions contained 0.5 mg/ml pyruvate kinase and 0.05 mg/ml lactate dehydrogenase (Sigma) and had an ionic strength of 180 mM, 5 mM free ATP, and 1 mM free magnesium, as determined by the Fabiato program (24). Pulldown Assay—His-tagged recombinant proteins, such as mouse C0-linker, C0-C1f, C0-C2, C0-C3, and C0mC1f, were expressed in E. coli and purified using nickel-nitrilotriacetic acid column chromatography at room temperature as described previ- ously (4, 15). Total tissue lysates from normal mouse left ventric- ular myocardium were prepared in radioimmune precipitation assay buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Nonidet P-40, 1% sodium deoxy- cholate, 2.5 mM sodium pyrophosphate, 1 mM -glycerophos- phate, 1 mM Na3VO4, and 1 g/ml leupeptin) at 1 mg protein/ml. 200 l of lysate was then incubated with 20 l of nickel-nitrilotri- acetic acid-agarose beads (Qiagen) at 4 °C for 2 h and spun down, and the pellet was discarded to remove proteins with nonspecific binding to the beads. The supernatant was then incubated with either His-tagged cMyBP-C protein fragments or SUMO-His- tagged -TM and 20 l of beads for 2 h at 4 °C, followed by wash- ing with Tris buffer (50 mM) containing 0.5% Triton X-100, 100 mMNaCl,10mMMgCl2,0.1mMPMSF,and1proteaseinhibitor. Proteins bound to the beads were eluted with Laemmli sample buffer (Bio-Rad) and subjected to Western blot analysis. SDS- PAGE and Western blot analyses were carried out as described previously (2). incubated with peptide for 5 min in relaxing solution and another 5 min in preactivating solution before measuring the force development. The peptide was also present in all working solutions thereafter. N-terminal Fragment of cMyBP-C Impairs Myofilament Function Muscle dimensions were determined using an ocular micrometer mounted in the dissection microscope (resolution, 10 m). Sarcomere length was measured in the passive relaxed condition by laser diffraction as previously described (21), adjusted to 2 m and then 2.3 m. The skinned fibers were X-ray Diffraction—Experiments were performed using the BioCAT undulator-based beamline at the Advanced Photon Source (Argonne National Labs, Lemont, IL) (20). The high x-ray flux density and low beam divergence delivered by this instrument are highly advantageous for small angle x-ray stud- ies of small specimens, such as those described here. Experi- ments were done using a 3-m distance between the sample and the detector and with the x-ray beam energy set to 12 keV (1.03 VOLUME 289•NUMBER 13•MARCH 28, 2014 8820 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 13•MARCH 28, 2014 N-terminal Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function FIGURE 1. hC0C1f incorporates into the human cardiac sarcomere. A, recombinant protein was run on SDS-PAGE and SYPRO-RUBY staining to show the purity of recombinant hC0C1f. B, electrospray ionization-Fourier transform mass spectrometry spectrum of recombinant hC0C1f. C, a skinned stretched cardiomyocyte stained with -actinin (red), a Z-line-specific protein, and incubated with IAF-labeled hC0C1f (green). The merged image highlights localization of hC0C1f in the A-band of the sarcomere, and the imaging was performed at SL of 2.1 m. The scale bars represent 5 m. g y y FIGURE 1. hC0C1f incorporates into the human cardiac sarcomere. A, recombinant protein was run on SDS-PAGE and SYPRO-RUBY staining to show the purity of recombinant hC0C1f. B, electrospray ionization-Fourier transform mass spectrometry spectrum of recombinant hC0C1f. C, a skinned stretched cardiomyocyte stained with -actinin (red), a Z-line-specific protein, and incubated with IAF-labeled hC0C1f (green). The merged image highlights localization of hC0C1f in the A-band of the sarcomere, and the imaging was performed at SL of 2.1 m. The scale bars represent 5 m. incubated with peptide for 5 min in relaxing solution and another 5 min in preactivating solution before measuring the force development. The peptide was also present in all working solutions thereafter. Isometric tension and ATPase activity were measured at various levels of Ca2 activation as previously described (22, 23). RESULTS This concentration approximates the EC50 of inhibition in force induced by hC0C1f. At 2-m SL, the fragment induced a 30% reduction in maximal force (Fmax) (Fig. 3, B and C) and a 50% increase in myofilament activation cooperativity, as reported by the Hill coefficient (nH) (Fig. 3D). Remarkably, hC0C1f did not affect Fmax or nH at long SL. Additionally, hC0C1f induced a rightward shift of the force-pCa relationship at both SLs, indicating decreased myofilament Ca2 sensitivity (20% reduction of EC50 at both SL) (Fig. 3E). Despite decreased myofilament Ca2 sensitivity, length-dependent activation was enhanced with an increased Fmax (difference between maximum force at long and short sarcomere lengths; 13.20  0.41 versus 20.56  1.70, control versus hC0C1f, p  0.05). Although the EC50 (difference between myofilament Ca2 sensitivity at long and short sarcomere lengths) increased, this change was not significant (0.28  0.06 versus 0.41  0.07, control versus hC0C1f, p 0.07). rescently labeled anti--actinin antibodies. IAF-labeled hC0C1f was present specifically between Z-discs, likely on the thin filaments (Fig. 1C). This is similar to the localization of endogenous cMyBP-C. To determine whether hC0C1f incor- poration disrupted sarcomere structure, x-ray diffraction experiments were performed on permeabilized human donor myocardium at short (2 m) and long (2.3 m) sarcomere length (SL) (Fig. 2, A and B). As expected, spacing between each thin and thick filament was reduced upon stretching. Further- more, incubation with hC0C1f did not significantly change interfilament spacing at either SL (Fig. 2B), suggesting that the presence of hC0C1f in the sarcomere does not adversely affect sarcomere lattice spacing. hC0C1f Disrupts Steady-state Force Development—To eval- uate the effect of hC0C1f on myocardial contractile properties, we developed a novel method to allow for the use of frozen donor heart tissue. The tissue was cut into thin strips to resem- ble trabeculae and then homogenized, permeabilized, and attached to a length controller and force transducer. The sam- ples were illuminated with a laser to reveal a diffraction pattern allowing SL determination (Fig. 2C). To establish a dose-re- sponse curve, permeabilized myocardium was incubated at pCa 5.85 (pCa50) with increasing concentrations of hC0C1f. As a control, the muscle was also incubated with buffer alone or a control protein, egg albumin, which has a molecular mass sim- ilar to that of hC0C1f (44 kDa). RESULTS Purification and Characterization of hC0C1f—Recombinant hC0C1f was generated, purified, and analyzed with SDS-PAGE showing a pure protein that migrates at 40 kDa (Fig. 1A). To further characterize the recombinant hC0C1f, high resolution mass spectrometry analysis was performed, confirming the amino acid sequences and a molecular mass of 31,989.20 dal- tons (Fig. 1B). To determine hC0C1f incorporation into the A-band of the sarcomere, permeabilized human ventricular myocytes were incubated with IAF-labeled hC0C1f and fluo- MARCH 28, 2014•VOLUME 289•NUMBER 13 MARCH 28, 2014•VOLUME 289•NUMBER 13 8821 MARCH 28, 2014•VOLUME 289•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY FIGURE 2. hC0C1f does not disrupt sarcomere structure. A, representative x-ray diffraction of skinned ventricular fibers with or without hC0C1f. B, thin and thick filament spacings indicated by d1,0 (thin filament spacing) and d1,1 (thick filament spacing) reflections, respectively. CTRL, control. C, representative laser diffraction pattern and intensity profile of skinned human ventricular muscle tissue at 2- and 2.3-m SL. Representative images are shown at 5 magnification. N-terminal Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function FIGURE 2. hC0C1f does not disrupt sarcomere structure. A, representative x-ray diffraction of skinned ventricular fibers with or without hC0C1f. B, thin and thick filament spacings indicated by d1,0 (thin filament spacing) and d1,1 (thick filament spacing) reflections, respectively. CTRL, control. C, representative laser diffraction pattern and intensity profile of skinned human ventricular muscle tissue at 2- and 2.3-m SL. Representative images are shown at 5 magnification. This concentration approximates the EC50 of inhibition in force induced by hC0C1f. At 2-m SL, the fragment induced a 30% reduction in maximal force (Fmax) (Fig. 3, B and C) and a 50% increase in myofilament activation cooperativity, as reported by the Hill coefficient (nH) (Fig. 3D). Remarkably, hC0C1f did not affect Fmax or nH at long SL. Additionally, hC0C1f induced a rightward shift of the force-pCa relationship at both SLs, indicating decreased myofilament Ca2 sensitivity (20% reduction of EC50 at both SL) (Fig. 3E). Despite decreased myofilament Ca2 sensitivity, length-dependent activation was enhanced with an increased Fmax (difference between maximum force at long and short sarcomere lengths; 13.20  0.41 versus 20.56  1.70, control versus hC0C1f, p  0.05). Although the EC50 (difference between myofilament Ca2 sensitivity at long and short sarcomere lengths) increased, this change was not significant (0.28  0.06 versus 0.41  0.07, control versus hC0C1f, p 0.07). RESULTS A dose-dependent reduction in force at pCa50 was observed with an EC50 (myofilament cal- cium sensitivity; Ca2 concentration at which half of Fmax is reached) of inhibition at 4.41 M and maximum inhibition occurring at 30 M of hC0C1f (Fig. 3A), with no effect on force development of vehicle or egg albumin. The effects of hC0C1f on tension development and cross-bridge cycling were determined by measuring force-pCa relationships in permeabi- lized human ventricular myocardium in the absence and pres- ence of 5 M hC0C1f. Given that length-dependent activation is characterized by an increase in both maximum Ca2-activated force and Ca2 sensitivity upon an increase in sarcomere length, all experiments were done at both short and long SL. hC0C1f Alters Cross-bridge Cycling Properties—We also eval- uated myocardium energy consumption by measuring the ATP hydrolysis rate in the absence and presence of 5 M hC0C1f. Although no significant difference in maximum ATP hydroly- sis was observed (Fig. 4A), the fragment significantly altered stretch-induced reduction of ATP hydrolysis, as suggested by reduced Max ATPase (Fig. 4B). We also observed a significant increase in the tension cost with hC0C1f at both SL (Fig. 4, C and D). The two-way analysis of variance determined that ten- sion cost decreased with sarcomere length (p 0.0005) and increased with hC0C1f treatment (p 0.0482; main effect). However, hC0C1f did not alter stretch-induced reduction in the tension cost (1.06  0.22 versus 1.07  0.26, control versus hC0C1f, p 0.9936). The rate of tension redevelopment (ktr) was also significantly increased in the presence of hC0C1f at both SLs (Fig. 4E). These data are consistent with increased tension cost and indicate a significant increase in cross-bridge cycling kinetics induced by hC0C1f. hC0C1f Interacts with the Thin Filament Proteins -TM and Actin—Previously, we demonstrated that cMyBP-C colocalized with -TM in the sarcomere (28), and many reports have VOLUME 289•NUMBER 13•MARCH 28, 2014 8822 8822 JOURNAL OF BIOLOGICAL CHEMISTRY URE 3. Altered force-Ca2 relationship and length-dependent activation in the presence of hC0C1f. A, dose-response curve of the inhibitory effect of 0C1f, compared with vehicle and egg albumin, on force development at pCa50 (pCa 5.85) for 2-m SL. CTRL, control. B, maximum force (Fmax) at pCa 4.5 in nned ventricular fibers treated with hC0C1f at 2- and 2.3-m SL. C, myofilament force-Ca2 relationship (panel i) and force-Ca2 relationship normalized to ximum force (panel ii). RESULTS D and E, normalized force-Ca2 relationships were fit to a modified Hill equation, and the Hill coefficient (D) and EC50 (E) were termined. *, p  0.05 control versus hC0C1f; †, p  0.05 2 versus 2.3-m SL within groups. N-terminal Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function g y p y FIGURE 3. Altered force-Ca2 relationship and length-dependent activation in the presence of hC0C1f. A, dose-response curve of the inhibitory effect of hC0C1f, compared with vehicle and egg albumin, on force development at pCa50 (pCa 5.85) for 2-m SL. CTRL, control. B, maximum force (Fmax) at pCa 4.5 in skinned ventricular fibers treated with hC0C1f at 2- and 2.3-m SL. C, myofilament force-Ca2 relationship (panel i) and force-Ca2 relationship normalized to maximum force (panel ii). D and E, normalized force-Ca2 relationships were fit to a modified Hill equation, and the Hill coefficient (D) and EC50 (E) were determined. *, p  0.05 control versus hC0C1f; †, p  0.05 2 versus 2.3-m SL within groups. actomyosin interactions, thereby increasing the probability of strong cross-bridge formation (Fig. 6, A and B). described its interaction with actin (9, 12). To demonstrate the direct interaction with -TM and actin, we generated several cMyBP-C N-terminal recombinant fragments (Fig. 5A) and used heart protein lysate to pull down interacting partners. Consistent with previous reports (9), our results show a direct interaction of hC0C1f with actin, but not with myosin (Fig. 5C). More interestingly and for the first time, we show a direct inter- action of cMyBP-C with -TM via the Pro-Ala-rich linker region (Fig. 5, C–E). This interaction, but not the interaction with actin, is aborted following mutation of five proline resi- dues to alanine (Fig. 5E), confirming that the interaction is mediated through the Pro-Ala-rich linker region. These results suggest a direct interaction of cMyBP-C N-region with both -TM and actin and that disruption of that interaction by C0-C1f leads to altered cross-bridge cycling. In the present study, we show that incubation with hC0C1f increased cooper- ativity, cross-bridge cycling, and ATP consumption. These results suggest that hC0C1f fragments constantly interact with thin filament proteins, such as actin and -TM, thereby altering DISCUSSION Catalytic cleavage of cMyBP-C, in human heart failure and post-MI, has been associated with abnormal contractility; how- ever, the underlying mechanisms have been unclear (2, 3, 29). Previously, we have shown that C0-C1f is released by catalytic cleavage of cMyBP-C at a -calpain site and that this release correlated with cardiac insults, such as I/R and MI (2, 30, 31). Further, overexpression of mouse C0C1f resulted in cell death, impaired Ca2 handling, altered contractile function of the sar- comere (4), and, finally, heart failure (3). Using in vitro motility and laser trap assays, we also previously determined that C0-C1f is sufficient to reduce actin sliding velocity in a manner similar to that of either C0-C2 or full-length cMyBP-C (13). Furthermore, we determined that the 17 residues of M domain in C0-C1f mediate interaction with actin (4, 13). In this study, we extend these findings to show, for the first time, that the MARCH 28, 2014•VOLUME 289•NUMBER 13 8823 JOURNAL OF BIOLOGICAL CHEMISTRY MARCH 28, 2014•VOLUME 289•NUMBER 13 FIGURE 4. hC0C1f increases cross-bridge cycling kinetics. A, maximum ATPase hydrolysis rate. B, difference in ATPasemax at short and long SL. C, force- ATPaserelationshipsinvarioustensionranges(binneddata)at2-m(paneli)and2.3-m(panelii)SL,andtheforce-ATPaserelationshipofeachindividualfiber was fit by linear regression. D, the slope was then calculated to determine the tension cost. E, the rate of force redevelopment (ktr) was determined following a rapid stretch-release-restretch maneuver. *, p  0.05 control versus hC0C1f. #, p  0.05, 2-m versus 2.3-m SL main effect. †, p  0.05 2-m versus 2.3-m SL within groups. N-terminal Fragment of cMyBP-C Impairs Myofilament Function minal Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function ent Function FIGURE 4. hC0C1f increases cross-bridge cycling kinetics. A, maximum ATPase hydrolysis rate. B, difference in ATPasemax at short and long SL. C, force- ATPaserelationshipsinvarioustensionranges(binneddata)at2-m(paneli)and2.3-m(panelii)SL,andtheforce-ATPaserelationshipofeachindividualfiber was fit by linear regression. D, the slope was then calculated to determine the tension cost. E, the rate of force redevelopment (ktr) was determined following a rapid stretch-release-restretch maneuver. *, p  0.05 control versus hC0C1f. #, p  0.05, 2-m versus 2.3-m SL main effect. †, p  0.05 2-m versus 2.3-m SL within groups. sue used in these experiments had normal levels of native cMyBP-C, and simple addition of hC0C1f was enough to dis- rupt the myofilament properties. DISCUSSION Of course, proteolysis of cMyBP-C would in itself cause additional dysfunction in the whole organ, and most likely, following I/R or MI, it is the addi- tive effect of less cMyBP-C and the presence of hC0C1f that will lead to contractile dysfunction in the heart. Furthermore, it is still unclear whether the fragment can penetrate neighboring cells. At this time, it is not possible to determine whether the contractile dysfunction seen in the whole heart following I/R or MI is solely due to hC0C1f acting within the cardiomyocyte or due to “diffusion” to remote areas. Along the same lines, it is unclear how long the fragment remains in the cells or attached to the myofilaments. hC0C1f can be detected in tissue several hours to several days following I/R and MI, and we do not know whether there are any mechanisms to actively remove the frag- ment from the cell. Additionally, it is very difficult to remove hC0C1f from the myofilaments in vitro because it does not wash off following removal. proteolytic fragment hC0C1f can incorporate into the human cardiac sarcomere and that this incorporation is sufficient to 1) decrease maximum force, 2) increase cooperativity, and 3) increase tension cost and cross-bridge cycling. We also show that these effects could be the result of a direct interaction of hC0C1f with both -TM and actin, inhibiting, in turn, the role of full-length cMyBP-C. This study provides a direct mecha- nism that explains, at least in part, the depressed contractile function observed post-MI in the noninfarcted myocardium. Although no attempt has been made to accurately measure the concentration of hC0C1f in the myofilaments following MI or I/R or in heart failure, Western blots done by us and others shown that hC0C1f is easily detectable in myofilaments and is of comparable intensity to the remaining cMyBP-C (2–4). Assuming that cMyBP-C is present at 15 M in cardiomyo- cytes, we estimate that hC0C1f is present at concentrations ranging from 3 to 15 M, depending on the conditions. In the present study, we used a concentration of 5 M hC0C1f to assay the effects of the fragment on myofilament properties. This concentration is well within the physiological range observed following MI or in heart failure. To provide direct support that N-specific fragments of cMyBP-C are sufficient to incorporate into the sarcomere, Her- ron et al. DISCUSSION hC0C1f interacts with the thick and thin filaments. A, SDS PAGE, followed by Coomassie staining of His tag recombinant proteins containing the N-terminal domains of cMyBP-C and SUMO-tagged -TM. B, pulldown assay with -TM (top panel) and C0C2 (bottom panel), followed by Western blot analysis for cMyBP-C and -TM, respectively. C, pulldown assay with dif- ferent N proteins of cMyBP-C, followed by Western blot analysis for myosin, actin, and -TM. D and E, pulldown assay with C0-C1f (D) and C0mC1f (with mutatedresiduesinthePro-Ala-richregion,E),followedbyWesternblotanal- ysis for -TM and actin. IB, immunoblotting; IP, immunoprecipitation. , , followed by Coomassie staining of His tag recombinant proteins containing the N-terminal domains of cMyBP-C and SUMO-tagged -TM. B, pulldown assay with -TM (top panel) and C0C2 (bottom panel), followed by Western blot analysis for cMyBP-C and -TM, respectively. C, pulldown assay with dif- ferent N proteins of cMyBP-C, followed by Western blot analysis for myosin, actin, and -TM. D and E, pulldown assay with C0-C1f (D) and C0mC1f (with mutatedresiduesinthePro-Ala-richregion,E),followedbyWesternblotanal- ysis for -TM and actin. IB, immunoblotting; IP, immunoprecipitation. interaction is lost, and constant interaction occurs. Indeed, we show that the C0-C1f region interacts with actin, but not myo- sin, because of the absence of M and C2 domains. Furthermore, part of the negative effects of hC0C1f could be due to disruption of the link between the thick and thin filament that native cMyBP-C provides. sarcomere. However, it remains unclear whether these frag- ments directly interact with myosin or actin, or both, in a man- ner that confers stability in the sarcomere. Recent studies dem- onstrate that cMyBP-C N-regions structurally reach thin filaments (32, 33) and dynamically interact with actin (12). In the present study, we confirmed that recombinant C0-C1f pro- teins were able to diffuse into the sarcomere and specifically localize on the A-band and I-band in the presence of endoge- nous cMyBP-C without changing the interfilament space at both short and longer SL. Interestingly, we did not see any staining of hC0C1f fragments on the Z-line, suggesting that interaction of C0-C1f is very specific to thin and thick filament proteins. However, further studies are needed to define the exact region of interaction that is necessary in the N-region on the thick and thin filaments. The N-region of cMyBP-C binds transiently to actin and the myosin S2 region in a phosphory- lation-dependent manner to regulate actomyosin interaction and force generation. DISCUSSION (7) overexpressed either C0-C1 or C0-C2 regions into neonatal cardiomyocytes and confirmed that these fragments of cMyBP-C could specifically incorporate in the A-band of the Based on the data presented in this study, it is clear that cleavage of cMyBP-C is not the most important/only mecha- nism leading to contractile dysfunction. Indeed, the human tis- 8824 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 13•MARCH 28, 2014 8824 FIGURE6.A,schematicdiagramofthesarcomerewiththick(titin,myosin,and cMyBP-C) and thin (-TM, cTn complex, and actin) filament proteins. cMyBP-C interacts with titin and myosin at its C-terminal, with actin at the C1 and M domains, myosin at the M domain, and -TM at the Pro-Ala-linker region. B, C0-C1f disrupts native cMyBP-C interaction with the thin filament by binding actin and -TM at multiple locations. Our data indicate that the presence of C0-C1f fragments in the normal sarcomere disrupts thin filament and thick filament protein interactions, thereby interfering with the regulatory role of native full-length cMyBP-C and, in turn, resulting in diminished force generation. l Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function N-terminal Fragment of cMyBP-C Impairs Myofilament Function FIGURE 5. hC0C1f interacts with the thick and thin filaments. A, SDS-PAGE, followed by Coomassie staining of His tag recombinant proteins containing the N-terminal domains of cMyBP-C and SUMO-tagged -TM. B, pulldown assay with -TM (top panel) and C0C2 (bottom panel), followed by Western blot analysis for cMyBP-C and -TM, respectively. C, pulldown assay with dif- ferent N proteins of cMyBP-C, followed by Western blot analysis for myosin, actin, and -TM. D and E, pulldown assay with C0-C1f (D) and C0mC1f (with mutatedresiduesinthePro-Ala-richregion,E),followedbyWesternblotanal- ysis for -TM and actin. IB, immunoblotting; IP, immunoprecipitation. N-terminal FIGURE6.A,schematicdiagramofthesarcomerewiththick(titin,myosin,and cMyBP-C) and thin (-TM, cTn complex, and actin) filament proteins. cMyBP-C interacts with titin and myosin at its C-terminal, with actin at the C1 and M domains, myosin at the M domain, and -TM at the Pro-Ala-linker region. B, C0-C1f disrupts native cMyBP-C interaction with the thin filament by binding actin and -TM at multiple locations. Our data indicate that the presence of C0-C1f fragments in the normal sarcomere disrupts thin filament and thick filament protein interactions, thereby interfering with the regulatory role of native full-length cMyBP-C and, in turn, resulting in diminished force generation. FIGURE 5. hC0C1f interacts with the thick and thin filam FIGURE 5. hC0C1f interacts with the thick and thin filaments. A, SDS-PAGE, FIGURE 5. REFERENCES Consistent with previous studies using various N fragments, such as mouse C0-C1 (5, 7), C1-C2 (6), and C0-C2 (7) of cMyBP-C, our results with the physiologically relevant hC0C1f show decreased Fmax and accelerated cross-bridge cycling. Although these studies previously showed increased myofila- ment Ca2 sensitivity (6, 7), we show decreased EC50 at both short and long SLs. This difference might be partly explained by the decreased phosphorylation status of endogenous cMyBP-C seen in failing hearts, compared with the healthy donor tissue used in this study. We and others have previously shown that cMyBP-C phosphorylation is greatly depressed in HF and fol- lowing MI (2, 34). Moreover, cMyBP-C binding to myosin (31) and actin is modulated by phosphorylation status, and N-ter- minal fragments do not bind actin when the cMyBP-C phos- phorylation level is high (10, 31). Although Herron et al. (7) determined an increase in Ca2 sensitivity following addition of C0-C2 fragment, our report shows that the C0-C1f fragment also contains the first 17 residues of the M-domain, which were sufficient to interact with actin (4, 13). As opposed to the hC0- Clf fragment, C0-C2 preserves the link between thick and thin filaments. Also, C0-C2 has been shown to bind to myosin (10), but hC0C1f does not, suggesting that cMyBP-C interaction with myosin S2 is mediated through M and C2 domains of cMyBP-C. 1. van der Velden, J., Merkus, D., Klarenbeek, B. R., James, A. T., Boontje, N. M., Dekkers, D. H., Stienen, G. J., Lamers, J. M., and Duncker, D. J. (2004) Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction. Circ. Res. 95, e85–95 2. Govindan, S., McElligott, A., Muthusamy, S., Nair, N., Barefield, D., Mar- tin, J. L., Gongora, E., Greis, K. D., Luther, P. K., Winegrad, S., Henderson, K. K., and Sadayappan, S. (2012) Cardiac myosin binding protein-C is a potential diagnostic biomarker for myocardial infarction. J. Mol Cell Car- diol. 52, 154–164 3. Razzaque, M. A., Gupta, M., Osinska, H., Gulick, J., Blaxall, B. C., and Robbins, J. (2013) An endogenously produced fragment of cardiac myo- sin-binding protein C is pathogenic and can lead to heart failure. Circ. Res. 113, 553–561 4. Govindan, S., Sarkey, J., Ji, X., Sundaresan, N. R., Gupta, M. P., de Tombe, P. P., and Sadayappan, S. (2012) Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro. J. Muscle Res. REFERENCES Cell Motil. 33, 17–30 5. Razumova, M. V., Shaffer, J. F., Tu, A. Y., Flint, G. V., Regnier, M., and Harris, S. P. (2006) Effects of the N-terminal domains of myosin binding protein-C in an in vitro motility assay. Evidence for long-lived cross- bridges. J. Biol. Chem. 281, 35846–35854 6. Harris, S. P., Rostkova, E., Gautel, M., and Moss, R. L. (2004) Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism. Circ. Res. 95, 930–936 7. Herron, T. J., Rostkova, E., Kunst, G., Chaturvedi, R., Gautel, M., and Kentish, J. C. (2006) Activation of myocardial contraction by the N-termi- nal domains of myosin binding protein-C. Circ. Res. 98, 1290–1298 8. Yasuda, M., Koshida, S., Sato, N., and Obinata, T. (1995) Complete pri- mary structure of chicken cardiac C-protein (MyBP-C) and its expression in developing striated muscles. J. Mol Cell Cardiol. 27, 2275–2286 y Recent studies in the literature report that the interaction of cMyBP-C N terminus with thin filaments requires the Pro-Ala- rich region (28, 32, 33, 35, 36). Herron et al. (7) proposed that the Pro-Ala-rich region may promote binding of cMyBP-C to thin filament proteins and thus enhance cross-bridge cycling. Here, we show, for the first time, that the hC0C1f region inter- acts with -TM via the Pro-Ala-rich region. As suggested by the increased Hill coefficient, hC0C1f interaction with -TM may alter -TM movement and indirectly modulate troponin posi- tion/structure leading to increased cooperativity. However, more experiments are required to investigate the impact of hC0C1f on -TM structure and regulation. We postulate that the effects of hC0C1f are primarily mediated by interactions with the thin filament through actin and -TM. Given the pro- posed role of cMyBP-C as a brake on actomyosin dynamics (31), we suggest that hC0C1f disables this brake by increasing ten- sion cost and ktr. This, in turn, mimics the absence of cMyBP-C, increases contraction velocity, and, in the working heart, reduces the systolic phase, similar to what is observed in cMyBP-C knock-out mice (37). This reduced efficiency of con- traction then further exacerbates dysfunction in energy-starved hearts. 9. Shaffer, J. F., Kensler, R. W., and Harris, S. P. (2009) The myosin-binding protein C motif binds to F-actin in a phosphorylation-sensitive manner. J. Biol. Chem. 284, 12318–12327 10. Kulikovskaya, I., McClellan, G., Flavigny, J., Carrier, L., and Winegrad, S. N-terminal Fragment of cMyBP-C Impairs Myofilament Function enhance length-dependent activation with increased Fmax and EC50. However, this effect is mostly caused by changes in Fmax and EC50 at short, but not long, SL, as seen with C0-C2 (7). Therefore, the hC0C1f fragment seems to interfere with thin filament function, as observed by changes in cooperativity and Ca2 sensitivity, at short SL. Conversely, hC0C1f impacts cross-bridge cycling at both SLs, suggesting that it acts on strong cross-bridge kinetics by increasing the off rate. This is supported by the colocalized immunofluorescence images, highlighting the presence of the fragment in both the A- and I-bands. In conclusion, we show, for the first time, that the cMyBP-C cleavage product hC0C1f is sufficient to cause the contractile dysfunction seen post-MI. We propose that its mechanism of action results from constant interaction with thin filament pro- teins, such as actin and -TM. Also, such an interaction dis- rupts the regulation of actomyosin interaction by affecting endogenous cMyBP-C regulation. Therefore, therapeutic interventions aimed at preventing cleavage of cMyBP-C and attenuating the effects of C0-C1f might confer protection fol- lowing MI. DISCUSSION When the region is cleaved off and C0-C1f is released from the full-length cMyBP-C, transient sarcomere. However, it remains unclear whether these frag- ments directly interact with myosin or actin, or both, in a man- ner that confers stability in the sarcomere. Recent studies dem- onstrate that cMyBP-C N-regions structurally reach thin filaments (32, 33) and dynamically interact with actin (12). In the present study, we confirmed that recombinant C0-C1f pro- teins were able to diffuse into the sarcomere and specifically localize on the A-band and I-band in the presence of endoge- nous cMyBP-C without changing the interfilament space at both short and longer SL. Interestingly, we did not see any staining of hC0C1f fragments on the Z-line, suggesting that interaction of C0-C1f is very specific to thin and thick filament proteins. However, further studies are needed to define the exact region of interaction that is necessary in the N-region on the thick and thin filaments. The N-region of cMyBP-C binds transiently to actin and the myosin S2 region in a phosphory- lation-dependent manner to regulate actomyosin interaction and force generation. When the region is cleaved off and C0-C1f is released from the full-length cMyBP-C, transient In the presence of C0-C2, studies show that maximal force is significantly reduced but that no changes occur in ktr, tension redevelopment, compared with the untreated control mouse myocytes (7). In contrast, when human myocytes were permea- bilized, myocardial preparations were treated with C0-C2, maximal force, and ktr values were decreased and increased, respectively, indicating that the difference in ktr largely resulted from the presence of -myosin heavy chain in adult human, compared with adult mouse, ventricle (7). Similarly, in our study, the presence of hC0C1f was able to reduce maximal force and increase ktr in human myocytes. It is unlikely that the increase in ktr was due to accelerated thin filament activation because this parameter was assessed at maximum saturating levels of activating Ca2, a condition where thin filament acti- vation kinetics is no longer rate-limiting for force development. Interestingly, our study determined that permeabilization of hC0C1f causes a decrease in myofilament Ca2 sensitivity in contrast to the C0-C2 effects. Furthermore, hC0C1f appears to MARCH 28, 2014•VOLUME 289•NUMBER 13 8825 JOURNAL OF BIOLOGICAL CHEMISTRY MARCH 28, 2014•VOLUME 289•NUMBER 13 N-terminal Fragment of cMyBP-C Impairs Myofilament Function 16. Chen, P. P., Patel, J. R., Rybakova, I. N., Walker, J. W., and Moss, R. L. (2010) Protein kinase A-induced myofilament desensitization to Ca2 as a result of phosphorylation of cardiac myosin-binding protein C. J. Gen. Physiol. 136, 615–627 27. Chandra, M., Rundell, V. L., Tardiff, J. C., Leinwand, L. A., De Tombe, P. P., and Solaro, R. J. (2001) Ca2 activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T. Am. J. Physiol. Heart Circ. Physiol. 280, H705–H713 17. Ge, Y., Rybakova, I. N., Xu, Q., and Moss, R. L. (2009) Top-down high- resolution mass spectrometry of cardiac myosin binding protein C re- vealed that truncation alters protein phosphorylation state. Proc. Natl. Acad. Sci. U.S.A. 106, 12658–12663 28. Barefield, D., and Sadayappan, S. (2010) Phosphorylation and function of cardiac myosin binding protein-C in health and disease. J. Mol. Cell. Car- diol. 48, 866–875 29. Sadayappan, S., and Robbins, J. (2008) The death of transcriptional chau- vinism in the control and regulation of cardiac contractility. Ann. N.Y. Acad. Sci. 1123, 1–9 18. Aït Mou, Y., Reboul, C., Andre, L., Lacampagne, A., and Cazorla, O. (2009) Late exercise training improves non-uniformity of transmural myocardial function in rats with ischaemic heart failure. Cardiovasc. Res. 81, 555–564 30. Decker, R. S., Decker, M. L., Kulikovskaya, I., Nakamura, S., Lee, D. C., Harris, K., Klocke, F. J., and Winegrad, S. (2005) Myosin-binding protein C phosphorylation, myofibril structure, and contractile function during low-flow ischemia. Circulation 111, 906–912 19. Fentzke, R. C., Buck, S. H., Patel, J. R., Lin, H., Wolska, B. M., Stojanovic, M. O., Martin, A. F., Solaro, R. J., Moss, R. L., and Leiden, J. M. (1999) Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart. J. Physiol. 517, 143–157 31. Previs, M. J., Beck Previs, S., Gulick, J., Robbins, J., and Warshaw, D. M. (2012) Molecular mechanics of cardiac myosin-binding protein C in na- tive thick filaments. Science 337, 1215–1218 20. Irving, T. C., Konhilas, J., Perry, D., Fischetti, R., and de Tombe, P. P. (2000) Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium. Am. J. Physiol. Heart Circ. Physiol. 279, H2568–H2573 32. Luther, P. K., and Craig, R. (2011) Modulation of striated muscle contrac- tion by binding of myosin binding protein C to actin. Bioarchitecture 1, 277–283 y y y 21. de Tombe, P. REFERENCES (2003) Effect of MyBP-C binding to actin on contractility in heart muscle. J. Gen. Physiol. 122, 761–774 11. Ratti, J., Rostkova, E., Gautel, M., and Pfuhl, M. (2011) Structure and interactions of myosin-binding protein C domain C0. Cardiac-specific regulation of myosin at its neck? J. Biol. Chem. 286, 12650–12658 12. Kensler, R. W., Shaffer, J. F., and Harris, S. P. (2011) Binding of the N-ter- minal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin. J Struct Biol 174, 44–51 13. Weith, A., Sadayappan, S., Gulick, J., Previs, M. J., Vanburen, P., Robbins, J., and Warshaw, D. M. (2012) Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhi- bition of actomyosin motility requires 17 amino acids of the motif domain. J. Mol Cell Cardiol. 52, 219–227 14. Gruen, M., Prinz, H., and Gautel, M. (1999) cAPK-phosphorylation con- trols the interaction of the regulatory domain of cardiac myosin binding protein C with myosin-S2 in an on-off fashion. FEBS Lett. 453, 254–259 15. Sadayappan, S., Osinska, H., Klevitsky, R., Lorenz, J. N., Sargent, M., Molk- entin, J. D., Seidman, C. E., Seidman, J. G., and Robbins, J. (2006) Cardiac myosin binding protein C phosphorylation is cardioprotective. Proc. Natl. Acad. Sci. U.S.A. 103, 16918–16923 8826 JOURNAL OF BIOLOGICAL CHEMISTRY 8826 VOLUME 289•NUMBER 13•MARCH 28, 2014 VOLUME 289•NUMBER 13•MARCH 28, 2014 N-terminal Fragment of cMyBP-C Impairs Myofilament Function P., and ter Keurs, H. E. (1990) Force and velocity of sarcom- ere shortening in trabeculae from rat heart. Effects of temperature. Circ. Res. 66, 1239–1254 33. Luther, P. K., Winkler, H., Taylor, K., Zoghbi, M. E., Craig, R., Padrón, R., Squire, J. M., and Liu, J. (2011) Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc. Natl. Acad. Sci. U.S.A. 108, 11423–11428 22. de Tombe, P. P., and Stienen, G. J. (1995) Protein kinase A does not alter economy of force maintenance in skinned rat cardiac trabeculae. Circ. Res. 76, 734–741 34. Sadayappan, S., Gulick, J., Osinska, H., Martin, L. A., Hahn, H. S., Dorn, G. W., 2nd, Klevitsky, R., Seidman, C. E., Seidman, J. G., and Robbins, J. (2005) Cardiac myosin-binding protein-C phosphorylation and cardiac function. Circ. Res. 97, 1156–1163 23. Rundell, V. L., Manaves, V., Martin, A. F., and de Tombe, P. P. (2005) Impact of -myosin heavy chain isoform expression on cross-bridge cy- cling kinetics. Am. J. Physiol. Heart Circ. Physiol. 288, H896–H903 24. van der Velden, J., Moorman, A. F., and Stienen, G. J. (1998) Age-depen- dent changes in myosin composition correlate with enhanced economy of contraction in guinea-pig hearts. J. Physiol. 507, 497–510 35. Squire, J. M., Luther, P. K., and Knupp, C. (2003) Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identifi- cation of a possible actin-binding domain. J. Mol. Biol. 331, 713–724 25. Güth, K., and Wojciechowski, R. (1986) Perfusion cuvette for the simul- taneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres. Pflugers Arch. 407, 552–557 36. Shaffer, J. F., and Harris, S. P. (2009) Species-specific differences in the Pro-Ala rich region of cardiac myosin binding protein-C. J. Muscle Res. Cell Motil. 30, 303–306 26. Sumandea, M. P., Pyle, W. G., Kobayashi, T., de Tombe, P. P., and Solaro, R. J. (2003) Identification of a functionally critical protein kinase C phos- phorylation residue of cardiac troponin T. J. Biol. Chem. 278, 35135–35144 37. Stelzer, J. E., Patel, J. R., and Moss, R. L. (2006) Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocar- dium is eliminated by ablation of cMyBP-C. Circ. Res. 99, 884–890 MARCH 28, 2014•VOLUME 289•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8827
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One-year outcomes of intravitreal brolucizumab injections in patients with polypoidal choroidal vasculopathy
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Arisa Ito1, Maiko Maruyama‑Inoue1*, Yoko Kitajima2, Shoko Ikeda1, Tatsuya Inoue1 & Kazuaki Kadonosono1 Arisa Ito1, Maiko Maruyama‑Inoue1*, Yoko Kitajima2, Shoko Ikeda1, Tatsuya Inoue1 & Kazuaki Kadonosono1 To evaluate the 1-year visual outcomes and anatomic responses of Japanese patients who received intravitreal brolucizumab (IVBr) injections for polypoidal choroidal vasculopathy (PCV). This was a retrospective study of 17 treatment-naïve eyes with PCV that were treated with IVBr. We evaluated the best-corrected visual acuity (BCVA), central macular thickness (CMT), central choroidal thickness (CCT) and number of injections for 1 year. The eradication of polypoidal lesions was also evaluated using by indocyanine green angiography during the 1-year follow-up. Non-infectious intraocular inflammation developed in two (11.8%) eyes; 15 eyes were assessed at the 1-year follow-up examination. The mean BCVA improved significantly from 0.28 at baseline to 0.13 (P < 0.05) at 1 year. The CMT and CCT decreased significantly after 1 year. The mean number of injections was 6.4 ± 0.13. The rate of complete resolution of polypoidal lesions at 1 year was 93.3%. A dry macula was achieved in 13 eyes (86.6%) after the loading phase and in 11 eyes (73.3%) at 1 year. The IVBr injections appeared to be effective for improving both functional and anatomic outcomes in Japanese patients with PCV, with a high regression rate of polypoidal lesions. Polypoidal choroidal vasculopathy (PCV) was originally described by Yannuzzi et al.1,2 as a distinct subtype of wet age-related macular degeneration (AMD). Recently, Spaide et al.3 described that PCV is a variant of type 1 macular neovascularization that is more prevalent in Asian individuals. Indocyanine green angiography (ICGA) visualized a branching vascular network and various numbers of aneurysmal dilations at the outer edge of the expanding lesion. In Japan, it has been reported that approximately half of the patients with wet AMD have ­PCV4.it In eyes with PCV, massive hemorrhages and significant loss of vision are evident after a long-term follow-up ­period5. Previous studies have reported that anti-vascular endothelial growth factor (VEGF) agents, such as ranibizumab (Lucentis, Genentech, Inc., South San Francisco, CA) and aflibercept (Eylea, Bayer Health Care, Berlin, Germany) have had been effective in the treatment of PCV, although numerous injections are needed to stabilize patients’ visual acuity (VA)6–13. Moreover, aflibercept is more effective than ranibizumab for achiev- ing eradication of polypoidal lesions. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Arisa Ito1, Maiko Maruyama‑Inoue1*, Yoko Kitajima2, Shoko Ikeda1, Tatsuya Inoue1 & Kazuaki Kadonosono1 However, the eradication rate of polypoidal lesions at 1 year in Japanese patients was not as high as the 13% to 39% reported with ­ranibizumab6–9 and the 39% to 55% reported with ­aflibercept10–12. l Recently, brolucizumab was sanctioned as a new anti-VEGF agent for the treatment of AMD. Brolucizumab is a roughly 26-kDa single-chain antibody ­fragment14. The HAWK and HARRIER ­studies15,16, worldwide phase 3 clinical trials, showed that intravitreal brolucizumab (IVBr) injections administered at every-12-week/every- 8-week intervals were effective for improving and stabilizing VA for 96 weeks and were not inferior to the every-8-week dosing interval for intravitreal aflibercept. Moreover, IVBr injections provided better intraretinal, subretinal, and sub-retinal pigment epithelial fluid control than intravitreal aflibercept. p g pll p However, the results of IVBr injections for treating PCV in a real-world clinical setting have not been reported. The purpose of this study was to evaluate the 1-year visual outcomes and anatomic responses of Japanese patients with PCV treated with IVBr injections. 1Department of Ophthalmology, Yokohama City University Medical Center, 4‑57 Urafune‑cho, Minami‑ku, Yokohama, Kanagawa  232‑0024, Japan. 2Department of Ophthalmology, Sakae Kyosai Hospital, Kanagawa, Japan. *email: maicoo@urahp.yokohama-cu.ac.jp Scientific Reports | (2022) 12:7987 | https://doi.org/10.1038/s41598-022-12216-2 www.nature.com/scientificreports/ ntificreports/ Figure 1. Changes in the best-corrected visual acuity (BCVA) during the 12-month follow-up period. The mean BCVAs at 4, 6, and 12 months improved significantly compared with the preoperative VA (*P < 0.05). Figure 1. Changes in the best-corrected visual acuity (BCVA) during the 12-month follow-up period. The i Figure 1. Changes in the best-corrected visual acuity (BCVA) during the 12-month follow-up period. The mean BCVAs at 4, 6, and 12 months improved significantly compared with the preoperative VA (*P < 0.05). Figure 2. Changes in the central macular thickness (CMT) during the 12-month follow-up period. The mean CMTs at 4, 6, and 12 months decreased significantly compared with baseline (**P < 0.01). Figure 2. Changes in the central macular thickness (CMT) during the 12-month follow-up period. The mean CMTs at 4, 6, and 12 months decreased significantly compared with baseline (**P < 0.01). Resultsh This study included 17 consecutive eyes of 17 Japanese patients with treatment-naïve PCV. Of the 17 patients enrolled in this study, two men developed intraocular inflammation (IOI) and were excluded. Therefore, the 1-year follow-up was completed in 15 eyes of 15 patients (13 men, 2 women; mean age, 77.8 ± 2.7 years; range, 53–90 years).hi y The best-corrected visual acuity (BCVA) improved significantly from 0.28 ± 0.05 (range; -0.079 to 0.69) before treatment to 0.17 ± 0.05 after the loading phase (4 months after starting treatment; P < 0.05). The BCVA was maintained at 0.12 ± 0.04 (P < 0.05) at 6 months and 0.13 ± 0.06 (P < 0.05) at 1 year (Fig. 1).hi y g The central macular thickness (CMT) decreased significantly from 421 ± 53 µm before the initial treatment to 219 ± 26 µm at 4 months (P < 0.01) and was maintained at 230 ± 22 µm (P < 0.01) at 6 months and 206 ± 18 µm (P < 0.01) at 1 year (Fig. 2).hi y g The central choroidal thickness (CCT) decreased significantly from 226 ± 35 µm before the initial treatment to 198 ± 32 µm at 4 months (P < 0.01) and was maintained at 197 ± 31 µm (P < 0.05) at 6 months and 181 ± 30 µm (P < 0.01) at 1 year (Fig. 3). Scientific Reports | (2022) 12:7987 | https://doi.org/10.1038/s41598-022-12216-2 www.nature.com/scientificreports/ ntificreports/ Figure 3. Changes in the central choroidal thickness (CCT) during the 12-month follow-up period. The mean CCTs at 4, 6, and 12 months decreased significantly compared with baseline (*P < 0.05, **P < 0.01). ntificreports/ Figure 3. Changes in the central choroidal thickness (CCT) during the 12-month follow-up period. The mean CCTs at 4, 6, and 12 months decreased significantly compared with baseline (*P < 0.05, **P < 0.01). Figure 3. Changes in the central choroidal thickness (CCT) during the 12-month follow-up period. The mean CCTs at 4, 6, and 12 months decreased significantly compared with baseline (*P < 0.05, **P < 0.01). During the 1-year follow-up, the mean number of injections administered was 6.38 ± 0.14. After the loading phase, nine patients (60.0%) continued maintenance treatment every 3 months. The treatment interval for four patients (26.6%) was shortened from every 3 months to every 2 months due to the appearance of fluid or new hemorrhage. Resultsh Two patients (13.3%) continued maintenance treatment every 2 months due to the residual fluid after the loading phase.ht t g p The proportion of patients with complete regression of polypoidal lesions after 1 year seen on ICGA was 93.3% (14 eyes). A dry macula was achieved in 13 eyes (86.6%) after the loading phase and 11 eyes (73.3%) at 1 year. One eye of four eyes without dry macular at 1 year was the eye without regression of the polypoidal lesion. Figure 4 shows a representative case.t g p Brolucizumab-related IOI developed in two (11.8%) of 17 eyes. A 72-year-old man had a vitreous opacity after the third injection. His visual acuity reduced from 0.22 logMAR units at baseline to 0.30 logMAR units. 0.1% betamethasone eye drops were started four times a day. A vitreous opacity resolved after two months, therefore he stopped using eye drops. 10 months after IOI was diagnosed, the patient’s visual acuity improved to 0.22 log- MAR units, and the fundus examination showed no recurrence of vitreous haze. OCT showed no recurrence of exudation. Another man, 63-year-old man, had vascular sheathing without retinal vascular occlusion after the fourth injection. His visual acuity did not reduce from 0.15 logMAR units at baseline to -0.079 logMAR units. The patients visited the hospital three months after the fourth injection without any symptoms. Therefore, we could not treat the prompt treatment. Four months after vascular sheathing was diagnosed, the patient’s visual acuity was maintained. The fundus examination showed vascular sheathing in the peripheral retina remained, however there was no enlargement of the lesions or the appearance of vascular occlusion. OCT showed recur- rence of exudation. Thereafter, the patient received additional intravitreal aflibercept injections. In both cases, the BCVA after 12 months did not deteriorate compared to the baseline BCVA. No other severe adverse events, such as infectious endophthalmitis, rhegmatogenous retinal detachment, cerebral infarction, or myocardial infarction, occurred. Discussion Koizumi et al.19 also reported changes in subfoveal choroidal thickness after intravitreal aflibercept injections in 86 patients with PCV at 1 year and reported a mean CCT decrease from 270 to 234 µm (13.3% decrease from baseline). Therefore, the reduction of the choroidal thickness in patients with PCV treated with IVBr injections tended to be greater than that achieved with aflibercept. Kim et al.20 reported decreased choroidal thickness was observed after anti-VEGF therapy for PCV. Choi et al.21 studied 88 patients with PCV who received anti-VEGF injections and reported that faster chorioretinal atrophy (CRA) growth was related significantly to a decreased choroidal thickness. A larger decrease in choroidal thickness might be a risk factor for long-term development of CRA in patients with PCV.Choroidal thinning might affect the CRA in the longer term and CRA might be related to the BCVA. Therefore, it is necessary to consider whether IVBr injections affect the VA for a longer follow-up period. h y jf g p p In the current study, 60.0% of the patients with PCV completed the 12-week interval injections for 1 year. A dry macula was achieved in 73.3% after 1 year. Ogura et al.17 reported the efficacy of brolucizumab in Japa- nese patients with PCV in a sub-analysis of the HAWK study. The authors reported that the probability of 12-week-interval injections after a loading phase through 48 weeks was 76%, which was similar to our result. The 12-week-interval injections were the longest intervals in the TAE regimen, which is widely used worldwide. The persistence of brolucizumab might derive from its high affinity for VEGF. Its low molecular weight allows more delivery of drug per injection compared with other available anti-VEGFs and offers the potential for more effective tissue penetration and increased duration of ­action14. Therefore, brolucizumab might reduce the treat- ment burden for patients with PCV. p In the current study, complete regression of polypoidal lesions at 12 months was achieved in 93.3%. Matsu- moto et al.22 and Fukuda et al.23 reported the short-term outcomes of IVBr for PCV. In these two reports, the regression rates of the polypoidal lesions after 3monthly IVBr injections were 78.9% (15eyes of 19eyes) and 78.6% (11 eyes of 14 eyes). There were no previous studies which reports the regression rate of polypoidal lesions regard- ing the 1-year outcomes. Discussion Twelve months after the first injection, the VA improved to 0.045 logMAR. (F) A color fundus photograph shows no reddish-orange lesion or hemorrhage at the macula. (G) FA shows staining with no leakage. (H) ICGA shows complete polyp regression, although an abnormal vascular network (arrow heads) remained. (I) OCT shows no polypoidal lesion or SRF. Irregular retinal pigment epithelium elevation was observed where the abnormal vascular network was located. Figure 4. The case of an 86-year-old man who presented with reduced visual acuity in his right eye. (A) A color fundus photograph shows reddish-orange polypoidal lesions, including his fovea, submacular hemorrhage, and a large area of subretinal fluid (SRF). (B) Fluorescein angiography (FA) demonstrates occult leakage. (C) Indocyanine green angiography (ICGA) shows two polypoidal lesions (white arrows) and an abnormal vascular network (arrow heads). (D, E) An optical coherence tomography (OCT) images obtained at baseline show SRF with polypoidal lesions. (D through the superior polypoidal lesion, E through the inferior polypoidal lesion involving the fovea) The visual acuity (VA) was 0.39 logarithm of the minimum angle of resolution (logMAR) in the right eye, and the patient was diagnosed with PCV. The patient received IVBr injections during the loading phase and during the maintenance phase, he was treated every 3 months IVBr injections. The exudative changes did not recur for 1 year. Twelve months after the first injection, the VA improved to 0.045 logMAR. (F) A color fundus photograph shows no reddish-orange lesion or hemorrhage at the macula. (G) FA shows staining with no leakage. (H) ICGA shows complete polyp regression, although an abnormal vascular network (arrow heads) remained. (I) OCT shows no polypoidal lesion or SRF. Irregular retinal pigment epithelium elevation was observed where the abnormal vascular network was located. our study and previous results is difficult, our visual outcomes using brolucizumab seem favorable, with fewer injections compared with aflibercept in Japanese patients with PCV.i j pl p p p In the current study, the CCT decreased significantly from 226 to 181 µm (19.9% decrease from baseline) at 1 year. We previously reported the 1-year results in treatment-naïve PCV treated with bimonthly intravitreal aflibercept that had a mean CCT decrease from 265 to 231 µm (12.8% decrease from baseline)11. Discussion We investigated the 1-year outcomes of IVBr injections in Japanese patients with treatment-naïve PCV and found that the BCVA improved significantly during the follow-up period. The CMT and CCT also decreased significantly after 1 year. The polypoidal lesions regressed completely after 1 year in 93.3% of eyes. To the best of our knowledge, no previous studies have evaluated the regression rate of polypoidal lesions regarding the 1-year outcomes following IVBr injections for patients with PCV.fi g j p In a sub-analysis of the HAWK study, Ogura et al.17 reported the efficacy of brolucizumab in Japanese patients with PCV. They reported brolucizumab (n = 39) was as good as aflibercept (n = 30) in improving vision. Yama- moto et al.12 conducted a multicenter study and reported the 1-year results in 90 treatment-naïve eyes with PCV treated with bimonthly intravitreal aflibercept and reported a mean BCVA improvement from 0.31 to 0.17 logMAR. The mean number of injections during the first year was 7.1. Morimoto et al.18 reported the 2-year results in 58 treatment-naïve eyes with PCV with good initial BCVA treated with intravitreal aflibercept using a treat-and-extend (TAE) regimen and found a mean BCVA improvement from 0.27 to 0.12 logMAR during the first year. The mean number of injections during the first year was 7.72. In the current study, the mean BCVA improved from 0.28 to 0.13 logMAR. The number of injections was 6.4. Although a direct comparison between Scientific Reports | (2022) 12:7987 | https://doi.org/10.1038/s41598-022-12216-2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. The case of an 86-year-old man who presented with reduced visual acuity in his right eye. (A) A color fundus photograph shows reddish-orange polypoidal lesions, including his fovea, submacular hemorrhage, and a large area of subretinal fluid (SRF). (B) Fluorescein angiography (FA) demonstrates occult leakage. (C) Indocyanine green angiography (ICGA) shows two polypoidal lesions (white arrows) and an abnormal vascular network (arrow heads). (D, E) An optical coherence tomography (OCT) images obtained at baseline show SRF with polypoidal lesions. (D through the superior polypoidal lesion, E through the inferior polypoidal lesion involving the fovea) The visual acuity (VA) was 0.39 logarithm of the minimum angle of resolution (logMAR) in the right eye, and the patient was diagnosed with PCV. The patient received IVBr injections during the loading phase and during the maintenance phase, he was treated every 3 months IVBr injections. The exudative changes did not recur for 1 year. Scientific Reports | (2022) 12:7987 | Methods Received: 7 February 2022; Accepted: 6 May 2022 Received: 7 February 2022; Accepted: 6 May 2022 www.nature.com/scientificreports/ ranibizumab monotherapy. In another of our previous studies, the regression rates of polypoidal lesions in patients treated with combination therapy with either intravitreal ranibizumab or intravitreal aflibercept were higher (78.2% and 78.9%) than either monotherapy with those ­drugs24. In the current study, the regression rate of polypoidal lesions with IVBr injections was much higher than with either ranibizumab or aflibercept mono- therapy or PDT combination therapy, which might be why 73.3% of the patients had a dry macula at 1 year. However, brolucizumab-related IOI is a major adverse event associated with intravitreal brolucizumab. In the current study, two (11.8%) of 17 eyes had IOI. The frequency of IOI was roughly the same compared with a sub-analysis of HAWK study which Ogura et al. reported (15.4%)15–17. Although IVBr injections for PCV are effective, we should be cautious about IOI and treat promptly if brolucizumab-related IOI develops.hh f p p y p This study had some limitations, a major one being the retrospective design. The second limitation was the small sample size. A large-scale randomized study should confirm the current results. Furthermore, the long-term outcomes are unknown. The results of this study must be evaluated with a longer follow-up period.f h In conclusion, IVBr injections effectively improved the vision and exudative changes in patients with PCV over a 1-year follow-up, along with higher resolution rate of polypoidal lesions as seen on ICGA. The results of this analysis suggested that brolucizumab could be useful to treat PCV with a lower treatment burden. Discussion In our previous study, when intraocular aflibercept monotherapy was used to treat PCV, complete regression of polypoidal lesions was achieved in 48.0% with a bimonthly fixed regimen and in 52.9% with an as-needed ­regimen11. In the EVEREST II ­study8, which compared the effectiveness between ranibizumab monotherapy with photodynamic therapy (PDT) combination therapy, the rates at 12 months were 69.3% in the patients treated with PDT/intravitreal ranibizumab therapy and 34.7% in patients treated with intravitreal Scientific Reports | (2022) 12:7987 | https://doi.org/10.1038/s41598-022-12216-2 www.nature.com/scientificreports/ References e e e ces 1. Yannuzzi, L. A., Sorenson, J., Spaide, R. F. & Lipson, B. Idiopathic polypoidal choroidal vasculopathy (IPCV). Retina 10, 1–8. https://​doi.​org/​10.​1097/​00006​982-​19900​1010-​00001 (1990). 1. Yannuzzi, L. A., Sorenson, J., Spaide, R. F. & Lipson, B. Idiopathic polypoidal choroidal vasculopathy (IPCV). Retina 10, 1–8. https://​doi.​org/​10.​1097/​00006​982-​19900​1010-​00001 (1990).h p g ( ) 2. Yannuzzi, L. A. et al. The expanding clinical spectrum of idiopathic polypoidal choroidal vasculopathy. Arch Ophthalmol. 115 478. https://​doi.​org/​10.​1001/​archo​pht.​1997.​01100​15048​0005 (1997). 3. Spaide, R. F. et al. Consensus nomenclature for reporting neovascular age-related macular degeneration data: consensus on neo- vascular age-related macular degeneration nomenclature study group. Ophthalmology 127, 616–636. https://​doi.​org/​10.​1016/j.​ ophtha.​2019.​11.​004 (2020). p 4. Maruko, I. et al. Clinical characteristics of exudative age-related macular degeneration in Japanese patients. Am. J. Ophthalmol 144, 15–22. https://​doi.​org/​10.​1016/j.​ajo.​2007.​03.​047 (2007). p g j j ( ) 5. Uyama, M. et al. Polypoidal choroidal vasculopathy: Natural history. Am. J. Ophthalmol. 133, 639–648. https://​doi.​org/​10.​1016/​ S0002-​9394(02)​01404-6 (2002). 6. Koh, A. et al. Polypoidal choroidal vasculopathy: Evidence-based guidelines for clinical diagnosis and treatment. Retina 33 686–716. https://​doi.​org/​10.​1097/​IAE.​0b013​e3182​852446 (2013). 7. Hikichi, T. et al. One-year results of three monthly ranibizumab injections and as-needed reinjections for polypoidal choroidal vasculopathy in Japanese patients. Am. J. Ophthalmol. 154, 117–124. https://​doi.​org/​10.​1016/j.​ajo.​2011.​12.​019 (2012).fii p y p p p p g j j 8. Koh, A. et al. Efficacy and safety of ranibizumab with or without verteporfin photodynamic therapy for polypoidal choroida vasculopathy: A randomized clinical trial. JAMA Ophthalmol. 135, 1206–1213. https://​doi.​org/​10.​1001/​jamao​phtha​lmol.​2017 4030 (2017). 8. Koh, A. et al. Efficacy and safety of ranibizumab with or without verteporfin photodynamic therapy for polypoidal choroidal vasculopathy: A randomized clinical trial. JAMA Ophthalmol. 135, 1206–1213. https://​doi.​org/​10.​1001/​jamao​phtha​lmol.​2017.​ 4030 (2017). ( ) 9. Mori, R., Yuzawa, M., Akaza, E. & Haruyama, M. Treatment results at 1 year of ranibizumab therapy for polypoidal choroidal vasculopathy in eyes with good visual acuity. Jpn. J. Ophthalmol. 57, 365–371. https://​doi.​org/​10.​1007/​s10384-​013-​0245-9 (2013).fil ( ) 9. Mori, R., Yuzawa, M., Akaza, E. & Haruyama, M. Treatment results at 1 year of ranibizumab therapy for polypoidal choroida vasculopathy in eyes with good visual acuity. Jpn. J. Ophthalmol. 57, 365–371. https://​doi.​org/​10.​1007/​s10384-​013-​0245-9 (2013) ( ) 9. Mori, R., Yuzawa, M., Akaza, E. & Haruyama, M. Treatment results at 1 year of ranibizumab therapy for polypoidal choroidal vasculopathy in eyes with good visual acuity. Jpn. J. Ophthalmol. 57, 365–371. https://​doi.​org/​10.​1007/​s10384-​013-​0245-9 (2013). 10. Lee, W. K. et al. Received: 7 February 2022; Accepted: 6 May 2022 Methods We retrospectively studied 17 consecutive eyes of 17 Japanese patients aged 50 years or older with previously untreated PCV. All patients received initial treatment using IVBr injections at Yokohama City University Medical Center between June 2020 and February 2021. The institutional review board of the Yokohama City University Medical Center approved this study, which followed the tenets of the Declaration of Helsinki. All patients pro- vided written informed consent before their medical record data were used in research. All patients received comprehensive ophthalmic examinations, including BCVA, slit-lamp biomicroscopy, optical coherence tomography (OCT) (Spectralis Product Family Version 5.3; Heidelberg Engineering, Heidel- berg, Germany), fluorescein angiography (FA) and ICGA (Spectralis Product Family Version 5.3; Heidelberg Engineering, Heidelberg, Germany). The diagnosis of PCV was based on previous ­reports4,6. Inclusion criteria included newly diagnosed PCV with the presence of polypoidal choroidal vascular lesion with branching vascular networks on ICGA. Exclusion criteria were previous history of AMD, history of laser photocoagulation, PDT, intravitreal injection of other anti-VEGF agents or intravitreal steroids. Patients with history of eye diseases such as uveitis, retinal vein occlusion, rhegmatogenous retinal detachment, diabetic retinopathy were also excluded. g g p y During the loading phase, the patients received three monthly IVBr injections. In the maintenance phase, patients received an IVBr injection every 12 weeks unless any fluid or a new hemorrhage was identified, in which case the interval between IVBr injections was shortened to every 8 weeks. The endpoints were the BCVA, CMT, CCT, number of intravitreal injections, regression of polypoidal lesions evaluated by ICGA after 1 year, and complications. These endpoints were evaluated retrospectively 1 year after the initial treatment. CMT and CCT were determined by using a caliper function of B-scan OCT. The CMT was defined as the thickness from the internal limiting membrane to the retinal pigment epithelium at the center of the fovea; the CCT was defined as the thickness between Bruch’s membrane and the inner surface of the choroidal-scleral junction at the fovea. The statistical analysis was performed with Ekuseru-Toukei (Social Survey Research Information, Tokyo, Japan). The BCVA measured on a Landolt chart was converted into the logarithm of the minimum angle resolution (logMAR) for statistical analyses. Wilcoxon signed-rank test was used to compare the changes of BCVA, CMT, and CCT between baseline and other time point. A value of P < 0.05 was considered statistically significant. p g The authors declare no competing interests. p g The authors declare no competing interests. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 2. Yamamoto, A. et al. One-year results of intravitreal aflibercept for polypoidal choroidal vasculopathy. Ophthalmology 122, 1866– 1872. https://​doi.​org/​10.​1016/j.​ophtha.​2015.​05.​024 (2015).l p g j p ( ) 13. Maruyama-Inoue, M., Sato, S., Yamane, S. & Kadonosono, K. Intravitreal injection of aflibercept in patients with polyp choroidal vasculopathy a 3-year follow-up. Retina 38, 2001–2009. https://​doi.​org/​10.​1097/​IAE.​00000​00000​001818 (2018). p y y p , p // g/ / ( ) 4. Nguyen, Q. D. et al. Brolucizumab: Evolution through preclinical and clinical studies and the implications for the management p y y p , p g 14. Nguyen, Q. D. et al. Brolucizumab: Evolution through preclinical and clinical studies and the implications for the m l l t d l d ti Ophth l l 127 963 976 htt //d i /10 1016/j hth 2019 1 14. Nguyen, Q. D. et al. Brolucizumab: Evolution through preclinical and clinical studies and the implications for the management of neovascular age-related macular degeneration. Ophthalmology 127, 963–976. https://doi.org/10.1016/j.ophtha.2019.12.031 (2020). 14. Nguyen, Q. D. et al. Brolucizumab: Evolution through preclinical and clinical studies and the implications for the management of l l t d l d ti Ophth l l 127 963 976 htt //d i /10 1016/j hth 2019 12 031 (2020) 14. Nguyen, Q. D. et al. Brolucizumab: Evolution through preclinical and clinical studies and the implications for the m neovascular age-related macular degeneration. Ophthalmology 127, 963–976. https://​doi.​org/​10.​1016/j.​ophtha.​2019.​1 g y g p p g neovascular age-related macular degeneration. Ophthalmology 127, 963–976. https://​doi.​org/​10.​1016/j.​ophtha.​2019.​12.​031 (2 15. Dugel, P. U. et al. HAWK and HARRIER: Phase 3, multicenter, randomized, double-masked trials of brolucizum age-related macular degeneration. Ophthalmology 127, 72–84. https://​doi.​org/​10.​1016/j.​ophtha.​2019.​04.​017 (2 U. et al. HAWK and HARRIER: Phase 3, multicenter, randomized, double masked trials of brolucizumab for neovascular d macular degeneration. Ophthalmology 127, 72–84. https://​doi.​org/​10.​1016/j.​ophtha.​2019.​04.​017 (2020). g age-related macular degeneration. Ophthalmology 127, 72–84. https://​doi.​org/​10.​1016/j.​ophtha.​2019.​04.​017 (2020). g g p gy p g j p 16. Dugel, P. U. et al. HAWK and HARRIER: Ninety-six-week outcomes from the phase 3 trials of brolucizumab for neovascula related macular degeneration. Ophthalmology 128, 89–99. https://doi.org/10.1016/j.ophtha.2020.06.028 (2021). g p gy , p g j p ( ) 17. Ogura, Y. et al. Efficacy and safety of brolucizumab versus aflibercept in eyes with polypoidal choroidal vasculopathy in participants of HAWK. Br. J. Ophthalmol. https://​doi.​org/​10.​1136/​bjoph​thalm​ol-​2021-​319090 (2021). k T l f d d h fl b p p p p g j p 18. Author contributionsh The authors were involved in the following aspects of the study: design and conduct (M.M-I.); collection of the data (A.I., Y.K., S.I.); management (M.M-I., A.I.); analysis (A.I., T.I.); preparation of article (A.I.); review and approval of the manuscript (M.M-I., K.K.). References Efficacy and safety of intravitreal aflibercept for polypoidal choroidal vasculopathy in the PLANET study a rand- p y y g y Jp J p , p g ( ) 0. Lee, W. K. et al. Efficacy and safety of intravitreal aflibercept for polypoidal choroidal vasculopathy in the PLANET study a rand- omized clinical trial. JAMA Ophthalmol. 136, 786–793. https://​doi.​org/​10.​1001/​jamao​phtha​lmol.​2018.​1804 (2018).l J p , p // g/ /j p ( ) 11. Inoue, M. et al. Aflibercept for polypoidal choroidal vasculopathy. Retina 36, 1527–1534. https://​doi.​org/​10.​1097/​IAE.​00000​00000​ 000933 (2016). 11. Inoue, M. et al. Aflibercept for polypoidal choroidal vasculopathy. Retina 36, 1527–1534. https://​doi.​org/​10.​1097/​IAE.​00000​00000​ 000933 (2016). https://doi.org/10.1038/s41598-022-12216-2 Scientific Reports | (2022) 12:7987 | www.nature.com/scientificreports/ Morimoto, M., Matsumoto, H., Mimura, K. & Akiyama, H. Two-year results of a treat-and-extend regimen with aflibercept for polypoidal choroidal vasculopathy. Graefe’s Arch. Clin. Exp. Ophthalmol. 255, 1891–1897. https://​doi.​org/​10.​1007/​s00417-​017-​ 3718-6 (2017).l ( ) 19. Koizumi, H. et al. Subfoveal choroidal thickness during aflibercept therapy for neovascular age-related macular degenera twelve-month results. Ophthalmology 123, 617–624. https://​doi.​org/​10.​1016/j.​ophtha.​2015.​10.​039 (2016).l p gy p g j p 20. Kim, J. H. et al. Short-term choroidal thickness changes in patients treated with either ranibizumab or aflibercept: A comparative study. Br. J. Ophthalmol. 100, 1634–1639. https://​doi.​org/​10.​1136/​bjoph​thalm​ol-​2015-​308074 (2016). 20. Kim, J. H. et al. Short-term choroidal thickness changes in patients treated with either ranibizumab or aflibercept: A comparative study. Br. J. Ophthalmol. 100, 1634–1639. https://​doi.​org/​10.​1136/​bjoph​thalm​ol-​2015-​308074 (2016). y J p , p g j p ( ) 21. Choi, E. Y. et al. Long-term incidence and growth of chorioretinal atrophy in patients with polypoidal choroidal vasculopathy. Ophthalmologica 243, 136–144. https://​doi.​org/​10.​1159/​00050​1724 (2020). 21. Choi, E. Y. et al. Long-term incidence and growth of chorioretinal atrophy in patients with polypoidal choroidal vasculop Ophthalmologica 243, 136–144. https://​doi.​org/​10.​1159/​00050​1724 (2020). 22. Matsumoto, H. et al. Short-term outcomes of intravitreal brolucizumab for treatment-naïve neovascular age-related macular degeneration with type 1 choroidal neovascularization including polypoidal choroidal vasculopathy. Sci. Rep. 11, 1–8. https://​doi.​ org/​10.​1038/​s41598-​021-​86014-7 (2021).l 22. Matsumoto, H. et al. Short-term outcomes of intravitreal brolucizumab for treatment-naïve neovascular age-related macular degeneration with type 1 choroidal neovascularization including polypoidal choroidal vasculopathy. Sci. Rep. 11, 1–8. https://​doi.​ org/​10.​1038/​s41598-​021-​86014-7 (2021).l g ( ) 23. Fukuda, Y. et al. Comparison of outcomes between 3 monthly brolucizumab and aflibercept injections for polypoidal chor vasculopathy. Biomedicines 9, 1164. https://​doi.​org/​10.​3390/​biome​dicin​es909​1164 (2021).l p y p g 24. Ito, A. et al. Comparison of one-year results of photodynamic therapy combined with ranibizumab or aflibercept for treating polypoidal choroidal vasculopathy. PLoS ONE 15, 1–11. https://​doi.​org/​10.​1371/​journ​al.​pone.​02352​13 (2020). 24. Ito, A. et al. Comparison of one-year results of photodynamic therapy combined with ranibizumab or aflibercept for treating polypoidal choroidal vasculopathy. PLoS ONE 15, 1–11. https://​doi.​org/​10.​1371/​journ​al.​pone.​02352​13 (2020). 4. Ito, A. et al. Comparison of one-year results of photodynamic therapy combined with ranibizumab or aflibercept for treating polypoidal choroidal vasculopathy. PLoS ONE 15, 1–11. https://​doi.​org/​10.​1371/​journ​al.​pone.​02352​13 (2020). Additional information Correspondence and requests for materials should be addressed to M.M.-I. Correspondence and requests for materials should be addressed to M.M.-I. © The Author(s) 2022 Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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A consistent discrete version of a non-autonomous SIRVS model
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1. Introduction to some particular properties of discrete epidemiological models obtained from continuous models by some NSFD scheme [16]. We note that while the papers cited above consider autonomous models, in the present work we discuss dynamical consistency for a nonautonomous model. To the best of our knowledge, this is the first work where the consistency of a discretized epidemiological model with the original continuous model is discussed in the nonau- tonomous context. Most of the epidemiological models in the literature are continuous models. In spite of this, recently, there has been a growing interest in discrete-time models [1–7]. In this work, we will use Mickens nonstandard difference (NSFD) scheme to achieve a discretization of a family of continuous epi- demiological models with vaccination and general incidence function considered in [8]. We have multiple objectives: firstly, we want to obtain conditions for extinction and per- manence of the disease for the discrete family; next, having a continuous and a corresponding discrete family of models, we wish to discuss the problem of consistency of the discrete models with the corresponding continuous ones; finally, we intend to present some simulation results.h We remark that, even in the very particular case of autonomous models with mass action incidence and assum- ing that there is no disease related death, it follows from results in [17] that, when R0 > 1, the continuous autonomous model has one or two endemic equilibrium points coexisting with the disease-free equilibrium. The existence and stability of the equilibriums are governed by R0 and three additional thresholds: R𝑞, RV, and R𝑞V. These thresholds determine the qualitative behaviour of the system. This very particular situation shows that, even in the autonomous context, the qualitative behaviour can be difficult to determine. The dynamical consistency of a numerical scheme with the associated continuous system is not a precise definition. By the expression “dynamical consistency,” it is meant that the numerical solutions replicate some of the properties of the continuous systems solutions. y We will consider the dynamical consistency regarding the permanence and extinction of the disease: whenever there is extinction (permanence) of the disease for the continuous- time model, the same holds for the discrete-time one. Several papers [9–15] discuss the dynamical consistency with respect One of the motivations for our work was that the difficulties increase considerably when we deal with a general nonautonomous situation. Hindawi Discrete Dynamics in Nature and Society Volume 2018, Article ID 8152032, 18 pages https://doi.org/10.1155/2018/8152032 Joaquim Mateus,1 César M. Silva ,2 and S. Vaz2 Joaquim Mateus, César M. Silva , and S. Vaz 1Unidade de Investigac¸˜ao para o Desenvolvimento do Interior (UDI), Instituto Polit´ecnico da Guarda, 6300-559 Guarda, Portugal 2Centro de Matem´atica e Aplicac¸˜oes e Departamento de Matem´atica, Universidade da Beira Interior, 6201-001 Covilh˜a, Portugal 1Unidade de Investigac¸˜ao para o Desenvolvimento do Interior (UDI), Instituto Polit´ecnico da Guarda, 6300-559 Guarda, Portugal 2Centro de Matem´atica e Aplicac¸˜oes e Departamento de Matem´atica, Universidade da Beira Interior, 6201-001 Covilh˜a, Portugal Correspondence should be addressed to C´esar M. Silva; csilva@ubi.pt Received 24 February 2018; Revised 27 May 2018; Accepted 29 May 2018; Published 24 June 2018 Academic Editor: Pilar R. Gordoa Copyright © 2018 Joaquim Mateus et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A family of discrete nonautonomous SIRVS models with general incidence is obtained from a continuous family of models by applying Mickens nonstandard discretization method. Conditions for the permanence and extinction of the disease and the stability of disease-free solutions are determined. Concerning extinction and persistence, the consistency of those discrete models with the corresponding continuous model is discussed: if the time step is sufficiently small, when we have extinction (permanence) for the continuous model, we also have extinction (permanence) for the corresponding discrete model. Some numerical simulations are carried out to compare the different possible discretizations of our continuous model using real data. 1. Introduction Thus, we decided to discuss an aspect of consistency that, nevertheless, is very important Discrete Dynamics in Nature and Society 2 infective class) from the susceptible individuals; the function 𝜎𝑛𝜓is the incidence (into the infective class) from the vac- cinated individuals; 𝜇𝑛are the natural deaths; 𝑝𝑛represents the susceptibles vaccination; 𝜂𝑛represents the immunity loss and consequence influx in the susceptible class; 𝛼𝑛are the deaths occurring in the infective class; 𝛾𝑛is the recovery. We will assume that (Λ 𝑛), (𝜇𝑛), (𝑝𝑛), (𝜂𝑛), (𝛼𝑛), (𝛽𝑛), (𝜎𝑛), and (𝛾𝑛) are bounded and nonnegative sequences and that there are positive constants 𝑤𝜇, 𝑤Λ, 𝑤𝑝, 𝑘𝜑, and 𝑘𝜓such that from the point of view of biomathematics: can we obtain consistency between continuous and discrete-time models from the point of view of persistence and extinction of the disease? Regarding our simulation results, we considered two different types of computational experiments. Our first set of simulation results are designed to compare different possible discretizations of our continuous models. After this discus- sion, we apply our model to a real situation, considering data from the incidence of measles in France in the period 2012- 2016. To the possible extent, this data is used to estimate our model parameters and the computational results obtained are compared with real data.h (H1) the functions 𝜑: R2 →R and 𝜓: R2 →R are nonnegative and differentiable in (R+ 0)2 and the func- tions R+ 0 ∋𝑥→𝜕2𝜑(𝑥, 0) and R+ 0 ∋𝑥→𝜕2𝜓(𝑥, 0) are nondecreasing and Lipschitz, with Lipschitz con- stants 𝑘𝜑and 𝑘𝜓, (H1) the functions 𝜑: R2 →R and 𝜓: R2 →R are nonnegative and differentiable in (R+ 0)2 and the func- tions R+ 0 ∋𝑥→𝜕2𝜑(𝑥, 0) and R+ 0 ∋𝑥→𝜕2𝜓(𝑥, 0) are nondecreasing and Lipschitz, with Lipschitz con- stants 𝑘𝜑and 𝑘𝜓, The law of mass action states that the rate of change in the disease incidence is directly proportional to the product of the number of susceptible and infective individuals and was the paradigm in the classic models in epidemiology. This is why classical models usually consider a bilinear incidence rate 𝛽𝑆𝐼, where 𝑆and 𝐼denote, respectively, the number of susceptible and infective individuals, to model the disease transmission. In spite of this, it is sometimes important to consider other forms of incidence functions. 1. Introduction Another usual assumption is the time independence of the parameters model parameters: in fact, the majority of the epidemiological models in the literature are given by a system of autonomous differential or difference equations. Neverthe- less, the assumption that the parameters are independent of time is not very realistic in many situations and it is useful to consider nonautonomous models that, for instance, allow the discussion of environmental and demographic effects that change with time [18, 19]. In this work the family of models considered is nonautonomous and the incidence rates are taken from a large class of functions. (H2) we have 𝜑(𝑥, 0) = 𝜓(𝑥, 0) = 𝜑(0, 𝑦) = 𝜓(0, 𝑦) = 0 for all 𝑥, 𝑦∈R+ 0, (H2) we have 𝜑(𝑥, 0) = 𝜓(𝑥, 0) = 𝜑(0, 𝑦) = 𝜓(0, 𝑦) = 0 for all 𝑥, 𝑦∈R+ 0, (H2) we have 𝜑(𝑥, 0) = 𝜓(𝑥, 0) = 𝜑(0, 𝑦) = 𝜓(0, 𝑦) = 0 for all 𝑥, 𝑦∈R+ 0, (H3) lim sup𝑛→+∞∏ 𝑛+𝜔𝜇 𝑘=𝑛(1/(1 + 𝜇𝑘)) < 1, (H3) lim sup𝑛→+∞∏ 𝑛+𝜔𝜇 𝑘=𝑛(1/(1 + 𝜇𝑘)) < 1, (H4) lim inf𝑛→+∞∑𝑛+𝜔Λ 𝑘=𝑛+1Λ 𝑘>0andlim inf𝑛→+∞∑ 𝑛+𝜔𝑝 𝑘=𝑛+1𝑝𝑘> 0, (H4) lim inf𝑛→+∞∑𝑛+𝜔Λ 𝑘=𝑛+1Λ 𝑘>0andlim inf𝑛→+∞∑ 𝑛+𝜔𝑝 𝑘=𝑛+1𝑝𝑘> 0, In this work, we prove, when our conditions prescribe extinction (permanence) for the continuous model we also have extinction (permanence) for the corresponding discrete model as long as the time step is smaller than some constant (that depends on some model parameters and on the thresh- old condition). We also consider a family of examples of the periodic system of period 1 such that the continuous and the discrete-time system with time step ℎ= 1/𝐿is not consistent, highlighting the importance of knowing that for time steps smaller than some explicit value we have consistency. Our model generalizes one obtained by Mickens nonstan- dard finite difference method from the continuous model [8] (see Section 2). In [20], a discrete nonautonomous epidemic model with vaccination and mass action incidence was obtained by Mickens method. We emphasize that, in the particular mass action case, our model is not exactly similar to the model in [20], although Mickens rules were considered in both. We briefly compare computationally these two slightly different models in Section 5.h Now, we need to make some definitions. We say that (i) the infectives (𝐼𝑛) are permanent if for any solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with initial conditions 𝑆0, 𝐼0, 𝑅0, 𝑉0 > 0 there are constants 0 < 𝑚< 𝑀such that (i) the infectives (𝐼𝑛) are permanent if for any solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with initial conditions 𝑆0, 𝐼0, 𝑅0, 𝑉0 > 0 there are constants 0 < 𝑚< 𝑀such that (i) the infectives (𝐼𝑛) are permanent if for any solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with initial conditions 𝑆0, 𝐼0, 𝑅0, 𝑉0 > 0 there are constants 0 < 𝑚< 𝑀such that Theorem 2 (Theorem 2 of [8]). Assume that conditions (C1), (C2), and (C3) hold. Then if there is a constant 𝜆> 0 such that 𝑅𝑢 𝐶(𝜆) < 0, then the infectives 𝐼go to extinction in system (2). 𝑚< lim inf 𝑛→∞𝐼𝑛⩽lim sup 𝑛→∞ 𝐼𝑛< 𝑀; (10) (10) (ii) the infectives (𝐼𝑛) go to extinction if for any solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with initial conditions 𝑆0, 𝐼0, 𝑅0, 𝑉0 ⩾0 we have lim𝑛→∞𝐼𝑛= 0. (ii) the infectives (𝐼𝑛) go to extinction if for any solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with initial conditions 𝑆0, 𝐼0, 𝑅0, 𝑉0 ⩾0 we have lim𝑛→∞𝐼𝑛= 0. In the literature, several models were discretized using Mickens NSFD schemes [22–35]. Next, we will apply Micken’s nonstandard method to obtain a discrete version of system (2). Let 𝜙: R+ 0 →R be a positive continuous function such that In the literature, several models were discretized using Mickens NSFD schemes [22–35]. Next, we will apply Micken’s nonstandard method to obtain a discrete version of system (2). Similar definitions can be made for the other compartments. For instance, if there exist constants 0 < 𝑚< 𝑀such that for any solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with initial conditions 𝑆0, 𝐼0, 𝑅0, 𝑉0 > 0 we have Let 𝜙: R+ 0 →R be a positive continuous function such that lim ℎ→0 𝜙(ℎ) = 0. (5) (5) 𝑚< lim inf 𝑛→∞𝑆𝑛⩽lim sup 𝑛→∞ 𝑆𝑛< 𝑀 (11) (11) Given ℎ∈R+, we let 𝑡= 𝑛ℎ, with 𝑛∈N, and identify 𝑆󸀠(𝑡) with we say that the susceptibles are permanent. 𝑆(𝑛ℎ+ ℎ) −𝑆(𝑛ℎ) 𝜙(ℎ) . (6) (6) 2. Discretization of the Continuous Model We start with a nonautonomous SIRVS model that is slightly less general than the one considered in [8] and generalizes the one in [21]. Namely, we consider the model: 𝑆󸀠= Λ (𝑡) −𝛽(𝑡) 𝜑(𝑆) 𝐼−(𝜇(𝑡) + 𝑝(𝑡)) 𝑆+ 𝜂(𝑡) 𝑉 𝐼󸀠= [𝛽(𝑡) 𝜑(𝑆) + 𝜎(𝑡) 𝜓(𝑉) −𝜇(𝑡) −𝛼(𝑡) −𝛾(𝑡)] 𝐼 𝑅󸀠= 𝛾(𝑡) 𝐼−𝜇(𝑡) 𝑅 (2) g yf The model we will consider is the following: 𝑆𝑛+1 −𝑆𝑛= Λ 𝑛−𝛽𝑛𝜑(𝑆𝑛+1, 𝐼𝑛) −(𝜇𝑛+ 𝑝𝑛) 𝑆𝑛+1 + 𝜂𝑛𝑉𝑛+1 (2) 𝐼𝑛+1 −𝐼𝑛= 𝛽𝑛𝜑(𝑆𝑛+1, 𝐼𝑛) + 𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) −(𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛) 𝐼𝑛+1 𝑉󸀠= 𝑝(𝑡) 𝑆−(𝜇(𝑡) + 𝜂(𝑡)) 𝑉−𝜎(𝑡) 𝜓(𝑉) 𝐼. (1) We assume that the functions Λ, 𝜇, 𝑝, 𝜂, 𝛼, 𝛽, 𝜎, and 𝛾 belong to the class 𝐶1(R+ 0) and are nonnegative and bounded. We also require that 𝑅𝑛+1 −𝑅𝑛= 𝛾𝑛𝐼𝑛+1 −𝜇𝑛𝑅𝑛+1 𝑉𝑛+1 −𝑉𝑛= 𝑝𝑛𝑆𝑛+1 −(𝜇𝑛+ 𝜂𝑛) 𝑉𝑛+1 −𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) , 𝑉𝑛+1 −𝑉𝑛= 𝑝𝑛𝑆𝑛+1 −(𝜇𝑛+ 𝜂𝑛) 𝑉𝑛+1 −𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) , (C1) the functions 𝜑: R →R and 𝜓: R →R are non- negative, nondecreasing, differentiable, and Lipschitz with Lipschitz constants 𝑘𝜑and 𝑘𝜓, respectively; 𝑛∈N, where the classes 𝑆, 𝐼, 𝑅, and 𝑉correspond, respec- tively, to susceptible, infective, recovered, and vaccinated individuals and the parameter functions have the following meanings: Λ 𝑛denotes the inflow of newborns in the sus- ceptible class; the function 𝛽𝑛𝜑is the incidence (into the 𝑛∈N, where the classes 𝑆, 𝐼, 𝑅, and 𝑉correspond, respec- tively, to susceptible, infective, recovered, and vaccinated individuals and the parameter functions have the following meanings: Λ 𝑛denotes the inflow of newborns in the sus- ceptible class; the function 𝛽𝑛𝜑is the incidence (into the (C2) 𝜑(0) = 𝜓(0) = 0; (C3) there is 𝜔> 0 such that lim inf𝑡→+∞∫ 𝑡+𝜔 𝑡 𝜇(𝑠)𝑑𝑠> 0. Discrete Dynamics in Nature and Society 3 Writing 𝑆𝑛= 𝑆(𝑛ℎ), 𝐼𝑛= 𝐼(𝑛ℎ), 𝑉𝑛= 𝑉(𝑛ℎ), Λ 𝑛= 𝜙(ℎ)Λ(𝑛ℎ), 𝛽𝑛= 𝜙(ℎ)𝛽(𝑛ℎ), 𝜇𝑛= 𝜙(ℎ)𝜇(𝑛ℎ), 𝑝𝑛= 𝜙(ℎ)𝑝(𝑛ℎ), and 𝜂𝑛= 𝜙(ℎ)𝜂(𝑛ℎ), we have Writing 𝑆𝑛= 𝑆(𝑛ℎ), 𝐼𝑛= 𝐼(𝑛ℎ), 𝑉𝑛= 𝑉(𝑛ℎ), Λ 𝑛= 𝜙(ℎ)Λ(𝑛ℎ), 𝛽𝑛= 𝜙(ℎ)𝛽(𝑛ℎ), 𝜇𝑛= 𝜙(ℎ)𝜇(𝑛ℎ), 𝑝𝑛= 𝜙(ℎ)𝑝(𝑛ℎ), and 𝜂𝑛= 𝜙(ℎ)𝜂(𝑛ℎ), we have In order to obtain threshold conditions for model (2), it was considered in [8] the following auxiliary system: 𝑥󸀠= Λ (𝑡) −[𝜇(𝑡) + 𝑝(𝑡)] 𝑥+ 𝜂(𝑡) 𝑦 𝑦󸀠= 𝑝(𝑡) 𝑥−[𝜇(𝑡) + 𝜂(𝑡)] 𝑦. (3) 𝑆𝑛+1 −𝑆𝑛= Λ 𝑛−𝛽𝑛𝜑(𝑆𝑛+1) 𝐼𝑛−(𝜇𝑛+ 𝑝𝑛) 𝑆𝑛+1 + 𝜂𝑛𝑉𝑛+1. 2. Discretization of the Continuous Model (8) (3) (8) And for each solution (𝑥∗(𝑡), 𝑦∗(𝑡)) of (3) with positive initial conditions, it was shown that the numbers Proceeding similarly for the other equations, we obtain the following discrete model: Proceeding similarly for the other equations, we obtain the following discrete model: 𝑅ℓ 𝐶(𝜆) = lim inf 𝑡→∞∫ 𝑡+𝜆 𝑡 𝛽(𝑠) 𝜑(𝑥∗(𝑠)) + 𝜎(𝑠) 𝜓(𝑦∗(𝑠)) 𝑆𝑛+1 −𝑆𝑛= Λ 𝑛−𝛽𝑛𝜑(𝑆𝑛+1) 𝐼𝑛−(𝜇𝑛+ 𝑝𝑛) 𝑆𝑛+1 + 𝜂𝑛𝑉𝑛+1 𝐼𝑛+1 −𝐼𝑛= 𝛽𝑛𝜑(𝑆𝑛+1) 𝐼𝑛+ 𝜎𝑛𝜓(𝑉𝑛+1) 𝐼𝑛 −(𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛) 𝐼𝑛+1 𝑅𝑛+1 −𝑅𝑛= 𝛾𝑛𝐼𝑛+1 −𝜇𝑛𝑅𝑛+1 𝑉𝑛+1 −𝑉𝑛= 𝑝𝑛𝑆𝑛+1 −(𝜇𝑛+ 𝜂𝑛) 𝑉𝑛+1 −𝜎𝑛𝜓(𝑉𝑛+1) 𝐼𝑛, (9) 𝑆𝑛+1 −𝑆𝑛= Λ 𝑛−𝛽𝑛𝜑(𝑆𝑛+1) 𝐼𝑛−(𝜇𝑛+ 𝑝𝑛) 𝑆𝑛+1 𝑆𝑛+1 −𝑆𝑛= Λ 𝑛−𝛽𝑛𝜑(𝑆𝑛+1) 𝐼𝑛−(𝜇𝑛+ 𝑝𝑛) 𝑆𝑛+1 + 𝜂𝑛𝑉𝑛+1 −𝜇(𝑠) −𝛼(𝑠) −𝛾(𝑠) 𝑑𝑠, 𝑅𝑢 𝐶(𝜆) = lim sup 𝑡→∞ ∫ 𝑡+𝜆 𝑡 𝛽(𝑠) 𝜑(𝑥∗(𝑠)) + 𝜎(𝑠) 𝜓(𝑦∗(𝑠)) −𝜇(𝑠) −𝛼(𝑠) −𝛾(𝑠) 𝑑𝑠 (4) −𝜇(𝑠) −𝛼(𝑠) −𝛾(𝑠) 𝑑𝑠, (4) 𝐼𝑛+1 −𝐼𝑛= 𝛽𝑛𝜑(𝑆𝑛+1) 𝐼𝑛+ 𝜎𝑛𝜓(𝑉𝑛+1) 𝐼𝑛 −(𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛) 𝐼𝑛+1 𝑅𝑛+1 −𝑅𝑛= 𝛾𝑛𝐼𝑛+1 −𝜇𝑛𝑅𝑛+1 𝑉𝑛+1 −𝑉𝑛= 𝑝𝑛𝑆𝑛+1 −(𝜇𝑛+ 𝜂𝑛) 𝑉𝑛+1 −𝜎𝑛𝜓(𝑉𝑛+1) 𝐼𝑛, (9) 𝑅𝑢 𝐶(𝜆) = lim sup 𝑡→∞ ∫ 𝑡+𝜆 𝑡 𝛽(𝑠) 𝜑(𝑥∗(𝑠)) + 𝜎(𝑠) 𝜓(𝑦∗(𝑠)) (9) 𝑅𝑛+1 −𝑅𝑛= 𝛾𝑛𝐼𝑛+1 −𝜇𝑛𝑅𝑛+1 are independent of the particular solution. are independent of the particular solution. Using the above numbers, the following results are con- tained in results obtained in [8]. 𝑛∈N0. We will consider a model that contains this one to obtain some of our results. Namely, based on model (9), in Sections 3 and 4 we will study model (1) that has a more general form for the incidence function.i Theorem 1 (Theorem 1 of [8]). Assume that conditions (C1), (C2), and (C3) hold. Then, if there is a constant 𝜆> 0 such that 𝑅ℓ 𝐶(𝜆) > 0, then the infectives 𝐼are permanent in system (2). Now, we need to make some definitions. We say that 3.1. Auxiliary Results. Consider the auxiliary system, 3.1. Auxiliary Results. Consider the auxiliary system, 3. Permanence and Extinction in the Discrete Model After deciding a nonlocal representation for the incidence function and that terms that do not correspond to an interaction will be considered in the 𝑛+ 1 time, the first equation in (2) becomes In this section, we will extend the results obtained for the model with the usual mass action incidence in [20] to our generalized family of models. Namely, suitable thresholds are defined and conditions for persistence and extinction of the disease are obtained. As a corollary of our results, we consider the periodic case where we have a unique number that establishes the boundary between the regions of permanence and extinction. Although the proofs of our results are inspired in [20], some difficulties must be dealt with. In particular, it was necessary to understand the right conditions to impose 𝑆((𝑛+ 1) ℎ) −𝑆(𝑛ℎ) = 𝜙(ℎ) [Λ (𝑛ℎ) −𝛽(𝑛ℎ) 𝜑(𝑆((𝑛+ 1) ℎ)) 𝐼(𝑛ℎ) 𝑆((𝑛+ 1) ℎ) −𝑆(𝑛ℎ) = 𝜙(ℎ) [Λ (𝑛ℎ) −𝛽(𝑛ℎ) 𝜑(𝑆((𝑛+ 1) ℎ)) 𝐼(𝑛ℎ) −(𝜇(𝑛ℎ) + 𝑝(𝑛ℎ)) 𝑆((𝑛+ 1) ℎ) −𝛽(𝑛ℎ) 𝜑(𝑆((𝑛+ 1) ℎ)) 𝐼(𝑛ℎ) (7) −(𝜇(𝑛ℎ) + 𝑝(𝑛ℎ)) 𝑆((𝑛+ 1) ℎ) + 𝜂(𝑛ℎ) 𝑉(𝑛ℎ+ ℎ)] . 4 Discrete Dynamics in Nature and Society to the incidence functions in order to overcome the technical difficulties. (v) when system (12) is 𝜔–periodic, it has a unique positive 𝜔–periodic solution which is globally uniformly attrac- tive. fi To lighten the reading, the proofs of our results are presented in the appendix. Lemma 4. Assume that condition (H5) holds. Then we have the following: 𝑥𝑛+1 = Λ 𝑛+ 𝜂𝑛𝑦𝑛+1 + 𝑥𝑛 1 + 𝜇𝑛+ 𝑝𝑛 𝑦𝑛+1 = 𝑝𝑛𝑥𝑛+1 + 𝑦𝑛 1 + 𝜇𝑛+ 𝜂𝑛 . (12) (i) all solutions (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with nonnegative initial conditions are nonnegative for all 𝑛∈N0; (12) (ii) all solutions (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (1) with positive initial conditions are positive for all 𝑛∈N0; Note that the auxiliary system describes the behaviour of the system in the absence of infection. If (Λ 𝑛), (𝜇𝑛), (𝑝𝑛), (𝜂𝑛), (𝛼𝑛), (𝜇𝑛), (𝜎𝑛), and (𝛽𝑛) are constant sequences, then the linear system (12) becomes autonomous and corresponds to the linearization of the equations for (𝑆𝑛) and (𝑉𝑛) in the classical (autonomous) SIRVS model. Note that the auxiliary system describes the behaviour of the system in the absence of infection. If (Λ 𝑛), (𝜇𝑛), (𝑝𝑛), (𝜂𝑛), (𝛼𝑛), (𝜇𝑛), (𝜎𝑛), and (𝛽𝑛) are constant sequences, then the linear system (12) becomes autonomous and corresponds to the linearization of the equations for (𝑆𝑛) and (𝑉𝑛) in the classical (autonomous) SIRVS model. (iii) there is a constant 𝑀> 0 such that, if (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) is a solution of (1) with nonnegative initial conditions, then lim sup 𝑛→+∞𝑆𝑛+ 𝐼𝑛+ 𝑅𝑛+ 𝑉𝑛< 𝑀. (16) (16) In order to proceed we need to recall some notions. A solution (𝑢𝑛) of some system of difference equations 𝑢𝑛+1 = 𝑓𝑛(𝑢𝑛) is said to be attractive if for all 𝑛0 ∈N and all 𝜀> 0 there is 𝜎(𝑛0) > 0 and 𝑇(𝜀, 𝑛0, 𝑢0) ∈N such that if (𝑢𝑛) is a solution with ‖𝑢0 −𝑢0‖ < 𝜎(𝑛0) then ‖𝑢𝑛−𝑢𝑛‖ < 𝜀, for all 𝑛⩾𝑛0+𝑇(𝜀, 𝑛0, 𝑢0). Additionally, if some solution is attractive and we can take 𝑇to be only dependent on 𝜀, we say that it is uniformly attractive.h Proof. See the appendix. For each 𝜆and each particular solution 𝜉∗ 𝑛= (𝑥∗ 𝑛, 𝑦∗ 𝑛) of (12) with 𝑥∗ 0 > 0 and 𝑦∗ 0 > 0 we define the numbers Rℓ 𝐷(𝜉∗, 𝜆) = lim inf 𝑛→∞ 𝑛+𝜆 ∏ 𝑘=𝑛 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 , (17) R𝑢 𝐷(𝜉∗, 𝜆) = lim sup 𝑛→∞ 𝑛+𝜆 ∏ 𝑘=𝑛 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 , (18) (17) The following theorem furnishes some simple properties of system (12). Lemma 3 (lemma 2.2 of [20]). Assume that conditions (H3) and (H4) hold. Then Lemma 3 (lemma 2.2 of [20]). Assume that conditions (H3) and (H4) hold. Then (i) all solutions (𝑥𝑛, 𝑦𝑛) of system (12) with initial condi- tion 𝑥0 ⩾0 and 𝑦0 ⩾0 are nonnegative for all 𝑛∈N0; where 𝜕𝑖𝑓denotes the partial derivative of 𝑓with respect to the 𝑖-th variable. Contrarily to what one could expect, the next lemma shows that the numbers above do not depend on the particular solution 𝜉𝑛= (𝑥𝑛, 𝑦𝑛) of (12) with 𝑥𝑛(0) > 0 and 𝑦𝑛(0) > 0. (ii) each fixed solution (𝑥𝑛, 𝑦𝑛) of (12) is bounded and globally uniformly attractive for all 𝑛∈N0; (iii) if (𝑥𝑛, 𝑦𝑛) is a solution of (12) and (̃𝑥𝑛, ̃𝑦𝑛) is a solution of the system Lemma 5. Assume that (H1), (H3), and (H4) hold. If (𝜉∗ 1 )𝑛= ((𝑥1)∗ 𝑛, (𝑦1)∗ 𝑛) and (𝜉∗ 2 )𝑛= ((𝑥2)∗ 𝑛, (𝑦2)∗ 𝑛) are two solutions of (12) with 𝑥∗ 𝑖(0) > 0 and 𝑦∗ 𝑖(0) > 0, 𝑖= 1, 2, then Lemma 5. Assume that (H1), (H3), and (H4) hold. If (𝜉∗ 1 )𝑛= ((𝑥1)∗ 𝑛, (𝑦1)∗ 𝑛) and (𝜉∗ 2 )𝑛= ((𝑥2)∗ 𝑛, (𝑦2)∗ 𝑛) are two solutions of (12) with 𝑥∗ 𝑖(0) > 0 and 𝑦∗ 𝑖(0) > 0, 𝑖= 1, 2, then 𝑥𝑛+1 = Λ 𝑛+ 𝜂𝑛𝑦𝑛+1 + 𝑥𝑛+ 𝑓𝑛 1 + 𝜇𝑛+ 𝑝𝑛 𝑦𝑛+1 = 𝑝𝑛𝑥𝑛+1 + 𝑦𝑛+ 𝑔𝑛 1 + 𝜇𝑛+ 𝜂𝑛 , (13) (13) Rℓ 𝐷(𝜉∗ 1 , 𝜆) = Rℓ 𝐷(𝜉∗ 2 , 𝜆) , R𝑢 𝐷(𝜉∗ 1 , 𝜆) = R𝑢 𝐷(𝜉∗ 2 , 𝜆) . (19) (19) with (̃𝑥0, ̃𝑦0) = (𝑥0, 𝑦0) then there is a constant 𝐿> 0, only depending on 𝜇𝑛, satisfying with (̃𝑥0, ̃𝑦0) = (𝑥0, 𝑦0) then there is a constant 𝐿> 0, only depending on 𝜇𝑛, satisfying Proof. We have the following lemma. 3.1. Auxiliary Results. Consider the auxiliary system, Therefore, we obtain the corollary. Therefore, we obtain the corollary. (23) Corollary 8 (periodic case). Assume that all coefficients are 𝜔-periodic in (1) and that conditions (H1) to (H5) hold. Then (a) if R𝑝𝑒𝑟 𝐷(𝜉∗) < 1 then the infectives (𝐼𝑛) go to extinction; (b) the disease-free solution (𝑥∗ 𝑛, 0, 0, 𝑦∗ 𝑛), where (𝑥∗ 𝑛, 𝑦∗ 𝑛)𝑛∈N is an disease-free 𝜔-periodic solution of (12), is globally attractive; Then, (a) if R𝑢 𝐶(𝜆) < 0, then R𝑢 𝐷(⌊𝜆/ℎ⌋, ℎ) < 1 for all ℎ∈]0, ℎ𝑢 𝑚𝑎𝑥[; (b) if Rℓ 𝐶(𝜆) > 0, then Rℓ 𝐷(⌊𝜆/ℎ⌋, ℎ) > 1 for all for all ℎ∈ ]0, ℎℓ 𝑚𝑎𝑥[. (c) if R𝑝𝑒𝑟 𝐷(𝜉∗) > 1, then the infectives (𝐼𝑛) are permanent. Proof. See the Appendix. Proof. Observe that (𝑥𝑛, 𝑦𝑛) = (𝑎, 𝑏), 𝑛∈N and (𝑥(𝑡), 𝑦(𝑡)) = (𝑎, 𝑏), 𝑡∈R, where Proof. Observe that (𝑥𝑛, 𝑦𝑛) = (𝑎, 𝑏), 𝑛∈N and (𝑥(𝑡), 𝑦(𝑡)) = (𝑎, 𝑏), 𝑡∈R, where (a) if there is a constant 𝜆> 0 such that R𝑢 𝐷(𝜆) < 1, then the infectives (𝐼𝑛) go to extinction; (a) if there is a constant 𝜆> 0 such that R𝑢 𝐷(𝜆) < 1, then the infectives (𝐼𝑛) go to extinction; (b) any solution (𝑥∗ 𝑛, 0, 0, 𝑦∗ 𝑛), where (𝑥∗ 𝑛, 𝑦∗ 𝑛) is a par- ticular solution of system (12), is globally uniformly attractive. (b) any solution (𝑥∗ 𝑛, 0, 0, 𝑦∗ 𝑛), where (𝑥∗ 𝑛, 𝑦∗ 𝑛) is a par- ticular solution of system (12), is globally uniformly attractive. Rℓ 𝐷(𝜆, ℎ) = lim inf 𝑛→∞ 𝑛+𝜆 ∏ 𝑘=𝑛 1 + 𝛽ℎ 𝑘𝜑(𝑥∗ 𝑘+1) + 𝜎ℎ 𝑘𝜓(𝑦∗ 𝑘+1) 1 + 𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘 , R𝑢 𝐷(𝜆, ℎ) = lim sup 𝑛→∞ 𝑛+𝜆 ∏ 𝑘=𝑛 1 + 𝛽ℎ 𝑘𝜑(𝑥∗ 𝑘+1) + 𝜎ℎ 𝑘𝜓(𝑦∗ 𝑘+1) 1 + 𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘 , (21) (21) Proof. See the Appendix. Proof. See the appendix. See the Appendix. Proof. See the Appendix. sup 𝑛∈N0 {󵄨󵄨󵄨󵄨̃𝑥𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨̃𝑦𝑛−𝑦𝑛󵄨󵄨󵄨󵄨} ⩽𝐿sup 𝑛∈N0 (󵄨󵄨󵄨󵄨𝑓𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑔𝑛󵄨󵄨󵄨󵄨) ; (14) By Lemma 5 we can drop the dependence of the partic- ular solution and simply write Rℓ 𝐷(𝜆) and R𝑢 𝐷(𝜆) instead of Rℓ 𝐷(𝜉∗, 𝜆) an R𝑢 𝐷(𝜉∗, 𝜆), respectively. By Lemma 5 we can drop the dependence of the partic- ular solution and simply write Rℓ 𝐷(𝜆) and R𝑢 𝐷(𝜆) instead of Rℓ 𝐷(𝜉∗, 𝜆) an R𝑢 𝐷(𝜉∗, 𝜆), respectively. (14) (iv) there exist constants 𝑚, 𝑀> 0 such that, for each solution (𝑥𝑛, 𝑦𝑛) of (12), we have (iv) there exist constants 𝑚, 𝑀> 0 such that, for each solution (𝑥𝑛, 𝑦𝑛) of (12), we have 3.2. Extinction and Permanence. We have the following result about the extinction of the disease. 𝑚⩽lim inf 𝑛→∞𝑥𝑛⩽lim sup 𝑛→∞ 𝑥𝑛⩽𝑀, (15) Theorem 6 (extinction of the disease). Assume that condi- tions (H1) to (H5) hold. Then 5 Discrete Dynamics in Nature and Society For a given time step ℎ> 0, the expressions Rℓ 𝐷(𝜆) and R𝑢 𝐷(𝜆) in (17) and (18) become, in our context, Proof. See the Appendix. We have the following result about the permanence of the disease. where (𝑥∗ 𝑘, 𝑦∗ 𝑘) is the solution of the (in our context autonomous) system (12). where (𝑥∗ 𝑘, 𝑦∗ 𝑘) is the solution of the (in our context autonomous) system (12). Theorem 7 (permanence of the disease). Assume that condi- tions (H1) to (H5) hold. If there is a constant 𝜆> 0 such that Rℓ 𝐷(𝜆) > 1 then the infectives (𝐼𝑛) are permanent in system (1). We have the following result. Theorem 9. For system (2), assume that Λ(𝑡) = Λ, 𝜇(𝑡) = 𝜇, 𝜂(𝑡) = 𝜂, and 𝑝(𝑡) = 𝑝for all 𝑡⩾0 and that the functions 𝛼(𝑡), 𝛾(𝑡), 𝛽(𝑡), and 𝜎(𝑡) are differentiable, nonnegative, and bounded and have bounded derivative. Assume also that conditions (C1) to (C3) hold and let Proof. See the Appendix. Proof. See the Appendix. We consider now the particular periodic case: assume that all parameters of system (1) are periodic with period 𝜔∈N. By (v) in Lemma 3, there is an 𝜔-periodic disease- free solution of (12), 𝜉∗= (𝑥∗ 𝑛, 𝑦∗ 𝑛)𝑛∈N. Thus, in the periodic setting, (17) and (18) become both equal to ℎ𝑢 𝑚𝑎𝑥= − 𝑅𝑢 𝐶(𝜆) sup𝑡⩾0 󵄨󵄨󵄨󵄨𝑓󸀠(𝑡)󵄨󵄨󵄨󵄨(𝜆+ 1), ℎℓ 𝑚𝑎𝑥= 𝑅ℓ 𝐶(𝜆) sup𝑡⩾0 󵄨󵄨󵄨󵄨𝑓󸀠(𝑡)󵄨󵄨󵄨󵄨(𝜆+ 1), (22) ℎ𝑢 𝑚𝑎𝑥= − 𝑅𝑢 𝐶(𝜆) sup𝑡⩾0 󵄨󵄨󵄨󵄨𝑓󸀠(𝑡)󵄨󵄨󵄨󵄨(𝜆+ 1), (22) R𝑝𝑒𝑟 𝐷(𝜉∗) = 𝜔−1 ∏ 𝑘=0 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 . (20) ℎℓ 𝑚𝑎𝑥= 𝑅ℓ 𝐶(𝜆) sup𝑡⩾0 󵄨󵄨󵄨󵄨𝑓󸀠(𝑡)󵄨󵄨󵄨󵄨(𝜆+ 1), where ( Λ (𝜇+ 𝜂) ) R𝑝𝑒𝑟 𝐷(𝜉∗) = 𝜔−1 ∏ 𝑘=0 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 . (20) (20) where where 𝑓(𝑡) = 𝛽(𝑡) 𝜑( Λ (𝜇+ 𝜂) 𝜇(𝜇+ 𝜂+ 𝑝)) + 𝜎(𝑡) 𝜓( 𝑝Λ 𝜇(𝜇+ 𝜂+ 𝑝)) −𝜇−𝛼(𝑡) −𝛾(𝑡) . (23)h 𝑓(𝑡) = 𝛽(𝑡) 𝜑( Λ (𝜇+ 𝜂) 𝜇(𝜇+ 𝜂+ 𝑝)) Therefore, we obtain the corollary. 4. Consistency Discrete Dynamics in Nature and Society 6 y contradiction, assume that We also have By contradiction, assume that By contradiction, assume that We also have By contradiction, assume that R𝑢 𝐶(𝜆) < 0, (26) and that there is a sequence (ℎ𝑚)𝑚∈N such that ℎ𝑚→0 as 𝑚→+∞and R𝑢 𝐷(⌊𝜆 ℎ𝑚 ⌋, ℎ𝑚) ( ) We also have 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 ⩽ℎ𝑚 ⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=2 (⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏))] 𝑘[𝜙(ℎ𝑚)] 𝑘 ⩽ℎ ⌊𝜆/ℎ𝑚⌋ ∑(⌊𝜆 ℎ⌋) [(𝛽𝑢( ) + 𝜎𝑢𝜓(𝑏))]𝑘[𝜙(ℎ)] 𝑘 (34) By contradiction, assume that R𝑢 𝐶(𝜆) < 0, (26) and that there is a sequence (ℎ𝑚)𝑚∈N such that ℎ𝑚→0 as 𝑚→+∞and We also have 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 ⩽ℎ𝑚 ⌊𝜆/ℎ𝑚⌋ ∑ 𝑘2 (⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏))] 𝑘[𝜙(ℎ𝑚)] 𝑘 R𝑢 𝐶(𝜆) < 0, (26) 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 R𝑢 𝐶(𝜆) < 0, (26) 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 R𝑢 𝐶(𝜆) < 0, (26) 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚 R𝑢 𝐶(𝜆) < 0, (26) and that there is a sequence (ℎ𝑚)𝑚∈N such that ℎ𝑚→0 as 𝑚→+∞and ⩽ℎ𝑚 ⌊𝜆/ℎ𝑚⌋ ∑(⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏))] 𝑘[𝜙(ℎ𝑚)] 𝑘 and that there is a sequence (ℎ𝑚)𝑚∈N such that ℎ𝑚→0 as 𝑚→+∞and ⩽ℎ𝑚 ⌊𝜆/ℎ𝑚⌋ ∑(⌊𝜆 ℎ𝑚 ⌋) [(𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏))] 𝑘[𝜙(ℎ𝑚)] 𝑘 and that there is a sequence (ℎ𝑚)𝑚∈N such that ℎ𝑚→0 as 𝑚→+∞and ⩽ℎ𝑚 ⌊𝜆/ℎ𝑚⌋ ∑(⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝛽𝑢𝜑(𝑎) R𝑢 𝐷(⌊𝜆 ℎ𝑚 ⌋, ℎ𝑚) R𝑢 𝐷(⌊𝜆 ℎ𝑚 ⌋, ℎ𝑚) = lim sup 𝑛→∞ 𝑛+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛 1 + 𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏) 1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘 ⩾1, (27) 𝑘=2 ( 𝑘 ) ⩽ℎ𝑚 ⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=0 (⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏))]𝑘[𝜙(ℎ𝑚)] 𝑘 = ℎ[1 + (𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏)) 𝜙(ℎ)] ⌊𝜆/ℎ𝑚⌋ (34) ⩽ℎ𝑚 ⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=0 (⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏))]𝑘[𝜙(ℎ𝑚)] 𝑘 (27) (27) = lim sup 𝑛→∞ 𝑛+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛 1 + 𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏) 1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘 ⩾1, = ℎ𝑚[1 + (𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏)) 𝜙(ℎ𝑚)] ⌊𝜆/ℎ𝑚⌋. = ℎ𝑚[1 + (𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏)) 𝜙(ℎ𝑚)] ⌊𝜆/ℎ𝑚⌋. for all 𝑚∈N. By (27), we conclude that, for each 𝑚∈N, there are sequences (ℎ𝑚)𝑚∈N and (𝑛𝑚,𝑟)𝑟∈N such that ℎ𝑚→0 as 𝑚→+∞, 𝑛𝑚,𝑟→+∞as 𝑟→+∞and Noting that, by (5), we have lim 𝑚→+∞[1 + (𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏)) 𝜙 for all 𝑚∈N. 4. Consistency By (27), we conclude that, for each 𝑚∈N, there are sequences (ℎ𝑚)𝑚∈N and (𝑛𝑚,𝑟)𝑟∈N such that ℎ𝑚→0 as 𝑚→+∞𝑛 →+∞as 𝑟→+∞and Noting that, by (5), we have Noting that, by (5), we have lim 𝑚→+∞[1 + (𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏)) 𝜙(ℎ𝑚)]⌊𝜆/ℎ𝑚⌋ , as 𝑚→+∞, 𝑛𝑚,𝑟→+∞as 𝑟→+∞and 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛𝑚,𝑟 (1 + 𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏)) 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛𝑚,𝑟 (1 + 𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏)) = lim 𝑚→+∞[(1 = lim 𝑚→+∞[(1 (28) (28) (35) [( + 𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏) 1/𝜙(ℎ𝑚) ) 1/𝜙(ℎ𝑚) ] 𝜙(ℎ𝑚)⌊𝜆/ℎ𝑚⌋ = e(𝛽𝑢𝜑(𝑎)+𝜎𝑢𝜓(𝑏))𝜆 (35) > (1 −ℎ𝑚) 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛𝑚,𝑟 (1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘) . > (1 −ℎ𝑚) 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛𝑚,𝑟 (1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘) . (28) 28), we have + 𝛽𝑢𝜑(𝑎) + 𝜎𝑢𝜓(𝑏) 1/𝜙(ℎ𝑚) ) 1/𝜙(ℎ𝑚) ] 𝜙(ℎ𝑚)⌊𝜆/ℎ𝑚⌋ = e(𝛽𝑢𝜑(𝑎)+𝜎𝑢𝜓(𝑏))𝜆 (35) By (28), we have By (28), we have = e(𝛽𝑢𝜑(𝑎)+𝜎𝑢𝜓(𝑏))𝜆 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 (𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏) −𝜇ℎ𝑚 𝑘−𝛼ℎ𝑚 𝑘−𝛾ℎ𝑚 𝑘) > (𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚) ℎ𝑚 , (29) and that a convergent sequence is bounded; by (34) there is 𝐶1 > 0 such that 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶1ℎ𝑚. (36) Similarly, we have and that a convergent sequence is bounded; by (34) there is 𝐶1 > 0 such that 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ (𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏) −𝜇ℎ𝑚 𝑘−𝛼ℎ𝑚 𝑘−𝛾ℎ𝑚 𝑘) and that a convergent sequence is bounded; by (34) there is 𝐶1 > 0 such that g 𝐶1 > 0 such that ) (29) 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶1ℎ𝑚. (36) 󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶1ℎ𝑚. (36) (29) (36) Similarly, we have 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 ⩽ ⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=2 (⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢)] 𝑘[𝜙(ℎ𝑚)] 𝑘 ⩽ ⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=0 (⌊𝜆 ℎ𝑚 ⌋ 𝑘 ) [(𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢)]𝑘[𝜙(ℎ𝑚)]𝑘 (37) where where 𝐴𝑛,𝐿,ℎfl−ℎ+ ℎ 𝑛+⌊𝐿/ℎ⌋ ∏ 𝑘=𝑛 (1 + 𝛽ℎ 𝑘𝜑(𝑎) + 𝜎ℎ 𝑘𝜓(𝑏)) −ℎ 𝑛+⌊𝐿/ℎ⌋ ∑ 𝑘=𝑛 (𝛽ℎ 𝑘𝜑(𝑎) + 𝜎ℎ 𝑘𝜓(𝑏)) , 𝐵𝑛,𝐿,ℎfl−ℎ+ ℎ 𝑛+⌊𝐿/ℎ⌋ ∏ 𝑘=𝑛 (1 + 𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘) −ℎ 𝑛+⌊𝐿/ℎ⌋ ∑ 𝑘=𝑛 (𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘) , 𝐶𝑛,𝐿,ℎ= ℎ 𝑛+⌊𝐿/ℎ⌋ ∏ 𝑘=𝑛 (1 + 𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘) . 𝐴𝑛,𝐿,ℎfl−ℎ+ ℎ 𝑛+⌊𝐿/ℎ⌋ ∏ 𝑘=𝑛 (1 + 𝛽ℎ 𝑘𝜑(𝑎) + 𝜎ℎ 𝑘𝜓(𝑏)) (30) (37) −ℎ 𝑛+⌊𝐿/ℎ⌋ ∑ 𝑘=𝑛 (𝛽ℎ 𝑘𝜑(𝑎) + 𝜎ℎ 𝑘𝜓(𝑏)) , 𝐵𝑛,𝐿,ℎfl−ℎ+ ℎ 𝑛+⌊𝐿/ℎ⌋ ∏ 𝑘=𝑛 (1 + 𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘) = ℎ𝑚[1 + (𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢) 𝜙(ℎ𝑚)] ⌊𝜆/ℎ𝑚⌋. = ℎ𝑚[1 + (𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢) 𝜙(ℎ𝑚)] ⌊𝜆/ℎ𝑚⌋. 4. Consistency In this section, under the additional assumption that the parameter functions Λ, 𝜇, 𝜂, and 𝑝are constant, we will get a result stating that when our integral conditions prescribe extinction (persistence) for the continuous-time model, then the discrete-time conditions prescribe extinction (persis- tence) for the corresponding discrete-time models, as long as the time step is less than some constant. Throughout this section, we assume that the parameter functions Λ, 𝜇, 𝜂, and 𝑝are constant functions and 𝜙(ℎ) will be the function used in the discretization of the derivative. (𝑎, 𝑏) = ( Λ (𝜇+ 𝜂) [𝜇(𝜇+ 𝜂+ 𝑝)], 𝑝Λ [𝜇(𝜇+ 𝜂+ 𝑝)]) , (24) (24) are, respectively, solutions of system (12) and system (3). Thus, are, respectively, solutions of system (12) and system (3). Thus, R𝑢 𝐶(𝜆) = lim sup 𝑡→∞ ∫ 𝑡+𝜆 𝑡 𝛽(𝑠) 𝜑(𝑎) + 𝜎(𝑠) 𝜓(𝑏) −[𝜇(𝑠) + 𝛼(𝑠) + 𝛾(𝑠)] 𝑑𝑠, R𝑢 𝐷(⌊𝜆 ℎ⌋, ℎ) (25) (25) We consider the continuous-time model (2) and, for a given time step ℎ, the corresponding discrete-time model, that is, the discrete-time model with parameters 𝛽ℎ 𝑘 = 𝜙(ℎ)𝛽(𝑘ℎ), 𝜎ℎ 𝑘= 𝜙(ℎ)𝜎(𝑘ℎ), Λℎ 𝑘= 𝜙(ℎ)Λ, 𝜇ℎ 𝑘= 𝜙(ℎ)𝜇, 𝑝ℎ 𝑘= 𝜙(ℎ)𝑝, 𝜂ℎ 𝑘= 𝜙(ℎ)𝜂, 𝛼ℎ 𝑘= 𝜙(ℎ)𝛼(𝑘ℎ), and 𝛾ℎ 𝑘= 𝜙(ℎ)𝛾(𝑘ℎ). = lim sup 𝑛→∞ 𝑛+⌊𝜆/ℎ⌋ ∏ 𝑘=𝑛 1 + 𝛽ℎ 𝑘𝜑(𝑎) + 𝜎ℎ 𝑘𝜓(𝑏) 1 + 𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘 . 4. Consistency (42) 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) + 𝑠𝑚,𝑟−∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 2 ⌊𝜆⌋ 2 (5 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶3ℎ𝑚. (42) (51) Thus, Thus, 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 ⩽󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 ⩽(𝐶1 + 𝐶2 + 𝐶3) ℎ𝑚, (43) < 𝐶ℎ2 𝑚⌊𝜆 ℎ𝑚 ⌋+ 𝐶ℎ2 𝑚< 𝐶(𝜆+ 1) ℎ𝑚, thus 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ ℎ𝑓(𝑘ℎ) < ∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑓(𝑠) 𝑑𝑠 𝑠 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 ⩽󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 ⩽(𝐶1 + 𝐶2 + 𝐶3) ℎ𝑚, (43) < 𝐶ℎ2 𝑚⌊𝜆 ℎ𝑚 ⌋+ 𝐶 thus 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ ℎ𝑓(𝑘ℎ 𝑛𝑚,𝑟,𝜆,ℎ𝑚 𝑛𝑚,𝑟,𝜆,ℎ𝑚 𝑛𝑚,𝑟,𝜆,ℎ𝑚 ⩽󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 ⩽(𝐶1 + 𝐶2 + 𝐶3) ℎ𝑚, (43) thus 𝑛𝑚,𝑟+⌊𝜆/ℎ ∑ ⩽󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 ( ) (43) thus 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) < ∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠−𝑠𝑚,𝑟 + 𝐶(𝜆+ 1) ℎ𝑚, (52) ⩽(𝐶1 + 𝐶2 + 𝐶3) ℎ𝑚, for all 𝑚⩾𝑀. Since the right hand side of (43) is independent of 𝑛𝑚,𝑟, we conclude that 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) < ∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠−𝑠𝑚,𝑟 + 𝐶(𝜆+ 1) ℎ𝑚, (52) for all 𝑚⩾𝑀. Since the right hand side of (43) is independent of 𝑛𝑚,𝑟, we conclude that ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) < ∫ 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠−𝑠𝑚,𝑟 𝐶(𝜆 1) ℎ (52) for all 𝑚⩾𝑀. Since the right hand side of (43) is independent of 𝑛𝑚,𝑟, we conclude that ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) < ∫ 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠 𝑠𝑚,𝑟 + 𝐶(𝜆+ 1) ℎ (52) for all 𝑚⩾𝑀. Since the right hand side of (43) is independent of 𝑛𝑚,𝑟, we conclude that (52) + 𝐶(𝜆+ 1) ℎ𝑚, 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚󳨀→0, (44) 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚󳨀→0, (44) and therefore (44) and therefore as 𝑚→+∞, uniformly in 𝑟. as 𝑚→+∞, uniformly in 𝑟. 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 𝜙(ℎ𝑚) ℎ𝑚𝑓(𝑘ℎ𝑚) < 𝜙(ℎ𝑚) [∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠+ 𝐶(𝜆+ 1) ℎ𝑚] . (53) y On the other hand, we note that the 𝐶1 function 𝑓: R+ 0 → R given by On the other hand, we note that the 𝐶1 function 𝑓: R+ 0 → R given by On the other hand, we note that the 𝐶1 function 𝑓: R+ 0 → R given by (53) 𝑓(𝑡) = 𝛽(𝑡) 𝜑(𝑎) + 𝜎(𝑡) 𝜓(𝑏) −𝜇(𝑡) −𝛼(𝑡) −𝛾(𝑡) (45) is Riemann-integrable on any bounded interval 𝐼⊂R+ 0. h h is Riemann-integrable on any bounded interval 𝐼⊂R+ 0. 4. Consistency (31) (31) (31) Using (5) again, we get lim 𝑚→+∞[1 + (𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢) 𝜙(ℎ𝑚)]⌊𝜆/ℎ𝑚⌋ ⩽e(𝜇𝑢+𝛼𝑢+𝛾𝑢)𝜆. (38) (38) 𝐶𝑛,𝐿,ℎ= ℎ 𝑛+⌊𝐿/ℎ⌋ ∏ 𝑘=𝑛 (1 + 𝜇ℎ 𝑘+ 𝛼ℎ 𝑘+ 𝛾ℎ 𝑘) . (32) ⩽e(𝜇𝑢+𝛼𝑢+𝛾𝑢)𝜆. There is 𝐶2 > 0 such that (32) ⩽e(𝜇𝑢+𝛼𝑢+𝛾𝑢)𝜆 There is 𝐶2 > 0 such that (32) 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶2ℎ𝑚. (39) and, multiplying both sides by ℎ𝑚, we get 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶2ℎ𝑚. (39) and, multiplying both sides by ℎ𝑚, we get and, multiplying both sides by ℎ𝑚, we get (39) 𝜙(ℎ𝑚) 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚[𝛽(𝑘ℎ𝑚) 𝜑(𝑎) + 𝜎(𝑘ℎ𝑚) 𝜓(𝑏) −𝜇(𝑘ℎ𝑚) −𝛼(𝑘ℎ𝑚) −𝛾(𝑘ℎ𝑚)] > 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 (33) Finally, we have 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨= ℎ𝑚 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛𝑚,𝑟 (1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘) (40 𝜙(ℎ𝑚) 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚[𝛽(𝑘ℎ𝑚) 𝜑(𝑎) + 𝜎(𝑘ℎ𝑚) 𝜓(𝑏) Finally, we have (33) (40) = ℎ𝑚(1 + 3𝜙(ℎ𝑚) max {𝜇𝑢, 𝛼𝑢, 𝛾𝑢})⌊𝜆/ℎ𝑚⌋+1 . −𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚. = ℎ𝑚(1 + 3𝜙(ℎ𝑚) max {𝜇𝑢, 𝛼𝑢, 𝛾𝑢})⌊𝜆/ℎ𝑚⌋+1 . −𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚. Discrete Dynamics in Nature and Society 7 Discrete Dynamics in Nature and Society Discrete Dynamics in Nature and Society 7 we obtain of size ℎ𝑚of the interval [𝑛𝑚,𝑟, 𝑛𝑚,𝑟+ 𝜆]. Note that According to (5), we obtain of size ℎ𝑚of the interval [𝑛𝑚,𝑟, 𝑛𝑚,𝑟+ 𝜆]. Note that of size ℎ𝑚of the interval [𝑛𝑚,𝑟, 𝑛𝑚,𝑟+ 𝜆]. Note that 𝑠𝑚,𝑟fl(𝜆−⌊𝜆 ℎ𝑚 ⌋ℎ𝑚) 𝑓(𝑛𝑚,𝑟ℎ𝑚+ ⌊𝜆 ℎ𝑚 ⌋ℎ𝑚) (49) (49) = lim 𝑚→+∞[(1 (41) 𝑚 𝑚 ⩽ℎ𝑚𝑓𝑢fl𝑠𝑚 (49) d 0 if l i = lim 𝑚→+∞[(1 ⩽ℎ𝑚𝑓𝑢fl𝑠𝑚 [( + 3 max {𝜇𝑢, 𝛼𝑢, 𝛾𝑢} 1/𝜙(ℎ𝑚) ) 1/𝜙(ℎ𝑚) ] 𝜙(ℎ𝑚)(⌊𝜆/ℎ𝑚⌋+1) (41) and 𝑠𝑚→0 as 𝑚→+∞, uniformly in 𝑟. Since 𝑓is 𝐶1 with bounded derivative, for any ℎ> 0 we have (41) + 3 max {𝜇𝑢, 𝛼𝑢, 𝛾𝑢} 1/𝜙(ℎ𝑚) ) 1/𝜙(ℎ𝑚) ] 𝜙(ℎ𝑚)(⌊𝜆/ℎ𝑚⌋+1) (41) and 𝑠𝑚→0 as 𝑚→+∞, uniformly in 𝑟. Since 𝑓is 𝐶1 with bounded derivative, for any ℎ> 0 we have 󵄨𝑓 𝑓 ℎ󵄨 ℎ 𝜙( 𝑚) ) ] = e3 max{𝜇𝑢,𝛼𝑢,𝛾𝑢}𝜆. 󵄨󵄨󵄨󵄨𝑓(𝑥) −𝑓(𝑥+ ℎ)󵄨󵄨󵄨󵄨⩽𝐶ℎ, (50) 󵄨󵄨󵄨󵄨𝑓(𝑥) −𝑓(𝑥+ ℎ)󵄨󵄨󵄨󵄨⩽𝐶ℎ, (50) (50) = e3 max{𝜇𝑢,𝛼𝑢,𝛾𝑢}𝜆. where 𝐶= sup𝑡⩾0|𝑓󸀠(𝑡)|. We conclude that There is 𝐶3 > 0 such that 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶3ℎ𝑚. (42) , 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚−𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 ⩽󵄨󵄨󵄨󵄨󵄨𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨 (43) 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) + 𝑠𝑚,𝑟−∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 < 𝐶ℎ2 𝑚⌊𝜆 ℎ𝑚 ⌋+ 𝐶ℎ2 𝑚< 𝐶(𝜆+ 1) ℎ𝑚, (51) thus 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) + 𝑠𝑚,𝑟−∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠 󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨󵄨 < 𝐶ℎ2 𝑚⌊𝜆 ℎ𝑚 ⌋+ 𝐶ℎ2 𝑚< 𝐶(𝜆+ 1) ℎ𝑚, (51) 󵄨󵄨󵄨󵄨󵄨𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚 󵄨󵄨󵄨󵄨󵄨⩽𝐶3ℎ𝑚. 4. Consistency In fact, noting that 𝑥∗(𝑡) + 𝑦∗(𝑡) = 1, we get Rℓ 𝐷(⌊𝜆 ℎ𝑚 ⌋, ℎ𝑚) = lim inf 𝑛→∞ 𝑛+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛 1 + 𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏) 1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘 ⩽1, (57) that 𝑥∗(𝑡) + 𝑦∗(𝑡) = 1, we get Rℓ C (1) = ∫ 1 0 𝛽(𝑠) 𝑥∗(𝑠) + 𝜎(𝑠) 𝑦∗(𝑠) −𝜇−𝛼− = ∫ 1 0 𝑑[1 + 𝑐sin2 (2𝜋𝐿𝑡) (1 + cos (2𝜋𝑡)) Rℓ 𝐷(⌊𝜆 ℎ𝑚 ⌋, ℎ𝑚) = lim inf 𝑛→∞ 𝑛+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛 1 + 𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏) 1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘 ⩽1, (57) Rℓ 𝐷(⌊𝜆 ℎ𝑚 ⌋, ℎ𝑚) ( ) Rℓ 𝐷(⌊𝜆 ℎ𝑚 ⌋, ℎ𝑚) Rℓ C (1) = ∫ 1 0 𝛽(𝑠) 𝑥∗(𝑠) + 𝜎(𝑠) 𝑦∗(𝑠) −𝜇−𝛼−𝛾𝑑𝑠 ∫ 1 Rℓ C (1) = ∫ 1 0 𝛽(𝑠) 𝑥∗(𝑠) + 𝜎(𝑠) 𝑦∗(𝑠) −𝜇−𝛼−𝛾𝑑𝑠 = ∫ 1 0 𝑑[1 + 𝑐sin2 (2𝜋𝐿𝑡) (1 + cos (2𝜋𝑡))] 𝑑𝑠 (63) (57) = lim inf 𝑛→∞ 𝑛+⌊𝜆/ℎ𝑚⌋ ∏ 𝑘=𝑛 1 + 𝛽ℎ𝑚 𝑘𝜑(𝑎) + 𝜎ℎ𝑚 𝑘𝜓(𝑏) 1 + 𝜇ℎ𝑚 𝑘+ 𝛼ℎ𝑚 𝑘+ 𝛾ℎ𝑚 𝑘 ⩽1, (63) −𝜇−𝛼−𝛾= 𝑑(1 + 𝑐 2) −𝜇−𝛼−𝛾. it is possible to conclude that it is possible to conclude that 𝜙(ℎ𝑚) 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚(𝛽(𝑘ℎ𝑚) 𝜑(𝑎) + 𝜎(𝑘ℎ𝑚) 𝜓(𝑏) −𝜇(𝑘ℎ𝑚) −𝛼(𝑘ℎ𝑚) −𝛾(𝑘ℎ𝑚)) < 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚 −𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚+ 𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚, We can also compute Rℓ 𝐷(1, 1/𝐿). Namely, we have ) (58) Rℓ 𝐷(1, 1 𝐿) = 1 + 𝑑/𝐿 1 + 𝜇+ 𝛼+ 𝛾. (64) Rℓ 𝐷(1, 1 𝐿) = 1 + 𝑑/𝐿 1 + 𝜇+ 𝛼+ 𝛾. (64) (64) (58) If we let 𝑑be sufficiently small so that 𝑑< (𝜇+ 𝛼+ 𝛾)𝐿, or in other words, 𝑑< (𝜇+ 𝛼+ 𝛾) and 𝑐be sufficiently large so that 𝑐> (2/𝑑)(𝜇+ 𝛾+ 𝛼−𝑑), we obtain −𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚+ 𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚, where 𝐴𝑛𝑚,𝑟,𝜆,ℎ𝑚, 𝐵𝑛𝑚,𝑟,𝜆,ℎ𝑚, and 𝐶𝑛𝑚,𝑟,𝜆,ℎ𝑚are given, respec- tively, by (30), (31), and (42) and still satisfy (36), (39), and (42). Consequently, given 𝛿> 0, there is 𝑟𝑚∈N such that, for all 𝑟⩾𝑟𝑚, Rℓ 𝐶(1) > 1 ⇐⇒ 1 + 𝑑(1 + 𝑐/2) 1 + 𝜇+ 𝛼+ 𝛾> 1, Rℓ 𝐷(1, 1 𝐿) < 1 ⇐⇒ (65) Rℓ 𝐶(1) > 1 ⇐⇒ 1 + 𝑑(1 + 𝑐/2) 1 + 𝜇+ 𝛼+ 𝛾> 1, Rℓ 𝐷(1, 1 𝐿) < 1 ⇐⇒ 1 + 𝑑/𝐿 1 + 𝜇+ 𝛼+ 𝛾< 1. (65) (65) 𝜙(ℎ𝑚) 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) > 𝜙(ℎ𝑚) [∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝑓(𝑠) 𝑑𝑠−𝛿+ 𝐶(𝜆+ 1) ℎ𝑚] . 4. Consistency In this context, (𝑥𝑛, 𝑦𝑛) = (𝑎, 𝑏), 𝑛∈N, and (𝑥(𝑡), 𝑦(𝑡)) = (𝑎, 𝑏), 𝑡∈R, where 4. Consistency (59) (59) So we conclude that we do not have consistency for time step 1/𝐿. Recalling that Rℓ 𝐶(𝜆) > 0, by assumption and since 𝛿> 0 is arbitrary, we obtain for sufficiently large 𝑚∈N, Let 𝐿= 6 and consider the continuous model with the following parameters 𝜇= Λ = 0.25, 𝛾= 0.3, 𝛼= 0.05, 𝜂= 0.05, 𝑝= 2/3, 𝑑= 0.6, and 𝑐= 1.5. In Figure 1, we plot function 𝛽(or similarly 𝜎) and the component 𝐼(𝑡) of the solution of system (2) given by the solver of Mathematica (that we take to represent the solution of the continuous-time model) and the solution of the discrete-time model (9) with time step 1/6. As can be seen, the infectives are persistent in the continuous-time model but go to extinction in the discrete-time model. We have inconsistency in this case. 0 ⩾𝜙(ℎ𝑚) 𝑛𝑚,𝑟𝑚+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟𝑚 𝑓(𝑘ℎ𝑚) > 0, (60) (60) which is a contradiction. We obtain (b) and the theorem follows. Next, for each 𝐿∈N, we give an example of a periodic system of period 1 such that the continuous and the discrete- time system with time step ℎ= 1/𝐿are not consistent; namely, we will have persistence for the continuous-time model and extinction for the discrete-time model with time step ℎ= 1/𝐿. Note that, changing 𝛽(𝑡) and 𝜎(𝑡) slightly, we can con- struct an example of a periodic system with period 1 where the infectives in the continuous-time model go to extinction but, in the discrete-time model with time step ℎ= 1/𝐿, the infectives are persistent. Example 10. Let 𝐿∈N. Consider in system (2) that 𝜙(𝑥) = 𝜓(𝑥) = 𝑥, that, with the exception of 𝜎and 𝛽, all parameters are constant, that Λ = 𝜇, and that Furthermore, we emphasize that this lack of consistency is not a result of the discretization method used but simply a result of the fact that the time steps lead to a situation where the points 𝑛/𝐿where the functions 𝛽and 𝜎are evaluated (in order to obtain the discrete-time parameters) correspond to minimums of 𝛽and 𝜎. 𝜎(𝑡) = 𝛽(𝑡) = 𝑑[1 + 𝑐sin2 (2𝜋𝐿𝑡) (1 + cos (2𝜋𝑡))] . (61) We obtain a periodic system of period 1. In this context, (𝑥𝑛, 𝑦𝑛) = (𝑎, 𝑏), 𝑛∈N, and (𝑥(𝑡), 𝑦(𝑡)) = (𝑎, 𝑏), 𝑡∈R, where We obtain a periodic system of period 1. 4. Consistency We have that g We have that By (53) we conclude that, given 𝛿> 0, there is 𝑟𝑚∈N such that, for all 𝑟⩾𝑟𝑚, 𝑛𝑚,𝑟+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟 ℎ𝑚𝑓(𝑘ℎ𝑚) + (𝜆−⌊𝜆 ℎ𝑚 ⌋ℎ𝑚) 𝑓(𝑛𝑚,𝑟ℎ𝑚+ ⌊𝜆 ℎ𝑚 ⌋ℎ𝑚) (46) that, for all 𝑟⩾𝑟𝑚, 𝜙(ℎ𝑚) 𝑛𝑚,𝑟𝑚+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟𝑚 ℎ𝑚𝑓(𝑘ℎ𝑚) < 𝜙(ℎ𝑚) [R𝑢 𝐶(𝜆) + 𝛿+ 𝐶(𝜆+ 1) ℎ𝑚] . (54) (54) < 𝜙(ℎ𝑚) [R𝑢 𝐶(𝜆) + 𝛿+ 𝐶(𝜆+ 1) ℎ𝑚] . is a Riemann sum of Finally, recalling that R𝑢 𝐶(𝜆) < 0, by assumption, by the arbitrariness of 𝛿> 0 and the fact that ℎ𝑚→0 as 𝑚→+∞, we obtain for sufficiently large 𝑚∈N, ∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝛽(𝑠) 𝜑(𝑎) + 𝜎(𝑠) 𝜓(𝑏) −[𝜇(𝑠) + 𝛼(𝑠) + 𝛾(𝑠)] 𝑑𝑠 (47) Finally, recalling that R𝐶(𝜆) < 0, by assumption, by the arbitrariness of 𝛿> 0 and the fact that ℎ𝑚→0 as 𝑚→+∞, we obtain for sufficiently large 𝑚∈N, 0 ⩽𝜙(ℎ𝑚) 𝑛𝑚,𝑟𝑚+⌊𝜆/ℎ𝑚⌋ ∑ ℎ𝑚𝑓(𝑘ℎ𝑚) < 0, (55) ∫ 𝑛𝑚,𝑟ℎ𝑚+𝜆 𝑛𝑚,𝑟ℎ𝑚 𝛽(𝑠) 𝜑(𝑎) + 𝜎(𝑠) 𝜓(𝑏) −[𝜇(𝑠) + 𝛼(𝑠) + 𝛾(𝑠)] 𝑑𝑠 (47) 0 ⩽𝜙(ℎ𝑚) 𝑛𝑚,𝑟𝑚+⌊𝜆/ℎ𝑚⌋ ∑ 𝑘=𝑛𝑚,𝑟𝑚 ℎ𝑚𝑓(𝑘ℎ𝑚) < 0, (55) (55) with respect to the partition which is a contradiction. We obtain (a). {𝑛𝑚,𝑟ℎ𝑚, 𝑛𝑚,𝑟ℎ𝑚+ ℎ𝑚, . . . , 𝑛𝑚,𝑟ℎ𝑚 ⌊𝜆⌋ } (48) which is a contradiction. We obtain (a). A similar argument allows us to prove (b). In fact, assuming by contradiction that {𝑛𝑚,𝑟ℎ𝑚, 𝑛𝑚,𝑟ℎ𝑚+ ℎ𝑚, . . . , 𝑛𝑚,𝑟ℎ𝑚 (48) ( ) A similar argument allows us to prove (b). In fact, assuming by contradiction that (48) ( ) A similar argument allows us to prove (b). In fact, assuming by contradiction that { , , , + ⌊𝜆 ℎ𝑚 ⌋ℎ𝑚, 𝑛𝑚,𝑟ℎ𝑚+ 𝜆} (48) A similar argument allows us to prove (b). In fact, assuming by contradiction that Rℓ 𝐶(𝜆) > 0, (56) (48) + ⌊𝜆 ℎ𝑚 ⌋ℎ𝑚, 𝑛𝑚,𝑟ℎ𝑚+ 𝜆} + ⌊𝜆 ℎ𝑚 ⌋ℎ𝑚, 𝑛𝑚,𝑟ℎ𝑚+ 𝜆} ( ) Rℓ 𝐶(𝜆) > 0, (56) + ⌊𝜆 ℎ𝑚 ⌋ℎ𝑚, 𝑛𝑚,𝑟ℎ𝑚+ 𝜆} Rℓ 𝐶(𝜆) > 0, Rℓ 𝐶(𝜆) > 0, (56) (56) Discrete Dynamics in Nature and Society 8 and that there is a sequence (ℎ𝑚)𝑚∈N such that ℎ𝑚→0 as 𝑚→+∞and are, respectively, solutions of system (12) and system (3). It is now possible to compute the number Rℓ 𝐶(1). 5. Simulation Our objective in this section is twofold. On the one hand, we want to consider different incidence functions 𝜑, correspond- ing to different discretizations of our continuous model, and (𝑎, 𝑏) = ( 𝜇+ 𝜂 𝜇+ 𝜂+ 𝑝, 𝑝 𝜇+ 𝜂+ 𝑝) , (62) (62) Discrete Dynamics in Nature and Society 9 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 R–K Mick 20 40 60 80 100 0.2 0.4 0.6 0.8 Figure 1: Left: function 𝛽; right: inconsistency (time step=1/6). Mick–Zhang Mickens 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 R–K Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Figure 2: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 4, 1, 0.5. Discrete Dynamics in Nature and Society 9 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 R–K Mick 20 40 60 80 100 0.2 0.4 0.6 0.8 Figure 1: Left: function 𝛽; right: inconsistency (time step=1/6). 9 Discrete Dynamics in Nature and Society 9 R–K Mick 20 40 60 80 100 0.2 0.4 0.6 0.8 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 Figure 1: Left: function 𝛽; right: inconsistency (time step=1/6). Mick–Zhang Mickens 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 R–K Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Figure 2: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 4, 1, 0.5. 0 10 20 30 40 50 0.2 0.4 0.6 0.8 10 Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Figure 2: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 4, 1, 0.5. than 0.05. Clearly, there is numerical evidence that there is consistency even for higher time steps. Figure 2 illustrates this situation. ℓ compare the several discrete models obtained. We do this in the first subsection. On the other hand, we want to use our model to describe a real situation. We do this in the second subsection where we consider data from the incidence of measles in France in the period 2012-2016. Changing 𝑏to 0.9 we obtain Rℓ 𝐶(4) = 3.4 > 0 and thus we conclude that we have persistence. 5. Simulation Taking time steps equal to 2, 1, and 0.5, we get Rℓ 𝐷(1, 2) = 3.201 > 1, Rℓ 𝐷(3, 1) = 5.9 > 1, and Rℓ 𝐷(7, 0.5) = 10.2 > 1 and we conclude that we have persistence for all these time steps. Figure 3 illustrates this situation. Figures 2 and 3 suggest that numerically our model is slightly better than Zhang’s model, at least for large time steps. 5.1. Simulation with Several NSFD Schemes. In this subsec- tion we do some simulation to illustrate our results. To begin, we compare our model (1) with mass action incidence (𝜑(𝑆, 𝐼) = 𝑆𝐼and 𝜓(𝑉, 𝐼) = 𝑉𝐼) with Zhang’s model [20]. We use the following set of parameters: 𝜙(ℎ) = ℎ+0.2ℎ2, Λ = 0.5, 𝜇(𝑡) = 𝛾(𝑡) = 𝛿(𝑡) = 0.3, 𝛼(𝑡) = 0.05, 𝜂= 0.05, 𝑝= 2/3 and Next, we compare our model with the discretized model obtained by Euler method and the output of the Mathematica solver ODE (that uses a Runge-Kutta method). Considering 𝑏= 0.3, we get extinction for the continuous-time model, as we already saw. Taking time steps equal to 2, 1, and 0.5, we can see in Figure 4 that for all methods considered and all time steps we have extinction, although the behaviour of our model shadows better the behaviour given by Mathematica’s solver, at least for these time steps. 𝛽(𝑡) = 𝜎(𝑡) = 𝑏(1 + 0.3 cos (𝑡𝜋 2 )) . (66) (66) Setting 𝑏= 0.3 we obtain R𝑢 𝐶(4) = −0.6 < 0 and thus we conclude that we have extinction for the continuous model. Taking time steps equal to 4, 1, and 0.5, we get R𝑢 𝐷(0, 4) = Rℓ 𝐷(0, 4) = 1, R𝑢 𝐷(3, 1) = 0.644 < 1, and R𝐷(7, 0.5) = 0.601 < 1 and we conclude that we have extinction for time steps 1 and 0.5. For these parameters, we have consistency in the sense of Theorem 9 as long as the time step is less Changing 𝑏to 0.9 we already saw that we get persistence for the continuous model. Figure 5 illustrates this situation. 5. Simulation 10 Discrete Dynamics in Nature and Society 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Figure 3: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 4: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 Discrete Dynamics in Nature and Society 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Figure 3: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 4: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. Euler Mickens 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 R–K Euler Mickens R–K Euler Mickens R–K Figure 5: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. Next, we change our incidence function and consider 𝜙(𝑆𝐼) = 𝑆𝐼/(1 + 0 7𝐼) maintaining the set of parameters extinction/persistence, relation to Zhang’s model and the model obtained by Euler method 10 Discrete Dynamics in Nature and Society 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 10 20 30 40 50 0.2 0.4 0.6 0.8 Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Mick–Zhang Mickens R–K Figure 3: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. Discrete Dynamics in Nature and Society 10 10 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 4: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. .5. 5. Simulation 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 7: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 20 30 40 50 0.2 0.4 0.6 0.8 Real Data Simulation 0 10 20 30 40 50 60 50 100 150 200 F 8 M l (2012 2016) i l ti our parameters estimation, we gathered information from several websites. We considered standard incidence functions 𝜓(𝑉𝑛+1, 𝐼𝑛) = 𝑉𝑛+1𝐼𝑛/𝑃𝑛and 𝜑(𝑆𝑛+1, 𝐼𝑛) = 𝑆𝑛+1𝐼𝑛/𝑃𝑛, where 𝑃𝑛is the total population. Inspired in the time series for the infectives (https://ecdc.europa.eu), we considered 𝜎𝑛= 0.03 and 𝛽𝑛given by our parameters estimation, we gathered information from several websites. We considered standard incidence functions 𝜓(𝑉𝑛+1, 𝐼𝑛) = 𝑉𝑛+1𝐼𝑛/𝑃𝑛and 𝜑(𝑆𝑛+1, 𝐼𝑛) = 𝑆𝑛+1𝐼𝑛/𝑃𝑛, where 𝑃𝑛is the total population. Inspired in the time series for the infectives (https://ecdc.europa.eu), we considered 𝜎𝑛= 0.03 and 𝛽𝑛given by 𝛽𝑛= { { { { { 3.8 + 10 sin ((𝑛+ 1) 𝜋 6 ) , if ⌊𝑛 12⌋⩽5 2.7, otherwise. (67) (67) The remaining parameters were considered time indepen- dent and were inspired in data contained in the web- sites http://www.worldbank.org, https://data.oecd.org, and http://www.geoba.se. Namely, we took the mortality rate 𝜇𝑛= 0.0007, the newborns Λ 𝑛 = 50000, the disease induced mortality 𝛼𝑛= 0.000375, the immunity loss 𝜂𝑛= 0.001, the vaccination rate 𝑝𝑛= 0.001, and the recovery rate 𝛾𝑛= 0.957. We used the initial conditions 𝑆0 = 7.20428 × 106, 𝐼0 = 106, 𝑉0 = 5.84372 × 107, and 𝑅0 = 1.81918 × 104. In Figure 8, we plot the real data for the infectives and the output given by our model. Figure 8: Measles (2012-2016), simulation. 5. Simulation 10 20 30 40 50 0.2 0.4 0.6 0.8 Euler Mickens R–K 5. 10 20 30 40 50 0.2 0.4 0.6 0.8 Euler Mickens R–K Figure 5: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 20 30 40 50 0.2 0.4 0.6 0.8 Figure 5: 𝑆𝐼; 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. extinction/persistence, relation to Zhang’s model and the model obtained by Euler method. extinction/persistence, relation to Zhang’s model and the model obtained by Euler method. Next, we change our incidence function and consider 𝜙(𝑆, 𝐼) = 𝑆𝐼/(1 + 0.7𝐼), maintaining the set of parameters. Letting 𝑏= 0.3 we have extinction for the continuous model and letting 𝑏= 0.9 we have persistence for the continuous- time model. Note that the thresholds R𝑢 𝐶, Rℓ 𝐶, R𝑢 𝐷, and Rℓ 𝐷 are similar to the mass action case. Figures 6 and 7 illustrate this situation. 5.2. Simulation with Real Data. In this subsection, we present some simulation regarding measles. This disease is endemic in some countries such as France. In that country, with the measles outbreak in 2011, a vaccination policy that lowered the number of reported cases was introduced. We will focus on measles in France, between 2012 and 2016. For a study concerning the period before 2012, see [36]. For Doing corresponding simulations and comparisons for our model with 𝜙(ℎ) = (1−e−0.002ℎ)/(0.002) instead of 𝜙(ℎ) = ℎ+ 0.2ℎ2, we can draw the same conclusions regarding Dynamics in Nature and Society 11 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 6: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 7: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. meters estimation, we gathered information from ebsites. We considered standard incidence functions 𝑛) = 𝑉𝑛+1𝐼𝑛/𝑃𝑛and 𝜑(𝑆𝑛+1, 𝐼𝑛) = 𝑆𝑛+1𝐼𝑛/𝑃𝑛, where total population. Inspired in the time series for the s (https://ecdc.europa.eu), we considered 𝜎𝑛= 0.03 ven by { { { { { 3.8 + 10 sin ((𝑛+ 1) 𝜋 6 ) , if ⌊𝑛 12⌋⩽5 2.7, otherwise. 5. Simulation (67) aining parameters were considered time indepen- d were inspired in data contained in the web- p://www.worldbank.org, https://data.oecd.org, and ww.geoba.se. Namely, we took the mortality rate 𝜇𝑛= he newborns Λ 𝑛 = 50000, the disease induced Real Data Simulation 0 10 20 30 40 50 60 50 100 150 200 Figure 8: Measles (2012-2016), simulation. Discrete Dynamics in Nature and Society 11 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 6: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 7: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 11 10 20 30 40 50 0.2 0.4 0.6 0.8 1.0 Discrete Dynamics in Nature and Society 11 11 Discrete Dynamics in Nature and Society 11 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 Euler Mickens R–K Euler Mickens R–K Euler Mickens R–K Figure 6: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 11 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 1.0 ns Euler Mickens R–K 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 Euler Mickens R–K ens 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. om ons ere he 03 67) en- eb- nd 𝑛= ed Real Data Simulation 0 10 20 30 40 50 60 50 100 150 200 Figure 8: Measles (2012-2016), simulation. 10 20 30 40 50 0.2 0.4 0.6 0.8 1.0 Figure 6: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. Figure 6: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 10 20 30 40 50 10 20 30 40 50 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 Euler Mickens R–K Euler Mickens R–K Figure 7: 𝑆𝐼/(1 + 0.7𝐼); 𝜙(ℎ) = ℎ+ 0.2ℎ2; step-size: 2, 1, 0.5. 6. Conclusions We considered a discretization procedure, based on Mick- ens NSFD scheme, to get a discrete-time model from a continuous time with vaccination and incidence given by a general function. For a family of models containing the previous discrete-time model, we achieved results on the persistence and the extinction of the disease (Theorems 6 and 7). They contain the results of Zhang [20] as a particular case. Can be seen, in a general way, that our model behaves in the same manner as the real data. It seems that if the vaccination policy in France continues to be very strict, it may decrease the number of cases. Discrete Dynamics in Nature and Society 12 and to carry out some numerical analysis in our general nonautonomous setting. and to carry out some numerical analysis in our general nonautonomous setting. Our threshold conditions depend on the parameters of the model and of the incidence function derivative, with respect to the infectives, computed on some disease-free solution. This agrees with the continuous counterparts of these results [8]. Appendix We also considered the problem of establishing the consistency of the continuous-time model and the discrete- time model for small time steps, in the sense that if the time step is small enough when we have persistence (extinction) for the continuous-time model we also have persistence (extinction) for the discrete-time model (at least for situations where Theorems 1 and 2 allow us to conclude that we have persistence or extinction). Assuming the differentiabil- ity of parameters, our result on this direction, Theorem 9, furnishes an interval [0, 𝑎], where 𝑎depends only on the parameters of the model and their derivatives, where there is consistency. Proof of the Results in Section 3 We begin this section by noting a simple consequence of our assumptions that will be used several times throughout the proofs: it follows from (H3) that there are constants 𝐾> 0 and 𝜃∈]0, 1[ such that 𝑛−1 ∏ 𝑘=𝑚 1 1 + 𝜇𝑘 < 𝐾𝜃𝑛−𝑚, (A.1)fi (A.1) for 𝑚, 𝑛∈N sufficiently large. Additionally, using (H1), (H2), and (H5), we have for 𝑚, 𝑛∈N sufficiently large. Additionally, using (H1), (H2), and (H5), we have 𝜑(𝑥, 𝑦) 𝑦 = 𝜑(𝑥, 𝑦) −𝜑(𝑥, 0) 𝑦−0 ⩽𝜑(𝑥, 𝑦) −𝜑(𝑥, 0) 𝑦−0 ⩽𝜕2𝜑(𝑥, 0) = 󵄨󵄨󵄨󵄨𝜕2𝜑(𝑥, 0) −𝜕2𝜑(0, 0)󵄨󵄨󵄨󵄨 ⩽𝑘𝜑𝑥 (A.2) We present an example of a periodic system of period 1 where the continuous and the discrete-time system with time step ℎ= 1/𝐿are not consistent. Namely, for that time step, we will have persistence for the continuous-time model and extinction for the discrete-time model. These examples show the importance of knowing that for time steps smaller than some explicit value we have consistency, a type of result like the one in Theorem 9. (A.2) and thus and thus 𝜑(𝑥, 𝑦) ⩽𝑘𝜑𝑥𝑦. (A.3) Finally, we carried out some simulations to illustrate our results. As one might expect our simulations furnish evidence that we may have consistency in intervals whose lengths are several times bigger than the length of the given interval in Theorem 9. Additionally, we used our model to describe a real situation, namely, the case of measles incidence in France in the period 2012-2016, and compared our results with the real- time series for the infectives. We found in general that the predictive behaviour of our model is very similar to the real data. (A.3) Similarly Similarly 𝜓(𝑥, 𝑦) ⩽𝑘𝜓𝑥𝑦. (A.4) (A.4) We will now proceed with the proof of the results in Section 3. Proof of Lemma 4. Let 𝑆𝑛> 0, 𝐼𝑛> 0, 𝑅𝑛> 0, and 𝑉𝑛> 0. By (1), (A.3), and (A.4), we obtain We remark that this work is far from giving a complete answer to the problem of consistency between our discrete and continuous-time models. Proof of the Results in Section 3 To show that Rℓ 𝐷(𝜉∗, 𝜆) is independent of the selection of 𝜉∗= (𝑥∗ 𝑛, 𝑦∗ 𝑛), a fixed solution of (12), it is important to note that according to (ii) in Lemma 3, for any 𝜀> 0 and any solution 𝜉= (𝑥𝑛, 𝑦𝑛) of system (12) with initial value 𝑥0 > 0, 𝑦0 > 0, there exists an 𝑁∈N+ such that, for 𝑘⩾𝑁, we have |𝑥𝑘−𝑥∗ 𝑘| ⩽𝜀and |𝑦𝑘−𝑦∗ 𝑘| ⩽𝜀. Hence, where where Θ𝜀= ( 𝜆+ 1 𝜆) 𝑟𝜆𝐶𝜀+ . . . + ( 𝜆+ 1 1 ) 𝑟𝐶𝜆𝜀𝜆 + 𝐶𝜆+1𝜀𝜆+1. (A.17) Θ𝜀= ( 𝜆+ 1 𝜆) 𝑟𝜆𝐶𝜀+ . . . + ( 𝜆+ 1 1 ) 𝑟𝐶𝜆𝜀𝜆 (A.17) (A.17) + 𝐶𝜆+1𝜀𝜆+1. 𝑥∗ 𝑘−𝜀⩽𝑥𝑘⩽𝑥∗ 𝑘+ 𝜀 𝑦∗ 𝑘−𝜀⩽𝑦𝑘⩽𝑦∗ 𝑘+ 𝜀. (A.9) Analogously Analogously (A.9) 𝑛+𝜆 ∏ 𝑘=𝑛 (𝑟𝑘− 𝐿𝑘𝜀 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 ) ⩾ 𝑛+𝜆 ∏ 𝑘=𝑛 𝑟𝑘−Θ𝜀. (A.18) (A.18) By (H1), we have 󵄨󵄨󵄨󵄨𝜕2𝜑(𝑥𝑘, 0) −𝜕2𝜑(𝑥∗ 𝑘, 0)󵄨󵄨󵄨󵄨⩽𝑘𝜑󵄨󵄨󵄨󵄨𝑥𝑘−𝑥∗ 𝑘 󵄨󵄨󵄨󵄨⩽𝑘𝜑𝜀, 󵄨󵄨󵄨󵄨𝜕2𝜓(𝑦𝑘, 0) −𝜕2𝜓(𝑦∗ 𝑘, 0)󵄨󵄨󵄨󵄨⩽𝑘𝜓󵄨󵄨󵄨󵄨𝑦𝑘−𝑦∗ 𝑘 󵄨󵄨󵄨󵄨⩽𝑘𝜓𝜀. (A.10) So, By (A.12), we obtain −Θ𝜀+ lim inf 𝑛→+∞ 𝑛+𝜆 ∏ 𝑘=𝑛 𝑟𝑘⩽Rℓ 𝐷(𝜉∗, 𝜆) (A 19) 󵄨󵄨󵄨󵄨𝜕2𝜑(𝑥𝑘, 0) −𝜕2𝜑(𝑥∗ 𝑘, 0)󵄨󵄨󵄨󵄨⩽𝑘𝜑󵄨󵄨󵄨󵄨𝑥𝑘−𝑥∗ 𝑘 󵄨󵄨󵄨󵄨⩽𝑘𝜑𝜀, 󵄨󵄨󵄨󵄨𝜕2𝜓(𝑦𝑘, 0) −𝜕2𝜓(𝑦∗ 𝑘, 0)󵄨󵄨󵄨󵄨⩽𝑘𝜓󵄨󵄨󵄨󵄨𝑦𝑘−𝑦∗ 𝑘 󵄨󵄨󵄨󵄨⩽𝑘𝜓𝜀. (A.10 By (A.12), we obtain By (A.12), we obtain (A.10) −Θ𝜀+ lim inf 𝑛→+∞ 𝑛+𝜆 ∏ 𝑘=𝑛 𝑟𝑘⩽Rℓ 𝐷(𝜉∗, 𝜆) So, So, (A.19) 𝜕2𝜑(𝑥∗ 𝑘, 0) −𝑘𝜑𝜀⩽𝜕2𝜑(𝑥𝑘, 0) ⩽𝜕2𝜑(𝑥∗ 𝑘, 0) + 𝑘𝜑𝜀, 𝜕2𝜓(𝑦∗ 𝑘, 0) −𝑘𝜓𝜀⩽𝜕2𝜓(𝑦𝑘, 0) ⩽𝜕2𝜓(𝑦∗ 𝑘, 0) + 𝑘𝜓𝜀. (A.11) ⩽Θ𝜀+ lim inf 𝑛→+∞ 𝑛+𝜆 ∏ 𝑘=𝑛 𝑟𝑘. (A.19) Thus, 𝜕2𝜑(𝑥∗ 𝑘, 0) −𝑘𝜑𝜀⩽𝜕2𝜑(𝑥𝑘, 0) ⩽𝜕2𝜑(𝑥∗ 𝑘, 0) + 𝑘𝜑𝜀, 𝜕2𝜓(𝑦∗ 𝑘, 0) −𝑘𝜓𝜀⩽𝜕2𝜓(𝑦𝑘, 0) ⩽𝜕2𝜓(𝑦∗ 𝑘, 0) + 𝑘𝜓𝜀. (A.1 (A.11) Thus, 󵄨󵄨󵄨󵄨󵄨Rℓ 𝐷(𝜉∗, 𝜆) −Rℓ 𝐷(𝜉, 𝜆)󵄨󵄨󵄨󵄨󵄨< Θ𝜀 (A.20) (A.20) Combining the previous computations, we get Combining the previous computations, we get 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝐿𝑘𝜀 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 and, by the arbitrariness of 𝜀, we obtain Rℓ 𝐷(𝜉, 𝜆) = Rℓ 𝐷(𝜉∗, 𝜆). Replacing lim inf by lim sup in the preceding argument, we reach a similar conclusion for R𝑢 𝐷(𝜉∗, 𝜆). The result follows. and, by the arbitrariness of 𝜀, we obtain Rℓ 𝐷(𝜉, 𝜆) = Rℓ 𝐷(𝜉∗, 𝜆). Replacing lim inf by lim sup in the preceding argument, we reach a similar conclusion for R𝑢 𝐷(𝜉∗, 𝜆). The result follows. ⩽1 + 𝛽𝑘𝜕2𝜑(𝑥𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦𝑘+1, 0) 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 (A.12) (A.12) Proof of Theorem 6. Proof of the Results in Section 3 Moreover, in addition to a further discussion of the qualitative behaviour of the con- tinuous and discrete systems, in future work it would be very interesting to discuss the convergence of the methods 𝑉𝑛+1 = 𝑉𝑛+ 𝑝𝑛𝑆𝑛+1 1 + 𝜇𝑛+ 𝜂𝑛+ 𝜎𝑛𝑘𝜓𝐼𝑛 (A.5) (A.5) and thus and thus 𝑆𝑛+1 ⩾ 𝜂𝑛(𝑉𝑛+ Λ 𝑛+ 𝑆𝑛) + (Λ 𝑛+ 𝑆𝑛) (1 + 𝜇𝑛+ 𝜎𝑛𝑘𝜓𝐼𝑛) (1 + 𝜇𝑛+ 𝛽𝑛𝑘𝜑𝐼𝑛) (1 + 𝜇𝑛+ 𝜂𝑛+ 𝜎𝑛𝑘𝜑𝐼𝑛) + 𝑝𝑛(1 + 𝜇𝑛+ 𝜎𝑛𝑘𝜓𝐼𝑛) . (A.6) (A.6) 𝐼𝑛> 0, 𝑅𝑛> 0, and 𝑉𝑛> 0 for all 𝑛∈N. In the same way, we can conclude that, if 𝑆0 ⩾0, 𝐼0 ⩾0, 𝑅0 ⩾0 and 𝑉0 ⩾0, then 𝑆𝑛⩾0. 𝐼𝑛⩾0, 𝑅𝑛⩾0 and 𝑉𝑛⩾0 for all 𝑛∈N. This proves (i) and (ii) in Lemma 4. By (1) we have Therefore, we conclude that 𝑆𝑛+1 > 0 and 𝑉𝑛+1 > 0. By the second and third equations in (1) we obtain 𝐼𝑛+1 ⩾ 𝐼𝑛 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 , 𝑅𝑛+1 = 𝛾𝑛𝐼𝑛+1 + 𝑅𝑛 1 + 𝜇𝑛 (A.7) ( ) By (1), we have (A.7) 𝑁𝑛+1 ⩽ Λ 𝑛 1 + 𝜇𝑛 + 𝑁𝑛 1 + 𝜇𝑛 , (A.8) (A.8) and we conclude that 𝐼𝑛+1 > 0 and 𝑅𝑛+1 > 0. The previous inequalities allow us to conclude by induction that 𝑆𝑛> 0, where 𝑁𝑛= 𝑆𝑛+𝐼𝑛+𝑅𝑛+𝑉𝑛is the total population. By Lemma 2 in [21] we obtain the result. where 𝑁𝑛= 𝑆𝑛+𝐼𝑛+𝑅𝑛+𝑉𝑛is the total population. By Lemma 2 in [21] we obtain the result. Discrete Dynamics in Nature and Society 13 Proof of Lemma 5. To show that Rℓ 𝐷(𝜉∗, 𝜆) is independent of the selection of 𝜉∗= (𝑥∗ 𝑛, 𝑦∗ 𝑛), a fixed solution of (12), it is important to note that according to (ii) in Lemma 3, for any 𝜀> 0 and any solution 𝜉= (𝑥𝑛, 𝑦𝑛) of system (12) with initial value 𝑥0 > 0, 𝑦0 > 0, there exists an 𝑁∈N+ such that, for 𝑘⩾𝑁, we have |𝑥𝑘−𝑥∗ 𝑘| ⩽𝜀and |𝑦𝑘−𝑦∗ 𝑘| ⩽𝜀. Hence, Proof of Lemma 5. Proof of the Results in Section 3 First note that the original system (1) can be rewritten as follows: Proof of Theorem 6. First note that the original system (1) can be rewritten as follows: ⩽1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) + 𝐿𝑘𝜀 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 , 𝑆𝑛+1 = 1 1 + 𝜇𝑛+ 𝑝𝑛 (Λ 𝑛+ 𝑆𝑛−𝛽𝑛𝜑(𝑆𝑛+1, 𝐼𝑛) + 𝜂𝑛𝑉𝑛+1) 𝐼𝑛+1 = 1 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 (𝛽𝑛𝜑(𝑆𝑛+1, 𝐼𝑛) + 𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) + 𝐼𝑛) 𝑅𝑛+1 = 1 1 + 𝜇𝑛 (𝛾𝑛𝐼𝑛+1 + 𝑅𝑛) 𝑉𝑛+1 = 1 1 + 𝜇𝑛+ 𝜂𝑛 (𝑝𝑛𝑆𝑛+1 −𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) + 𝑉𝑛) , (A.21) where 𝐿𝑘= 𝛽𝑘𝑘𝜑+ 𝜎𝑘𝑘𝜓. Let where 𝐿𝑘= 𝛽𝑘𝑘𝜑+ 𝜎𝑘𝑘𝜓. Let 𝑘 Let 𝑟𝑘= 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 . (A.13) 𝐼𝑛+1 = 1 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 (𝛽𝑛𝜑(𝑆𝑛+1, 𝐼𝑛) + 𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) + 𝐼𝑛) 𝑅𝑛+1 = 1 1 + 𝜇𝑛 (𝛾𝑛𝐼𝑛+1 + 𝑅𝑛) 𝑉𝑛+1 = 1 1 + 𝜇𝑛+ 𝜂𝑛 (𝑝𝑛𝑆𝑛+1 −𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) + 𝑉𝑛) , (A.21) (A.21) Using (H1) and (iv) in Lemma 3, it is easy to see that Using (H1) and (iv) in Lemma 3, it is easy to see that 𝑘⩽ 1 + 2𝛽𝑢𝑘𝜑𝑀+ 2𝜎𝑢𝑘𝜓𝑀 1 + 𝜇𝑙+ 𝛼𝑙+ 𝛾𝑙 š 𝑟, (A.14) large 𝑘∈N, and that 𝑅𝑛+1 = 1 1 + 𝜇𝑛 (𝛾𝑛𝐼𝑛+1 + 𝑅𝑛) 𝑉 = 1 (𝑝𝑆 −𝜎 𝑟𝑘⩽ 1 + 2𝛽𝑢𝑘𝜑𝑀+ 2𝜎𝑢𝑘𝜓𝑀 1 + 𝜇𝑙+ 𝛼𝑙+ 𝛾𝑙 š 𝑟, (A.14) 𝑟𝑘⩽ 1 + 2𝛽𝑢𝑘𝜑𝑀+ 2𝜎𝑢𝑘𝜓𝑀 1 + 𝜇𝑙+ 𝛼𝑙+ 𝛾𝑙 š 𝑟, (A.14) ntly large 𝑘∈N, and that 𝑅𝑛+1 = 1 1 + 𝜇𝑛 (𝛾𝑛𝐼𝑛+1 + 𝑅𝑛) 𝑉𝑛+1 = 1 1 + 𝜇𝑛+ 𝜂𝑛 (𝑝𝑛𝑆𝑛+1 −𝜎𝑛𝜓(𝑉𝑛+1, 𝐼 for sufficiently large 𝑘∈N, and that for sufficiently large 𝑘∈N, and that 𝐿= 𝐿𝑘 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 ⩽ 𝛽𝑢𝑘𝜑+ 𝜎𝑢𝑘𝜓 1 + 𝜇𝑙+ 𝛼𝑙+ 𝛾𝑙š 𝐶. (A.15) o, for sufficiently large 𝑛, 𝑛= 0, 1, . . .. Firstly, we will establish (a). Since R𝑢 𝐷(𝜆) < 1, we can choose 𝜀0 > 0, 𝜀∈]0, 1[ and a sufficiently large integer 𝑁1 ∈N such that 𝐿= 𝐿𝑘 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 ⩽ 𝛽𝑢𝑘𝜑+ 𝜎𝑢𝑘𝜓 1 + 𝜇𝑙+ 𝛼𝑙+ 𝛾𝑙š 𝐶. (A.15) o, for sufficiently large 𝑛, 𝑛= 0, 1, . . .. Firstly, we will establish (a). Proof of the Results in Section 3 Since R𝑢 𝐷(𝜆) < 1, we can choose 𝜀0 > 0, 𝜀∈]0, 1[ and a sufficiently large integer 𝑁1 ∈N such that So, for sufficiently large 𝑛, So, for sufficiently large 𝑛, 𝑛+𝜆 ∏ 𝑘=𝑛 (𝑟𝑘+ 𝐿𝑘𝜀 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 ) ⩽ 𝑛+𝜆 ∏ 𝑘=𝑛 (𝑟𝑘+ 𝐶𝜀) 6) 𝑛+𝜆 ∏ 𝑘=𝑛 1 + 𝛽𝑘𝜕2𝜑(𝑥𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦𝑘+1, 0) + (𝛽𝑢𝑘𝜑+ 𝜎𝑢𝑘𝜓) 𝜀0 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 < 𝜀 (A.22) for all 𝑛⩾𝑁1. (A.22) (A.16) (A.16) = 𝑛+𝜆 ∏ 𝑘=𝑛 𝑟𝑘+ Θ𝜀 for all 𝑛⩾𝑁1. 14 14 Discrete Dynamics in Nature and Society For any solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) of (A.21) with initial con- ditions 𝑆0 > 0, 𝐼0 > 0, 𝑅0 > 0 and 𝑉0 > 0, we have 𝜕2𝜓(𝑉𝑛+1, 0) ⩽𝜕2𝜓(𝑦∗ 𝑛+1, 0) + 𝑘𝜓𝜀0. (A.30)h erefore by (A 22) (A 26) (A 27) (A 29) and (A 30) we 𝜕2𝜓(𝑉𝑛+1, 0) ⩽𝜕2𝜓(𝑦∗ 𝑛+1, 0) + 𝑘𝜓𝜀0. (A.30) (A.30) 𝑆𝑛+1 ⩽Λ 𝑛+ 𝜂𝑛𝑉𝑛+1 + 𝑆𝑛 1 + 𝜇𝑛+ 𝑝𝑛 𝑉𝑛+1 ⩽𝑝𝑛𝑆𝑛+1 + 𝑉𝑛 1 + 𝜇𝑛+ 𝜂𝑛 . (A.23) (A.23) (A.23)h have 𝐼𝑛+1 By the comparison principle, we obtain 𝑆𝑛⩽𝑥𝑛and 𝑉𝑛⩽ 𝑦𝑛for all 𝑛∈N, where (𝑥𝑛, 𝑦𝑛) is the solution of (12) with initial condition (𝑥0, 𝑦0) = (𝑆0, 𝑉0). According to Lemma 3, the solution (𝑥∗ 𝑛, 𝑦∗ 𝑛) is globally uniformly attractive and thus, for the aforementioned 𝜀0 > 0, there exists an 𝑁2 ∈N such that ⩽𝜀𝐼𝑛, for all 𝑛⩾𝑁2. We conclude that 𝐼𝑚⩽𝜀𝑚−𝑁2𝐼𝑁2 →0 as 𝑚→ ∞. This completes the proof of (a). for all 𝑛⩾𝑁2. We conclude that 𝐼𝑚⩽𝜀𝑚−𝑁2𝐼𝑁2 →0 as 𝑚→ ∞. This completes the proof of (a). h p p Next, to establish (b), let us consider two arbitrary solu- tions of the original system (𝑆(1) 𝑛, 𝐼(1) 𝑛, 𝑉(1) 𝑛, 𝑅(1) 𝑛) and (𝑆(2) 𝑛, 𝐼(2) 𝑛, 𝑉(2) 𝑛, 𝑅(2) 𝑛) and 𝜆a constant such that 𝑅𝑢 0(𝜆) < 1. Let 𝜄𝑛= 𝐼(1) 𝑛− 𝐼(2) 𝑛 and 𝜌𝑛= 𝑅(1) 𝑛−𝑅(2) 𝑛. By (9), we have 󵄨󵄨󵄨󵄨𝑥𝑛−𝑥∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, 󵄨󵄨󵄨󵄨𝑦𝑛−𝑦∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0 󵄨󵄨󵄨󵄨𝑥𝑛−𝑥∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, 󵄨󵄨󵄨󵄨𝑦𝑛−𝑦∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0 (A.24) (A.24) ∀𝑛⩾𝑁2 𝜌𝑛+1 −𝜌𝑛= (𝑅(1) 𝑛+1 −𝑅(1) 𝑛) −(𝑅(2) 𝑛+1 −𝑅(2) 𝑛) = 𝛾𝑛(𝐼(1) 𝑛+1 −𝐼(2) 𝑛+1) −𝜇𝑛(𝑅(1) 𝑛+1 −𝑅(2) 𝑛+1) = 𝛾𝑛𝜄𝑛+1 −𝜇𝑛𝜌𝑛+1. (A.32) 𝜌𝑛+1 −𝜌𝑛= (𝑅(1) 𝑛+1 −𝑅(1) 𝑛) −(𝑅(2) 𝑛+1 −𝑅(2) 𝑛) From this, it may be concluded that From this, it may be concluded that 𝑆𝑛⩽𝑥∗ 𝑛+ 𝜀0, 𝑉𝑛⩽𝑦∗ 𝑛+ 𝜀0 ∀𝑛⩾𝑁2. Proof of the Results in Section 3 (A.25) 𝑆𝑛⩽𝑥∗ 𝑛+ 𝜀0, 𝑉𝑛⩽𝑦∗ 𝑛+ 𝜀0 𝑉𝑛⩽𝑦∗ 𝑛+ 𝜀0 (A.25) = 𝛾𝑛𝜄𝑛+1 −𝜇𝑛𝜌𝑛+1. ∀𝑛⩾𝑁2. Because 𝑅𝑢 0(𝜆) < 1, we conclude that 𝜄𝑛→0 as 𝑛→+∞and therefore, given 𝜀> 0, there is 𝑁∈N sufficiently large such that, for 𝑛⩾𝑁, Because 𝑅𝑢 0(𝜆) < 1, we conclude that 𝜄𝑛→0 as 𝑛→+∞and therefore, given 𝜀> 0, there is 𝑁∈N sufficiently large such that, for 𝑛⩾𝑁, By the second equation of (1) we get By the second equation of (1) we get 𝐼𝑛+1 = 1 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 (𝛽𝑛𝜑(𝑆𝑛+1, 𝐼𝑛) + 𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) + 𝐼𝑛) = 1 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 (𝛽𝑛 𝜑(𝑆𝑛+1, 𝐼𝑛) 𝐼𝑛 + 𝜎𝑛 𝜓(𝑉𝑛+1, 𝐼𝑛) 𝐼𝑛 + 1) 𝐼𝑛. (A.26) 𝜌𝑛+1 + 𝜇𝑛𝜌𝑛+1 = 𝛾𝑛𝜄𝑛+1 + 𝜌𝑛< 𝜀+ 𝜌𝑛 (A.33) Thus, since 𝜇𝑛> 0, we get 𝜌𝑛+1 < 𝜀 1 + 𝜇𝑛 + 𝜌𝑛 1 + 𝜇𝑛 . (A.34) and proceeding by induction ( 𝑛−1 ∏ 1 ) 𝑛−1 ∑( 𝑛−1 ∏ 1 ) (A ) 𝜌𝑛+1 + 𝜇𝑛𝜌𝑛+1 = 𝛾𝑛𝜄𝑛+1 + 𝜌𝑛< 𝜀+ 𝜌𝑛 (A.33) Thus, since 𝜇𝑛> 0, we get 𝜌𝑛+1 + 𝜇𝑛𝜌𝑛+1 = 𝛾𝑛𝜄𝑛+1 + 𝜌𝑛< 𝜀+ 𝜌𝑛 (A.33) (A.33) Thus, since 𝜇𝑛> 0, we get 𝜌𝑛+1 < 𝜀 1 + 𝜇𝑛 + 𝜌𝑛 1 + 𝜇𝑛 . (A.34) and proceeding by induction and proceeding by induction + 𝜎𝑛 𝜓(𝑉𝑛+1, 𝐼𝑛) 𝐼𝑛 + 1) 𝐼𝑛. 𝜌𝑛+1 < ( 𝑛−1 ∏ 𝑚=0 1 1 + 𝜇𝑚 ) 𝜌0 + 𝜀 𝑛−1 ∑ 𝑚=0 ( 𝑛−1 ∏ 𝑘=𝑚 1 1 + 𝜇𝑘 ) . (A.35) (A.35) By (H5), we have 𝜑(𝑆𝑛+1, 𝐼𝑛) 𝐼𝑛 ⩽𝜕2𝜑(𝑆𝑛+1, 0) , 𝜓(𝑉𝑛+1, 𝐼𝑛) 𝐼𝑛 ⩽𝜕2𝜓(𝑉𝑛+1, 0) . (A.27) By (H3) and (A.1), we conclude that By (H3) and (A.1), we conclude that lim sup 𝑛→+∞𝜌𝑛= 0. (A.36) (A.27) lim sup 𝑛→+∞𝜌𝑛= 0. (A.36) (A.27) (A.36) Thus, 𝜌𝑛= 𝑅(1) 𝑛−𝑅(2) 𝑛 →0 as 𝑛→+∞. Similar computations show that 𝑆(1) 𝑛−𝑆(2) 𝑛 →0 and 𝑉(1) 𝑛 −𝑉(2) 𝑛 →0. This proves (b) and the result follows. By (H1), 𝑥󳨃→𝜕2𝜑(𝑥, 0) and 𝑥󳨃→𝜕2𝜓(𝑥, 0) are non- decreasing and also Lipschitz, so, using (A.25) we obtain Proof of Theorem 7. Proof of the Results in Section 3 (A.40) 𝑆𝑛⩾𝑥𝑛⩾𝑥𝑛 (A.48) By (1), (A.39), and (A.40), we have By (1), (A.39), and (A.40), we have By (1), (A.39), and (A.40), we have for all 𝑛⩾𝑁2. Thus, since 𝑆𝑛⩽𝑥∗ 𝑛and 𝑉𝑛⩽𝑦∗ 𝑛for all 𝑛∈N, we have 𝑆𝑛+1 ⩾ Λ 𝑛+ 𝜂𝑛𝑉𝑛+1 + 𝑆𝑛−𝛽𝑢𝑘𝜑𝑀𝜀1 1 + 𝜇𝑛+ 𝑝𝑛 𝑆𝑛+1 ⩾ Λ 𝑛+ 𝜂𝑛𝑉𝑛+1 + 𝑆𝑛−𝛽𝑢𝑘𝜑𝑀𝜀1 1 + 𝜇𝑛+ 𝑝𝑛 𝑉𝑛+1 ⩾ 𝑝𝑛𝑆𝑛+1 + 𝑉𝑛−𝜎𝑢𝑘𝜓𝑀𝜀1 1 + 𝜇𝑛+ 𝜂𝑛 . 𝑆𝑛+1 ⩾ Λ 𝑛+ 𝜂𝑛𝑉𝑛+1 + 𝑆𝑛−𝛽𝑢𝑘𝜑𝑀𝜀1 1 + 𝜇𝑛+ 𝑝𝑛 𝑉𝑛+1 ⩾ 𝑝𝑛𝑆𝑛+1 + 𝑉𝑛−𝜎𝑢𝑘𝜓𝑀𝜀1 1 + 𝜇𝑛+ 𝜂𝑛 . (A.41) 2h 𝑛 𝑛 𝑛 𝑦𝑛 we have 󵄨󵄨󵄨󵄨𝑆𝑛−𝑥∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, 󵄨󵄨󵄨󵄨𝑉𝑛−𝑦∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, (A.49) (A.41) 󵄨󵄨󵄨󵄨𝑆𝑛−𝑥∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, 󵄨󵄨󵄨󵄨𝑉𝑛−𝑦∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, (A.49) 1 + 𝜇𝑛+ 𝑝𝑛 𝑉𝑛+1 ⩾ 𝑝𝑛𝑆𝑛+1 + 𝑉𝑛−𝜎𝑢𝑘𝜓𝑀𝜀1 1 + 𝜇𝑛+ 𝜂𝑛 . (A.41) 󵄨󵄨󵄨󵄨𝑆𝑛−𝑥∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, 󵄨󵄨󵄨󵄨𝑉𝑛−𝑦∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, (A.49) (A.41) (A.49) l 𝑛⩾𝑁1. Given 𝜀1 > 0, consider the auxiliary system 𝑥𝑛+1 = Λ 𝑛+ 𝜂𝑛𝑦𝑛+1 + 𝑥𝑛−𝛽𝑢𝑘𝜑𝑀𝜀1 1 + 𝜇+ 𝑝 for all 𝑛⩾𝑁2. By (H1), we have, for all 𝑛⩾𝑁2, 󵄨󵄨󵄨󵄨𝜕2𝜑(𝑆𝑘+1, 0) −𝜕2𝜑(𝑥∗ 𝑘+1, 0)󵄨󵄨󵄨󵄨⩽𝑘𝜑󵄨󵄨󵄨󵄨𝑆𝑘+1 −𝑥∗ 𝑘+1 󵄨󵄨󵄨󵄨 ⩽𝑘𝜑𝜀0 (A.50) ll 𝑛⩾𝑁1. Given 𝜀1 > 0, consider the auxiliary system 𝑥𝑛+1 = Λ 𝑛+ 𝜂𝑛𝑦𝑛+1 + 𝑥𝑛−𝛽𝑢𝑘𝜑𝑀𝜀1 1 + 𝜇𝑛+ 𝑝𝑛 𝑦𝑛+1 = 𝑝𝑛𝑥𝑛+1 + 𝑦𝑛−𝜎𝑢𝑘𝜓𝑀𝜀1 1 + 𝜇𝑛+ 𝜂𝑛 . (A.42) for all 𝑛⩾𝑁2. By (H1), we have, for all 𝑛⩾𝑁2, 󵄨󵄨󵄨󵄨𝜕2𝜑(𝑆𝑘+1, 0) −𝜕2𝜑(𝑥∗ 𝑘+1, 0)󵄨󵄨󵄨󵄨⩽𝑘𝜑󵄨󵄨󵄨󵄨𝑆𝑘+1 −𝑥∗ 𝑘+1 󵄨󵄨󵄨󵄨 ⩽𝑘𝜑𝜀0 (A.50) and thus 𝜕2𝜑(𝑥∗ 𝑘+1, 0) −𝑘𝜑𝜀0 ⩽𝜕2𝜑(𝑆𝑘+1, 0) 𝜕 ( ∗ ) 𝑘 (A.51) for all 𝑛⩾𝑁1. 1 Given 𝜀1 > 0, consider the auxiliary system 1 Given 𝜀1 > 0, consider the auxiliary system (A.50) 𝑥𝑛+1 = Λ 𝑛+ 𝜂𝑛𝑦𝑛+1 + 𝑥𝑛−𝛽𝑢𝑘𝜑𝑀𝜀1 1 + 𝜇𝑛+ 𝑝𝑛 𝑦𝑛+1 = 𝑝𝑛𝑥𝑛+1 + 𝑦𝑛−𝜎𝑢𝑘𝜓𝑀𝜀1 1 + 𝜇𝑛+ 𝜂𝑛 . (A.42) and thus (A.42) and thus (A.42) 𝜕2𝜑(𝑥∗ 𝑘+1, 0) −𝑘𝜑𝜀0 ⩽𝜕2𝜑(𝑆𝑘+1, 0) ⩽𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝑘𝜑𝜀0. (A.51) 𝜕2𝜑(𝑥∗ 𝑘+1, 0) −𝑘𝜑𝜀0 ⩽𝜕2𝜑(𝑆𝑘+1, 0) ⩽𝜕𝜑(𝑥∗ 0) + 𝜕2𝜑(𝑥∗ 𝑘+1, 0) −𝑘𝜑𝜀0 ⩽𝜕2𝜑(𝑆𝑘+1, 0) −𝑘𝜑𝜀0 ⩽𝜕2𝜑(𝑆𝑘+1, 0) ⩽𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝑘𝜑𝜀0. (A.51) (A.51) (A.51) ⩽𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝑘𝜑𝜀0. (A.51 For any 𝑛0 ∈N and 𝑥0, 𝑦0 ∈R+, let (𝑥𝑛, 𝑦𝑛) be the solution of (12) with initial condition (𝑥𝑛0, 𝑦𝑛0) = (𝑥0, 𝑦0) and let (𝑥𝑛0, 𝑦𝑛0) = (𝑥0, 𝑦0) be the solution of (A.42) with the same initial condition. Proof of the Results in Section 3 Since Rℓ 0(𝜆) > 1, there are 𝜀, 𝜀0 > 0 such that 𝜕2𝜑(𝑆𝑛+1, 0) −𝜕2𝜑(𝑥∗ 𝑛+1, 0) ⩽𝜕2𝜑(𝑥∗ 𝑛+1 + 𝜀0, 0) −𝜕2𝜑(𝑥∗ 𝑛+1, 0) = 󵄨󵄨󵄨󵄨𝜕2𝜑(𝑥∗ 𝑛+1 + 𝜀0, 0) −𝜕2𝜑(𝑥∗ 𝑛+1, 0)󵄨󵄨󵄨󵄨⩽𝑘𝜑𝜀0 (A.28) 𝜕2𝜑(𝑆𝑛+1, 0) −𝜕2𝜑(𝑥∗ 𝑛+1, 0) (A.28) lim inf 𝑛→+∞ 𝑛+𝜆 ∏ 𝑘=𝑛 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝑢 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 > 1 + 𝜀, (A.37) lim inf 𝑛→+∞ 𝑛+𝜆 ∏ 𝑘=𝑛 1 + 𝛽𝑘𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑘𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝑢 1 + 𝜇𝑘+ 𝛼𝑘+ 𝛾𝑘 (A.37) (A.37) and thus and thus > 1 + 𝜀, 𝜕2𝜑(𝑆𝑛+1, 0) ⩽𝜕2𝜑(𝑥∗ 𝑛+1, 0) + 𝑘𝜑𝜀0. (A.29) for all 𝑢∈[0, 𝜀0]. Discrete Dynamics in Nature and Society 15 Noting that (A.41) can be written as We claim that there is 𝜀1 > 0 such that Noting that (A.41) can be written as 𝑆𝑛+1 ⩾ 𝜂𝑛𝑝𝑛(1 + 𝜇𝑛) (1 + 𝜇𝑛+ 𝑝𝑛+ 𝜂𝑛) (Λ 𝑛+ 𝑆𝑛−𝛽𝑢𝑀𝑘𝜑𝜀1) (1 + 𝜇𝑛) (1 + 𝜇𝑛+ 𝑝𝑛) (1 + 𝜇𝑛+ 𝑝𝑛+ 𝜂𝑛) + (𝑉𝑛−𝜎𝑢𝑀𝑘𝜓𝜀1) (1 + 𝜇𝑛+ 𝑝𝑛) (1 + 𝜇𝑛) (1 + 𝜇𝑛+ 𝑝𝑛) (1 + 𝜇𝑛+ 𝑝𝑛+ 𝜂𝑛) (A.47) lim sup 𝑛→∞ 𝐼𝑛> 𝜀1 (A.38) 𝑆𝑛+1 (A.38) ⩾ 𝜂𝑛𝑝𝑛(1 + 𝜇𝑛) (1 + 𝜇𝑛+ 𝑝𝑛+ 𝜂𝑛) (Λ 𝑛+ 𝑆𝑛−𝛽𝑢𝑀𝑘𝜑𝜀1) (1 + 𝜇𝑛) (1 + 𝜇𝑛+ 𝑝𝑛) (1 + 𝜇𝑛+ 𝑝𝑛+ 𝜂𝑛) for every solution with positive initial conditions of system (1). + (𝑉𝑛−𝜎𝑢𝑀𝑘𝜓𝜀1) (1 + 𝜇𝑛+ 𝑝𝑛) (1 + 𝜇𝑛) (1 + 𝜇𝑛+ 𝑝𝑛) (1 + 𝜇𝑛+ 𝑝𝑛+ 𝜂𝑛) (A.47) ( ) We proceed by contradiction. Assume that (A.38) does not hold. Then, for each 𝜀1 there is 𝑁1 ∈N and a solution (𝑆𝑛, 𝐼𝑛, 𝑅𝑛, 𝑉𝑛) with positive initial conditions such that 𝐼𝑛⩽ 𝜀1 for all 𝑛⩾𝑁1. By (iii) in Lemma 4, we can assume that 𝑆𝑛, 𝑉𝑛< 𝑀for all 𝑛⩾𝑁1. By (A.3) we have (A.47) 𝑉𝑛+1 ⩾ 𝑝𝑛(Λ 𝑛+ 𝑆𝑛−𝛽𝑢𝑀𝑘𝜑𝜀1) + (𝑉𝑛−𝜎𝑢𝑀𝑘𝜓𝜀1) (1 + 𝜇𝑛+ 𝑝𝑛) (1 + 𝜇𝑛) (1 + 𝜇𝑛+ 𝑝𝑛+ 𝜂𝑛) 𝜑(𝑆𝑛+1, 𝐼𝑛) = 𝑘𝜑𝑆𝑛+1𝐼𝑛⩽𝑘𝜑𝑀𝜀1 (A.39) (A.39) and, using (A.42) and (A.46), we conclude that and likewise, by (A.4), we get 𝜓𝑀𝜀1. (A.40) 𝑆𝑛⩾𝑥𝑛⩾𝑥∗ 𝑛−𝜀0, 𝑉𝑛⩾𝑦𝑛⩾𝑦∗ 𝑛−𝜀0, (A.48) 𝜓(𝑉𝑛+1, 𝐼𝑛) = 𝑘𝜓𝑉𝑛+1𝐼𝑛⩽𝑘𝜓𝑀𝜀1. (A.40) 𝑆𝑛⩾𝑥𝑛⩾𝑥∗ 𝑛−𝜀0, 𝑉⩾ ⩾ ∗ 𝜓(𝑉𝑛+1, 𝐼𝑛) = 𝑘𝜓𝑉𝑛+1𝐼𝑛⩽𝑘𝜓𝑀𝜀1. (A.40) (A.39), and (A.40), we have 𝑆𝑛⩾𝑥𝑛⩾𝑥∗ 𝑛−𝜀0, 𝑉𝑛⩾𝑦𝑛⩾𝑦∗ 𝑛−𝜀0, (A.48) 𝜓(𝑉𝑛+1, 𝐼𝑛) = 𝑘𝜓𝑉𝑛+1𝐼𝑛⩽𝑘𝜓𝑀𝜀1. Proof of the Results in Section 3 Letting 𝜀2 > 0 in (A.69) be sufficiently small and (𝑥𝑛, 𝑦𝑛) be the solution of (12) with 𝑥𝑠𝑛,𝑘+1 = 𝑆𝑠𝑛,𝑘+1 and 𝑦𝑠𝑛,𝑘= 𝑉𝑠𝑛,𝑘, we have by (iii) in Lemma 3 Recall that, since Rℓ 0(𝜆) > 1, there are 𝜀, 𝜀0 > 0 such that (A.37) holds for all 𝑢∈[0, 𝜀0]. Recall that, since Rℓ 0(𝜆) > 1, there are 𝜀, 𝜀0 > 0 such that (A.37) holds for all 𝑢∈[0, 𝜀0]. If (A.59) does not hold, then, given 𝜀0 > 0, there must be a sequence of solutions of (1), ((𝑆𝑛,𝑘, 𝐼𝑛,𝑘, 𝑅𝑛,𝑘, 𝑉𝑛,𝑘)𝑛∈N)𝑘∈N, with initial conditions (𝑆0,𝑘, 𝐼0,𝑘, 𝑅0,𝑘, 𝑉0,𝑘) such that 󵄨󵄨󵄨󵄨𝑥𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦𝑛−𝑦𝑛 󵄨󵄨󵄨󵄨⩽𝜀0 2 (A.70) (A.70) lim inf 𝑛→+∞𝐼𝑛,𝑘< 𝜀0 𝑘2 . (A.60) (A.60) (A.60) for all 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘] ∩N. We conclude that for all 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘] ∩N. We conclude that From (A.38), for each 𝑘∈N, there must be two sequences (𝑠𝑚,𝑘)𝑚∈N and (𝑡𝑚,𝑘)𝑚∈N such that 𝑠𝑚,𝑘→+∞as 𝑚→+∞, 𝑆𝑛⩾𝑥𝑛⩾𝑥𝑛−𝜀0 2 > 𝑥∗ 𝑛−𝜀0, 𝑉𝑛⩾𝑦𝑛⩾𝑦𝑛−𝜀0 2 > 𝑦∗ 𝑛−𝜀0 (A.71) s 1) 𝑆𝑛⩾𝑥𝑛⩾𝑥𝑛−𝜀0 2 > 𝑥∗ 𝑛−𝜀0, 𝑉𝑛⩾𝑦𝑛⩾𝑦𝑛−𝜀0 2 > 𝑦∗ 𝑛−𝜀0 (A.71) (A.71) 0 < 𝑠1,𝑘< 𝑡1,𝑘< 𝑠2,𝑘< 𝑡2,𝑘< ⋅⋅⋅< 𝑠𝑚,𝑘< 𝑡𝑚,𝑘 < (A.61) 0 < 𝑠1,𝑘< 𝑡1,𝑘< 𝑠2,𝑘< 𝑡2,𝑘< ⋅⋅⋅< 𝑠𝑚,𝑘< 𝑡𝑚,𝑘 (A.61) (A.61) < . . . , 𝐼𝑠𝑚,𝑘,𝑘> 𝜀0 𝑘, 𝐼𝑡𝑚,𝑘,𝑘< 𝜀0 𝑘2 , (A.62) 𝜀0 𝑘2 ⩽𝐼𝑛,𝑘⩽𝜀0 𝑘, ∀𝑛∈[𝑠𝑚,𝑘+ 1, 𝑡𝑛,𝑘−1] ∪N. (A.63) Given 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘−1] ∪N, we have for all 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘] ∩N. Proceeding like before, we obtain (A.51) and (A.52) with 𝑆𝑘and 𝑉𝑘replaced by 𝑆𝑛+1,𝑘and 𝑉𝑛+1,𝑘, respectively, and, for sufficiently large 𝑛∈N, 𝜑(𝑆𝑛+1,𝑘, 𝐼𝑛,𝑘) 𝐼𝑛,𝑘 ⩾𝜕2𝜑(𝑥∗ 𝑘+1, 0) −𝑘𝜑𝜀0 −𝜃1 (𝜀1) , 𝜓(𝑉𝑛+1,𝑘, 𝐼𝑛,𝑘) 𝐼𝑘 ⩾𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝑘𝜓𝜀0 −𝜃2 (𝜀1) , (A.72) for all 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘] ∩N. for all 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘] ∩N. 𝑚,𝑘 𝑚,𝑘 Proceeding like before, we obtain (A.51) and (A.52) with 𝑆𝑘and 𝑉𝑘replaced by 𝑆𝑛+1,𝑘and 𝑉𝑛+1,𝑘, respectively, and, for sufficiently large 𝑛∈N, 𝑚,𝑘 𝑚,𝑘 Proceeding like before, we obtain (A.51) and (A.52) with 𝑆𝑘and 𝑉𝑘replaced by 𝑆𝑛+1,𝑘and 𝑉𝑛+1,𝑘, respectively, and, for sufficiently large 𝑛∈N, (A.62) 𝐼𝑡𝑚,𝑘,𝑘< 𝑘2 , 𝜀0 𝑘2 ⩽𝐼𝑛,𝑘⩽𝜀0 𝑘, ∀𝑛∈[𝑠𝑚,𝑘+ 1, 𝑡𝑛,𝑘−1] ∪N. (A.63) Given 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘−1] ∪N, we have 𝜑(𝑆𝑛+1,𝑘, 𝐼𝑛,𝑘) 𝐼𝑛,𝑘 ⩾𝜕2𝜑(𝑥∗ 𝑘+1, 0) −𝑘𝜑𝜀0 −𝜃1 (𝜀1) , 𝜓(𝑉𝑛+1,𝑘, 𝐼𝑛,𝑘) 𝐼𝑛,𝑘 ⩾𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝑘𝜓𝜀0 −𝜃2 (𝜀1) , (A.72) (A.72) 𝐼𝑛+1,𝑘 𝐼𝑛,𝑘 where 𝜃1(𝜀1) →0 as 𝜀1 →0. Therefore, 𝐼𝑛+1,𝑘 where 𝜃1(𝜀1) →0 as 𝜀1 →0. Therefore, where 𝜃1(𝜀1) →0 as 𝜀1 →0. Proof of the Results in Section 3 By (iii) in Lemma 3 we obtain For any 𝑛0 ∈N and 𝑥0, 𝑦0 ∈R+, let (𝑥𝑛, 𝑦𝑛) be the solution of (12) with initial condition (𝑥𝑛0, 𝑦𝑛0) = (𝑥0, 𝑦0) and let (𝑥𝑛0, 𝑦𝑛0) = (𝑥0, 𝑦0) be the solution of (A.42) with the same initial condition. By (iii) in Lemma 3 we obtain Reasoning similarly we obtain, for all 𝑛⩾𝑁2, Reasoning similarly we obtain, for all 𝑛⩾𝑁2, 𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝑘𝜓𝜀0 ⩽𝜕2𝜓(𝑉𝑘+1, 0) ⩽𝜕2𝜓(𝑦∗ 𝑘+1, 0) + 𝑘𝜓𝜀0. (A.52) (A.52) sup 𝑛∈N0 {󵄨󵄨󵄨󵄨𝑥𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦𝑛−𝑦𝑛󵄨󵄨󵄨󵄨} ⩽𝐿𝑀(𝛽𝑢𝑘𝜑+ 𝜎𝑢𝑘𝜓) 𝜀1 (A.43) (A.43) By (H1) and (H2), we conclude that By (H1) and (H2), we conclude that ⩽𝐿𝑀(𝛽𝑢𝑘𝜑+ 𝜎𝑢𝑘𝜓) 𝜀1 𝜑(𝑆𝑛+1, 𝐼𝑛) = 𝜑(𝑆𝑛+1, 0) + 𝜕2𝜑(𝑆𝑛+1, 𝜀𝑛) (𝐼𝑛−0) = 𝜕2𝜑(𝑆𝑛+1, 𝜀𝑛) 𝐼𝑛, (A.53) and thus we can take 𝜀1 > 0 small enough such that (A.53) sup 𝑛∈N0 {󵄨󵄨󵄨󵄨𝑥𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦𝑛−𝑦𝑛󵄨󵄨󵄨󵄨} ⩽𝜀0 2 . (A.44) for some 𝜀𝑛∈[0, 𝜀1], and all 𝑛⩾𝑁2. Thus, by continuity of 𝜕2𝜑, On the other hand, by (ii) in Lemma 3, there is 𝑁2 ⩾𝑁1 sufficiently large such that 𝜑(𝑆𝑛+1, 𝐼𝑛) 𝐼𝑛 = 𝜕2𝜑(𝑆𝑛+1, 𝜀𝑛) (A.54) 𝜑(𝑆𝑛+1, 𝐼𝑛) 𝐼𝑛 = 𝜕2𝜑(𝑆𝑛+1, 𝜀𝑛) ⩾𝜕2𝜑(𝑆𝑛+1, 0) −𝜃1 (𝜀1) , (A.54) 󵄨󵄨󵄨󵄨𝑥∗ 𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦∗ 𝑛−𝑦𝑛󵄨󵄨󵄨󵄨⩽𝜀0 2 , (A.45)h 𝜑(𝑆𝑛+1, 𝑛) 𝐼𝑛 = 𝜕2𝜑(𝑆𝑛+1, 𝜀𝑛) ⩾𝜕2𝜑(𝑆𝑛+1, 0) −𝜃1 (𝜀1) , (A 󵄨󵄨󵄨󵄨𝑥∗ 𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦∗ 𝑛−𝑦𝑛󵄨󵄨󵄨󵄨⩽𝜀0 2 , (A.45) 𝐼𝑛 󵄨󵄨󵄨󵄨𝑥∗ 𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦∗ 𝑛−𝑦𝑛󵄨󵄨󵄨󵄨⩽𝜀0 2 , (A.45) 𝐼𝑛 = 𝜕2𝜑(𝑆𝑛+1, 𝜀𝑛) ⩾𝜕2𝜑(𝑆𝑛+1, 0) −𝜃1 (𝜀1) , 󵄨󵄨󵄨󵄨𝑥∗ 𝑛−𝑥𝑛󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦∗ 𝑛−𝑦𝑛󵄨󵄨󵄨󵄨⩽𝜀0 2 , (A.45) (A.54) (A.45) ⩾𝜕2𝜑(𝑆𝑛+1, 0) −𝜃1 (𝜀1) , for all 𝑛⩾𝑁2. Therefore, for all 𝑛⩾𝑁2. Therefore, Therefore, 󵄨󵄨󵄨󵄨𝑥𝑛−𝑥∗ 𝑛 󵄨󵄨󵄨󵄨+ 󵄨󵄨󵄨󵄨𝑦𝑛−𝑦∗ 𝑛 󵄨󵄨󵄨󵄨⩽𝜀0, (A.46) with 𝜃1(𝜀1) →0 as 𝜀1 →0, for all 𝑛⩾𝑁2. Thus, by (A.51) 𝜑(𝑆𝑛+1, 𝐼𝑛) 𝐼𝑛 ⩾𝜕2𝜑(𝑥∗ 𝑘+1, 0) −𝑘𝜑𝜀0 −𝜃1 (𝜀1) , (A.55) (A.55) for all 𝑛⩾𝑁2. Discrete Dynamics in Nature and Society 16 where 𝜃1(𝜀1) →0 as 𝜀1 →0. Similarly, for all 𝑛⩾𝑁2, we h b (A 52) and therefore where 𝜃1(𝜀1) →0 as 𝜀1 →0. Similarly, for all 𝑛⩾𝑁2, we have, by (A.52) and therefore 𝜀0 𝑡 𝜎𝑡𝑛,𝑘−𝑠𝑛,𝑘𝜀0 (A ) where 𝜃1(𝜀1) →0 as 𝜀1 →0. Similarly, for all 𝑛⩾𝑁2, we have, by (A.52) and therefore 𝜀 where 𝜃1(𝜀1) →0 as 𝜀1 →0. Similarly, for all 𝑛⩾𝑁2, we have, by (A.52) (𝑉 𝐼) and therefore 𝜀0 𝑘2 > 𝐼𝑡𝑛𝑘,𝑘⩾𝜎𝑡𝑛,𝑘−𝑠𝑛,𝑘𝐼𝑠𝑛𝑘,𝑘> 𝜎𝑡𝑛,𝑘−𝑠𝑛,𝑘𝜀0 𝑘 , (A.65) y 𝜓(𝑉𝑛+1, 𝐼𝑛) 𝐼𝑛 ⩾𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝑘𝜓𝜀0 −𝜃2 (𝜀1) , (A.56) where 𝜃2(𝜀1) →0 as 𝜀1 →0. Proof of the Results in Section 3 From the second equation in (1), we have 𝜀0 𝑘2 > 𝐼𝑡𝑛,𝑘,𝑘⩾𝜎𝑡𝑛,𝑘−𝑠𝑛,𝑘𝐼𝑠𝑛,𝑘,𝑘> 𝜎𝑛,𝑘 𝑛,𝑘𝜀0 𝑘 , (A.65) where 𝜎= 1 1 + 𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢. (A.66) 𝜓( 𝑛+1 𝑛) 𝐼𝑛 ⩾𝜕2𝜓(𝑦𝑘+1, 0) −𝑘𝜓𝜀0 −𝜃2 (𝜀1) , (A.56) where 𝜃2(𝜀1) →0 as 𝜀1 →0. From the second equation in (1), we have where 𝜎= 1 1 + 𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢. (A.66) →0. From the second equation in (1), 𝜎= 1 1 + 𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢. (A.66) where 𝜃2(𝜀1) →0 as 𝜀1 →0. From the second equation in (1), we have 𝜎= 1 1 + 𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢. (A.66) (A.66) Thus, by (A.62) and (A.65), 𝐼𝑛+1 = 1 + 𝛽𝑛𝜑(𝑆𝑛+1, 𝐼𝑛) /𝐼𝑛+ 𝜎𝑛𝜓(𝑉𝑛+1, 𝐼𝑛) /𝐼𝑛 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 𝐼𝑛 ⩾1 + 𝛽𝑛𝜕2𝜑(𝑥∗ 𝑘+1, 0) + 𝜎𝑛𝜕2𝜓(𝑦∗ 𝑘+1, 0) −𝑢 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 𝐼𝑛, (A.57) Thus, by (A.62) and (A.65), 𝑡𝑛,𝑘−𝑠𝑛,𝑘⩾ ln 𝑘 ln (1/𝜎) 󳨀→+∞ as 𝑘󳨀→+∞. (A.67) In view of (A.67), we can choose 𝑘0 ∈N such that (A.67) In view of (A.67), we can choose 𝑘0 ∈N such that In view of (A.67), we can choose 𝑘0 ∈N such that 𝑡𝑛,𝑘−𝑠𝑛,𝑘> 𝑁+ 𝜆+ 1, (A.68) where (A.68) 𝑢= 𝛽𝑢(𝑘𝜑𝜀0 + 𝜃1 (𝜀1)) + 𝜎𝑢(𝑘𝜓𝜀0 + 𝜃2 (𝜀1)) , (A.58) for all 𝑘⩾𝑘0. Letting 𝑚and 𝑘⩾𝑘0 be sufficiently large and 𝜀2 > 0 be sufficiently small, we may assume that for all 𝑛⩾𝑁2. Letting 𝜀1 be sufficiently small, we conclude, according to (A.37) and (A.57), that 𝐼𝑛→+∞as 𝑛→+∞, a contradiction. Thus, we conclude that (A.38) holds. 𝑆𝑛,𝑘⩾ Λ 𝑛+ 𝜂𝑛𝑉𝑛,𝑘+ 𝑆𝑛,𝑘−𝛽𝑢𝑘𝜑𝑀𝜀2 1 + 𝜇𝑛+ 𝑝𝑛 𝑉𝑛,𝑘⩾ 𝑝𝑛𝑆𝑛,𝑘+ 𝑉𝑛,𝑘−𝜎𝑢𝑘𝜓𝑀𝜀2 1 + 𝜇𝑛+ 𝜂𝑛 (A.69) h Next we will prove the permanence of the infectives. 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Proof of the Results in Section 3 Therefore, = 1 + 𝛽𝑛𝜑(𝑆𝑛+1,𝑘, 𝐼𝑛,𝑘) /𝐼𝑛,𝑘+ 𝜎𝑛𝜓(𝑉𝑛+1,𝑘, 𝐼𝑛,𝑘) /𝐼𝑛,𝑘 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 (A.64) 1( 𝐼𝑛+1,𝑘 = 1 + 𝛽𝑛𝜑(𝑆𝑛+1,𝑘, 𝐼𝑛,𝑘) /𝐼𝑛,𝑘+ 𝜎𝑛𝜓(𝑉𝑛+1,𝑘, 𝐼𝑛,𝑘) /𝐼𝑛,𝑘 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 (A.64) 1 1 1h 𝐼𝑛+1,𝑘 1 𝛽𝜕 ( ∗ 0) 𝜕 ( ∗ 0) (A (A.73) ⩾1 + 𝛽𝑛𝜕2𝜑(𝑥∗ 𝑛+1, 0) + 𝜎𝑛𝜕2𝜓(𝑦∗ 𝑛+1, 0) −𝑢 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 𝐼𝑛,𝑘, (A.73) ⋅𝐼𝑛,𝑘⩾ 1 1 + 𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢𝐼𝑛,𝑘 ⩾1 + 𝛽𝑛𝜕2𝜑(𝑥∗ 𝑛+1, 0) + 𝜎𝑛𝜕2𝜓(𝑦∗ 𝑛+1, 0) −𝑢 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 𝐼𝑛,𝑘, (A.73) ⋅𝐼𝑛,𝑘⩾ 1 1 + 𝜇𝑢+ 𝛼𝑢+ 𝛾𝑢𝐼𝑛,𝑘 Discrete Dynamics in Nature and Society 17 for all 𝑛∈[𝑠𝑚,𝑘, 𝑡𝑚,𝑘] ∩N, where 𝑢is given by (A.58). Thus, [10] Q. Cui and Q. Zhang, “Global stability of a discrete SIR epidemic model with vaccination and treatment,” Journal of Difference Equations and Applications, vol. 21, no. 2, pp. 111–117, 2015. 𝜀0 𝑘2 > 𝐼𝑡𝑚,𝑘,𝑘 ⩾𝐼𝑡𝑚,𝑘−𝜆,𝑘 𝑡𝑚,𝑘 ∏ 𝑛=𝑡𝑚,𝑘−𝜆 1 + 𝛽𝑛𝜕2𝜑(𝑥∗ 𝑛+1, 0) + 𝜎𝑛𝜕𝑛𝜓(𝑦∗ 𝑛+1, 0) −𝑢 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 > 𝜀0 𝑘2 (A.74) [11] D. Ding, Q. Ma, and X. Ding, “A non-standard finite difference scheme for an epidemic model with vaccination,” Journal of Difference Equations and Applications, vol. 19, no. 2, pp. 179–190, 2013. 𝑘2 𝑚,𝑘, ⩾𝐼𝑡𝑚,𝑘−𝜆,𝑘 𝑡𝑚,𝑘 ∏ 𝑛=𝑡𝑚,𝑘−𝜆 1 + 𝛽𝑛𝜕2𝜑(𝑥∗ 𝑛+1, 0) + 𝜎𝑛𝜕𝑛𝜓(𝑦∗ 𝑛+1, 0) −𝑢 1 + 𝜇𝑛+ 𝛼𝑛+ 𝛾𝑛 (A.74) [12] D. Ding and X. Ding, “Dynamic consistent non-standard numerical scheme for a dengue disease transmission model,” Journal of Difference Equations and Applications, vol. 20, no. 3, pp. 492–505, 2014. which is a contradiction. The theorem follows. [13] S. Jang and S. Elaydi, “Difference equations from discretization of a continuous epidemic model with immigration of infec- tives,” Canadian Applied Mathematics Quarterly, vol. 11, no. 1, pp. 93–105, 2003. Conflicts of Interest The authors declare that they have no conflicts of interest. [16] R. E. Mickens, “Nonstandard finite difference schemes for differential equations,” Journal of Difference Equations and Applications, vol. 8, no. 9, pp. 823–847, 2002. Acknowledgments [17] C. Sun and W. Yang, “Global results for an SIRS model with vaccination and isolation,” Nonlinear Analysis: Real World Applications, vol. 11, no. 5, pp. 4223–4237, 2010. Joaquim Mateus, C´esar M. Silva, and S. Vaz were partially supported by FCT through CMA-UBI (Project UID/MAT/00212/2013). [18] A. A. Khasnis and M. D. Nettleman, “Global warming and infectious disease,” Archives of Medical Research, vol. 36, no. 6, pp. 689–696, 2005. References 119, no. 1, pp. 97–114, 1994. [8] E. Pereira, C. M. Silva, and J. A. da Silva, “A generalized nonautonomous SIRVS model,” Mathematical Methods in the Applied Sciences, vol. 36, no. 3, pp. 275–289, 2013. [26] K. Hattaf, A. A. Lashari, B. El Boukari, and N. Yousfi, “Effect of discretization on dynamical behavior in an epidemiological model,” Differential Equations and Dynamical Systems, vol. 23, no. 4, pp. 403–413, 2015. [9] Q. Cui, X. Yang, and Q. Zhang, “An NSFD scheme for a class of SIR epidemic models with vaccination and treatment,” Journal of Difference Equations and Applications, vol. 20, no. 3, pp. 416– 422, 2014. [27] V. Kaitala, M. Heino, and W. M. Getz, “Host-parasite dynamics and the evolution of host immunity and parasite fecundity Discrete Dynamics in Nature and Society 18 strategies,” Bulletin of Mathematical Biology, vol. 59, no. 3, pp. 427–450, 1997. [28] G. Di Lena and G. Serio, “A discrete method for the iden- tification of parameters of a deterministic epidemic model,” Mathematical Biosciences, vol. 60, no. 2, pp. 161–175, 1982. [29] C. Lefevre and M.-P. Malice, “A discrete time model for an S-I-S Infectious disease with a random number of contacts between individuals,” Applied Mathematical Modelling: Simulation and Computation for Engineering and Environmental Systems, vol. 7, no. 5-8, pp. 785–792, 1986. [30] C. Lefevre and P. Picard, “On the formulation of discrete-time epidemic models,” Mathematical Biosciences, vol. 95, no. 1, pp. 27–35, 1989. [31] J. Longini, “The generalized discrete-time epidemic model with immunity: a synthesis,” Mathematical Biosciences, vol. 82, no. 1, pp. 19–41, 1986. [32] R. E. Mickens, “A discrete-time model for the spread of periodic diseases without immunity,” BioSystems, vol. 26, no. 3, pp. 193– 198, 1992. [33] C. C. Spicer, “The mathematical modeling of influenza,” British Medical Bulletin, vol. 35, pp. 23–28, 1979. [34] D. Pierre-Loti-Viaud, “Large deviations for discrete-time epi- demic models,” Mathematical Biosciences, vol. 117, no. 1-2, pp. 197–210, 1993. [35] R. Webster West and J. R. Thompson, “Models for the simple epidemic,” Mathematical Biosciences, vol. 141, no. 1, pp. 29–39, 1997. [36] N. Bacar and E. H. Ait Dads, “On the probability of extinction in a periodic environment,” Journal of Mathematical Biology, vol. 68, no. 3, pp. 533–548, 2014. References Hindawi www.hindawi.com Volume 2018 Mathematics Journal of Hindawi www.hindawi.com Volume 2018 Mathematical Problems in Engineering Applied Mathematics Journal of Hindawi www.hindawi.com Volume 2018 Proba Hindawi www.hindawi.com Journa Hindawi www.hindawi.com Math Advanc Complex Analysis Journal of Hindawi www.hindawi.com Volume 2018 Opt Journa Hindawi www.hindawi.com Hindawi www.hindawi.com Volume 2018 Engineering Mathematics International Journal of Hindawi www.hindawi.com Volume 2018 Operations Research Advances in Journal of Hindawi www.hindawi.com Volume 2018 Function Spaces Abstract and Applied Analysis Hindawi www.hindawi.com Volume 2018 International Journal of Mathematics and Mathematical Sciences Hindawi www.hindawi.com Volume 2018 Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Hindawi www.hindawi.com Volume 2018 Volume 2018 Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Advances in Advances in Discre Natur Hindawi www.hindawi.com Hindawi www.hindawi.com Diferential Equations International Journal of Volume 2018 Hindawi www.hindawi.com Volume 2018 Decision Sciences Advances in Hindawi www.hindawi.com Ana Interna Hindawi www.hindawi.com Volume 2018 Stochastic Analysis International Journal of Submit your manuscripts at www.hindawi.com Applied Mathematics Journal of Hindawi www.hindawi.com Volume 2018 Probability and Statistics Hindawi www.hindawi.com Volume 2018 Journal of Optimization Journal of Hindawi www.hindawi.com Volume 2018 International Journal of Hindawi www.hindawi.com Volume 2018 Operations Research Advances in International Journal of Mathematics and Mathematical Sciences Hindawi www.hindawi.com Volume 2018 Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Hindawi www.hindawi.com Volume 2018 Decision Sciences Advances in Submit your manuscripts at www.hindawi.com Applied Mathematics Journal of Hindawi www.hindawi.com Volume 2018 Probability and Statistic Hindawi www.hindawi.com Volu Journal of Hindawi www.hindawi.com Volume 2018 Operations Research Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Hindawi www.hindawi.com Volume 2018 Decision Sciences Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2013 Hindawi www.hindawi.com The Scientific World Journal Volume 2018 Probability and Statistics Hindawi www.hindawi.com Volume 2018 Journal of Hi d i P bli hi C i Hi d i The Scientific World Journal Hindawi www hindawi com Volume 2018 Decision Sciences Advances in HindawiOperations Research Advances in Probability and Statistics Hindawi www.hindawi.com Volume 2018 Journal of Applied Mathematics Journal of Hindawi Optimization Journal of Hindawi www.hindawi.com Volume 2018 Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 Analysis International Journal of Submit your manuscripts at www.hindawi.com Hindawi www.hindawi.com Volume 2018 Mathematics Journal of Hindawi www.hindawi.com Volume 2018 Mathematical Problems in Engineering Hindawi www.hindawi.com Volume 2018 Mathematical Physics Advances in Complex Analysis Journal of Hindawi www.hindawi.com Volume 2018 Journal of Hindawi www.hindawi.com Volume 2018 Function Spaces Abstract and Applied Analysis Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Volume 2018 Volume 2018 Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Advances in Advances in Discrete Dynamics in Nature and Society Hindawi www.hindawi.com Volume 2018 Hindawi www.hindawi.com Diferential Equations International Journal of Volume 2018 Hindawi www.hindawi.com Volume 2018 Stochastic Analysis International Journal of Discrete Dynamics in Nature and Society Hindawi www.hindawi.com Volume 2018 Hindawi www hindawi com Volume 2018 Mathematical Problems in Engineering Hindawi hi d i V l 2018 V l 2018 Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Numerical Analysis Advances in Advances in Hindawi www.hindawi.com Volume 2018 Mathematics Journal of
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Causal Effects of Prenatal Exposure to PM2.5 on Child Development and the Role of Unobserved Confounding
International journal of environmental research and public health/International journal of environmental research and public health
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Causal Effects of Prenatal Exposure to PM2.5 on Child Development and the Role of Unobserved Confounding Viola Tozzi 1, Aitana Lertxundi 2,3,4, Jesus M. Ibarluzea 2,3,5,6 and Michela Baccini 1,* 1 Department of Statistics, Computer Science, Applications, University of Florence, 59 50134 Florence, Viale Morgagni, Italy, viola.t@hotmail.it Faculty of Medicine, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain 5 Sub-Directorate for Public Health of Guipúzcoa, Department of Health, Government of the Basque Country, 20013 San Sebastian, Spain 6 Faculty of Psychology, University of the Basque Country (UPV/EHU), 20018 San Sebastian, Spain * Correspondence: michela.baccini@unifi.it; Tel.: +39 055 2751563 y y gy y q y p * Correspondence: michela.baccini@unifi.it; Tel.: +39 055 2751563 Received: 12 September 2019; Accepted: 6 November 2019; Published: 9 November 2019 ceived: 12 September 2019; Accepted: 6 November 2019; Published: 9 November 2019   Abstract: Prenatal exposure to airborne particles is a potential risk factor for infant neuropsychological development. This issue is usually explored by regression analysis under the implicit assumption that all relevant confounders are accounted for. Our aim is to estimate the causal effect of prenatal exposure to high concentrations of airborne particles with a diameter < 2.5 µm (PM2.5) on children’s psychomotor and mental scores in a birth cohort from Gipuzkoa (Spain), and investigate the robustness of the results to possible unobserved confounding. We adopted the propensity score matching approach and performed sensitivity analyses comparing the actual effect estimates with those obtained after adjusting for unobserved confounders simulated to have different strengths. On average, mental and psychomotor scores decreased of −2.47 (90% CI: −7.22; 2.28) and −3.18 (90% CI: −7.61; 1.25) points when the prenatal exposure was ≥17 µg/m3 (median). These estimates were robust to the presence of unmeasured confounders having strength similar to that of the observed ones. The plausibility of having omitted a confounder strong enough to drive the estimates to zero was poor. The sensitivity analyses conferred solidity to our findings, despite the large sampling variability. This kind of sensitivity analysis should be routinely implemented in observational studies, especially in exploring new relationships. Keywords: child development; airborne particles; propensity score matching; sensitivity analysis; bias analysis; Monte Carlo simulations International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health 1. Introduction Many studies around the world documented long term and short term effects of air pollution on population mortality and morbidity [1,2]. Recently, several researches focused on the effects of prenatal exposures to air pollution on children’s neuropsychological development [3]. Harris et al. [4] analyzed 1109 mother–child pairs from Eastern Massachusetts (USA) concluding that parental proximity to major roadways may negatively influence performance across a range of mental domains in childhood. Guxens et al. [5] found significant associations between high levels of nitrogen dioxide (NO2) and benzene and infant neurodevelopment in the Spanish cohorts participating in the INMA (Infancia y Medio Ambiente, the Spanish for Childhood and Environment) Project. High levels of NO2 were Int. J. Environ. Res. Public Health 2019, 16, 4381; doi:10.3390/ijerph16224381 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2019, 16, 4381 2 of 12 found to be associated also with delayed psychomotor development in children between one and six years of age [6]. Lertxundi et al. [7,8] investigated the effect of prenatal exposure to airborne particles with a diameter < 2.5 µm (PM2.5) and NO2 on infant cognitive and psychomotor development and on the cognitive functions in children of 4–6 years of age, reporting the existence of negative associations. All these studies accounted for the confounding effect of several mother and child characteristics through the specification of regression models, under the implicit assumption that all relevant confounders were known and observed (assumption of unconfoundedness [9]). However, given the complex nature of the problem, the presence of residual confounding due to unknown or unmeasured factors related to the familiar and environmental context where children live should not be excluded. In the literature, the problem related to the presence of unobserved confounders and the evaluation of the associated bias has been addressed through sensitivity analyses that check whether and to what extent the estimated effect or association is robust to possible deviations from unconfoundedness [10–18]. Usually, the aim is to assess how strong an unobserved confounder would have to be to substantially change the study results. However, despite the importance of this issue, quantitative evaluations of the potential bias associated with residual confounding have rarely received consideration in epidemiological applications (see chapter 19 in [10]). 1. Introduction In the present paper, with reference to a birth cohort of the province of Gipuzkoa [7], we estimated the causal effect of high PM2.5 exposures during maternal pregnancy on child mental and psychomotor outcomes at 15 months of age, within a potential outcomes approach to causal inference [19]. Then, we checked the robustness of the results to the possible presence of unobserved confounders through a sensitivity analysis based on simulations. In particular, we compared the actual causal effect estimates obtained by using propensity score (PS) matching [9], to those obtained after controlling also for simulated unobserved confounders of different strengths, according to the method proposed by Ichino et al. [11] and implemented in the sensatt function of STATA (StataCorp LP., College Station, TX, USA) [20,21]. 2.1. Data As part of the Environment and Childhood Research Network (INMA network) [22], the birth cohort of Gipuzkoa was recruited between May 2006 and January 2008 among the residents of a study area of 519 km2 spanning three narrow valleys: high Urola-Goierri, mid Urola and high Oria. The total population of the area was approximately 88,000 inhabitants, spread across 25 small localities. Data on exposures, outcomes and potential confounders were available for 438 mother–child pairs. Children’s mental and psychomotor developments were assessed at around 15 months of age (range 13–18 months), according to the Bayley Scales of Infant Development [23], by one of two trained neuropsychologists, blinded to the child’s exposure status. The raw scores were standardized to a mean of 100 points with a standard deviation of 15 points, with higher scores indicating better developments. Data on the mother’s characteristics and habits were obtained through questionnaires administered during the first and third trimesters of pregnancy. The daily levels of PM2.5 in the area were measured from the beginning of the study until the last birth by three fixed-site samplers belonging to the Air Quality Network of the Basque Government and three Digitel DHA-80 high-volume aerosol samplers, two of which were monthly rotated in different locations (Figure 1). Due to the presence of moving monitors, daily measurements were characterized by the presence of design-missing values. With the aim to assess the average exposure of the mother–child couples during pregnancy, we used a multiple imputation (MI) procedure starting from the weekly data. First, we derived weekly means from the daily levels of PM2.5 measured in each location. Considering a weekly mean as missing when daily measurements were missing for more than two days during the week, the percentage of missing weekly averages during the study period was around 30%. Then, under the Missing At Random (MAR) assumption, we applied an MI procedure [24] on the incomplete data set including weekly averages concentrations of the air pollutants measured by the monitors, 3 of 12 Int. J. Environ. Res. Public Health 2019, 16, 4381 meteorological variables and seasonality indicators. In this way, we generated five imputed data sets, thus five imputed values for each missing weekly mean of PM2.5. Finally, we averaged over weekly means, obtaining five imputed values of the average level of PM2.5 during the pregnancy for each mother–child couple. Figure 1 2.2. Notation 2.2. Notation Unless otherwise specified the notation and the methods described hereinafter refer to a single Unless otherwise specified, the notation and the methods described hereinafter refer to a single imputed data set. 2.2. Notation U l th i ifi d th t ti d th th d d ib d h i ft f t i l Unless otherwise specified, the notation and the methods described hereinafter refer to a single imputed data set. Unless otherwise specified, the notation and the methods described hereinafter refer to a single imputed data set. Being i = 1, N the indicator of the child, let Ei be a binary exposure indicator, which was equal to 0 if the average level of PM2.5 during the pregnancy was <17 µg/m3 (control) and equal to 1 if it was ≥17 µg/m3 (treatment). The threshold of 17 µg/m3, which approximated the median exposure of the it th i t d d t t fi d i i i d t id t h d t d d ti [25] Being i = 1, N the indicator of the child, let Ei be a binary exposure indicator, which was equal to 0 if the average level of PM2.5 during the pregnancy was <17 µg/m3 (control) and equal to 1 if it was ≥17 µg/m3 (treatment). The threshold of 17 µg/m3, which approximated the median exposure of the units across the imputed data sets, was fixed a priori in order to avoid post-hoc data dredation [25]. Let Yi be the observed mental or psychomotor score for child i and Xi a vector of observed covariates. units across the imputed data sets, was fixed a priori in order to avoid post-hoc data dredation [25]. Let Yi be the observed mental or psychomotor score for child i and Xi a vector of observed covariates. According to the potential outcome approach to causal inference, under the Stable Unit Treatment Value Assumption, we associated with each child two potential outcomes Yi(1) and Yi(0), representing the score under treatment and under control [9]. 2.1. Data We assumed that the mother–child couples were exposed to PM2.5 levels measured at the monitoring site located in their town or in the closest one. For more detail on the MI procedure see [7]. generated five imputed data sets, thus five imputed values for each missing weekly mean of PM2.5. Finally, we averaged over weekly means, obtaining five imputed values of the average level of PM2.5 during the pregnancy for each mother–child couple. We assumed that the mother–child couples were exposed to PM2.5 levels measured at the monitoring site located in their town or in the closest one. For more detail on the MI procedure see [7]. The study was approved by the hospital ethics committee and all participating mothers provided informed consent. The study was approved by the hospital ethics committee and all participating mothers provided informed consent. Figure 1. Study area and location of the particles with a diameter < 2.5 µm (PM2.5) air samplers. Figure 1. Study area and location of the particles with a diameter < 2.5 µm (PM2.5) air samplers. Figure 1 2.2. Notation For each child it was possible to observe only one of the two potential outcomes: for children belonging to the treated group we observed only According to the potential outcome approach to causal inference, under the Stable Unit Treatment Value Assumption, we associated with each child two potential outcomes Yi(1) and Yi(0), representing the score under treatment and under control [9]. For each child it was possible to observe only one of the two potential outcomes: for children belonging to the treated group we observed only Y(1) (Yi(1) = Yi if Ei = 1); for children belonging to the control group we observed only Y(0) (Yi(0) = Yi if Ei = 0). 2.4. Assumption and ATT estimation We included in the PS model not only pre-treatment variables, as usually recommended [27], but also some post-treatment variables (smoking and diet during pregnancy, child care mode, breastfeeding) which were not affected by the exposure and could be thought of as proxies for unmeasured pre-treatment covariates, mainly related to the socio-economic status of the family. We expect that adjusting for them could reduce the bias without inflating the variance [28,29]. (1) For each child, we estimated the PS as predicted value from a logistic model defined on the exposure Ei, given Xi [26]. The model accounted for the following variables: child’s sex, year of birth, nursery attendance (yes, no), main caregiver (only mother, both parents, other relative, other caregiver), maternal education (secondary school or less, university), maternal work (non-manual worker, manual worker), mother’s age (<25, 25–34, 35+), mother’s body mass index (underweight, normal, overweight, obese), mother’s parity (0, 1+), mother’s vegetable and fruit intake during pregnancy (≤405 g/day, >405 g/day), smoking during pregnancy (yes, no), smoking at 32 weeks of pregnancy (yes, no), breastfeeding (yes, no), indicator of the neuropsychologist who evaluated the child (first, second evaluator). We included in the PS model not only pre-treatment variables, as usually recommended [27], but also some post-treatment variables (smoking and diet during pregnancy, child care mode, breastfeeding) which were not affected by the exposure and could be thought of as proxies for unmeasured pre-treatment covariates, mainly related to the socio-economic status of the family. We expect that adjusting for them could reduce the bias without inflating the variance [28,29]. Regarding the need of adjusting for the neuropsychologist indicator, it is worth noting that the probability of exposures ≥17 µg/m3 was very low (from 1% to 5%) for the children examined by the second neuropsychologist, who substituted the first one at the end of the study period. The observed decrease in the exposures over time was mainly due to the substantial reduction of the atmospheric level of PM2.5 in 2007, likely attributable to the high level of precipitations (58.8 mm in 2006 versus 126.5 mm in 2007) and to the closure of a polluting industry in the area in July of the same year (Figure S1) [30]. This, coupled with the fact that the scores assigned by the second psychologist were systematically higher than those assigned by the first one, made crucial to adjust for the neuropsychologist indicator. y p Y(1) (Yi(1) = Yi if Ei = 1); for children be if E 0) 2.3. Research Question and Estimands if Ei = 0). 2.3. Research Question and Estimands The research question we were interested in was the following: what would have been the mental and psychomotor development of the children with prenatal exposure ≥17 µg/m3, if their exposure were been <17 µg/m3? In order to reply to this questions, we focused, separately for h d l h l ff f h d (ATT) hi h The research question we were interested in was the following: what would have been the mental and psychomotor development of the children with prenatal exposure ≥17 µg/m3, if their exposure were been <17 µg/m3? In order to reply to this questions, we focused, separately for psychomotor and mental score, on the average causal effect of treatment on the treated (ATT), which compares the average score observed among the “treated” children, with the average score that we would have observed on them if they were not “treated”: Int. J. Environ. Res. Public Health 2019, 16, 4381 4 of 12 ATT = PN i=1 Yi(1)Ei PN i=1 Ei − PN i=1 Yi(0)Ei PN i=1 Ei = PN i=1 YiEi PN i=1 Ei − PN i=1 Yi(0)Ei PN i=1 Ei . (1) (1) 2.4. Assumption and ATT estimation In order to estimate the ATT, we imputed the missing potential outcome Yi(0) in (1) through a matching procedure based on the PS [9]. This required two assumptions [26]: (i) Unconfoundedness for controls: Ei⊥Yi(0)|Xi. This assumption states that treatment assignment is independent of the potential outcome under control, conditional on the observed covariates. In other words, this assumption requires that there are no unobserved confounders. (i) Unconfoundedness for controls: Ei⊥Yi(0)|Xi. This assumption states that treatment assignment is independent of the potential outcome under control, conditional on the observed covariates. In other words, this assumption requires that there are no unobserved confounders. p q (ii) Weak positivity assumption: P(Ei = 1|Xi = x) < 1 for each i, for each x. This assumption implies that for each treated unit there is a similar untreated unit that can be used as a matched control. p q (ii) Weak positivity assumption: P(Ei = 1|Xi = x) < 1 for each i, for each x. This assumption implies that for each treated unit there is a similar untreated unit that can be used as a matched control. (ii) Weak positivity assumption: P(Ei = 1|Xi = x) < 1 for each i, for each x. This assumption implies for each treated unit there is a similar untreated unit that can be used as a matched control. Being the PS a balancing score, the conditions (i) and (ii) are valid also conditionally to the PS [26]. Under (i) and (ii), we applied a PS matching procedure for AT estimation which consisted of two steps: p (1) For each child, we estimated the PS as predicted value from a logistic model defined on the exposure Ei, given Xi [26]. The model accounted for the following variables: child’s sex, year of birth, nursery attendance (yes, no), main caregiver (only mother, both parents, other relative, other caregiver), maternal education (secondary school or less, university), maternal work (non-manual worker, manual worker), mother’s age (<25, 25–34, 35+), mother’s body mass index (underweight, normal, overweight, obese), mother’s parity (0, 1+), mother’s vegetable and fruit intake during pregnancy (≤405 g/day, >405 g/day), smoking during pregnancy (yes, no), smoking at 32 weeks of pregnancy (yes, no), breastfeeding (yes, no), indicator of the neuropsychologist who evaluated the child (first, second evaluator). 2.5. Sensitivity Analysis The sensatt function generates U starting from four tuning parameters: pij = Pr(U = 1 E = i, Y∗= j), i = 0, 1, j = 0, 1, and calculates the odds ratios Γ and Λ from the simulations. In our study, we focused on unobserved confounders with a distribution similar to the empirical distribution of relevant observed confounders, by deriving the pij from the observed data. Successively, we explored the whole space of the hypothetical Us by setting pij = 0.1, 0.25, 0.5, 0.75, 0.9, i = 0, 1, j = 0, 1, in order to search for the so-called “killer” confounder, i.e., the confounder U able to explain away the estimated effect, driving the estimated ATT to zero. In this second case, it is crucial to assess the plausibility of the configurations of Γ and Λ which lead to the killer confounder: if the killer configuration is unlikely, the ATT estimate can be considered robust to the violation of the unconfoundedness assumption. All the sensitivity analyses were separately performed on each of the five multiple-imputed data sets and the results combined according to Rubin [32]. 2.4. Assumption and ATT estimation Once the relevant covariates for the PS estimation have been defined, the key criterion for driving the specification of the PS model was to obtain PS estimates that balanced the covariate distribution between treated and controls [9]. According to the procedure implemented in the STATA command pscore, we checked the balancing property of the PS by comparing the covariate distribution between the two groups of exposure within strata of units with similar estimated PS [31]. We also checked for the weak positivity condition (ii), by the inspection of the PS distributions among treated and controls. (2) On the basis of the estimated PS, we matched each treated child with the child in the control group having closest PS, Yc i . The same matched control could be used for different treated units, in order to reduce the bias due to inappropriate matching. Finally, the ATT was estimated as: ˆ ATT = PN i=1 YiEi PN i=1 Ei − PN i=1 Yc i Ei PN i=1 Ei . (2) (2) The standard error of ˆ ATT was estimated without accounting for the uncertainty around the estimated PS [31]. 5 of 12 Int. J. Environ. Res. Public Health 2019, 16, 4381 The estimation procedure was separately performed on each of the five multiple-imputed data sets, and the five results combined according to Rubin [32]. It is worth noticing that, for each multiple-imputed data set, the ATT was calculated on different treated populations. This required a cautionary interpretation of the ATT, which referred to different sets of children depending on the imputation. 2.5. Sensitivity Analysis The Ichino’s sensitivity analysis, which is mainly employed in social science and economics, is based on Monte Carlo simulations [11]. Let us suppose that the unconfoundedness assumption (i) is not valid because of the presence of a binary unobserved confounder U. A value of U is attributed to each subject by sampling from a hypothetical distribution, then U is included in the set of variables used to estimate the ATT. This procedure can be repeated many times (e.g., 1000) and, at the end, an average ATT is estimated. Comparing this average ATT with the causal estimate obtained without adjusting for U, the robustness of the result to violation of the unconfoundedness assumption can be checked: if the ATT estimates before and after adjusting for U are close, the result can be considered robust to the presence of an unobserved confounder similar to U. In the present paper, we explored the space of the possible unobserved confounders by generating different versions of U, which covered different hypotheses on confounding, as expressed by the associations of U with the outcome and with the exposure. According to Ichino et al. [11], indicating with Y∗a binary version of the outcome (for example Y∗= 1 if Y > 75th percentile, Y∗= 0 otherwise), the association of U with the outcome (outcome effect) is measured by the odds ratio Γ comparing the odds of Y∗= 1 when U is equal to 1 with the odds of Y∗= 1 when U is equal to 0, among the controls: Γ = P(Y∗= 1|E = 0,U = 1,X) P(Y∗= 0|E = 0,U = 1,X) P(Y∗= 1|E = 0,U = 0,X) P(Y∗= 0|E = 0,U = 0,X) (3) (3) The association of U with the exposure (selection effect) is measured by the odds ratio Λ comparing the odds of being exposed when U is equal to 1 with the odds of being exposed when U is equal to 0: Λ = P(E = 1|U = 1,X) P(E = 0|U = 1,X) P(E = 1|U = 0,X) P(E = 0|U = 0,X) (4) (4) The sensatt function generates U starting from four tuning parameters: pij = Pr(U = 1 E = i, Y∗= j), i = 0, 1, j = 0, 1, and calculates the odds ratios Γ and Λ from the simulations. 3. Results The analyses were conducted on the subset of the 391 children without missing covariates. The covariates distribution in this subset did not substantially differ from that observed on the entire data set (Table 1). 6 of 12 Int. J. Environ. Res. Public Health 2019, 16, 4381 Table 1. Distribution of the characteristics of the child–mother couples in the entire data set and in the subset of the complete observations by exposure. N. of Missing Values Entire Data Set (n = 438) Complete Cases (n = 391) Percentage/Mean Percentage/Mean Treated Controls Treated Controls Gender 0 Male 45.7 44.7 47.6 45.4 Female 54.3 55.3 52.4 54.6 Neuropsychologist 0 First 98.4 47.8 97.8 50.6 Second 1.6 52.2 2.2 49.4 Maternal fruit and veg. intake 5 >405 g/day 76.2 70.6 77.7 70.4 ≤405 g/day 23.8 29.4 22.3 29.6 Maternal smoke 13 Yes 18.2 28.3 18.4 27.4 No 81.8 71.7 81.6 72.6 Smoke at 32 weeks of pregnancy 34 Yes 11.3 11.0 11.0 11.1 No 88.7 89.0 89.0 88.9 Nursery attendance 26 Yes 48.6 44.6 48.7 44.9 No 51.4 55.4 51.3 55.1 Caregiver 28 Mother 55.6 48.2 55.2 48.0 Other 44.4 51.8 44.8 52.0 Maternal education 2 University 47.6 53.9 44.8 53.0 Secondary school or less 52.4 46.1 55.2 47.0 Maternal work 0 Manual 45.6 38.3 46.0 36.6 Non manual 54.4 61.7 54.0 63.4 Mother’s age 0 (mean) 31.2 31.7 31.3 31.7 Mother’s Body Mass Index 1 (mean) 23.1 22.7 23.2 22.8 Parity 0 1+ 46.4 44.4 45.6 42.8 0 53.6 55.6 54.4 57.2 Breastfeeding 24 No 46.6 47.6 47.1 45.5 Yes 53.4 52.4 52.9 54.5 The reported proportions (for the categorical variables) and means (for the continuous variables) come from the combination of the same quantities arising from the 5 imputed data sets [32]. The reported proportions (for the categorical variables) and means (for the continuous variables) come from the combination of the same quantities arising from the 5 imputed data sets [32]. The reported proportions (for the categorical variables) and means (for the co combination of the same quantities arising from the 5 imputed data sets [32]. The procedure for the balancing check implemented in the pscore function did not find a relevant difference between the covariate distribution of treated and controls within strata of children with similar estimated PS (results not reported). 3. Results Moreover, the distributions of the estimated PS among treated and controls substantially overlapped (Table S1), indicating that it was possible to find, for each treated, a matched control with similar PS, as stated by the positivity condition (ii). We would like to stress that the fact that the probability of being treated was close to zero for the children evaluated by the second neuropsychologist (Table 1) did not affect the assumption (ii). The number of subjects in the high exposure group varied from 183 to 213 depending on the imputed data set. Similarly, the number of matched controls varied from 62 to 69. The population of the treated units, which the ATT referred to, was quite stable across imputed data sets: for more than 90% of the 391 analyzed children the exposure status did not change across the imputed data sets: 172 children were “always exposed” and 181 were “always unexposed” across the five imputed data sets (Figure S2). Likely, the exposure status was uncertain only for children with actual average prenatal exposure close to the threshold of 17 µg/m3. Combining the estimates of the ATT from the five imputations, we found that the expected scores under PM2.5 ≥17µg/m3 were lower than the average score under PM2.5< 17 µg/m3, with final ATT estimates equal to −2.47 (90% CI: −7.22, 2.28) for the mental score and −3.18 (90% CI: −7.61, 1.25) for the psychomotor score. In all the analyses, the statistical variability was due to both the within and between-imputation variances, with the second being less than 10% of the first one (Table S2). 7 of 12 Int. J. Environ. Res. Public Health 2019, 16, 4381 Tables 2 and 3 report the results of the sensitivity analyses conducted by simulating several versions of U mimicking the behavior of the observed confounders. In doing this we selected some relevant categories of the confounders and simulated U according to the corresponding indicators (this was necessary because the pscore function permits only for the definition of binary unobserved confounders). The odds ratios Γ and Λ were calculated defining Y* = 1 if Y > 75th percentile and 0 otherwise. Even if in principle the selection effect Λ should be the same in the mental and psychomotor scores analyses, the values reported in Tables 1 and 2 are different because they arise from different simulations. 3. Results The odds ratio Γ ranged from 0.20 to 2.25 for mental score and from 0.31 to 4.30 for psychomotor score; Λ ranged from 0.57 to very large values (>103). This indicates that we were exploring a fairly wide class of possible unobserved confounders. Table 2. Results of the simulation-based sensitivity analysis for mental score. Observed Confounder Mimicked by U ATT 90% CI Outcome Effect (Γ) Selection Effect (Λ) Gender: male, female (ref) −1.88 −7.12; 3.35 0.57 1.07 Neuropsychologist: first, second (ref) 0.78 −7.02; 8.57 0.20 >103 Maternal fruit and vegetable intake: ≤405 g/day (ref), >405 g/day −1.89 −7.24; 3.47 1.04 1.55 Smoke: yes, no (ref) −1.73 −7.11; 3.65 1.18 0.62 Smoke at 32 weeks of pregnancy: yes, no (ref) −2.07 −7.35; 3.22 1.76 1.19 Nursery attendance: yes (ref), no −1.88 −7.13; 3.37 1.05 0.89 Caregiver: both parents, other (ref) −1.81 −7.16; 3.55 1.75 0.90 Caregiver: mother, other (ref) −1.85 −7.14; 3.44 0.85 1.42 Caregiver: relative, other (ref) −2.11 −7.28; 3.07 0.39 0.74 Maternal education: secondary school or less, university (ref) −1.74 −7.13; 3.65 0.74 1.43 Maternal work: non-manual worker (ref), manual worker −1.75 −7.12; 3.61 0.63 1.52 Mother’s age: <25, 25+ (ref) −2.12 −7.29; 3.06 0.31 3.00 Mother’s age: <35 (ref), 35+ −1.92 −7.25; 3.41 0.70 0.58 Body Mass Index: Normal weight, other (ref) −1.89 −7.22; 3.43 2.25 1.00 Parity: 0 (ref), 1+ −1.92 −7.16; 3.32 0.97 1.15 Breastfeeding: no, yes (ref) −1.88 −7.18; 3.42 1.67 1.16 Results of the simulation-based sensitivity analysis on the average causal effect of treatment on the treated (ATT) for the mental score. Unobserved confounders U were simulated to mimic the observed covariates. The estimated ATTs (90% CI) after adjusting for the different versions of U are reported, as well as the odds ratios for outcome and selection effects (Γ and Λ) calculated from the simulations. All the reported results arose from the combination of the results from 5 multiple imputed data sets, according to Rubin [32]. Table 2. Results of the simulation-based sensitivity analysis for mental score. Results of the simulation-based sensitivity analysis on the average causal effect of treatment on the treated (ATT) for the mental score. Unobserved confounders U were simulated to mimic the observed covariates. The estimated ATTs (90% CI) after adjusting for the different versions of U are reported, as well as the odds ratios for outcome and selection effects (Γ and Λ) calculated from the simulations. 3. Results All the reported results arose from the combination of the results from 5 multiple imputed data sets, according to Rubin [32]. Overall, including among the confounders in the PS matching, the simulated Us also did not bring to qualitatively different ATT estimates. Only when U mimicked the neuropsychologist indicator, generating low values of Γ and very large values of Λ, the estimated effects were fully explained away. It is worth noticing that extremely high values of Λ when U mimicked the neuropsychologist indicator originated from the fact that the probability of being treated for the children evaluated by the second neuropsychologist ranged, in the imputed data sets, from 1% to 5%. The results obtained in searching for the killer confounder are reported in Figure 2. Unobserved confounders positively associated with the outcome and negatively associated with the exposure, or vice versa, could kill the effect if at least one of the odds ratios Λ and Γ was approximately lower than 1:10 or larger than 10:1. However, the plausibility of having omitted a confounder of such strength was poor in this context, especially considering that many relevant covariates had already been included in the PS model. 8 of 12 Int. J. Environ. Res. Public Health 2019, 16, 4381 Table 3. Results of the simulation-based sensitivity analysis for psychomotor score. 3. Results Public Health 2019, 16, x 9 Results of the simulation-based sensitivity analysis on the average causal effect of treatment on the treated (ATT) for the psychomotor score. Unobserved confounders U were simulated to mimic the observed covariates. The estimated ATTs (90% CI) after adjusting for the different version of U are reported, as well as the odds ratios for outcome and selection effects (Γ and Λ), calculated from the simulations. All the reported results arose from the combination of the results from 5 multiple imputed data sets, according to Rubin [32]. J. Environ. Res. Public Health 2019, 16, x 9 Figure 2. Configurations of the outcome and selection effects (Γ and Λ) driving the causal effect estimate to zero, as obtained from the simulation-based sensitivity analysis a. Bold points correspond to unobserved confounders that could kill the effect. a Simulations were performed setting the tuning parameters 𝑝ଵ଴, 𝑝ଵଵ, 𝑝଴ଵ, 𝑝଴଴ to 0.1, 0.25, 0.5, 0.75, 0.9. 4. Discussion Our results suggest that prenatal exposure to average levels of PM2 5 ≥17 µg/m3 had a negative Figure 2. Configurations of the outcome and selection effects (Γ and Λ) driving the causal effect estimate to zero, as obtained from the simulation-based sensitivity analysis a. Bold points correspond to unobserved confounders that could kill the effect. a Simulations were performed setting the tuning parameters p10, p11, p01, p00 to 0.1, 0.25, 0.5, 0.75, 0.9. Selecting the 50th percentile of Y for the definition of Y* did not lead to substantially ifferent conclusions. Figure 2. Configurations of the outcome and selection effects (Γ and Λ) driving the causal effect estimate to zero, as obtained from the simulation-based sensitivity analysis a. Bold points correspond to unobserved confounders that could kill the effect. a Simulations were performed setting the tuning parameters 𝑝ଵ଴, 𝑝ଵଵ, 𝑝଴ଵ, 𝑝଴଴ to 0.1, 0.25, 0.5, 0.75, 0.9. Figure 2. Configurations of the outcome and selection effects (Γ and Λ) driving the causal effect estimate to zero, as obtained from the simulation-based sensitivity analysis a. Bold points correspond to unobserved confounders that could kill the effect. a Simulations were performed setting the tuning parameters p10, p11, p01, p00 to 0.1, 0.25, 0.5, 0.75, 0.9. 4. Discussion Our results suggest that prenatal exposure to average levels of PM ≥17 µg/m3 had a negative Selecting the 50th percentile of Y for the definition of Y* did not lead to substantially different conclusions. 4. 3. Results Observed Confounder Mimicked by U ATT 90% CI Outcome Effect (Γ) Selection Effect (Λ) Gender: male, female (ref) −2.59 −8.13; 2.95 0.58 1.12 Neuropsychologist: first, second (ref) −0.88 −9.38; 7.61 0.31 >103 Maternal fruit and vegetable intake: ≤405 g/day (ref), >405 g/day −2.70 −8.32; 2.91 1.60 1.65 Smoke: yes, no (ref) −2.33 −7.97; 3.31 2.42 0.63 Smoke at 32 weeks of pregnancy: yes, no (ref) −2.89 −8.35; 2.57 3.77 1.27 Nursery attendance: yes (ref), no −2.69 −8.28; 2.89 1.07 0.90 Caregiver: both parents, other (ref) −2.67 −8.19; 2.84 1.62 0.90 Caregiver: mother, other (ref) −2.60 −8.22; 3.02 1.03 1.44 Caregiver: relative, other (ref) −2.88 −8.31; 2.54 0.36 0.83 Maternal education: secondary school or less, university (ref) −2.47 −8.19; 3.25 0.93 1.48 Maternal work: non-manual worker (ref), manual worker −2.44 −8.14; 3.26 0.71 1.53 Mother’s age: <25, 25+ (ref) −2.91 −8.28; 2.45 4.30 3.18 Mother’s age: <35 (ref), 35+ −2.70 −8.39; 2.99 0.71 0.57 Body Mass Index: Normal weight, other (ref) −2.77 −8.2; 2.67 0.84 0.95 Parity: 0 (ref), 1+ −2.69 −8.26; 2.89 1.80 1.23 Breastfeeding: no, yes (ref) −2.66 −8.16; 2.85 0.76 1.07 Results of the simulation-based sensitivity analysis on the average causal effect of treatment on the treated (ATT) for the psychomotor score. Unobserved confounders U were simulated to mimic the observed covariates. The estimated ATTs (90% CI) after adjusting for the different version of U are reported, as well as the odds ratios for outcome and selection effects (Γ and Λ), calculated from the simulations. All the reported results arose from the combination of the results from 5 multiple imputed data sets, according to Rubin [32]. nt. J. Environ. Res. Public Health 2019, 16, x 9 o Table 3. Results of the simulation-based sensitivity analysis for psychomotor score. Results of the simulation-based sensitivity analysis on the average causal effect of treatment on the treated (ATT) for the psychomotor score. Unobserved confounders U were simulated to mimic the observed covariates. The estimated ATTs (90% CI) after adjusting for the different version of U are reported, as well as the odds ratios for outcome and selection effects (Γ and Λ), calculated from the simulations. All the reported results arose from the combination of the results from 5 multiple imputed data sets, according to Rubin [32]. J. Environ. Res. 4. Discussion Our results suggest that prenatal exposure to average levels of PM2.5 ≥17 µg/m3 had a negative effect on a child’s mental and psychomotor development at around 15 months of age, even if the confidence intervals of the effects estimates were wide, reflecting large sampling variability. Unobserved confounders moderately associated with both outcome and exposure could kill the effects in the sense of driving the point estimates of the causal effects to zero. However, the required associations were stronger than those calculated for most of the observed confounders, making the omission of such relevant factors quite implausible, in particular if the sensitivity analysis results are interpreted “in context”, i.e., accounting for number and relevance of the observed covariates which we already adjusted for [13]. In fact, the sensitivity analysis explores the robustness of the results to the existence of unmeasured confounders that act through pathways independent of the observed covariates. Therefore, the same strength of association required to an unobserved confounder to kill the effect can be indicative of large robustness if the original model accounts for many relevant covariates, as in the present paper, or of poor robustness if the original model accounts just for few confounding factors. g In interpreting the results of the sensitivity analysis, we focused on the point estimates of the effects and not on the confidence intervals limits as sometimes suggested (searching for possible unobserved confounders able to explain away the upper or lower limit of the interval, depending on the sign of effect estimate) [11,12]. This choice was motivated by our will to distinguish between the evaluation of bias and evaluation of uncertainty. In particular, we think that the sensitivity analysis should be performed regardless the wideness of the confidence interval and its position in respect to the null hypothesis, because its aim is to evaluate the possible presence of major bias due to omission of relevant confounders, which is different from evaluating the “significance” of the result. When the confidence interval of the estimate is wide as in our analysis, the robustness of the point estimate, if demonstrated, can confer a certain strength to the conclusions, despite the large sampling variability. As other kinds of selective reporting, limiting the sensitivity analysis to the subset of “significant” results may produce bias in the literature, with only studies with narrow confidence intervals evaluated for their robustness in respect to unobserved confounding. 3. Results Discussion Our results suggest that prenatal exposure to average levels of PM2 5 ≥17 µg/m3 had a negativ Selecting the 50th percentile of Y for the definition of Y* did not lead to substantially different conclusions. Int. J. Environ. Res. Public Health 2019, 16, 4381 9 of 12 4. Discussion This issue is crucial especially in a perspective where, as desirable, this kind of procedure can become standard in observational studies. Finally, even if it refers to a specific case study, the sensitivity analysis may provide useful insights about the confounders to be accounted for or collected in future studies on the same research question, contributing to enhance the knowledge about the phenomenon of interest. Ichino’s sensitivity analyses rely on the definition of a single binary unobserved confounder U, and on the assumption of no interaction between the effects of the exposure and U on the outcome. A sensitivity analysis that relaxes these assumptions has been recently proposed by Ding and VanderWeele [12,13], but it was not considered in this paper. A second drawback of the simulation method is its high computational demand. In fact, it requires that the statistical procedure used to estimate the effect is repeated n times in order to generate a distribution of the effect estimate adjusted for the hypothetical unobserved confounder. This is particularly expensive when complex analyses are required, as in our application, where an MI procedure is used. The computational burden of the simulation approach is particularly evident when the aim is to search for the killer confounder over the whole space of the hypothetical Us. Our findings qualitatively agree with those obtained for the same cohort by Lertxundi et al. [7] using a regression approach. In Lertxundi et al. [7], the linearity assumption on the exposure-outcome relationship, together with the parametric specification of the exposure-confounders-outcome relationship, contributed to increase the precision of the effect estimates, but, at the same time, could have generated a certain amount of bias due to problems of inappropriate model specification. On the contrary, the PS matching protected us from possible bias due to nonlinearity or inappropriate modeling of the exposure-confounders-outcome relationship, but at the prize of a lower precision of the effect estimates. Anyway, the fact that different statistical methods brought to consistent results strengthens our conclusions regarding the negative effect of the exposure. Int. J. Environ. Res. Public Health 2019, 16, 4381 10 of 12 10 of 12 Study Limitations Our study has several limitations. First, we limited the analyses to the subset of children for which all covariates included in the PS model were observed. This affected the power of the analysis and could have brought a certain amount of bias, although it has been suggested that in several situations estimating the PS on the complete cases can yield a valid estimate of the causal effect in the subjects without missing values [33]. Second, exposure during pregnancy was not based on personal exposure, that, if available, could provide more accurate results. Always in relation with the exposure definition, because of to the small dimension of the area and the small number of available monitors, we preferred to associate to each mother–child couple the level of PM2.5 measured by the monitor located in the municipality of residence instead of using more complex exposure assessment procedure, e.g., kriging interpolation, to predict the level of the exposure at the residence address. Finally, we treated continuous exposure as a binary one, after having defined an arbitrary threshold. Focusing on a binary version of the exposure allowed us to apply the sensitivity analysis as implemented in the sensatt function without complex adaptations, but at the same time led to a certain degree of arbitrariness related to the choice of the threshold for the exposure, and to loss of information which resulted in large sampling variability. A future promising extension of the analysis could use methods based on the generalized propensity score, which, treating the exposure as continuous, allows for estimating the whole exposure-response relationship [34,35]. References 1. WHO. Health Risks of Air Pollution in Europe—HRAPIE Project: Recommendations for Concentration–Response Functions for Cost–Benefit Analysis of Particulate Matter, Ozone and Nitrogen Dioxide; World Health Organization Regional Office for Europe: Copenhagen, Denmark, 2013. 2. WHO. Review of Evidence on Health Aspects of Air Pollution—REVIHAAP Project; World Health Organization Regional Office for Europe: Copenhagen, Denmark, 2013. 3. Suades-González, E.; Gascon, M.; Guxens, M.; Sunyer, J. Air Pollution and Neuropsychological Development: A Review of the Latest Evidence. Endocrinology 2015, 156, 3473–3482. [CrossRef] [PubMed] 4. Harris, M.H.; Gold, D.R.; Rifas-Shiman, S.L.; Melly, S.J.; Zanobetti, A.; Coull, B.A.; Schwartz, J.D.; Gryparis, A.; Kloog, I.; Koutrakis, P.; et al. Prenatal and Childhood Traffic-Related Pollution Exposure and Childhood Cognition in the Project Viva Cohort (Massachusetts, USA). Environ. Health Perspect. 2015, 123, 1072–1078. [CrossRef] [PubMed] 5. Guxens, M.; Aguilera, I.; Ballester, F.; Marisa Estarlich, M.; Fernández-Somoano, A.; Lertxundi, A.; Lertxundi, N.; Mendez, M.A.; Tardón, A.; Vrijheid, M.; et al. Prenatal Exposure to Residential Air Pollution and Infant Mental Development: Modulation by Antioxidants and Detoxification Factors. Environ. Health Perspect. 2012, 120, 144–149. [CrossRef] [PubMed] 6. Guxens, M.; Garcia-Esteban, R.; Giorgis-Allemand, L.; Forns, J.; Badaloni, C.; Ballester, F.; Beelen, R.; Cesaroni, G.; Chatzi, L.; de Agostini, M.; et al. Air pollution during pregnancy and childhood cognitive and psychomotor development: Six European birth cohorts. Epidemiology 2014, 25, 636–647. [CrossRef] 7. Lertxundi, A.; Baccini, M.; Lertxundi, N.; Fano, E.; Aranbarri, A.; Martínez, M.D.; Ayerdi, M.; Alvarez, J.; Santa-Marina, L.; Dorronsoro, M.; et al. Exposure to fine particle matter, nitrogen dioxide and benzene during pregnancy and cognitive and psychomotor developments in children at 15 months of age. Environ. Int. 2015, 80, 33–40. [CrossRef] 8. Lertxundi, A.; Andiarena, A.; Martínez, M.D.; Ayerdi, M.; Murcia, M.; Estarlich, M.; Sunyer, J.; Julvez, J.; Ibarluzea, J. Prenatal exposure to PM2.5 and NO2 and sex-dependent infant cognitive and motor development. Environ. Res. 2019, 174, 114–121. [CrossRef] 9. Imbens, G.W.; Rubin, D.B. Causal Inference: For Statistics, Social, and Biomedical Sciences: An Introduction, 1st ed.; Cambridge University Press: New York, NY, USA, 2015. [CrossRef] 10. Rothman, K.J.; Greenland, S.; Lash, T.L. Modern Epidemiology, 3rd ed.; Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2008. 11. Ichino, A.; Mealli, F.; Nannicini, T. From temporary help jobs to permanent employment: What can we learn from matching estimators and their sensitivity? J. Appl. Econom. 2008, 23, 305–327. [CrossRef] 12. Ding, P.; VanderWeele, T.J. Sensitivity Analysis Without Assumptions. Epidemiology 2016, 27, 368–377. 5. Conclusions Although the variability of the estimates was large, our findings indicated that prenatal exposure to high levels of PM2.5 had a negative effect on a child’s mental and psychomotor development in the analyzed cohort. The causal effect estimates appeared substantially robust to the possible presence of unmeasured confounders, even considering that the causal estimates were already adjusted for many relevant factors. Checking the robustness of the estimates to the presence of unobserved confounders is very important in observational studies and should not be considered less relevant than quantifying the sampling uncertainty around the estimates. Especially when new relationships are investigated, sensitivity analyses aimed to evaluate the robustness of the results to the omission of relevant factors in the analysis should become a standard. In this paper, we proposed a simulation approach to be employed after PS matching that, even if computationally expensive, is implemented in standard statistical software and provides results easy to be interpreted. The use of other methods for sensitivity analysis, which eventually require less computational effort and rely on less stringent assumptions, should be explored as well. Supplementary Materials: The following are available online at http://www.mdpi.com/1660-4601/16/22/4381/s1, Figure S1: Time series of monthly average PM2.5 exposures and monthly average mental and psychomotor scores. The red line indicates the time when the second neuropsychologist substituted the first one; Figure S2: Distribution of the average prenatal exposure to PM2.5, by the imputed data set; Table S1: Range of the estimated propensity score among treated and controls, by imputation; Table S2: Between imputation variance, within imputation variance and their ratio, calculated for the average causal effect of treatment on the treated (ATT) for mental and psychomotor scores. Author Contributions: M.B. and A.L. designed the study. M.B. designed and supervised the statistical analyses. M.B. and V.T. performed the statistical analyses and wrote the first version of the manuscript. A.L. acquired data and controlled their quality. M.B., A.L., J.M.I. discussed the results. All the authors critically reviewed and approved the final version of the manuscript. Funding: This study was partially supported by local research funds (ex 60% funding program University of Florence 2018), by Basque Government funds (reference: 2009111069) and Spanish Health Ministry funds (reference: PI06/0867). Conflicts of Interest: The authors declare no conflict of interest. 11 of 12 11 of 12 Int. J. Environ. Res. Public Health 2019, 16, 4381 References The cost of dichotomising continuous variables. BMJ 2006, 332, 1080. [CrossRef 25. Altman, D.G.; Royston, P. The cost of dichotomising continuous variables. BMJ 2006, 332, 1080. [CrossRef] 26. Rosenbaum, P.R.; Rubin, D.B. The central role of the propensity score in observational studies for causal effects. Biometrika 1983, 70, 41–55. [CrossRef] 26. Rosenbaum, P.R.; Rubin, D.B. The central role of the propensity score in observational studies for causal effects. Biometrika 1983, 70, 41–55. [CrossRef] 27. Brookhart, M.A.; Schneeweiss, S.; Rothman, K.J.; Glynn, R.J.; Avorn, J.; Stürmer, T. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. References f 11. Ichino, A.; Mealli, F.; Nannicini, T. From temporary help jobs to permanent employment: What can we learn from matching estimators and their sensitivity? J. Appl. Econom. 2008, 23, 305–327. [CrossRef] 12. Ding, P.; VanderWeele, T.J. Sensitivity Analysis Without Assumptions. Epidemiology 2016, 27, 368–377. [CrossRef] 13. VanderWeele, T.J.; Ding, P. Sensitivity Analysis in Observational Research: Introducing the E-Value. Ann. Intern. Med. 2017, 167, 268–274. [CrossRef] 14. Cornfield, J.; Haenszel, W.; Hammond, E.C.; Lilienfeld, A.M.; Shimkin, M.B.; Wynder, E.L. Smoking and lung cancer: Recent evidence and a discussion of some questions. J. Natl. Cancer Inst. 1959, 22, 173–203. [CrossRef] 15. Rosenbaum, P.R.; Rubin, D.B. 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Generalized Cornfield conditions for the risk difference. Biometrika 2014, 1–13. [CrossRef] 19. Hernán, M.A. Invited commentary: Hypothetical interventions to define causal effects—Afterthought or prerequisite? Am. J. Epidemiol. 2005, 162, 618–620. [CrossRef] 20. Nannicini, T. A Simulation-Based Sensitivity Analysis for Matching Estimators. Stata J. 2007, 7, 334–350. [CrossRef] 12 of 12 Int. J. Environ. Res. Public Health 2019, 16, 4381 21. StataCorp. Stata Statistical Software: Release 13; StataCorp LP.: College Station, TX, USA, 2013. 22. Ramón, R.; Ballester, F.; Rebagliato, M.; Ribas, N.; Torrent, M.; Fernández, M.; Sala, M.; Tardón, A.; Marco, A.; Posada, M.; et al. The Environment and Childhood Research Network (“INMA” network): Study protocol. Rev. Española Salud Publica 2005, 79, 203–220. [CrossRef] 3. Bayley. Bayley Scales of Infant Development; Psychological Corporation: San Antonio, TX, USA, 1977. 24. Li, F.; Baccini, M.; Mealli, F.; Zell, E.R.; Frangakis, C.E.; Rubin, D.B. Multiple imputation by ordered monotone blocks with application to the anthrax vaccine research program. J. Comput. Graph. Stat. 2014, 23, 877–892. [CrossRef] Altman, D.G.; Royston, P. References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Aspirin has little additional anti‐platelet effect in healthy volunteers receiving prasugrel
Journal of thrombosis and haemostasis
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Introduction The use of dual anti-platelet therapy comprising aspirin and a P2Y12 inhibitor, such as clopidogrel or prasugrel, has become a standard therapy in patients at risk of thrombotic vascular events [1–4]. The rationale of this approach is to target the primary drivers of platelet aggregation: thromboxane A2 (TXA2) and ADP. Aspirin reduces platelet TXA2 production and P2Y12 receptor blockers prevent binding of the ligand ADP. The observation that strong P2Y12 inhibition alone can reduce TXA2-dependent platelet aggregation in both in vitro and in vivo models [5–8] has raised the question of whether aspirin provides an additional anti-platelet effect in the presence of strong P2Y12 receptor blockade [9,10]. In addition to its anti-platelet effects, aspirin also reduces the formation of other intravascular prostanoids, such as the vasoprotective hormone prostacyclin (PGI2) [11]. PGI2 may be important in controlling platelet responses to pro-aggregatory stimuli and we have proposed that the administration of aspirin in the setting of strong P2Y12 receptor blockade removes this protective factor, while the reduction of TXA2 adds little to the anti-platelet effect [9]. The production of each prostanoid within the body can be assessed by measurement of the stable urinary metabolites: 2,3-dinor-6-keto PGF1a (PGI-M) for PGI2 and 11-dehydro-TXB2 (TX-M), for TXA2. In the present study, we aimed to investigate, in healthy male volunteers, whether co-administration of either low or high doses of aspirin with prasugrel provide any additional bene- ficial effects on platelet reactivity and intravascular prostanoid levels, over those seen with prasugrel alone. Correspondence: Tim D. Warner, The William Harvey Research Institute, Barts & the London School of Medicine & Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Tel.: +44 20 7882 2100; fax: +44 20 7882 8251. Tel.: +44 20 7882 2100; fax: +44 20 7882 8251. E-mail: t.d.warner@qmul.ac.uk E-mail: t.d.warner@qmul.ac.uk Aspirin has little additional anti-platelet effect in healthy volunteers receiving prasugrel P. D. M. LEADBEATER*, N. S. KIRKBY*, S. THOMAS, A.-R. DHANJI, A. T. TUCKER§, G. L. MILNE, J. A. MITCHELL* and T. D. WARNER *Department of Cardiothoracic Pharmacology, Imperial College London, National Heart and Lung Institute; The William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK; Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, TN, USA; and §The Ernest D Cooke Clinical Microvascular Unit, St Bartholomews Hospital, London, UK London, UK To cite this article: Leadbeater PDM, Kirkby NS, Thomas S, Dhanji A-R, Tucker AT, Milne GL, Mitchell JA, Warner TD. Aspirin has little additional anti-platelet effect in healthy volunteers receiving prasugrel. J Thromb Haemost 2011; 9: 2050–6. Keywords: aspirin, PGI-M, platelets, prasugrel, TX-M. Summary. Background: Strong P2Y12 blockade, as can be achieved with novel anti-platelet agents such as prasugrel, has been shown in vitro to inhibit both ADP and thromboxane A2- mediated pathways of platelet aggregation, calling into question the need for the concomitant use of aspirin. Objective: The present study investigated the hypothesis that aspirin provides little additional anti-aggregatory effect in a group of healthy volunteers taking prasugrel. Study participants/methods: In all, 9 males, aged 18 to 40 years, enrolled into the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg until day 21. At day 8, aspirin 75 mg was introduced and the dose increased to 300 mg on day 15. On days 0, 7, 14 and 21, platelet function was assessed by aggregometry, response to treatments was determined by VerifyNowTM and urine samples were collected for quantification of prostanoid metabolites. Results: At day 7, aggregation responses to a range of platelet agonists were reduced and there was only a small further inhibition of aggregation to TRAP-6, collagen and epinephrine at days 14 and 21, when aspirin was included with prasugrel. Urinary prostanoid metabolites were unaffected by prasugrel, and were reduced by the addition of aspirin, independent of dose. Conclusions: In healthy volunteers, prasugrel produces a strong anti-aggregatory effect, which is little enhanced by the addition of aspirin. The addition of aspirin as a dual-therapy with potent P2Y12 receptor inhibitors warrants further inves- tigation.  2011 International Society on Thrombosis and Haemostasis DOI: 10.1111/j.1538-7836.2011.04450.x DOI: 10.1111/j.1538-7836.2011.04450.x Journal of Thrombosis and Haemostasis, 9: 2050–2056 Methods Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen# OnlineOpen_Terms  2011 International Society on Thrombosis and Haemostasis Blood collection for platelet aggregation studies Blood was collected by venepuncture into tri-sodium citrate (3.2%, 1:9 v/v; Sigma, Poole, Dorset, UK). Platelet-rich plasma (PRP) was obtained by centrifugation at 175 · g for 15 min at 25 C. Platelet-poor plasma (PPP) was obtained by centrifu- gation of PRP at 15 000 · g for 2 min. All experiments were completed within 2 h of blood collection. 96-well plate light transmission aggregometry PGI-M (2,3-dinor-6-keto PGF1a) and TXB-M (11-dehydro- TXB2) were quantified using stable isotope dilution assays with gas chromatography/mass spectrometry, as previously described [13]. To assess the aggregation of platelets in 96-well plates a modified light transmission method was used. Briefly, 100 lL samples of PRP were placed into the individual wells of a 96- well microtiter plate (Nunc, Lutterworth, Leicestershire, UK) containing 10 lL of vehicle or agonist: ADP (0.1–30 lM; LabMedics, Salford, Manchester, UK), arachidonic acid (AA; 0.03–1 mM; Sigma), Horm collagen (0.1–30 lg mL)1; Ny- comed, Linz, Austria), epinephrine (0.001–100 lM; LabMed- ics), TRAP-6 amide specific for PAR1 (SFLLRN; 0.1–30 lM; VerifyNowTM P2Y12 and aspirin cartridge assays Blood was collected by venepuncture into 2-mL partial fill vacuum tubes containing tri-sodium citrate (3.2% final concentration; Griener Bio-One, Stonehouse, UK). Verify- NowTM assays for effects of P2Y12 receptor blockade and aspirin activity were performed in accordance with the manufacturers instructions (VerifyNowTM; Accumetrics, Eli- tech, UK). A positive response to aspirin was taken as an aspirin response unit (ARU) score < 550, as described in the cartridge package insert. Study participants Study participants In all, 9 healthy male volunteers, aged 18–40 years, were recruited and participated in the present study. The volunteers Received 28 February 2011; accepted 7 July 2011 Leadbeater: prasugrel and aspirin 2051 Bachem, Bubendorf, Switzerland) and the stable TXA2- mimetic U46619 (0.1–30 lM; Cayman Chemical Company, Ann Arbor, MI, USA). The plate was then placed into a 96- well plate reader (Tecan Sunrise, Tecan, Reading, UK) at 37 C, and absorbance was measured at 595 nm every 15 s for 16 min with vigorous shaking between readings. Percentage aggregation was calculated with reference to the absorbance of PPP as a surrogate for 100% aggregation. Graphs shown are for aggregation responses at 16 min. health statuses were determined through their medical histories and physical examination including blood pressure, pulse rate, blood chemistry and urinalysis. Volunteers with normal clinical profiles were included in the study. The study was approved by the St Thomass Hospital Research Ethics Committee (Ref. 07/ Q0702/24), conducted according to the Declaration of Helsinki and all volunteers gave written informed consent before entering the study. Urine collection and storage A fresh mid-stream urine sample was collected into sterile containers and the preservative chlorhexidine-propyl gallate (1:1, 20% (wt/v) aqueous chlorhexidine digluconate and 20% (wt/v) n-propyl gallate in methanol) added at 1:500 parts. Samples were then stored at )40 C for later assay of urinary prostanoid metabolites. All samples were obtained 3–6 h after dosing on each study day. Thromboxane B2 assay Before starting this 21-day study, all volunteers had abstained from aspirin, non-steroid anti-inflammatory drugs (NSAIDs), paracetamol or any other anti-platelet therapy for 14 days. The volunteers received a 60-mg loading dose of prasugrel on day 1 of the study and a maintenance dose of 10 mg prasugrel per day on days 2–21 (Efient, Eli Lilly and Company, Basingstoke, UK). On day 8 of the study, volunteers began taking 75 mg aspirin and on day 15 the aspirin dose was increased to 300 mg per day until day 21 (Angettes 75; Bristol-Myers Squibb, Uxbridge, UK). Compliance was assessed by interview. Blood and urine samples were collected on day 0, before commencing drug treatment, and on days 7, 14 and 21 of treatment. At the end of the PRP aggregation monitoring (i.e. 16 min), cyclo-oxygenase activity was halted by the addition of 1 mM diclofenac (Sigma), the samples were centrifuged at 1300 · g for 10 min at 5 C, and the supernatants removed and frozen. In samples from PRP stimulated with either arachidonic acid or collagen, plasma thromboxane B2 (TXB2) levels, as a surrogate for TXA2 production, were determined using a selective, competitive EIA (Cayman Chemicals) in accordance with the package insert. In our previous studies we have found readily detectable levels of TXB2 after platelet activation by either collagen or AA, but assessments to be less robust for other agonists [7,8,10,12]. Samples were diluted between 1:10 and 1:1000 in diluent and assayed parallel to known TXB2 standards and a maximum binding control. The percentage binding of known standards was calculated in reference to the maximum binding (zero TXB2) control wells, plotted against the logarithm of concentration and analyzed by non-linear regression using a four-parameter logistical fit model. Unknown samples were expressed in a similar fashion, interpolated from this standard curve and corrected for dilution. All statistical analyzes were performed in Prism v5.0 (GraphPad software, La Jolla, CA, USA).  2011 International Society on Thrombosis and Haemostasis Stastical analysis Although the present study included only nine subjects we are confident that this is sufficient to detect differences between the  2011 International Society on Thrombosis and Haemostasis 2052 P. D. M. Leadbeater et al 100 A B 800 600 400 200 80 60 40 P2Y12 reactivity (% inhibition) Asprin reactivity units 20 0 0 - - - - - - 7 14 21 0 7 14 21 Day Prasugrel Aspirin Day Prasugrel Aspirin 10 mg o.d. 10 mg o.d. 10 mg o.d. 10 mg o.d. 75 mg o.d. ↑ Aspirin non-responder ↓ Aspirin responder 300 mg o.d. 75 mg o.d. 300 mg o.d. 10 mg o.d. 10 mg o.d. 100 A B 800 600 400 200 80 60 40 P2Y12 reactivity (% inhibition) Asprin reactivity units 20 0 0 - - - - - - 7 14 21 0 7 14 21 Day Prasugrel Aspirin Day Prasugrel Aspirin 10 mg o.d. 10 mg o.d. 10 mg o.d. 10 mg o.d. 75 mg o.d. ↑ Aspirin non-responder ↓ Aspirin responder 300 mg o.d. 75 mg o.d. 300 mg o.d. 10 mg o.d. 10 mg o.d. Fig. 1. VerifyNowTM aspirin and P2Y12 cartridge reactivity. Upper panel, percent inhibition of P2Y12 cartridge reactivity, normalized to baseline for values for each of nine study participants. Lower panel, Aspirin Reactivity Units (ARUs) for each of nine study participants at baseline, and study days 7, 14 and 21. ARU values < 550 indicate a response to aspirin (abnormal platelet response to arachidonic acid). In each panel, group means are indicated by a horizontal line. o.d., once daily. conditions that we have investigated. The variability of platelet aggregation responses between healthy volunteers in this study was 12% for AA, 8% for ADP, 5% for collagen, 8% for TRAP-6 and 6% for U46619 and we propose this low level of variability allowed changes in platelet reactivity to be detected readily, as we have found in previous studies [8,12]. Indeed, power calculations demonstrate that with our sample size (n = 9) in healthy volunteers and standard deviation (SD) of response (< 0.45) we had a > 98% power to detect after analysis of data by two-way ANOVA and Bonferronis post tests [14]. Similarly, others have detected effects in volunteer groups of 8–12 subjects [15,16]. A All statistical analyzes were conducted using GraphPad Prism v5 (GraphPad). Results Upper panel, percent inhibition of P2Y12 cartridge reactivity, normalized to baseline for values for each of nine study participants. Lower panel, Aspirin Reactivity Units (ARUs) for each of nine study participants at baseline, and study days 7, 14 and 21. ARU values < 550 indicate a response to aspirin (abnormal platelet response to arachidonic acid). In each panel, group means are indicated by a horizontal line. o.d., once daily. were also moderately inhibited by prasugrel alone, and at the highest concentrations of these agonists there was a further effect seen with aspirin, although there was no variation between the effects of the two aspirin doses (Fig. 2, panels C, D and E). Stastical analysis Agonist concentration response curves were plotted and analyzed according to the four parameter logistic equation: Y = Bottom + (Top ) Bot- tom)/[1 + 10^((LogEC50-X) · HillSlope)]. B Concentration-response curves were compared using two- way ANOVA and Bonferronis post-tests whereas urinary metab- olite data were compared by one-way ANOVA with Bonferronis post-tests. In each case statistical significance was taken as P < 0.05. Other analyzes are stated in text as appropriate. Asprin reactivity units Effects of prasugrel alone and in combination with aspirin on ex vivo platelet reactivity, in 96-well plate aggregometry Effects of prasugrel alone and in combination with aspirin on ex vivo platelet reactivity, in 96-well plate aggregometry Platelet aggregation to AA, ADP and U46619 was strongly inhibited at all concentrations by prasugrel alone, and prasu- grel in combination with aspirin, although there was no further reduction with aspirin at either dose (Fig. 2, panels A, B and F). Responses to collagen, epinephrine and TRAP-6 amide Thromboxane production in response to AA and collagen by 96-well plate aggregometry Prasugrel administration reduced the production of TXA2 in response to both AA and collagen by platelets in 96-well aggregometry (Fig. 3A and B, respectively). Co-administration of either dose of aspirin with prasugrel resulted in the complete abolition of any remaining TXA2 production.  2011 International Society on Thrombosis and Haemostasis Results Responses to prasugrel alone and in combination with aspirin in the VerifyNowTM P2Y12 and aspirin cartridge assays Responses to prasugrel alone and in combination with aspirin in the VerifyNowTM P2Y12 and aspirin cartridge assays To confirm the response of the study subjects to both prasugrel and aspirin we used the VerifyNowTM cartridge assay system. None of the subjects had positive tests for either P2Y12 inhibitors or aspirin at baseline (Fig. 1). With prasugrel alone, eight of the nine study participants had > 95% inhibition of baseline results on the P2Y12 assay, whereas the ninth participant had only 50% inhibition on this assay, and these results were maintained with the addition of both low- and high-dose aspirin (Fig. 1A). Although this subject was an outlier from the group, based on this assay, all data relating to this subject were included in further analyzes. It would have been interesting to test the efficacy of the prasugrel active metabolite in a PRP sample of this individual to explore reasons for this difference, but we could not arrange further blood sampling. After the addition of low-dose aspirin (75 mg), all subjects were identified as aspirin responders, and these responses were maintained at the higher dose of aspirin (300 mg) (Fig. 1B). Aspirin responsiveness was determined as an aspirin response units (ARU) score of < 550. Fig. 1. VerifyNowTM aspirin and P2Y12 cartridge reactivity. Upper panel, percent inhibition of P2Y12 cartridge reactivity, normalized to baseline for values for each of nine study participants. Lower panel, Aspirin Reactivity Units (ARUs) for each of nine study participants at baseline, and study days 7, 14 and 21. ARU values < 550 indicate a response to aspirin (abnormal platelet response to arachidonic acid). In each panel, group means are indicated by a horizontal line. o.d., once daily. Fig. 1. VerifyNowTM aspirin and P2Y12 cartridge reactivity. Upper panel, percent inhibition of P2Y12 cartridge reactivity, normalized to baseline for values for each of nine study participants. Lower panel, Aspirin Reactivity Units (ARUs) for each of nine study participants at baseline, and study days 7, 14 and 21. ARU values < 550 indicate a response to aspirin (abnormal platelet response to arachidonic acid). In each panel, group means are indicated by a horizontal line. o.d., once daily. Fig. 1. VerifyNowTM aspirin and P2Y12 cartridge reactivity.  2011 International Society on Thrombosis and Haemostasis Production of urinary prostanoid metabolites 100 75 50 25 0 ** * Day 0 Day 7 Day 21 Day 14 % aggregation –5 –4 –3 AA (log M) A 100 75 50 25 0 *** Day 0 Day 7 Day 21 Day 14 % aggregation –8 –7 –6 –5 –4 ADP (log M) B B A 100 75 50 25 0 100 75 50 25 0 * * ** *** ** 1 4 1 4 1 4 Day 0 Day 7 Day 21 Day 14 Day 0 Day 7 Day 21 Day 14 Day 0 Day 7 Day 21 Day 14 % aggregation % aggregation 100 75 50 25 0 % aggregation U46619 (log M) –8 –7 –6 –5 –4 ADP (log M) Epinephrine (log M) –10 –8 –6 –4 –8 –7 –6 –5 –4 B D F * ** Day 0 Day 7 Day 21 Day 14 100 75 50 25 0 % aggregation –5 –4 –3 AA (log M) Collagen (log g mL–1) –8 –7 –6 –5 –4 C 100 75 50 25 0 * ** Day 0 Day 7 Day 21 Day 14 % aggregation ADP (log M) Epinephrine (log M) –10 –8 –6 –4 D D C * ** D D D D 100 75 50 25 0 % aggregation Collagen (log g mL–1) TRAP-6 amide (log M) –8 –7 –6 –5 –4 E E Fig. 2. Platelet aggregation induced by arachidonic acid (0.03–1 mM; panel A), ADP (0.1–30 lM; panel B), collagen (0.1–30 lg mL)1; panel C), epi- nephrine (0.001–100 lM; panel D), TRAP6 (0.1–30 lM; panel E) and U46619 (0.1–30 lM; panel F) at baseline and on days 7, 14 and 21 (prasugrel, prasugrel + low aspirin and prasugrel + high aspirin, respectively). Data shown are mean ± standard error of the mean (SEM) of responses measured by 96-well plate aggregometry in citrated platelet-rich plasma (PRP) prepared from nine different individuals. All data points were analyzed by two-way ANOVA with Bonferronis post test. *Represents a significant difference, P-value < 0.05, compared with baseline; represents a significant difference, P- value < 0.05, between prasugrel and prasugrel + low aspirin. Symbols at the end of rows indicate a difference between all points; symbols at individual points signify particular differences. production of vascular PGI2. Production of urinary prostanoid metabolites To assess the changes in urinary prostanoid levels we studied the stable urinary metabolites of thromboxane, 11-dehydro-TXB2 (TX-M), and PGI2, 2,3 dinor-6-keto-PGF1a (PGI-M), at each time point of the study. Neither urinary PGI-M nor TX-M were reduced from baseline values with prasugrel alone (Fig. 4, panel A and B, respectively, P > 0.05). When prasugrel and low-dose aspirin were co-administered, the levels of both urinary prostanoid metabolites decreased (Day 0 or Day 7 vs. Day 14, PGI-M, P < 0.01; TX-M, P < 0.001), although there was no further decrease seen with prasugrel in combination with high-dose aspirin (Day 14 vs. Day 21, PGI-M, P > 0.05; TX-M, P > 0.05). production of vascular PGI2. To assess the changes in urinary prostanoid levels we studied the stable urinary metabolites of thromboxane, 11-dehydro-TXB2 (TX-M), and PGI2, 2,3 dinor-6-keto-PGF1a (PGI-M), at each time point of the study. Neither urinary PGI-M nor TX-M were reduced from baseline values with prasugrel alone (Fig. 4, panel A and B, respectively, P > 0.05). When prasugrel and low-dose aspirin were co-administered, the levels of both urinary prostanoid metabolites decreased (Day 0 or Day 7 vs. Day 14, PGI-M, P < 0.01; TX-M, P < 0.001), although there was no further decrease seen with prasugrel in combination with high-dose aspirin (Day 14 vs. Day 21, PGI-M, P > 0.05; TX-M, P > 0.05). dosing protocol, prasugrel inhibited platelet aggregation to a range of agonists, with additional effects of aspirin seen only against the highest concentrations of collagen, epinephrine and TRAP-6. The urinary levels of TXA2 and PGI2 metabolites were unaffected by prasugrel alone, but were both reduced by aspirin. Higher doses of aspirin in combination with prasugrel did not have any additional effects on either platelet reactivity or urinary prostanoid metabolites, compared with those seen with prasugrel and low-dose aspirin. The effects of prasugrel alone, and in combination with aspirin, were similar to those seen in a recent in vitro study reported by our group [10], although we observed a slightly larger effect of aspirin ex vivo. The observations presented here confirm that P2Y12 receptor blockade by prasugrel strongly inhibits platelet aggregation to ADP as measured by 96-well plate aggregometry, although a small residual response was seen to the highest concentrations of this agonist. In addition, platelet production of TXA2 and TXA2 (TP) receptor-depen- dent platelet aggregation was reduced. Production of urinary prostanoid metabolites In contrast to our recent in vitro findings [10], TP receptor-dependent aggregation was  2011 International Society on Thrombosis and Haemostasis Production of urinary prostanoid metabolites In addition to reducing platelet-derived TXA2 production, aspirin can, by inhibition of cyclooxygenases, reduce the  2011 International Society on Thrombosis and Haemostasis Leadbeater: prasugrel and aspirin 2053 100 75 50 25 0 100 75 50 25 0 100 75 50 25 0 ** * * * * ** * * ** ** ** ** Day 0 Day 7 Day 21 Day 14 Day 0 Day 7 Day 21 Day 14 Day 0 Day 7 Day 21 Day 14 Day 0 Day 7 Day 21 Day 14 Day 0 Day 7 Day 21 Day 14 Day 0 Day 7 Day 21 Day 14 % aggregation 100 75 50 25 0 % aggregation 100 75 50 25 0 % aggregation % aggregation % aggregation 100 75 50 25 0 % aggregation –5 –4 –3 AA (log M) Collagen (log g mL–1) TRAP-6 amide (log M) U46619 (log M) –8 –7 –6 –5 –4 ADP (log M) Epinephrine (log M) –8 –10 –8 –6 –4 –7 –6 –5 –4 –8 –7 –6 –5 –4 –8 –7 –6 –5 –4 A B C D E F Fig. 2. Platelet aggregation induced by arachidonic acid (0.03–1 mM; panel A), ADP (0.1–30 lM; panel B), collagen (0.1–30 lg mL)1; panel C), epi- nephrine (0.001–100 lM; panel D), TRAP6 (0.1–30 lM; panel E) and U46619 (0.1–30 lM; panel F) at baseline and on days 7, 14 and 21 (prasugrel, prasugrel + low aspirin and prasugrel + high aspirin, respectively). Data shown are mean ± standard error of the mean (SEM) of responses measured by 96-well plate aggregometry in citrated platelet-rich plasma (PRP) prepared from nine different individuals. All data points were analyzed by two-way ANOVA with Bonferronis post test. *Represents a significant difference, P-value < 0.05, compared with baseline; represents a significant difference, P- value < 0.05, between prasugrel and prasugrel + low aspirin. Symbols at the end of rows indicate a difference between all points; symbols at individual points signify particular differences. Discussion *Shows P < 0.01 difference from Day 0 by one-way ANOVA plus Bonferronis post test. grel + high aspirin, respectively). Data shown are mean ± standard error of the mean (SEM) of responses collected from 96-well plate aggregometry in citrated platelet-rich plasma (PRP) prepared from nine study particpants. *Shows P < 0.05 difference from baseline by two-way ANOVA plus Bonferronis post test; Shows P < 0.05 difference between prasugrel and prasugrel + low aspirin. Symbols at the end of lines signify a difference in set; symbols at individual points signify particular differ- ences. by the residual potential for TP receptor-induced platelet aggregation, as described above. not entirely abolished by prasugrel. As TP-mediated platelet aggregation relies upon the ADP-P2Y12 axis [5,6,10] this may be explained by the residual responses to ADP observed in the present study. Alternatively, this may reflect a P2Y12-indepen- dent response to TP receptor stimulation, although it is unclear why this may be apparent ex vivo after prasugrel treatment, although not after in vitro treatment with prasugrel active metabolite. not entirely abolished by prasugrel. As TP-mediated platelet aggregation relies upon the ADP-P2Y12 axis [5,6,10] this may be explained by the residual responses to ADP observed in the present study. Alternatively, this may reflect a P2Y12-indepen- dent response to TP receptor stimulation, although it is unclear why this may be apparent ex vivo after prasugrel treatment, although not after in vitro treatment with prasugrel active metabolite. Although the combination of prasugrel and aspirin severely limits the platelet aggregation response to soluble mediators (ADP and TXA2) there is still a reasonable aggregation response to high concentrations of platelet agonists that act through different signaling pathways. For instance, PAR1 activation by TRAP-6 amide is able to induce approximately 50% aggregation, even when ADP is rendered inactive and TXA2 formation is inhibited. Additionally, high concentrations of collagen induced a moderate level of aggregation in the absence of soluble mediator activities. This is consistent with strong signaling from the G-protein coupled receptors, a topic that has been addressed in two informative reviews [17,18], and from platelet integrins [19]. Measurable residual platelet reac- tivity is unsurprising given the wealth of clinical data that reportstheoccurrenceofocclusivethromboticeventsinpatients taking both aspirin and P2Y12 receptor blockers [20–23]. When aspirin is used in combination with prasugrel there is a complete inhibition of platelet TXA2 production, although the further reduction of platelet aggregation, compared with prasugrel alone, is limited. Discussion In this healthy volunteer study we looked at the effects of a strong P2Y12 receptor blocker, prasugrel, alone and in combination with low- and high-dose aspirin, on platelet reactivity and urinary prostanoid metabolites. After a standard 2054 P. D. M. Leadbeater et al by the residual potential for TP receptor-induced platelet 800 Day 0 Day 7 * * * * Day 14 Day 21 Day 0 Day 7 Day 14 Day 21 600 400 200 300 200 100 0 0 –5 –8 –7 –6 –5 –4 Arachidonic acid (log M) Collagen (log g mL–1) Thromboxane B2 (ng mL–1) Thromboxane B2 (ng mL–1) –4 –3 A B Fig. 3. Platelet production of TXA2 induced by arachidonic acid (0.03– 1 mM; panel A) and collagen (0.1–30 lg mL)1; panel B) at baseline and days 7, 14 and 21 (prasugrel, prasugrel + low aspirin and prasu- grel + high aspirin, respectively). Data shown are mean ± standard error of the mean (SEM) of responses collected from 96-well plate aggregometry in citrated platelet-rich plasma (PRP) prepared from nine study particpants. *Shows P < 0.05 difference from baseline by two-way ANOVA plus Bonferronis post test; Shows P < 0.05 difference between prasugrel and prasugrel + low aspirin. Symbols at the end of lines signify a difference in set; symbols at individual points signify particular differ- ences. 0.20 0.4 0.3 0.2 0.1 0.0 0.15 0.10 0.05 2,3 dinor-6-keto PGF 1α (ng mg–1 creatinine) 11-dehydro-TXB2 (ng mg–1 creatinine) 0.00 0 7 14 21 0 7 14 21 Day Prasugrel Aspirin Day Prasugrel Aspirin 10 mg o.d. 10 mg o.d. 10 mg o.d. 300 mg o.d. 75 mg o.d. - - - 10 mg o.d. 10 mg o.d. 10 mg o.d. 300 mg o.d. 75 mg o.d. - - - A B * * * * Fig. 4. Urinary PGI-M (panel A) and TX-M (panel B) production at days 0, 7, 14 and 21 (baseline, prasugrel, prasugrel + low aspirin and prasu- grel + high aspirin, respectively). Values for urinary prostanoid metab- olites (PGI-M or TX-M) are corrected against urinary creatinine concentration. Data shown are mean ± standard error of the mean (SEM) of nine study participants. *Shows P < 0.01 difference from Day 0 by one-way ANOVA plus Bonferronis post test. Discussion - - - A B * * * * Fig. 4. Urinary PGI-M (panel A) and TX-M (panel B) production at days 0, 7, 14 and 21 (baseline, prasugrel, prasugrel + low aspirin and prasu- grel + high aspirin, respectively). Values for urinary prostanoid metab- olites (PGI-M or TX-M) are corrected against urinary creatinine concentration. Data shown are mean ± standard error of the mean (SEM) of nine study participants. *Shows P < 0.01 difference from Day 0 by one-way ANOVA plus Bonferronis post test. 800 Day 0 Day 7 * * Day 14 Day 21 600 400 200 0 –5 Arachidonic acid (log M) Thromboxane B2 (ng mL–1) –4 –3 A A 5 Arachidonic acid (log M) 4 3 B * * Day 0 Day 7 Day 14 Day 21 300 200 100 0 –8 –7 –6 –5 –4 ( g ) Collagen (log g mL–1) Thromboxane B2 (ng mL–1) B B Collagen (log g mL–1) Fig. 3. Platelet production of TXA2 induced by arachidonic acid (0.03– 1 mM; panel A) and collagen (0.1–30 lg mL)1; panel B) at baseline and days 7, 14 and 21 (prasugrel, prasugrel + low aspirin and prasu- l + hi h i i ti l ) D t h ± t d d Fig. 3. Platelet production of TXA2 induced by arachidonic acid (0.03– 1 mM; panel A) and collagen (0.1–30 lg mL)1; panel B) at baseline and days 7, 14 and 21 (prasugrel, prasugrel + low aspirin and prasu- grel + high aspirin, respectively). Data shown are mean ± standard error of the mean (SEM) of responses collected from 96-well plate aggregometry in citrated platelet-rich plasma (PRP) prepared from nine study particpants. *Shows P < 0.05 difference from baseline by two-way ANOVA plus Bonferronis post test; Shows P < 0.05 difference between prasugrel and prasugrel + low aspirin. Symbols at the end of lines signify a difference in set; symbols at individual points signify particular differ- ences. Fig. 4. Urinary PGI-M (panel A) and TX-M (panel B) production at days 0, 7, 14 and 21 (baseline, prasugrel, prasugrel + low aspirin and prasu- grel + high aspirin, respectively). Values for urinary prostanoid metab- olites (PGI-M or TX-M) are corrected against urinary creatinine concentration. Data shown are mean ± standard error of the mean (SEM) of nine study participants.  2011 International Society on Thrombosis and Haemostasis Discussion 0.20 0.4 0.3 0.2 0.1 0.0 0.15 0.10 0.05 2,3 dinor-6-keto PGF 1α (ng mg–1 creatinine) 11-dehydro-TXB2 (ng mg–1 creatinine) 0.00 0 7 14 21 0 7 14 21 Day Prasugrel Aspirin Day Prasugrel Aspirin 10 mg o.d. 10 mg o.d. 10 mg o.d. 300 mg o.d. 75 mg o.d. - - - 10 mg o.d. 10 mg o.d. 10 mg o.d. 300 mg o.d. 75 mg o.d. - - - A B * * * * Fig. 4. Urinary PGI-M (panel A) and TX-M (panel B) production at days 0, 7, 14 and 21 (baseline, prasugrel, prasugrel + low aspirin and prasu- grel + high aspirin, respectively). Values for urinary prostanoid metab- olites (PGI-M or TX-M) are corrected against urinary creatinine concentration. Data shown are mean ± standard error of the mean (SEM) of nine study participants. *Shows P < 0.01 difference from Day 0 by one-way ANOVA plus Bonferronis post test. 800 Day 0 Day 7 * * * * Day 14 Day 21 Day 0 Day 7 Day 14 Day 21 600 400 200 300 200 100 0 0 –5 –8 –7 –6 –5 –4 Arachidonic acid (log M) Collagen (log g mL–1) Thromboxane B2 (ng mL–1) Thromboxane B2 (ng mL–1) –4 –3 A B Fig. 3. Platelet production of TXA2 induced by arachidonic acid (0.03– 1 mM; panel A) and collagen (0.1–30 lg mL)1; panel B) at baseline and days 7, 14 and 21 (prasugrel, prasugrel + low aspirin and prasu- grel + high aspirin, respectively). Data shown are mean ± standard error of the mean (SEM) of responses collected from 96-well plate aggregometry in citrated platelet-rich plasma (PRP) prepared from nine study particpants. *Shows P < 0.05 difference from baseline by two-way ANOVA plus Bonferronis post test; Shows P < 0.05 difference between prasugrel and prasugrel + low aspirin. Symbols at the end of lines signify a difference in set; symbols at individual points signify particular differ- ences. 0.20 0.4 0.3 0.2 0.1 0.0 0.15 0.10 0.05 2,3 dinor-6-keto PGF 1α (ng mg–1 creatinine) 11-dehydro-TXB2 (ng mg–1 creatinine) 0.00 0 7 14 21 0 7 14 21 Day Prasugrel Aspirin Day Prasugrel Aspirin 10 mg o.d. 10 mg o.d. 10 mg o.d. 300 mg o.d. 75 mg o.d. - - - 10 mg o.d. 10 mg o.d. 10 mg o.d. 300 mg o.d. 75 mg o.d. Discussion For instance, the abolition of TXA2 in collagen-induced aggregation results in a further reduction of platelet aggregation only at high concentrations of this agonist. Aspirin also has a small additional effect on the platelet aggregation induced by strong PAR1 activation (by TRAP-6 amide; SFLLRN) and the highest concentrations of epinephrine. In these circumstances, it is likely that the effect of TXA2 (that is lost by the addition of aspirin) is partly mediated The predominant choice of which aspirin dose to use in dual anti-platelet therapy varies with geographic location [24] and so Leadbeater: prasugrel and aspirin 2 2055 it was useful to include both a low- and a high dose of aspirin in combination with prasugrel in the present study. Consistent with meta-analysis of aspirin as a monotherapy, which show that the efficacy of aspirin is not enhanced at higher doses [1], there was no evidence that the higher dose of aspirin produced any further anti-platelet effects than a strong P2Y12 inhibitor together with low-dose aspirin. The present study did not determine the effects of aspirin alone on platelet aggregation using the 96-well plate assay method, although previous work by our group has demonstrated that this assay readily detects the effects of aspirin in healthy volunteers [12]. contribute to the efficacy of drugs targeting the P2Y12 receptor pathway. Clearly, this can only occur if the production of vascular prostacyclin is preserved, whereas the co-administra- tion of aspirin with P2Y12 receptor blockers results in reduced PGI2 formation and a potential limitation of the anti-throm- botic effect. Furthermore, loss of vascular PGI2 has been proposed as the underlying mechanism of cardiovascular risk that is associated with NSAIDs based on data derived from urinary PGI-M measurements [29]. A limitation of the present study was the continuous administration of treatments without any wash-out periods to differentiate the effects of each drug combination from a potential effect of time. An alternative study design could have also included a treatment period of aspirin alone. Clearly, such a study would have been a much larger undertaking and would have provided further levels of information. However, it is our belief that for the mechanisms of drug action that we wished to explore the study design chosen was appropriate. In addition to platelet reactivity, we measured the urinary metabolites of both TXA2 and PGI2, which are considered a marker of in vivo prostanoid levels [11]. Discussion Urinary metabolites were measured within 6 h of dosing on the relevant study day, which is within the time period in which the effects of aspirin are maintained in healthy subjects taking consecutive daily doses of this drug [11,25]. In healthy volunteers PGI-M and TX-M were unaffected by prasugrel alone, although these results contrast with the effects of clopidogrel, which we have previously reported in a similar study [8]. Urinary TX-M formation was reduced after 7 days of clopidogrel dosing in healthy volunteers, although in this case the samples were measured by enzyme immunoassay rather than mass spectros- copy [8] and we have greater confidence in the data we present here; although the small number of individuals included in each study makes a definitive analysis difficult, especially because of the large inter-individual variability in urinary PGI-M and TX- M levels. The failure of prasugrel to reduce TX-M excretion seems contradictory to the reduction of ex vivo TXA2 formation that is associated with clopidogrel [8,26] and now prasugrel; however, TX-M is used as a marker of physiological TXA2 levels whereas ex vivo TXA2 formation describes the production of this lipid mediator in response to strong thrombotic stimuli, which are absent in young healthy volun- teers. In patients with vascular inflammation [13] and athero- sclerosis [27], urinary TXA2 formation is increased and the effects of prasugrel on urinary TX-M may be different in these pathologic states where in vivo platelet activation may contrib- ute more to urinary TX-M levels. In conclusion, the present study indicates that strong P2Y12 receptor blockade produces an inhibition of platelets that is only slightly enhanced by aspirin, as assessed ex vivo. Aspirin also reduces the levels of PGI2 in vivo, an effect that is suggested to increase the risk of thrombosis. As no clinical trial has tested the effects of aspirin in the presence of strong P2Y12 receptor blockade it is unclear whether or not aspirin enhances or limits the anti-thrombotic effect that can be achieved with drugs such as prasugrel. We would therefore suggest that dedicated clinical trials should be considered to ascertain the true benefits, and rates of adverse events, when using aspirin alongside an effective, strong P2Y12 receptor blocker. Acknowledgements This research was supported by the William Harvey Research Foundation, the National Heart and Lung Institute Founda- tion and the Wellcome Trust (085255/Z/08/Z). This work forms part of the research themes contributing to the translational research portfolio of Barts and the London Cardiovascular Biomedical Research Unit, which is supported and funded by the National Institute of Health Research. In the present study, both prostanoids were reduced, independently of dose, when aspirin was co-administered. It has been previously suggested in a similarly sized study that the reduction of PGI-M by aspirin is dose dependent, although when scrutinizing these earlier data we found a large overlap between the values for groups taking 75 and 325 mg aspirin similar to the results we report here [11]. It is unclear from our data but it is possible that in larger studies we may have seen a dose-dependent relationship between aspirin and urinary PGI-M. Disclosure of Conflict of Interest The authors state that they have no conflict of interest.  2011 International Society on Thrombosis and Haemostasis 2 Saw J, Madsen EH, Chan S, Maurer-Spurej E. The ELAPSE (Eval- uation of Long-Term Clopidogrel Antiplatelet and Systemic Anti- Inflammatory Effects) study. J Am Coll Cardiol 2008; 52: 1826–33. 3 Mehta SR, Yusuf S, Peters RJ, Bertrand ME, Lewis BS, Natarajan MK, Malmberg K, Rupprecht H, Zhao F, Chrolavicius S, Copland I, Fox KA. Effects of pretreatment with clopidogrel and aspirin followed 1 Antiplatelet Trialists Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002; 324: 71–86. References 1 Antiplatelet Trialists Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002; 324: 71–86. The effect of vascular-derived PGI2 cannot be observed in ex vivo aggregation studies owing to the short half-life of this prostanoid. However, in vitro studies suggest that in the presence of strong P2Y12 receptor blockade platelets may be more sensitive to the inhibitory effects of this hormone [28], and it is plausible that such a phenomenon has the potential to 2056 P. D. M. Leadbeater et al by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet 2001; 358: 527–33. by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet 2001; 358: 527–33. by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet 2001; 358: 527–33. 17 Kahner BN, Shankar H, Murugappan S, Prasad GL, Kunapuli SP. Nucleotide receptor signaling in platelets. J Thromb Haemost 2006; 4: 2317–26. 4 Anderson JL, Adams CD, Antman EM, Bridges CR, CaliffRM, Casey DE Jr, Chavey WE 2nd, Fesmire FM, Hochman JS, Levin TN, LincoffAM, Peterson ED, Theroux P, Wenger NK, Wright RS, Smith SC Jr. ACCF/AHA focused update incorporated into the acc/aha 2007 guidelines for the management of patients with unstable angina/ non-st-elevation myocardial infarction: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation 2011; 123: e426–579. 18 Offermanns S. Activation of platelet function through g protein-cou- pled receptors. Circ Res 2006; 99: 1293–304. 19 Moers A, Wettschureck N, Gruner S, Nieswandt B, Offermanns S. Unresponsiveness of platelets lacking both Galpha(q) and Galpha(13). Implications for collagen-induced platelet activation. J Biol Chem 2004; 279: 45354–9. 20 Wallentin L, Becker RC, Budaj A, Cannon CP, Emanuelsson H, Held C, Horrow J, Husted S, James S, Katus H, Mahaffey KW, Scirica BM, Skene A, Steg PG, Storey RF, Harrington RA, Freij A, Thorsen M. Ticagrelor versus clopidogrel in patients with acute coronary syn- dromes. N Engl J Med 2009; 361: 1045–57. 5 Li Z, Zhang G, Le Breton GC, Gao X, Malik AB, Du X. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases. J Biol Chem 2003; 278: 30725–31. 6 Paul BZS, Jin J, Kunapuli SP. Molecular mechanism of thromboxane a2-induced platelet aggregation. Essential role for P2Tac and alpha 2a receptors. References J Biol Chem 1999; 274: 29108–14. 21 Wiviott SD, Braunwald E, McCabe CH, Montalescot G, Ruzyllo W, Gottlieb S, Neumann FJ, Ardissino D, De Servi S, Murphy SA, Riesmeyer J, Weerakkody G, Gibson CM, Antman EM. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2007; 357: 2001–15. 7 Armstrong PC, Truss NJ, Ali FY, Dhanji AA, Vojnovic I, Zain ZN, Bishop-Bailey D, Paul-Clark MJ, Tucker AT, Mitchell JA, Warner TD. Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of throm- boxane A2. J Thromb Haemost 2008; 6: 1933–43. 22 Mehta SR, Yusuf S. Short- and long-term oral antiplatelet therapy in acute coronary syndromes and percutaneous coronary intervention. J Am Coll Cardiol 2003; 41: 79S–88S. 8 Armstrong PC, Dhanji AR, Tucker AT, Mitchell JA, Warner TD. Reduction of platelet thromboxane A2 production ex vivo and in vivo by clopidogrel therapy. J Thromb Haemost 2010; 8: 613–5. 23 Diener HC, Bogousslavsky J, Brass LM, Cimminiello C, Csiba L, Kaste M, Leys D, Matias-Guiu J, Rupprecht HJ. Aspirin and clopidogrel compared with clopidogrel alone after recent ischaemic stroke or transient ischaemic attack in high-risk patients (MATCH): randomised, double-blind, placebo-controlled trial. Lancet 2004; 364: 331–7. 9 Warner TD, Armstrong PC, Curzen NP, Mitchell JA. Dual antiplat- elet therapy in cardiovascular disease: does aspirin increase clinical risk in the presence of potent P2Y12 receptor antagonists? Heart 2010; 96: 1693–4. 10 Armstrong PC, Leadbeater PD, Chan MV, Kirkby NS, Jakubowski JA, Mitchell JA, Warner TD. In the presence of strong P2Y(12) receptor blockade, aspirin provides little additional inhibition of platelet aggregation. J Thromb Haemost 2011; 9: 552–61. 24 Warner TD, Nylander S, Whatling C. Anti-platelet therapy: cycloox- ygenase inhibition and the use of aspirin with particular regard to dual anti-platelet therapy. Br J Clin Pharmacol 2011; 72: 619–33. 25 Capone ML, Tacconelli S, Sciulli MG, Grana M, Ricciotti E, Minuz P, Di Gregorio P, Merciaro G, Patrono C, Patrignani P. Clinical phar- macology of platelet, monocyte, and vascular cyclooxygenase inhibi- tion by naproxen and low-dose aspirin in healthy subjects. Circulation 2004; 109: 1468–71. 11 Fitzgerald GA, Oates JA, Hawiger J, Maas RL, Roberts LJ 2nd, Lawson JA, Brash AR. Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man. J Clin Invest 1983; 71: 676–88.  2011 International Society on Thrombosis and Haemostasis References 26 Bhavaraju K, Georgakis A, Jin J, Gartner TK, Tomiyama Y, Nurden A, Nurden P, Kunapuli SP. Antagonism of P2Y reduces physiological thromboxane levels. Platelets 2010; 21: 604–9. 12 Armstrong PC, Dhanji AR, Truss NJ, Zain ZN, Tucker AT, Mitchell JA, Warner TD. Utility of 96-well plate aggregometry and measure- ment of thrombi adhesion to determine aspirin and clopidogrel effec- tiveness. Thromb Haemost 2009; 102: 772–8. 27 Belton O, Byrne D, Kearney D, Leahy A, Fitzgerald DJ. Cyclooxy- genase-1 and -2-dependent prostacyclin formation in patients with atherosclerosis. Circulation 2000; 102: 840–5. 13 McAdam BF, Byrne D, Morrow JD, Oates JA. Contribution of cyclooxygenase-2 to elevated biosynthesis of thromboxane A2 and prostacyclin in cigarette smokers. Circulation 2005; 112: 1024–9. 28 Cattaneo M, Lecchi A. Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacy- clin. J Thromb Haemost 2007; 5: 577–82. 14 Lenth RV. Statistical power calculations. J Anim Sci 2007; 85: E24–9. 15 Madsen EH, Schmidt EB, Gehr N, Johannesen NL, Kristensen SR. Testing aspirin resistance using the Platelet Function Analyzer-100: some methodological caveats and considerations. J Thromb Haemost 2008; 6: 386–8. 29 Grosser T, Fries S, FitzGerald GA. Biological basis for the cardio- vascular consequences of COX-2 inhibition: therapeutic challenges and opportunities. J Clin Invest 2006; 116: 4–15. 16 Madsen EH, Schmidt EB, Maurer-Spurej E, Kristensen SR. Effects of aspirin and clopidogrel in healthy men measured by platelet aggrega- tion and PFA-100. Platelets 2008; 19: 335–41.  2011 International Society on Thrombosis and Haemostasis
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The Future and Journey of Social Media Towards Virtual World
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The Future and Journey of Social Media Towards Virtual World Raju Sitaram Nandkar, BSc Agri., MBA(BII) , MA(RD), ISTD Indira Gandi National Open University ,New Delhi Raju Sitaram Nandkar, BSc Agri., MBA(BII) , MA(RD), ISTD Indira Gandi National Open University ,New Delhi Abstract:- Social media has covered and permeated the entire human life and also existence of human beings. Sociability is a basic human characteristic and gossiping is an integral part of social dialogue. Social Media is a highly developed form of social gossiping. In one click we can interacts with any person from any country in the world through social media. Friendship and interaction with many people at a time is an important feature of social media. Social media allows sharing of feelings. Ideas and information is exchanged through social media. Social contact continues to growing day by day. There are lots of examples people help each other through Social contact. Despite these many benefits, social media cannot demonstrate spatial presence. Although social media enhances interaction, it represents a virtual existence. Obviously, virtual world issues also come into focus on social media. Hence, an anonymous fear seems to be forming that social media is not moving towards the virtual world. Many problems arise in the virtual world, whether it is family or social, its consequences can be felt by the entire human race. The virtual world will remain a superficial and imperfect world. The virtual world is not perfect and how to deal with it is a huge challenge. Good and bad effects of social media also affect your emotions. Social media creates problems with properly controlling and channeling emotions because it creates emotional hanging. Mental pressure builds up as emotions go up and down. Mentally, the person also becomes depressed. The use of social media reduces efficiency. As the time that should be spent on productive work is not available, it has a far-reaching impact on overall growth. Due to social media there is a huge increase in cyber crime. Overall, people are still experimenting in the virtual world of social media and facing lot of issues in adjustment. In this article we try to find out in which direction this journey of the virtual world going? What are its possible side effects? And information about how all mankind will find a way out of it has been made available. revolutionized human communication. Volume 8, Issue 1, January – 2023 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 II. Social media is now spread everywhere in the world. One who said ‘no one can survive without social media. It is the fastest medium of communication. Social media encompasses every sphere and corner of mankind. The first recognizable social media site Six Degrees was created in 1997. It helps users to upload a profile and make friends with other users. The first blogging sites became popular in 1999, creating a social media sensation. After the invention of blogging, social media began in popularity. Sites like The Future and Journey of Social Media Towards Virtual World Script is a distinctive writing system, based on a repertoire of specific elements or symbols, or that repertoire. Language is the system of communication in speech and writing that is used by people of a particular country. There is two types of communication one is verbal and another is non verbal. In ancient period we can found some of the oldest forms of human communication include making sounds, drawing or painting, dancing, acting, and using symbols. The journey of communication includes Cave Paintings, Smoke Signals, Carrier Pigeons, Postal System, Newspapers, Radios, Telegraph, Telephone, Television, Internet, Email, Text Message and Social media. The Internet has made evolution of communication more effective. We can send messages with just one click. Computers, mobile phones, laptops, radios, etc, all help us to communicate. The latest mode of communication in the digital world is the social media. People share their entire life events on social media. Social media platforms help people share pictures, videos, and almost everything on the internet. Social media has reduced the geographical boundaries and distances between two people and the time gap has been reduced to a fraction of a second. All in all, social media has captured the entire human life. Just as the force of gravity works, the social media worked to connect the bonds of contact and communication between two or more people. Social media is emerging as a new innovation in human communication. However question marks have been always raised about this. Fake accounts, cyber crime, cheating, pornography, blackmailing, defamation etc. now became common type fraud on social media. Therefore all users are in pressure however the cant able to keep away from social media because there are lot of benefits they are enjoying from social media platform. However there is urgent need to balance the use of social media in our daily life and need to avoid virtual world in life. In this article, we will try to explain the future of social media and the journey towards the virtual world. We also try to shed light on the emergence of the social media and its use and its future. II. LITERATURE REVIEW Keywords:- Social, Virtual, Media, Communication, Interaction. Keywords:- Social, Virtual, Media, Communication, Interaction. I. INTRODUCTION Food, clothing, and shelter are the basic human needs. Now, social media has become the fourth basic need. Some day before, some important applications in social media were shut down and life seemed to come to a standstill. Communication has been a natural gift from immemorial times. The discovery of scripts and languages has www.ijisrt.com IJISRT23JAN498 604 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 MySpace and LinkedIn gained prominence in the early 2000s, and sites like Photobucket and Flickr facilitated online photo sharing. YouTube came out in 2005, creating an entirely new way for people to communicate and share with each other across great distances. In 2006, Facebook and Twitter both became available to users throughout the world. These sites remain some of the most popular social networks on the Internet. Other sites like Tumblr, Spotify, Foursquare and Pinterest began popping up to fill specific social networking niches. Today, there is a tremendous variety of social networking sites, and many of them can be linked to allow cross-posting. This creates an environment where users can reach the maximum number of people without sacrificing the intimacy of person-to-person communication. and a considerable space for the virtual interactions of intellectual and popular society categories; through the worldwide known media and interactive gates. Gogolin, et al.(2014) critically discussed about the use of virtual worlds and social media has grown to the point that more than one-quarter of the world's population utilize it in some manner. Security and privacy concerns regarding the use and capabilities of current and emerging technologies such as gaming, blogging, podcasting, virtual meetings, virtual worlds and Web 3.0 is examined. Security and privacy concerns are investigated in the context of exploits, vulnerabilities, and related security risks; confidential access control; communication trends and patterns in the use of massive communication strategies; intellectual property and product risk management; resource management; financial considerations and accountability; and safety. Several technologies and personal practices are reviewed, as well as ways to mitigate or eliminate their associated risks. The core principles of information security-confidentiality, integrity, and availability-provide an overall framework. Mohammed et al., 2021; observed in research that the social media is becoming a critical part of everyone’s life. Social media has numerous platforms including Fecebook, Twitter, Instagram, and LinkedIn. I. INTRODUCTION Impersonation is a common phenomenon found nearly on all social media platforms; it is the act of attempting to deceive someone by pretending that he is another person. Impersonators always try to hide a real account by making another similar profile to spread the fake contents on social media platforms making it very difficult to know the real accounts from the fake ones. The article explains on the social media impersonation, impersonation types, how to identify the social media impersonation, cases of social media impersonation, how to prevent impersonation, and how to protect the security of a social media user. Social media impersonation is the act of pretending that a person is another person which usually occurs on all social media platforms. Eraslan, et al., (2019), illustrated about the living in the age of constant technology developments shifted social communication patterns and shifted social relations to virtual environments. The socialization process that takes place in digital platforms also transferred many negative elements experienced in social life to the virtual environment. That is, the aggression behaviours concerning these negative processes have also been transferred to the virtual communication. The current study examines the effects of social media aggression (SMA) regarding digital platforms on the social relations in human life in the context of various variables. Results of the study revealed that the counter- comments towards participants’ values have a significant effect on participants’ demonstration of aggressive tendencies. Messinger,et al.,(2009), discussed about the virtual worlds, where thousands of people can interact simultaneously within the same environment, represent a frontier in social computing with critical implications for business, education, social sciences, and our society at large. In this paper, they first trace the history of virtual worlds back to its antecedents in electronic gaming and on-line social networking. They provide an overview of extant virtual worlds, including education-focused, theme-based, community-specific, children-focused, and self-determined worlds and they analyze the relationship among these worlds according to an initial taxonomy for the area. Papp, Raymond;(2010), discussed thoroughly about virtual worlds and social networking. As online communication and collaboration becomes more commonplace, universities are exploring the educational possibilities of online virtual environments for reaching the Millennial. III. DISCUSSION, ANALYSIS AND INTERPRETATION Three million years ago, a truly wise man started roaming the earth. For many years, everything went smoothly, such as living in the same territory, roaming for food, and reproducing. Gradually, as man began to comprehend, analyze, and reflect on the information he received, he became involved in the process of evolution. Such an evolution had begun not only on a physical but also on a mental level. As a result, he had to adapt to the environment in which he lived, as his body adapted. However, these changes were taking place at a very subtle and very slow pace. On the other hand, mental changes were advancing at a slower pace, and so human evolution was advancing faster than other animals. In the past, we used to express these feelings on occasion, but now they are completely broken. Therefore, emotions and thoughts are harmonized. Everyone is dependent on this social media. The world is constantly changing and science and technology are accelerating this change. As the changes took place, we became more and more aware of that change. But the big question for researchers is whether the current change has brought about mental and physical harmony. Among them, it will be necessary to see whether the massive use of social media and the mental and physical changes it requires are taking place. Mobile and its electromagnetic waves cover the whole world. The mobile revolution happened, but with it came the social media revolution. Social media has shocked many superpowers globally and social media has done a disservice to the family. This pace of evolution began with the discovery of fire, increased with the discovery of the wheel, speeded up due with discovery of agriculture, and the discovery of script and language gained real momentum for human evolution and development. Printing, Radio, Internet, and Mobile Phones have brought the world closer together. Social media brought man closer and the geographical distance of man-to-man became zero. Evolutionary changes are very slow, while revolutions happen in a few years and suddenly. In simple terms, evolution means change and getting used to that change, while revolution is sudden change. Whether social media is evolution or revolution? When changes happen and those changes are not accustomed, it has physical and mental trauma. As the man sat up and started working, he started getting back and neck ailments. I. INTRODUCTION Both virtual worlds and social networking constitute a large part of the Millennial' time and incorporating these technologies into the classroom can foster a more collaborative and diverse learning atmosphere.Virtual campus tours, recruiting, advising, simulations and classes are all part of the growing virtual environment. Corporate and educational institutions are exploring their use in cutting costs, delivering higher customer satisfaction and catering to a more tech-savvy clientele. Madouni, et al., (2020) elaborated about the virtual media emerged and disseminated immensely ; specifically in the last ten years of the twenty first Century, through innumerable channels and virtually broadcasting pages, as strong equivalently as the traditional mass media in almost life critical domains and areas ; as a result and feature of the technological progress. The technology of the twenty-first Century gave to hands a wide reach and availability of information, it allows people and communities to participate even in producing and making influential public opinions towards local and international issues and topical; as ways of social interaction behind devices screens. Technology and changes create a sort of circumstantial adaptation which did not exist before. Furthermore and notably, the traditional mass media amid this advance; they specify regular corners Pradeep,et al.,(2016), shade light on the Social Networking Sites (SNSs) such as Fecebook have made inroads in the life of users. The current study used a quantitative approach to explore adolescents usage of SNS in Mumbai and their perceptions and experiences of the same. Easily accessible Internet and availability of devices such as smart phones influenced access to SNS. Computer-mediated- communication was the preferred means of communication. www.ijisrt.com IJISRT23JAN498 605 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 Selective self-representation and online social comparison were pertinent themes seen through the study. Gender and age differences were found in the overall usage and experiences of SNS. Although social media is not as physically and mentally accustomed as it is, the attraction of the information available and the interaction and communication that takes place is so great that man is drawn to it like a force of gravity. This power does not allow him to sit still and think. As a result, information, data, language, scripts, videos, audio and pictures are simply exploding. I. INTRODUCTION How to store so much information is a challenge in front of the brain, so it immediately began to put the information in the dustbin. There is so much information that it takes a lot of time and energy for the brain to grasp and analyze it, so the brain may be stopped grasping and analyzing. It is certainly not desirable for a wise person to accept that the brain is moving in the right direction. Naturally, social media will have far- reaching effects on human comprehension and analysis. Naturally, the connection between the brain and the mind is disrupted. The confusion between what the mind tells the brain and what the brain tells the mind has created confusion and roughness in the emotions that arise from the mind. Therefore, there is an upheaval in happiness, sorrow, anger, fear, hatred, surprise, love. III. DISCUSSION, ANALYSIS AND INTERPRETATION As the man gained weight, he began to develop diabetes and high blood pressure. Of course, social media is a part of human evolution or revolution is a subject of research, but the radical changes brought about by social media did not take many years to get used to physically and mentally, and suddenly we started riding on social media. Eventually, many of its good and bad consequences are beginning to emerge. The above mentioned changes, which do not get used to the physical and mental, certainly cause physical and mental disorders. No one should be upset that social media has increased the physical complaints of man. Eye strain, strain on the cervical spine, the effect of mobile light on the skin of the face is some of the complaints that need to be mentioned deliberately. No one should be offended by the fact that mental problems are exacerbated by the slowness of thought processes, constant distractions due to incoming messages, persistence of thought processes, grief and frustration, fear and irritability by mobile and social media as a whole. Everyone has a situation where they understand but do not turn around. We are all connected to the virtual relationship and the virtual world, rather than personal and interpersonal relationships. In a virtual relationship, even though the connection of contacts and communication is fast, they cannot create intimacy. As we aware eyes contact and touches and actual emotional and mental support play an important role in creating intimacy. Virtual relationships seem to lack this. Although social media has accelerated communication and interaction, the disadvantages outweigh the benefits. The benefits of social media are obvious at the business level and at the administrative level; however social media has not been able to work beyond virtual communication at the individual level. Social media has also contributed to the decline in human efficiency and effectiveness. There is a fear that we will lose creativity and innovation. Although social media is attractive, it is not conducive to long-term human development. The time that you have is so precious that it is spent in the virtual world and just listening to, viewing and reading information so no one else has time left to be for constructive work. III. DISCUSSION, ANALYSIS AND INTERPRETATION There are also some gentlemen who are IJISRT23JAN498 IJISRT23JAN498 www.ijisrt.com 606 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 spending more than ten hours out of twenty four hours on mobile and social media. The power of human capital as a whole is weakening and diminishing. As a result, its problems are becoming more prevalent in the business, social spheres and at the family level. Going even further, some organized cybercriminals are on the rise, increasing the number of people being deceived in various ways. Fake accounts, cyber crime, cheating, pornography, blackmailing, defamation etc. now became common type fraud on social media. Instead of that the Fake friends, Free app downloads, Quizzes ,Hidden URLS, Lottery and Free Gift Card Scam, Gossip Scam, Healthcare Scam, Cat fishing, Photo of You Scam, Account Cancelled Scam, Nigerian Scam, Stuck Abroad Scam, IQ Scam, See Who Viewed Your Profile Scam etc type of frauds now became very common on social media. permanent. Emotions cannot be controlled and flowed properly in virtual world because the reality is different. Taking all these factors and things for granted, there is a global need for a comprehensive effort to stop the society polarization towards a virtual world. We need to realize that social media is a necessity not a must. Anything in this world is difficult but not impossible. REFERENCES [1]. Eraslan, Levent & Kukuoğlu, Ahmet. (2019). Social relations in virtual world and social media aggression. World Journal on Educational Technology: Current Issues. 11. 140-149. 10.18844/wjet.v11i2.4145. [2]. Gogolin, Greg & Gogolin, Erin & Kam, Hwee-Joo. (2014). Virtual Worlds and Social Media. International Journal of Artificial Life Research. 4. 30-42. 10.4018/ijalr.2014010103. Man is such an intelligent creature on earth that he is always looking for reuse from waste. Naturally, even though the world is occupied by social media, a lot of changes are expected in the future. Also, the use of social media needs to be as efficient and effective as it needs to be. Otherwise the possibility of human beings running away from this social media cannot be ruled out. There is nothing that can defeat a human being, deprive him, because at the same time the human race has many options in front of it and it is capable of choosing the right option at the right time. Although it is not possible to say how long social media will control and cover to human being, many more good and bad effects of social media are yet to come. Naturally, the four generations that exist on the surface of the earth need to use social media in a timely, limitedly, efficiently, effectively and to make human life simpler, easier, straightforward, convenient and enjoyable. [3]. Messinger, Paul & Stroulia, Eleni & Lyons, Kelly & Bone, Michael & Niu, Run & Smirnov, Kristen & Perelgut, Stephen. (2009). Virtual worlds past, present, and future: New directions in social computing. Decision Support Systems. 47. 10.1016/j.dss.2009.02.014. [4]. Mohammed, Gharawi & Badawy, Ahmed & Ramadan, Doaa & Elsayed, Shaymaa. (2021). SOCIAL MEDIA IMPERSONATION IN THE VIRTUAL WORLD. Al Hikmah International Journal of Islamic Studies and Human Sciences. [5]. Papp, Raymond. (2010). Virtual worlds and social networking: Reaching the millennials. Journal of Technology Research. [6]. Pradeep, Pooja & Sriram, Sujata. (2016). The Virtual World of Social Networking Sites: Adolescents Use and Experiences. Psychology & Developing Societies. 28. 10.1177/0971333615622911. IV. The changes are inevitable we can’t deny it. The social media is one of the major changes in our daily life. It is now became a part and parcel of our routine life. No one can avoid it and no one can deny it. Social media has touched every aspect of human life and made it easy and comfortable. Smart phones and social media are the inventions of science and technology. Social media has definitely revolutionized for human life. Social media works on the three goals that are communication, relationship and interaction. Social media brings two or more people together so the exchange of feelings and thoughts takes place at a faster pace. Useful information is also available quickly through social media. Social media has permeated and covered the entire life of human being although it has also been created some serious problems for mankind. This will include unnecessary information bombardment, spreading false and misleading information, inflammatory emotions, wastage of time, and absenteeism from work and cyber crime, family conflict. These all problems are the due to virtual feature of social media. The entry and spread of this virtual world is a challenge created by social media for the entire world. Since the virtual world is mostly superficial, nothing substantial happens in it. The virtual world is shallow in nature. Relationships in this virtual world are not that much 607 www.ijisrt.com www.ijisrt.com IJISRT23JAN498
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Geistige Stromungen der Gegenwart.
˜The œPhilosophical review
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% BOOK 190.EU22G 1913 c. EUCKEN # GEISTIGE STROEMUNGEh 1 DERGEGENWART 3 T153 000t,ME73 fl (B Geistige Strömungen Geistige Strömungen ^^- DER Gegenwart VON RUDOLF EUCKEN DER GRUNDBEGRIFFE DER GEGENWART VIERTE UMGEARBEITETE AUFLAGE Neue Ausgabe VERLAG VON VEIT&COMP. IN LEIPZIG 1913 Druck von Fr. Richter, O. m. b. H., Leipzig. Vorwort zur dritten Auflage ----- Die dritte Auflage ist gegen die zweite noch mehr verändert S als diese C: legung den Grundstock, den die sachliche Erörterung nur um- gegen die erste; bildete bei ihr die geschichtliche Dar- säumte, so diese in der zweiten Auflage weit selbständiger ist geworden und hat in der dritten die volle Herrschaft erlangt; das Buch nun vor allem ein Ausdruck einer eigentümlichen ist ß philosophischen Gesamtüberzeugung und will 4 würdigt sein. j als solcher ge- Das mußte auch die Darstellung wesentlich ver- ändern, das verlangte namentlich eine präzisere Anordnung und Einteilung des Stoffes bis in die einzelnen Abschnitte hinein. j Den Grundgedanken der früheren Behandlungen: die Ver- <5^ knüpfung von Historischem und Sachlichem einerseits, die Zer- -V legung in einzelne Abschnitte andererseits glaubte ich dabei festhalten zu können. Daß das Geschichtliche mir mehr ist i als ein Gegenstand gelehrter Beschäftigung, daß es, freilich f^ unter bestimmten Voraussetzungen, zur Erhöhung der eignen -J Arbeit kräftig beizutragen vermag, dafür kämpft das Buch ebenso als Ganzes wie in besonderen Erörterungen, die sich hier nicht vorwegnehmen lassen. Das Ausgehen von einzelnen Problemen aber gewährte den Vorteil greifbarer Angriffspunkte, von denen l/l sich rasch Allerdings y -^ zu verblieb Überzeugung sich in irgendwelcher Entscheidung dabei der Mißstand, vordringen ließ. daß das Ganze der nicht als solches volle Rechenschaft geben und einem fortlaufenden Zusammenhange darlegen kann. Vorwort VI Dieser Mangel sei bereitwillig zugestanden, dieser Behandlungsweise verbunden, als helfen ist zu eng mit daß sich ihm hier ab- Gewisse Ergänzungen bieten ließe. er dieser Hinsicht in meine früheren Bücher, die größte Lücke liegt in dem Mangel genügenden einer erkenntnistheoretischen Fundamentierung, mein nächstes Buch wird einer prinzipiellen Erörterung dieses Problems gewidmet sein. Mehr noch die Art als der Behandlung aber ist es eine durchgehende Grundüberzeugung, welche verschiedenen die Auflagen zusammenhält, die Überzeugung von der Unsicherheit des Bodens, dem auf unser ganzes Kulturleben und mit ihm auch unsere wissenschaftliche Arbeit steht, die Überzeugung, daß dieses Leben nicht nur einzelne Probleme in Hülle und Fülle enthalte, sondern daß es auch als Ganzes einer energischen und Revision einer gründlichen Erneuerung Streben danach aber schien mir auch teiligen zu müssen, Mitarbeit berufen. ja sie bedürfe. Am die Philosophie sich be- besonders schien hier zu eifriger Das brachte mich in Gegensatz zum Haupt- zuge der heutigen deutschen Philosophie, der seine wissenschaftliche Arbeit unbeirrt können meint. fortführen zu namentlich durch jene Fragen und Zweifel ruhig Wie viel Wertvolles diese Arbeit, der genaueren Durchbildung der einzelnen Er- in kenntnisgebiete, geleistet hat und dankbar anerkannt. und weiter leistet, das sei freudig Aber zugleich sei auch auf dem Rechte und der Notwendigkeit jenes allgemeineren Problems mit aller Entschiedenheit bestanden; wir werden uns in der Arbeit dafür in keiner Weise durch die Sorge um die Stellung anderer dazu beirren lassen, sondern lediglich und allein der inneren Not- wendigkeit der Sache vertrauen. Aber Fülle es sprechen neuerdings dafür, eintreten, ihre unserer auch Zeichen in Hülle und daß weitere Kreise den Problemen, für die wir Teilnahme zuwenden. Die inneren Verwicklungen Kultur, ja unserer gesamten geistigen Lage werden Vorwort VII immer augenscheinlicher, mehr und mehr empfinden wir darin schwere Unwahrheiten, Phrasen, wo wir Brot suchten. Sinn unseres Nun wo wir Wirklichkeiten, Steine, das Glück und der dabei steht dem Spiele; so erhebt sich eignen Daseins auf immer dringender das Verlangen nach Klärung wie nach Befestigung, so wird auch die Philosophie immer zwingender zur Arbeit an diesen Lebensfragen aufgerufen. Neue Wogen des Lebens steigen auf, neue Stimmungen ergreifen die Gemüter und heißen sie neue Ziele suchen. Diese inneren Wandlungen haben auch meinen Büchern mehr und mehr Freunde zugeführt und mir das Bewußtsein engen geistigen Kontaktes mit der Zeit gegeben, das ich eines früher nicht haben konnte. Mit besonderer Freude begrüße ich die unerwartet rasch wachsende Teilnahme des aufsteigenden jüngeren Geschlechts; möchte solche Teilnahme auch diesem Buche zugute kommen, und möchte Weiterführung der hier bloß sehr entworfenen die Idee eines und sicherlich oft ist schließlich nichts Denn was geringeres neuen Menschen und einer neuen Kultur; nur ein Zusammenschluß der alles namentlich zu einer unvollkommen behandelten Probleme wirken. uns gemeinsam vorschwebt, als sie Kräfte, nur eine Überwindung bloß Individuellen, nur das Entstehen einer durchgehenden Bewegung kann uns bei einer so gewaltigen Frage weiter- bringen. Jena, im Februar 1Q04. Rudolf Eucken Vorwort zur vierten Auflage Die vierte Auflage ist gegen die dritte nicht so umge- wandelt worden wie diese gegen die zweite, immerhin bringt sie Mehrere Abschnitte sind völlig um- manche Veränderung. einer gearbeitet, (über den Wert des Lebens) neu hinzugefügt worden; durchgängig aber war das Bemühen, die Darstellung flüssiger, stalten, dem den Inhalt gesättigter, die Hauptthesen präziser zu ge- die allen Probleme der Zeit unmittelbarer zu das Ganze anschaulicher und erfassen, mit eindringlicher zu machen; auch wurden diesmal weit mehr die Bewegungen des Auslandes in die Betrachtung hineingezogen. So hoffe ich; daß die neue Auflage als Ganzes einen merklichen Fortschritt bringt. Jena, Ende August 1Q08. Rudolf Eucken Inhaltsübersicht Einleitung. Seite 'Die Zeitlage A. und unsere Aufgabe ihr gegenüber 1 Zum Grundbegriff des Geisteslebens. 1. Subjektiv -Objektiv. a) Geschichtliches b) Das c) Die positive Behauptung. a. • . 19. Jahrhundert 18 Einführung 27 Der Grundbegriff des Geisteslebens Y. Das Verhältnis des Menschen zum Geistesleben 30 ß. o. 2. 10 .... Ergebnisse für den Wahrheitsbegriff 33 35 Theoretisch - praktisch (Intellektualismus -Voluntarismus). a) 37 Geschichtliches Behauptung des Voluntarismus c) Der Pragmatismus d) Die eigene Behauptung: der Aktivismus e) Intellekt und Intellektualismus a. Die Überflutung des modernen Lebens durch den Intellekb) Die 43 . y. o. £. Die Begründung des Erkennens im Lebensprozesse Die bewegende Kraft im Wahrheitsstreben Konsequenzen Konsequenzen Philosophie 51 52 53 tualismus ß. 47 ... für die Erkenntnisarbeit für die 56 59 63 Behandlung der Geschichte der 65 Inhaltsübersicht 3. B. Seite Idealismus- Realismus. a) Die Ausdrücke b) Zum Kampf der Lebensgestaltungen 68 70 a. Der Realismus des ß. Die Schranken des neuen Realismus y. Kritik der o. Erörterung des Wirklichkeitsproblemes 79 £. Die Forderungen eines neuen Idealismus 81 19. Jahrhunderts überkommenen Formen des Idealismus 72 74 ... 76 Zum Erkenntnisproblem. 1. Denken und Erfahrung (Metaphysik). a) Geschichtliches b) Das Recht einer selbständigen Philosophie 84 Die Wendung zur Metaphysik d) Der Gesamtanblick der menschlichen Erkenntnisarbeit 93 104 c) e) 2. Würdigung des Rationalismus und des Empirismus. . 111 ... 117 . . Mechanisch - organisch (Teleologie). Zur Geschichte der Ausdrücke und Begriffe Problems c) Erwägungen zum Kampf der Gegenwart a. Das Problem im Gebiet der Philosophie ß. Das Problem in der Naturwissenschaft y. Das Problem auf gesellschaftlichem Gebiet 3. a) 126 b) Zur Geschichte des 130 142 144 149 Gesetz. a) Zur Geschichte b) Der 154 Kampf um das Gesetz in der Neuzeit 159 C. Zum Weltproblem. 1. 2. Monismus und Dualismus. a) Zur Geschichte und Kritik der Begriffe b) Der Monismus der Gegenwart 170 183 Entwicklung. Zur Geschichte des Ausdrucks Zur Geschichte des Begriffs und Problems c) Die Verwicklungen und Schranken der bloßen Entwicklungs- a) 192 b) 193 d) lehre 206 Forderungen für einen neuen Lebenstypus 221 D. Zu den Problemen des Menschenlebens. 1. Kultur. a) Zur Geschichte des Ausdrucks und Begriffs 228 Inhaltsübersicht XI Erwägung. Das Problem des Wesens und Wertes der Kultur Das Problem des Inhalts der Kultur Das unsichere Verhältnis des Menschen zur Kultur Seite b) Kritische a. . . . . . 240 a. Die Notwendigkeit einer tieferen Begründung 243 ß. Die Notwendigkeit einer inneren Weiterbildung der Kultur 247 ß. y. c) 2. 237 Forderungen für ein wahrhaftiges Kulturleben. Geschichte. Zur Entwicklung des Problems Forderungen und Ausblicke Anhang: zum Begriff des Modernen 3. 235 . a) 253 b) 262 273 und Individuum (Sozialismus). Das Verhältnis von Gesellschaft und Individuum. Gesellschaft a) a. Geschichtliches ß. Die Probleme der Gegenwart. , 283 aa. Die Unzulänglichkeit einer bloßgesellschaftlichen Kultur 292 ßß. Die Unzulänglichkeit einer bloßen Individualkultur 302 ff. Die Notwendigkeit einer inneren . Überwindung des Gegensatzes 312 » b) Die sozialdemokratische 4. Bewegung 313 Probleme der Moral. Die unsichere Stellung der Moral in der Gegenwart und Metaphysik c) Moral und Kunst (ethische und ästhetische Weltanschauung) a) . b) Moral 5. a. Die Geschichte des Problems ß. Die Probleme der Gegenwart. . 322 324 329 aa. Der moderne Ästhetizismus ßß. Die Stellung der Kunst im modernen Leben Persönlichkeit . 336 .... 339 und Charakter. a) Persönlichkeit. Zur Geschichte des Ausdrucks Zur Geschichte des Begriffs 343 ß. y. Untersuchung des Problems 348 a. 345 b) Charakter. 6. a. Geschichtliches zum Ausdruck ß. Die Lage der Zeit und Begriff 355 358 Freiheit des Willens. a) Einleitung b) Erwägungen zur Behauptung des Determinismus 363 .... 366 Inhaltsübersicht XII E. Letzte 1. 2. Seite Probleme. Der Wert des Lebens. a) Einleitendes 376 b) Die Verwicklung der Gegenwart 378 Das Problem der Religion (Immanenz und Transzendenz). Zur Geschichte der Ausdrücke b) Die Bewegung der Neuzeit zur Immanenz c) Die Verwicklungen im Begriffe der Immanenz d) Das Wiedererwachen des religiösen Problems 390 392 a) e) Forderungen für die gegenwärtige Lage der Religion . 394 396 . . 398 Schlußwort 404 Sachregister 405 Einleitung. Die Zeitiage und unsere Aufgabe ihr gegeniiber. VV /er • '^ die Lage der Zeit überblickt und geistige der wird prüft, vor allem eine starke Verworrenheit und eine peinliche Un- sicherheit über das Hauptziel des Strebens empfinden; überall ein Auseinandergehen spaltensein Menschheit der des Menschen bei sich unsichere Stand Überlieferung in Parteien, selbst. oft auch ein Ge- Dieser verworrene und mag zunächst als eine Wirkung der geschichtlichen Denn von der Vergangenheit her um- erscheinen. fangen uns verschiedenartige, ja einander feindliche Strömungen, ein Erbe und eine Last aus tausendjähriger Arbeit; nichts unterscheidet moderne Kultur mehr von der einfacheren des Altertums als solches Durchtränktsein von Gegensätzen. Das Mittelalter überlieferte die ein Lebensganzes, das die grundverschiedene antike und liche, künstlerische und religiöse, weltfreudige und altchrist- weltfeindliche Denkweise weniger gegenseitig ausglich als geschickt zusammenfügte. Diesem Gefüge setzte die Neuzeit einen neuen Lebensdrang, ein Ver- langen nach unbegrenzter Entfaltung der Kraft und voller Beherrschung der Dinge entgegen, aber die nähere Ausführung dessen entzweite das Neue alsbald bei sich selbst: einerseits verlangte das Seelenleben mit seinem Denken, andererseits die Natur mit ihrem Mechanismus die Herrschaft über Leben mus). Alle historischen und Welt (Intellektualismus und Naturalis- solche Gegensätze läßt das 19. Jahrhundert mit seiner Bildung und seiner grüblerischen Reflexion in greller Deutlichkeit sehen und mit voller Stärke empfinden, es drängt zwingend zu einer schärferen Scheidung der verschlungenen Gedankenmassen, es verbietet ihnen immer strenger ein friedliches Zusammengehen. Und wie viel hat das 1 9. Jahrhundert bei sich selbst erlebt, wie eingreifende Eucken, Grundbegriffe. 4. Aufl. 1 Einleitung. 2 hat es durchgemacht, Wandlungen äußerer Zurückdrängung uns deren einzelne Phasen auch bei innerlich nahe bleiben und nach wider- streitenden Richtungen ziehen: die künstlerische Geisteskultur unserer klassischen Literaturepoche, ein kraftvoller und selbstbewußter Realis- mus, ein Rückschlag gegen diesen Realismus in einem Subjektivismus seelischen Fürsichseins, schwebender Stimmung! Wie viele Gegen- wir aus altem und neuem Bestände in uns, sätze tragen haben wir zu tun, wie viel um ihrer innerlich Herr zu werden! Um diese verschiedenartigen Anregungen zu verarbeiten und mit einander auszugleichen, bedürfte es überlegener geistiger Kraft. Da diese so fehlt, erscheinen alle Mißstände, welche das Be- Menschen von seinen eignen Erlebnissen, das des wältigtwerden Unterliegen unter die Zerstreutheit des Daseins herbeiführen muß. Keine festen Ziele beherrschen das Streben, keine einfachen Ideen dem Chaos und entwinden sich befreien von seinen Wirren und Zweifeln. Vielmehr bewältigen uns die unmittelbaren Eindrücke und zerreiben das Leben unsicher auf den unter ihren Widersprüchen. Wogen der Zeit einher, So treiben wehrlos gegen alles, mr was uns mit starkem Bewußtsein und kecker Behauptung naht, wehrlos auch gegen die eigenen Einfälle und Leidenschaften, ein Spiel von wechselnden Lagen und Launen. Diese Lage wird namentlich dadurch gespannt, daß die Wandlungen, die wir erfahren, sich schließlich zu Einer Frage verbinden und uns vor ein einziges Entweder-Oder stellen, das keine Verschleierung duldet und eine Entscheidung des ganzen Menschen Das stille, aber unablässige und unwiderstehliche Wirken der modernen Arbeit hat an der überkommenen Lebensführung nicht verlangt nur alle einzelnen Punkte verändert, es hat sie als Ganzes unter- graben und unhaltbar gemacht offen oder versteckt, Die ältere Denkweise behandelte, gröber oder feiner, sinnlicher oder geistiger, den Menschen als das Maß und den Mittelpunkt des Alls, verwandelte die Wirklichkeit in ein Reich von menschenartigen Größen machte das Wohlergehen des Menschen zum Ziele und alles Geschehens. Diesen Anthropismus hat das Ganze der modernen Arbeit gründlich zerstört; nicht nur die unermeßliche Erweiterung der äußeren Welt, auch die Aufdeckung innerer Notwendigkeiten und sachlicher Zusammenhänge im eigenen Bereich der Menschheit, ein weites Hinauswachsen des geistigen Schaffens über das bloße Subjekt machen jenes Sicheinspinnen in das Menschliche zu einer unerträglichen Enge, sie Einleitung. erwecken zugleich glühendes Verlangen nach einem weiteren, ein freieren, gehaltvolleren 3 Durst nach einem Leben Sein, einen starken mit der Unendlichkeit und der Wahrheit des lungen treten immer mehr auch und verlangen stürmisch Alls. Diese Wand- das Bewußtsein der Menschheit in ihr Recht. Aber aus dem Nein entspringt dabei keineswegs rasch und der Erschütterung entspricht nicht eine Befestigung. neue Lage eröffnet zwei Möglichkeiten, ein Ja, Denn die die, als schroffe Gegensätze, Hat jene weltgeschichtliche Bewegung keinerlei Ausgleichung dulden. gegeii das Beharren beim Bloßmenschlichen den Sinn, daß der Mensch sich als ein bloßes Naturwesen Tun dem Rahmen zu verstehen und all sein Sinnen und Dann wäre habe? der Natur einzufügen alles unterscheidend und auszeichnend Menschliche als ein verderblicher Wahn zu entfernen, Größen und Güter unseres Lebens hätten empfangen. Oder besagt jene Bewegung, daß innerhalb des Menschen selbst eine neue Weib eine geistige Welt emporsteigt und ihn wie über die Natur auch über sich selbst hinaushebt? Beginnt mit ihm eine neue Stufe der Wirklichkeit, und kann sein Seelenleben sich von innen her zu einer Welt erweitern? Dann würde zur Hauptaufgabe die Ergreifung, Aneignung und Ausbildung dieser Welt, dann müßte der Mensch vor allem hier sich befestigen und all sein Sinnen und Streben nicht sowohl rückwärts als vorwärts richten. So ist der Mensch alle ihr Gesetz und ihre Gestalt von der Natur zu entweder weniger oder mehr, je als er sich heute einzuschätzen pflegt; nach der Entscheidung für dieses oder jenes muß aber das Leben sich vom Größten bis zum Kleinsten verschieden gestalten. Aber so unerläßlich die hier gebotene Entscheidung ist, jene Schwäche des Einheitsstrebens läßt die Zeit unsicher zögern wechselnden Eindrücken neigt sie sie im Gesamturteil das eine billigt, bald und schwanken, nach hieher bald dahin; indem will sie zugleich von dem anderen nicht lassen; so bejaht sie hier, was sie dort verneint, so setzt sie an keiner Stelle ihr ganzes Wollen und Wesen ein. Oft genug ist diese Lage geschildert worden, ihr rascher Wechsel der Strömungen und Stimmungen, lichkeit ihr Mangel an Logik, wie ihn sowohl die Unempfind- auch für die härtesten Widersprüche und das Ineinander- schieben verschiedenartigster Gedankenmassen, als die Schwäche des Ausdenkens, des Verfolgens der Behauptungen in ihre Voraussetzungen wie ihre Konsequenzen zeigt. In allem Sinken des inneren Lebensstandes, ja diesem erscheint ein starkes eine innere Verarmung des Einleitung. Lebens, das aber inmitten staunenswerter Fortschritte an seiner Peripherie, inmitten nie gesehener, nie geahnter Virtuosität von technischen Leistungen, inmitten überströmenden Reichtums an äußeren Erfolgen Augenscheinlich befinden wir uns uns zu übermannen droht. heit Aber es in ist einer geistigen Krise, die diese Krise nicht der Bos- oder der Zweifelsucht Einzelner, sondern der weltgeschichtlichen Lage entsprungen. dem Ganzen ist Sollten wir nicht hoffen daß die Notwendigkeit, welche eine solche Krise erzeugte, dürfen, uns sie irgendwelche Mittel auch und Hülfen gewährt, die über sie Widerständen und Gegen- hinausführen könnten? Wahrheit In fehlt es nicht an wirkungen gegen jene chaotische Lage, an Versuchen, ihr eine ein- heitliche Gestaltung des Lebens, ein einheitliches Bild der Wirklich- keit entgegenzusetzen; leider bleiben aber diese Versuche meist unter dem Einfluß dessen, das sie überwinden möchten. Die Zeit des selbst- bewußten Spezialismus, den die Arbeit an der endlosen Breite der Dinge alle hinter sich. meist so, Sorge um das Ganze vergessen ließ, Aber das Streben zur Einheit hat ihren Gipfel gestaltet sich zunächst daß die einzelnen Lebens- und Wissensgebiete die Sache an sich reißen und das Bild Eindrücken, vom Ganzen nach ihren besonderen Zwecken entwerfen. Erfahrungen, Mehr als es sonst geschah, erzeugen sie innerhalb ihres besonderen Kreises geschlossene Gedanken massen, dringen damit kühnlich über die Grenzen jenes Ihre Kreises hinaus und möchten die ganze Wirklichkeit meistern. besonderen Aufgaben werden vor alle übrigen gestellt, ihre Begriffe, Maßstäbe, Methoden sollen schlechtweg gelten, ihr Gebiet wird ihnen zum beherrschenden Mittelpunkt der gesamten Wirklichkeit. So bildet sich die Religion, so oft auch die Kunst ihre eigene Welt, so erzeugt die soziale Bewegung ihre besondere Weltanschauung, so erweitern sich auf intellektuellem Gebiet namentlich oft die Naturwissenschaften Das tat zunächst die Zoologie zu einer allumfassenden Philosophie. unter dem Einfluß des Darwinismus, Physikern, Physiologen Weltgedankens ist jetzt gewandert, und es u. s. das sehen wir w. unternommen. jetzt auch von Die Kühnheit des von den Philosophen zu den Naturforschern fehlt hier nicht an kecken Husarenritten in das Land der Wahrheit; die Verquickung der philosophischen Behauptung mit tüchtiger Forschungsarbeit läßt dabei manche das Ungeheuerliche des Wagnisses kaum empfinden. So entstehen eigentümliche Durchblicke, Partialweltbilder, deren Einleitung. sinnliche Nähe und leichte Faßlichkeit die Geister gewinnt gutes Stück mit sich fortreißt. schließlich wird 5 Wahrheit der Dinge Widerstand die und Jedoch immer nur ein Stück. zu knappe Maß zersprengen; ein Denn und leisten wird es um das aufgedrängte, viel so eher, als jene Bewegung die verschiedenen Ansprüche bald zu- sammenstoßen und und daß samtwahrheit Nun vom Teil zum Ganzen bauen sich ihr Recht gegenseitig bestreiten läßt. wird offenbar, daß sich nicht wohl läßt, Überspannung zur Ge- die Teilwahrheiten mit ihrer sich sie in Soweit aber jene Partial- Irrung verkehren. bewegungen Macht behaupten, einander hemmen und durchkreuzen, müssen sie die Verwirrung, die sie bekämpfen, vielmehr steigern; kaum etwas so sehr zur Entzweiung als jenes vielleicht wirkt heute unzulängliche Streben nach Einheit. Nie war so viel die Rede von Monismus wie heute, und nie ging die Menschheit so weit auseinander wie heute. Aber so unzulänglich jene Versuche sind, sie bleiben wertvoll durch ihre Lehren. Namentlich zeigt ihr Scheitern mit voller Klar- daß sich von den einzelnen Punkten her ausrichten läßt, heit, nichts sondern es eine der Zerstreuung überlegene Einheit zu suchen ohne eine Erhebung über das Ganze der Zeitlage, ohne gilt; ein Er- greifen neuer Anfänge gibt es keine Hoffnung, der Krise gewachsen zu Aber warum werden. schichte ist für sollte jenes unmöglich sein? das Innere des Lebens kein Die Ge- fortlaufender Anstieg zur Höhe; da wesenhaftes Geistesleben sich in der Geschichte nicht nur entwickelt, sondern sich auch in ihr auslebt, so kommen immer wieder Zeiten, wo es aus der Wirkung im menschlichen Dasein zu sich selbst zurückkehren und die Wurzeln seiner Kraft neu beleben muß. So allein kann es der Zeit überlegen werden und dahin wirken, das Wahre in ihr von dem Problematischen zu befreien, das uns beirrt und gilt entzweit. Eine solche Zeit eine Selbstbesinnung auf die unser Grundverhältnis zur Welt, bloßen Zeit an das Ewige die überlegenen Gewalten in ist einmal wieder gekommen, es Grundlagen unseres Daseins, auf es eine gilt der Zeit, Berufung von der vom bloßen Menschen an die aus dem Menschen und Ordnungen, mehr als ein bloßes Naturwesen machen. Bei solcher Lage hat jeder, der den Notstand durchschaut, nach dem Maße seines Vermögens für jenes Ziel der Vertiefung des Lebens und der Erneuerung der Kultur zu wirken. Der Weg, den unser Werk dabei einschlagen soll, wird namentlich durch drei Merkmale bestimmt. Einleitung. Unsern nächsten Vorwurf sollen die der Zeit charakteristischen 1. Hauptbewegungen, die geistigen Strömungen, Wir sprechen von heißen mag. um oder Ideen, bei nicht als ob um bloß intellektuelle Vorgänge handle und Mag sich diesen die Entscheidung liege. auf Kürze von Begriffen von Anfang an die Meinung fernzuhalten, es sich an erster Stelle vorwiegend bilden, wie es in und solchen Gebiet intellektuellem der Streit äußerlich dahinter abspielen, stehen Lebensbewegungen aus dem Ganzen, dahinter steht eine eigentümAbsteckung der Wirklichkeit, liche des Lebens; inmitten vielfachen Probleme hindurch kann Gestaltung eigentümliche eine und durch verschiedene Streites bei diesen Voraussetzungen der Zeit eine Gemeinschaft walten; so Heraushebung besonders geeignet, ihre ist zu einem Gesamtbilde der Zeit zu verhelfen und das Eigentümliche der Zeit klar erkennen zu lassen. Das Ausgehen von der Vielheit aber hat den Vorteil, uns die Behauptungen und Probleme der Zeit und anschaulicher zu machen; greifbarer die Erörterung dem rasch Notwendigkeiten sachliche Wege Denken zeigen, zu es hat den weiteren Vorteil, bestimmten Punkt zu führen, einen auf hervorzubrechen daß wir überall auf dieselben Fragen kommen, ja an unserem Untersuchung Die vermögen. weisen und wird daß ein und dasselbe Hauptproblem durch alle Mannigfaltigkeit wirkt, auch wird sie wird, so zeigen, die daß, wie an jeder Stelle um das Ganze gekämpft Entscheidung über das Ganze in alle Verzweigung Der eignen Behauptung darüber dürfen wir uns aber hineinreicht. um so sicherer fühlen, je mehr die Erfahrungen und Forderungen der einzelnen Punkte zu ihr drängen und sie als die einzige Möglichkeit einer glücklichen 2. Lösung zeigen. Was wir aber näher bei den einzelnen Strömungen ermitteln und wonach wir sie prüfen wollen, das ist der von ihnen behauptete oder doch in die Frage beschäftigen, ihnen enthaltene Lebensprozeß; namentlich ob dieser Lebensprozeß Geistesleben möglich macht. lebens pflegt, wenn auch werden; wie viel aber in ein soll uns selbständiges Ein gewisser Tatbestand des Geistes- oft widerwillig, ihm liegt, von jedem anerkannt zu und was er über die nächste Erscheinung hinaus verlangt, an welche Voraussetzungen und Be- dingungen er geknüpft das ist, meist in völligem Dunkel. bleibt Wie sich die Bewegungen der Zeit zu diesem Problem, zum Problem der Möglichkeit des Geisteslebens, darauf sei stellen, vornehmlich das Augenmerk und was sie dafür leisten, gerichtet. So soll uns nicht Einleitung^ die Breite der Leistungen sondern wir streben rasch zu festhalten, dem sie durchwaltenden Leben, als dem letzten Punkt, der erreichbar ist und von dem aus sich unsere Gedankenwelt aufzubauen hat; es bringt uns solche Wendung zum Lebensprozesse wohl am sichersten an den Punkt, wo die Probleme dem Einzelnen zum eignen wo Erlebnis werden, er am eigene Erfahrungen einsetzen ehesten und am wenigsten eine eigne Entscheidung ablehnen kann. 3. Wo der Gehalt der Zeit den Ausgangs- wie den Endpunkt da empfiehlt sich ein Heranziehen der geschichtlichen Be- bildet, trachtung zur Jene Be- Unterstützung der philosophischen Arbeit. trachtung helfe zunächst dazu, die geistige Art der Gegenwart durch Aufdeckung ihres beleuchten und Werdens und deutlicher Zusammenhänge ihrer Für abzugrenzen. die heller Fassung zu und Schätzung dessen, was die Zeit beherrscht, kann es nicht gleichgiltig ihm eine Woge des bloßen Augenblickes oder einen bleibenden Lebensstrom erkennen, ob das heutige Erlebnis schon öfter erlebt wurde und einem wiederkehrenden Rhythmus anob wir sein, gehört, Neues und Eigenartiges aufeine Gegenwirkung, oder ob in ihm etwas völlig ob es mehr Wirkung oder auch steigt, in eine mehr einen Vorstoß oder einen Rückschlag schichtliche Betrachtung hat schieden weit in Bewegung Oft wird die Hauptphasen durch das Ganze der europä- Kulturentwicklung ischen den verschiedenen Punkten ver- bei die Vergangenheit zurückzugreifen. ihren in Diese ge- bedeutet. zu verfolgen sein, an anderen Stellen wird die nächstvorangehende Epoche zur Aufklärung der Gegenwart genügen. Hand der mag eine selbständige Untersuchung vorbereiten, wenn Solche hellere Beleuchtung des Tatbestandes an der Geschichte anders ein Ding in seiner Eigentümlichkeit schärfer sehen, zugleich seine Grenzen deutlicher sehen und ein Problem in ihm erkennen Aber nicht nur die Behauptung der Gegenwart, auch die heißt geschichtlichen Zusammenhänge, wandeln bei sich ja Aufdeckung des die Geschichte als in Ganzes ver- ihnen wirksamen Lebenspro- zesses in ein Problem; der Lebensprozeß mit seiner Bewegung läßt sich aus dem Chaos der Erscheinungen nicht wohl heraussehen ohne eine Versetzung aus der geschichtlichen Betrachtung zeitlose Seiner Wahrheit in eine Aufnehmen der Frage nach und seinem Rechte; das Ganze läßt sich nicht und unmittelbare, ohne ein durchleuchten, ohne daß sich ursprüngliche, eigene, letzte Tatsachen Einleitung. von den übermittelten scheiden. Wendung zu eine und Erörterung Umkehrung mit ihrer Verwandlung der Sache vollziehen; erst diese der Geschichte mag sich so eine Umkehrung, Es einer unmittelbaren Betrachtung Lebens macht Entfaltung eines zeitlosen die in es den Befund des Daseins von innen her zu durchschauen, möglich, von der Erscheinung zur Tatsache, vom bloßen Faktum zur be- gründenden Wahrheit vorzudringen, erkennen, ja ihrem Ganzen den Bewegungen der Ge- in und durchgehende Richtungen zu innere Notwendigkeiten schichte irgendwelchen Sinn zu entringen. Auf Grund solcher Betrachtung aus bleibender Wahrheit erst läßt sich Bedeutung der einzelnen Epochen ermessen, sowie an der die der Leistung Gegenwart eine immanente werde an dem hauptung der Zeit Die Be- weltgeschichtlichen Stande der üben. Kritik mehr Gehalt und Tiefe erschlossen, als jene Behauptung in sich aufnehmen kann, hat die Geschichte schon geistigen Evolution geprüft; so wird das Streben notwendig über sie hinausgetrieben, auch es zugleich es weiter eine Anweisung über zu suchen mit Bei solcher Verflechtung hat. ischen Arbeit Kritik nicht zurückschauend der die Richtung empfangen, weltgeschichttichen und die sie fordert, von der philosoph- Erfahrung braucht reflektierend zu bleiben, produktiv und vordringend werden, sie mag in der sie die kann kann die Weiterbewegung, sich aus fördern. Eine solche Untersuchung hat darauf vor allem ihr Augenmerk zu richten, die Selbstverständlichkeit zu zerstören, mit der sich die Bestrebungen einer Zeit zu geben gewohnt sind, und zugleich von dem Dogmatismus zu befreien, Bedingung dafür bildet unternimmt und erreicht, der jenen anzuhaften präziseres ein präzise Umfang der Leistung sehen, und Die pflegt. erste Sehen dessen, was die Zeit sehen heißt hier zugleich den das allein macht es möglich, zu einem Urteil zu gelangen, das selbständiger und kräftiger Art ist, ohne ungerecht zu werden und Paradoxie für Unabhängigkeit zu geben. Darauf vornehmlich der Mannigfaltigkeit und sei dem also das scheinbaren Augenmerk gerichtet, in Durcheinander der Be- wegungen durchgehende Züge, einfache Grundlinien aufzudecken; von hier aus am ehesten werden wir hoffen dürfen, den Wahrheitsgehalt der Zeit, ihre inneren Notwendigkeiten von der entstellenden Zutat menschlicher Irrung und Leidenschaft zu befreien, aber Anhaltspunkte für das eigne Streben zu gewinnen. zutreffend beurteilen zugleich Die Zeit kann nur, wer sie innerlich mitzuerleben ver- Einleitung. mag; wer sich ihr von vornherein 9 krittelnd und nörgelnd entgegen- dessen Urteil hat keinen Wert. stellt, Endlich sei noch hinzugefügt, daß, wie in den früheren Auf- lagen des Buches, so auch in dieser die Bezeichnungen der Hauptbegriffe sorgfältig Gebrauch verschuldet zum Indem einmal Gegenwart. strengere Fassungen Ihr schwankender nicht geringen Teil die Verwirrung der werden beachtet oft bei sollen. demselben Ausdruck laxere und durcheinanderlaufen, erschleichen hauptungen mehr Sicherheit und mehr Gehalt, heit indem gebührt; ferner verschiedene Bedeutungen Sache und versteckt sich nicht hat, selten verwirrt als dasselbe sich leicht leicht Be- ihnen in Wahr- Wort wesentlich der Anblick der der Punkt der Entscheidung. Zu allen Zeiten decken die Ausdrücke und die Begriffe sich nur in annähernder Weise, heute Bekämpfung aber gehen sie besonders weit auseinander. Zur eines solchen Mißstandes bedarf es auch eines Blickes auf die Geschichte der Ausdrücke; so sei auch dieser ein bescheidner Platz gewährt. Zum Grundbegriff des Geisteslebens. A. Subjektiv 1. a) - Objektiv. Geschichtliches. |as Verhältnis von Subjekt und Objekt I *—^ punkte der Arbeit und des Kampfes; des einen oder des anderen die Begriffe gestalten Hauptbewegung des Lebens entweder vom Men- zum Menschen. schen zur Welt, oder von der Welt Stand müssen In alle die übrigen Probleme einmünden, Behandlung Phasen werden wir Lösung erkennen und zugleich Wie so trägt sein Wirkungen der gesamten Geschichte bei seiner diesen Bilder die von der Wirklichkeit, die Fassungen der Wahrheit ver- schieden, geht die Problem dem Vorwiegen vom Leben, nach je sich heute im Mittel- steht in dies in heutiger und sich, es ihre Hauptphasen gegenwärtig sein. die wichtigsten eine Möglichkeiten der durchgehende Bewegung ge- wahren, welche, die Arbeit in eine gewisse Richtung drängt. Eigentümliche Verwicklungen der Sache verrät schon die merk- würdige Geschichte der Ausdrücke subjektiv und objektiv; sie im Lauf vertauscht. Bei Duns Scotus (f 1308), der zuerst der Jahrhunderte ander gegenüberstellte, das Subjekt der ihre geradezu sie als also auf die haben Kunstausdrücke ein- subjectivum dasjenige, was »/hieß Urteile, Bedeutung sich auf konkreten Gegenstände des Denkens bezieht; hingegen objectivum jenes, was im bloßen obicere, d. h. im Vorstelligmachen, liegt stellenden fällt" IIL 208). In diesem Sinne sophen stück (s. Prantl, und hiemit auf Rechnung des Vor- gehen die Ausdrücke bis in das 17. und 18. Jahrhundert, des gebräuchlicheren Logik Geschichte der objektive im Abendlande, bei den Philo- doch bildet das Gegen- öfter formaliter oder auch Subjektiv — Objektiv. schon U Auch zeigen die Ausläufer der Scholastik realiter. 1 Schwanken, ein gebrauch vorbereitet. das die Wendung zum objectivus bei neueren Sprach- 2 Die völlige Umkehrung der Bedeutung erfolgte aber Übergang schen in die deutsche Sprache, so Schule, z. erst beim und zwar innerhalb der Woffi- bei A. F. Müller (Einleitung B. sophische Wissenschaft, 1733), die philo- Baumgarten und Gottsched. Doch in bleiben die Ausdrücke (man sagt übrigens «subjektivisch" an zunächst jektivisch") gebunden, Schule die und » ob- so erscheinen sie dem Streit zwischen Lessing und Goetze als gelehrte Termini; erst die Kantische Philosophie hat sie dem allgemeinen Sprachgebrauch zugeführt, in dem sie zu Beginn des 19. Jahrhunderts einen breiten Raum erlangen. Von Deutschland aus ist die z. noch B. in neue Bedeutung zu den anderen Völkern gewandert und von ihnen zunächst oft als fremdartig empfunden worden. Der heutige Sprachgebrauch so ist, deutlich er vom sich Schwankung und Un»Subjektiv bezeichnet zunächst das, was der bloßen mittelalterlichen abhebt, bei sich selbst sicHerheit. voll Vorstellung des Individuums angehört, nicht selten aber auch, namentich Naturforschem, bei Wesen fühlende bei und alles sich selbst jedes, erlebt, was das denkende auch wird wohl alle und Über- zeugung, die den unmittelbaren Tatbestand überschreitet, subjektiv und damit genannt und für eine erscheinen Flachstes nachträgliche damit als Zutat erklärt. gleichen Tiefstes »Objektiv Rechtes. leidet namentlich an der Zweideutigkeit, daß es das Gegenständliche bald als ihrer befindlich aller seelischen Betätigung innerhalb moderne Naturalismus sein. In den Erörterungen subjective = formaliter in se ^ als Gegenständlich wollte Goethe, gegen- bezeichnet. ständlich will auch der gegenüber, bald unterscheidet (oeuv. div. 1727, zwischen Descartes und Qassendi ipsis, III. findet sich objective =: idealiter in intellectu. Bayle 334a) objectivement dans notre esprit und und noch bei Berkeley heißt es (Ausgabe von phaenomena are only natural appearances. They such as we see and perceive them. They real and objective reellement hors de notre esprit, Fräser are, II. 477): „Natural therefore, nature ' are, therefore, the same." So heißt es z. B. in Chauvins lexicon rationale (1692) unter certitudo: objectiva nonnullis est ipsa necessitas objecti, seu propositio necessaria objectiva. AHis autem intellectus per nihil aliud est quem objectum nimmt ratio objectiva quam denominatio quae sumitur ab actu repraesentatur. Goclen (lex. philos. = res ipsa quatenus definitioni respondet. 1613) Zum Grundbegriff des Geisteslebens. 12 In der Sache handelt es sich augenscheinHch um das Verhältnis des Menschen und seines Gedankenkreises zur Welt, der er angehört. Soweit das Denken selbständig wird, mit ihr unablässig beschäftigen sich Scheidung zu zugleich selbst Welt gegen- es sich der aber zugleich kann es nie vergessen, über, und stellt daß muß; zu ihr gehört es so entsteht mit der unabweisbares Verlangen, ein sie irgend überwinden, das Geschiedene wieder zusammenzubringen zu verwickeln, mehr wir uns mit ihr befassen. je und Diese Aufgabe aber scheint sich immer mehr fest zusammenzuhalten. Die Verwick- lungen hat schon das griechische Altertum stark empfunden, aber es Die Lösung, ist. versuchte, noch mit ihnen hat sich möglich ist am meisten abgefunden, leichter als uns Späteren Höhe der klassischen Zeit zu geschichtlicher Wirkung gelangt; was welche hier die leitenden Denker, ein Plato die und ein Aristoteles, an Lehren entwickeln, das schöpft vornehmlich daraus eine Überzeugungskraft, daß es ein Ganzes der Lebensführung hinter sich hat. griechische Lebensführung hat ihre Eigentümlichkeit und ihre Stärke Diese alt- darin, das naive Verhältnis des Menschen zur Natur ohne einen schroffen Bruch ins Geistige zu heben und zugleich zu veredeln, den Menschen in die Welt hineinzusehen, zu sich selbst Äußeres, haben aber aus der Spiegelung ihn geläutert zurückzuführen. die anfängliche Mensch und Welt, Inneres und Vermengung überwunden, aber sie sind noch nicht so schroff geschieden, um sich nicht durch geistige Arbeit rasch wieder zusammenzufinden. ander wesensverwandt seiner eigenen und Denn innerlich zugetan, beides scheint ein- jedwedes bedarf zu Vollendung der Ergänzung durch das Gegenstück: von innerem Leben erfüllte Natur erreicht ihre Höhe in der Aneignung durch den Menschen; was aber in diesem an Kräften die schlummert, das wird erst vollem Leben geweckt. durch die Berührung mit der Welt zu In der Einigung, wie sie Anschauung und Liebe bewirken, gewinnt das Leben die Höhe und Seligkeit geistigen Schaffens. Eine solche Überzeugung kann unbedenklich die Wahr- heit als eine Übereinstimmung unseres Denkens mit dem Gegenstande (adaequatio intellectus et rei) fassen. Diese Fassung genügt aber nur für einen Stand des Lebens, wo die schien, Natur noch seelischer und der Mensch noch wo weder jene eine volle Selbständigkeit in natürlicher eigentümlichen und Gesetzen gewonnen, noch das Innenleben sich zu einer eignen Welt vertieft hatte. So gewiß jene größere Nähe und jene Kräften Subjektiv — Objektiv. 13 fruchtbare Wechselwirkung beider Seiten eine großgesinnte, frohe, künstlerische Kultur hat bilden helfen, lebens- jener enge Anschluß des Geisteslebens an das naive Weltbild war für die Dauer nicht zu erhalten. So hat denn schon das Neuplatonismus neue spätere Altertum in der Stoa Wege versucht, und im aber stärker als diese hat auf die Neuzeit jene ältere Art gewirkt, da sie eine nicht unbedeutende Nachblüte in der mittelaltedichen Scholastik fand und durch diese unmittelbar die Neuzeit berührte; namentlich in Auseinandersetzung mit ihr hat diese ihre Eigentümlichkeit gefunden. Es erscheint aber die neue Art zunächst des Subjektes, einer trotzigen Losreißung von der Entfaltung und das Leben zu her die Welt zu bilden Welt zu empfangen und an sie sie gestalten, statt Anschluß zu suchen. aus der Gewaltiger hat die Wissenschaft den Anblick der Dinge verändert; alles ausscheidet, was Um- vom Menschen und seinem Denken gebung, einem kühnen Versuch, als je einer kräftigeren in Prüfung nicht besteht, ihre indem das Ver- bleibende aber durchleuchtet und enger verbindet, wird das ganze Dasein des Menschen das Element des Gedankens getaucht und in Das Innere erkennt seine Einheit und befestigt sich sicher im eigenen Kreise, die Außenwelt weicht davor zurück, sie verliert alles innere Leben, indem sie Ideelle gehoben. ins Gedankenhafte, Begriffliche, zur räumlichen Bewegung sie verliert alle bunte Farbe, indem die ganze Fülle der sinnlichen keiner Seele mehr zu bedürfen scheint, Eigenschaften aus einem eigenen Besitz der Dinge zu einem bloßen Gewände wird, womit sie die fremd; innerlich und mit So wird nunmehr von leblosen Massen und Bewegungen der die Natur als ein Reich Seele Seele umkleidet. als auf sich selbst gestellt die Unendlichkeit bezwingend, fühlt sich die Seele ihrer Denkkraft aber, ihr weitaus überlegen. Das ein Hauptstück, ist der Neuzeit, nicht aber ist vielleicht es das Hauptstück des Lebens das Ganze dieses Lebens. Denn unverkennbar hat die Neuzeit neben dem Drange zur Steigerung des Subjektes auch den entgegengesetzten Zug, des Menschen heraus zur gegenüber dem menschlichen wirren Kreises ein aus der Kleinheit Größe der umgebenden Welt zu flüchten, Getriebe und der dumpfen Enge des weiteres, gehaltvolleres, reineres Leben dem unermeßlichen All zu schöpfen. So ein Trieb zum Objekt, ein Streben, sich in sein Wesen zu versenken, seinen Gehalt ohne aus Zum Grundbegriff des Geisteslebens. 14 Trübung aufzunehmen. sich in Dinge, der Erfahrung, Hier wird von der Mitteilung der der Mensch darf seine Heil erwartet; alles Art der Welt nicht irgendwie aufdrängen wollen, er dienstwillig einfügen, die Verstärkung Denn indem selbst alle des Subjekts das Subjekt Ende bereitet, heit entfalten, Ganzen bei alten verketten; jetzt erst, Reich von die Natur auf sich Das Gesetzen. klammern, Vermenschlichung des Weltbildes ein leerer wie ein dem Men- Wahn, und es wird Von zu unterwerfen suchen. alles Fürsichsein des Subjekts gefordert, daß das Leben sich anschmiege und lediglich ihren Geboten folge. Zusammenschluß des Menschen mit der Umgebung, So ein engerer ein neuer Lebenstypus unter der Herrschaft des Objekts. Demnach sie unverbrüchlichen zunächst gegenüber ganz und gar sich willig Schleier ge- muß es sich zu ihm zurückwenden, ihn um- aus erscheint mehr und mehr den Dingen zu einem Autonomie und wird volle alles entwickelt sich ihn fester nachdem jener trübende eine Zusammenhängen lückenlosen schen, aber schließlich ein Selbst Wendung. Konzentration kräftigerer enger verbinden und seine Vielheit erlangt als diese kann das Objekt seine eigene Natur mit voller Rein- fallen, hier indirekt unterstützt den Dingen geliehenen Eigenschaften von ihnen zurück- und damit der fordert muß sich ihr um seinem Leben Wahrheit zu sichern. es ist nicht eine einzige, sondern es sind zwei Be- wegungen, die durch die Neuzeit gehen und auf ihrem Boden ein gleiches Recht zweit, und ihr und Unruhe. die meisten behaupten. Leben Diesen der Solcher Zwiespalt So enthält ist sie innerlich bei sich selbst ent- von Haus aus eine starke Spannung Doppelcharakter der Neuzeit werden von uns behandelten Probleme erkennen stellt auch lassen. der geistigen Arbeit eine schwere, aber un- abweisbare Aufgabe; nicht aus der unmittelbaren Lage heraus, sondern nur durch ihre Weiterbildung, nur durch Aufdeckung eines neuen Grundgefüges der Wirklichkeit läßt sich eine überlegene Einheit und zugleich eine sichere Wahrheit erhoffen. Daher war es kein bloßer Eigensinn der Spekulation, es war eine innere Notwendigkeit, welche große Forscher auf neue Bahnen trieb und sie eine vom Denken getragene Wirklichkeit dem ersten Lebens- und Weltbilde entgegenstellen hieß. sind als von Spinoza und Kant; jener Von diesen Versuchen Ausdrücke eines neuen Lebenstypus besonders wichtig die das Objekt, um den Gegensatz zu überwinden, verstärkt dieser das Subjekt, jenem dringt das Objekt in Subjektiv — Objektiv. 15 das Subjekt, diesem das Subjekt in das Objekt vor. und den Menschen bindet die Spinoza ver- Welt durch die Aufdeckung einer Weltkraft im Menschen und ihre scharfe Abhebung von aller bloß- menschlichen Art; diese Weltkraft sinnliche Umgebung befreite, ist das von aller Bindung an die auf sich selbst gestellte und lediglich von seiner eigenen Notwendigkeit getriebene Denken, wie z. B. die Mathematik es zeigt, das Kleinmenschliche dagegen besteht in dem bloßsubjektiven Fürsichsein mit seinen Affekten und Zwecken. Die Wendung von und heit der Dinge und keit Die damit So ,waren welche es Bewegung zugleich die Wahrund Unendlich- unmittelbar ihre Ewigkeit und Lebens die Wissenschaft, Vollendung zu Klarheit einem auch die Dinge tragenden Allleben in teilt des Seele zur Befangenheit so erfaßt es in seiner ist, wird und den Menschen ein Weltleben gewinnen; läßt denn wie das Denken gegründet Enge seiner Weite des Denkens von Erlöserin die die Wissenschaft, die und künstlerischer Kontemplation religiöser Nöten allen sich ihrer in gestaltet. auch vor allem künstlerische und kontemplative Naturen, Lebenstypus dieser mit herben seiner und Größe stillen anzog; aber weit über den Kreis der Anhänger hinaus wirkte diese Denkweise durch das Scheiden von Kosmischem und Kleinmenschlichem innerhalb des Menschen durch selbst, die energische Be- kämpfung des Anthropismus des Denkens nicht nur, sondern auch der Gesinnung, der sich im so Mittelalter eingenistet fest hatte. Die Kleinheit des gewöhnlichen Glückverlangens und die Enge des kommen Vorstellungskreises landläufigen zu jetzt Emp- deutlicher findung; einmal empfunden und als unzulänglich befunden, können sie die alte Selbstverständlichkeit in nun und nimmer wieder erlangen. Das aber bleibt eine Frage, ob unser geistiges Leben gänzlich Denken aufgeht, ob nicht die Wendung vom Schein der Sinne zur Wahrheit des Denkens verlangt, die jenseit selbst eine Tat des bloßen Denkens des ganzen Auch liegt. Menschen die Voraus- setzung dieser Lösung: das Zusammenstimmen unseres Denkens mit der Welt um uns, Leben des Alls, ist das Umfangensein beider von einem einzigen keineswegs zweifellos; unseres Denkens unsicher wird, da ist wo aber der Weltcharakter sofort die Wahrheit des ge- botenen Lebens erschüttert. Dieses Bedenken entgegengesetzten der Dinge in wirkte auch Weg einschlug. eine unzugängliche Jbei Denn Kant, bei als er einen ihm weicht Feme zurück, und es gerade die fällt Welt alle Zum Grundbegriff des Geisteslebens. 16 Möglichkeit, Übereinstimmung mit ihnen gewiß zu werden. einer irgendwelche Wahrheit für uns bestehen bleiben, Soll also sie innerhalb des Subjektes zum Objekt zu suchen. von diesem Nein nicht selbst, in so ist einem Verhältnis Das bedeutet ein entschiedenes Nein, aber findet sich für Kant ein er innerhalb unseres Lebenskreises Weg zu einem Ja, indem große Gesamtleistungen aufzeigt, vornehmlich die Bildung einer wissenschaftlichen Erfahrung und die Was immer an diesen Leistwird vom Subjekt geleistet; so eines Reichs des sittlichen Handelns. ungen geistiger Art von ist, das muß es auch bei sich selbst der herkömmlichen Fassung entwachsen. Es nun ist sowohl Einzelpunkt, geistiges Gewebe; was nicht geistige Struktur, Tätigkeit erfaßt, individuelle Existenz, von und es sich- gewinnt damit eine Giltigkeit für alle als seiner Einzelnen; so entsteht eine neue Art von Objektivität,^ ein neuer Begriff der Wahrheit. Sein näherer Inhalt bestimmt sich nach der Beschaffen- heit und der Bedeutung der Tätigkeit, bei der und theoretischen menschliche Erkenntnis bleibt bei er ist daher grundverschieden der Vernunft. praktischen Alle nach Kant an eine undurchsichtige Welt gebunden; die Gedankenwelt, die wir auf ihre Anregung hin entwickeln, gilt nur für uns selbst und unser Vorstellen, unser Weltbild reicht nicht über uns hinaus, nicht nur die Formen der sinnlichen bloßmenschlicher Art. das Denkens sind und bleiben Anders auf praktischem Gebiet. Hier erlangt Anschauung, Tun des Menschen auch die des eine volle Ursprünglichkeit und vermag es aus sich selbst eine Welt zu erzeugen; hier, wo das Unterscheidende in der Unterordnung aller menschlichen Besonderheit unter allgemeine Normen liegt, ist die Wahrheit nicht bloßmenschlicher, sondern abArt. Der Mensch steht hier unmittelbar in den tiefsten Gründen der Dinge, als moralisches Wesen wächst das Subjekt bei sich selbst zum Träger einer Welt. So wird hier die Moral zu soluter ' Dieser neue Begriff der Objektivität ist freilich voller Verwicklung und wurde von den Gegnern Kants hart angegriffen. So sagt z. B. Plattner, Philosophische Aphorismen I, § 6Q9 Anmerkung: „Wenn nun aber damit bewiesen werden soll, daß unsere Erkenntnis objektive Giltigkeit hat: so übt man doch fürwahr an dem Worte Objektiv eine Gewalttätigkeit aus, die bisher in dem philosophischen Sprachgebrauche unerhört war; denn man deutet damit gerade den entgegengesetzen Begriff Subjektiv an. Wirklich Schmid, der nie von seiner Liebe zur Wahrheit abweicht, in hat sich Herr der Notwendigkeit gesehen, die Kantische Objektivität subjektive Objektivität zu nennen. Wörterb., Art. Objektiv." Subjektiv — Objektiv. 17 einem selbständigem Reich und zugleich zum Kern des Lebens, die und bekommt zur höchsten Erkenntnisarbeit rückt in die Peripherie Welt vor Störungen moralische Aufgabe, die entsteht damit ein zu bewahren. zu dem Spinozas: dort die Ruhe der Kontemplation, ruf zur Aktivität; dort Vordringen ein vorhandenen Welt, hier Ausgleichung Gegensätze aller das Schaffen in eine Spaltung der Wirklichkeit sätze. Es neuer Typus des Lebens in vollem Gegensatz hier der Auf- zu den Grundlagen einer einer neuen Welt; dort die einer allumfassenden Einheit, hier und eine Verschärfung aller Gegen- Beiden ist aber das Streben gemeinsam, unserem Leben irgend- wie einen Weltcharakter zu geben, den Menschen aus sich selbst herauszureißen und ihn zu neuen Tiefen zu führen. Eine Erörterung des Kantischen Denk- und Lebenstypus bleibe der Betrachtung der Gegenwart Leben erweckt hat. an dem Belassen eines Dinges menschlichen Vermögens, sich fiel die die Scheidung neuem zu ihn als Söhne und freudigem Lebensgefühl, schweren An- einer Zeit von kräftigem stoß- vorbehalten, Die unmittelbaren Nachfolger nahmen, an sich und der Begrenzung die daraus hervorgeht Mit dem Ding an von theoretischer uud praktischer Vernunft, mehr im Wege, das Leben in einen einzigen Zusammenhang zu verwandeln. So wurde denn kühnen Mutes unternommen, alle Wirklichkeit aus unserer geistigen Tätigkeit, im besonderen aus dem mit innerer Bewegung ausgestatteten Denken hervorzubringen. Das Denken, so hatte schon Plotin gezeigt, vermag im eigenen Kreise den Gegensatz von Subjekt und Objekt zu überwinden, indem es sich gegen sich selbst kehrt, das Denken selbst und es stand zum Vorwurf nichts Denkens macht Das brauchte nur mit voller Konsequenz entwickelt, vom bloßen Individuum abgelöst und auf das Ganze der weltgeschichtlichen Arbeit übertragen zu werden, und es entstand des das Hegeische System, eine Selbstentwicklung ein Selbstbewußtwerden des Denkens des das die ganze Wirklichkeit in Wahrheit als verwandelt, Geistes versteht die und den Menschen an dieser absoluten Wahrheit vollauf teilnehmen läßt alle Eigenwilligkeit eines subjektiven Nur muß er Meinens aufgeben und allein den Notwendigkeiten des Denkprozesses folgen. Dies Unternehmen ergriff nicht nur wie ein brausender Sturm seine eigne Zeit, es hat sich mit seinem Flüssigmachen aller Größen und seinem Zusammenschweißen aller Bestand des Geisteslebens eingegraben. Eucken Orundbegriffe. 4. Aufl. , Mannigfaltigkeit tief in den Aber auch ein Rückschlag 2 Zum Grundbegriff des Geisteslebens. 18 konnte nicht fehlen, sobald jener kühne Aufschwung nachließ, und das Ausleben selbst die Grenzen des Unternehmens zur Empfindung Unabweisbar wurde nun die Frage, ob der Prozeß nicht brachte. über sich selbst will und es hinausweist, da er als geistiger wieder erlebt sein dazu eines überlegenen Punktes bedarf; unabweisbar die andere, ob die ausschließliche Verwandlung des Lebens in Denken der Wirklichkeit nicht Gewebe von ist weniger durch als durch worden. und aus ihr — Wie immer dem bloßes Wendung des Lebens wir den Boden, betreten der dem 9. Jahrhundert hat das Problem unmittelbarer zur Empfindung wirken und den Gegensatz mehr erfährt es läßt sie und sich breiter entfalten. Überwindung aber gebracht, wie schon das stete Das Problem 19. Jahr- Das 19. Jahrhundert. zum Bewußtsein gebracht als irgendwelche frühere Zeit, einer überwunden zurückgedrängt ist. b) den Versuch ein sein mag, das Ganze wissenschaftliche Gegenarbeit tatsächliche Damit aber 1 ist, eine hundert eigentümlich Das raubt hier nicht viel zu rasch die menschliche Geistigkeit zur absoluten erhoben eine Inhalt Formen und Formeln macht; unabweisbar logischen ob endlich die, allen hat es Für kaum etwas neues Zurückgehen auf Kant bekundet. zunächst den Einfluß der Wendung von den Zielen der inneren Bildung zur Beherrschung der sichtbaren Welt durch Naturwissenschaft, Technik und politisch-soziales Wirken. Solche Richtung des Lebens heißt den Menschen stets den engsten Anschluß an die Dinge suchen und nur von der Bindung seiner Kräfte an sie eine Realität und Wahrheit erwarten, während ein davon abgelöstes Leben zu einem bloßen Schattenreich, einer leeren Einbildung sinkt. So verlegt das Leben seinen Schwerpunkt ins Objektive und findet seinen Kern in der mit den Gegenständen befaßten und durch ihre Natur bedingten Arbeit; diese Arbeit vollzieht eine Emanzipation von den bloßen Individuen, entwickelt bei sich Zusammenhänge ausgedehntester Art und macht mit ihrem unaufhörlichen Anschwellen den Menschen mehr und mehr zu einem bloßen Diener und Werkzeug. So zunächst in der technischen Arbeit mit ihren Fabriken, so mehr und mehr auch in den anderen Lebensgebieten. Je mehr überall das Sinnen und Streben sich auf gemeinsame und sichtbare Leistungen richtet, desto nebensächlicher selbst Subjektiv — Objektiv. was wird, der Seele des Einzelnen vorgeht, desto gleichgültiger in und Befinden, desto mehr wird das Subjekt erscheint sein Ergehen am Eimer, zu einer Größe, die sich ohne zu einem bloßen Tropfen Schaden zurückstellen und ausschalten läßt. Ausdruck findet das Einen wissenschaftlichen der Theorie des Positivismus, in Grundsätze folgerichtig durchführt, ihre 19 sie soweit sie nicht mit andersartigen Gedankenmassen verquickt Dieser Zug überwiegt noch immer im Leben der Aber Zeit. stärker und stärker empfinden wir die Grenzen eines solchen Strebens, und immer deutlicher steigt das Gefühl der Leere auf; bekundet nicht schon das ein Wiedererwachen des Subjekts und die Unmöglich- sich nach dieser Richtung damit nahe, das Subjekt beginnt tritt seiner Zuständlichkeit in das Erste und als zusehen, es entwickelt die Neigung, zugeben, Ein jäher Um- Verzichtes auf alle innere Befriedigung? keit eines schlag alles Geschehen alle Überlegene an- Bindung nach außen auf- nach der Wirkung auf das eigne Befinden zu messen, schließlich das Leben möglichst in freischwebende Stimmung zu verwandeln. Literatur, So geht es neuerdings in breiten Wogen durch die das künstlerische Schaffen und auch durch das gesellschaft- um die Seele zu befriedigen und Aber Zusammensein. liche Gegner zu besiegen, dazu entbehrt Gehalts. eines ihr All Aufrufen diese der Bewegung individuellen viel den zu sehr ergibt Kräfte keineswegs eine zusammenhängende Innenwelt und eine gemeinsame Wahrheit; von der schließlich er befreien Subjektivismus darf mittelbar dieser wollte. Weg in dieselbe Leere zurück, Als wissenschaftlicher Vertreter dieses am ehesten der Psychologismus gelten, der un- von der Seele des Einzelnen her zu einer Gedankenwelt Zeitweise hat dies viele überwältigend fortgerissen, aber der strebte. Rückschlag daß führt sich ist rasch gekommen und immer wird deutlicher uns, von dieser schwankenden Grundlage aus zu einer Wissen- schaft, zu einem Reich der Wahrheiten nun und nimmer gelangen läßt^ Auch mehr die jenseit Schranken der Wissenschaften des Subjektivismus. erkennen mehr und wir Aber da wir zugleich zum Objektivismus im geschilderten Sinne unmöglich zurückkehren können, so verbleiben wir * in peinlichem Zwiespalt, und es drohen Die durchschlagendste Widerlegung des Psychologismus enthalten die Logischen Untersuchungen von Husserl, 1900 und 1901; im besonderen wird hier überzeugend erwiesen, wie tief der Psychologismus auch in die Ge- dankenwelt solcher Forscher eingedrungen ist, die ihn prinzipiell verwerfen. 2* Zum Grundbegriff des Geisteslebens. 20 und Arbeit sich immer mehr zu einander Seele des Lebens läßt sich unmöglich solcher Zerfall Ein verfeinden. endgültig hin- als muß die Kluft überwunden werden. An Bemühungen darum fehlt es nicht, am meisten verbindet nehmen, irgendwie von innen her so zu verstärken die Geister ein Streben, das Subjekt und auszuweiten, daß damit ein neuer Weltdurchblick und ein neues Leben gewonnen wird. Es geschieht das aber in engem, wenn auch keineswegs völligem Anschluß an Kant. sowohl die Theologie und dort — es handelt um die von Ritschi ausgehende Bewegung — hier namentlich sich es gestaltet sich aber hier In der Theologie verschiedener Weise. in Ein derartiges Streben zeigt als die Philosophie, wird unternommen, die religiöse Wahrheit von der Unsicherheit der Spekulation und der Metaphysik zu Wesen der Seele einen und befreien Grund zu festen im innersten ihr Vornehmlich geben. in der Moral, in der Ausbildung der sittlichen Persönlichkeit, scheint das Geistesleben über sicher und zu erzeugen eignes Reich ein alles ihm sich mit Nach diesem Ge- andere Dasein hinauszuheben. dankengange bedarf, was zur geistigen Selbstbehauptung nötig keiner Bestätigung von außen her, Vermögen, durch eignes sein Lebens. es erweist seine Wahrheit die Steigerung ist, durch des ethisch-religiösen Die nähere Entwicklung der Gedankenwelt wird hier vor- nehmlich durch die „Werturteile" bestimmt, die jene Beziehung zum Kern des Lebens vertreten und damit werden; das moralischreligiöse führung überlegen nach theoretischen aller ihm innewohnenden Notwendigkeiten den nur in steter Ganzes ein Überzeugungen, das freilich keine Welterklärung sein Geltung Beweis- Leben entwirft will von und seine Zurückbeziehung auf die Grundwahrheiten jenes Lebens hat. Dieses Streben, verschiedene Färbungen es die barer begründen schehen kann, denken hervor. handelt Gefühl will, die nähere Als sich selbst insofern gewiß in ist". III, Kern (Ritschi, 142). als des Menschen fester und unmittel- manche Be- Lösung dieser Aufgabe ruft des Lebens das Gefühl be- ein pflegt Christi. Aber hier Aufbau versucht zu werden. nun einmal die geistige Funktion, Versöhnung gutem Recht, Leben in höherem Grade einen Tatcharakter und von ihm aus ist ist das durch theoretische Erwägungen ge- als als es dem Aber verleiht. hat, Überzeugungen letzten Durchführung übrigens recht nähere dessen in „Das welcher das Ich bei Lehre von der Rechtfertigung und läßt in Wahrheit das Gefühl schon Subjektiv - Objektiv. ein Beisichselbstsein des Lebens erreichen? und flach leer Es erzeugt doch sein? sondern es gewinnt ihn nur Inhalt, des Lebens. artigsten nicht von weiteren nicht sich aus einen Zusammenhängen Wie das Gefühl in stetem Fluß und der verschieden- Deutung dem Leben in 21 Kann das Gefühl ein fähig so läßt sich von ihm aus unmöglich ist, wie Halt ein Inhalt geben. Der Versuch eines Aufbaues einer Gedankenwelt vom Gefühl und damit vom Subjekt würde sich vom bloßen Subjektivismus kaum unterscheiden, wenn sich nicht das Gefühl als als der notwendig und der von ihm bejahte Inhalt Besonderheit natürlichen des Menschen dartun überlegen Wie aber sollte das von der bloßen Tatsächlichkeit des Seelen- ließe? Mag ein Gefühl noch so unabweismag lebens her geschehen können? bar scheinen, es ist das zunächst nur für das besondere Subjekt; einem bestimmten Inhalt noch so verwachsen scheinen, diese Verbindung besagt mehr als der unmittelbare Eindruck enthält, sie ist das Ergebnis einer Deutung, und eine solche Deutung kann irre es mit So gehen. leistet die Stärke eines Gefühls nicht die mindeste Ge- währ für die Wahrheit des aus ihm entwickelten Gedankengehaltes. Das bekundet u. a. die Vielheit und der Streit der Religionen. Jede von ihnen glaubt ihres sein als die anderen, Grundgefühles mindestens ebenso sicher zu und doch gelangen sie zu grundverschiedenen notwendig einer höheren Instanz, um über das Recht der verschiedenen Ansprüche zu entscheiden, und Wahrheiten. diese So bedarf es kann nicht das Gefühl Menschen aus zu Überhaupt aber läßt sich vom sein. einer Wahrheit nur gelangen, wenn in ihm ein seiner natürlichen Besonderheit überlegenes Leben durchbricht; eine an jene Besonderheit geknüpfte Wahrheit ist keine Wahrheit. Schon das bringt es mit sich, daß der Mensch das Problem seines Grundverhältnisses zur Wirklichkeit nur zurückschieben kann. träglich Art aufgedrängt, Das Leben es nun und nimmer aufgeben oder auch Dies Problem wird ihm nicht gehört von erst nach- vornherein zu seiner geistigen eines geistigen Wesens erschöpft sich einmal nicht in die bloße Zuständlichkeit, es umspannt auch das Gegenständliche und muß sich mit ihm auseinandersetzen, es muß auf einer Überwindung jener Spaltung zwingend bestehen, während ihm die Beschränkung auf die bloße Zuständlichkeit zu einer unerträglichen Enge wird. Was hier an Verwicklung vorliegt, das verdunkelt und vergißt sich namentlich deshalb leicht, weil die Regung des Gefühls durch Zum Grundbegriff des Geisteslebens. 22 eine geschichtlich überlieferte Gedankenwelt ergänzt zu werden pflegt, der Halt scheint damit fester und der Inhalt reicher zu werden. ist zu und das kann erweisen, Gefühl geschehen; her In die Wahrheit der geschichtlichen Überlieferung erst der Tat aber diesem Zusammenhange nur in ebenso könnte auch nur dieses vom darüber entscheiden, was vom Gehalt jener Überlieferung gelten hätte. So kommen wir hier auf Umwegen immer wieder auf als wertvoll das Gefühl zurück und bleiben an seinen Kreis gebannt. zu mehr Je es aber auf sich selbst gestellt wird, desto weniger Inhalt gewährt es, desto mehr droht es sich in einzelne zerstreute und sinnlose Vorgänge aufzulösen. So führt dieser Weg mehr in die Verwicklung hinein als aus ihr heraus. Was immer aber uns von der hier gebotenen Gedanken- entwicklung trennt, es kann uns nicht verhindern, die entschiedene Kräftigung des ethisch-religiösen Lebens anzuerkennen, die mit jenem Streben erreicht ist; nur können wir die wissenschaftliche Fassung dessen nicht glücklich finden. dem Gebiet der wo der Begriff des Wertes^ den Mittelpunkt einer be- Wesentlich anders gestaltet die Sache sich auf Philosophie, deutenden und fruchtbaren Bewegung bildet ^ Über den Begriff und die Diese Bewegung vertritt Bedeutung des Wertes ist in den letzten deren Betrachtung und Würdigung hier nicht wohl möglich ist; nur das Werk Meinongs „Psychologisch-ethische Untersuchungen zur Wert-Theorie", 1894, möge hier angeführt Wünschenswert wäre eine Gesamtgeschichte des Wertproblems und sein. Jahrzehnten eine ausgedehnte Literatur entstanden, Wertbegriffes; nur aus Höffdings Religionsphilosophie folgende „Der Philosophie Kants verdanken wir die Selbständig- hier seien Stellen hervorgehoben : keit des Wertproblems dem Erkenntnisproblem gegenüber. Er lehrte uns die Schätzung von der Erklärung unterscheiden." (S. IL) Femer „Kant redet häufiger von Zwecken von Werten. Es ist aber klar (obschon Kant dies weder noch in seiner Ethik recht beachtete), daß der Begriff des Zweckes den Begriff des Wertes voraussetzt, da ich zum Zwecke nur das machen kann, dessen Wert ich erfahren habe. Wenn Kant von dem Reiche der Zwecke' im Gegensatz zur kausalen Naturordnung redet, so meint er hiermit dasselbe, was spätere Philosophen ,das Reich der Werte' nannten. Der Kantianer Fries geht von dem Begriff des Wertes aus (System der Philoals in seiner Psychologie, , sophie, Leipzig 1804; §§ 238, 255, 330. berg 1807. III. S. 14). — Neue Kritik der Vernunft. Heidel- Vorzüglich sind es aber Herbart und Lotze, die dem Begriffe des Wertes in weiteren Kreisen Eingang verschafften. Nach Lotze nahmen der Theolog Albrecht Ritschi und dessen Schüler denselben auf." Über den Wertbegriff bei Fries ist neuerdings eine Schrift von Pöschmann erschienen. lich modern Es ist aber der Begriff mit seinem Problem keineswegs ausschließ; er tritt hervor, sobald das Subjekt eine größere Selbständigkeit Subjektiv - Objektiv. 23 Ganzes angesehen die moderne Denkweise gegenüber der antiken, Wo den Hauptgegen- als namentlich sofern diese durch Plato bestimmt ist. satz der Wirklichkeit der des beharrenden Seins und des flüchtigen Wer- dens bildet, wie bei diesem, da liegt es nahe, das wesenhafte Sein zugleich als das Gute und Wertvolle zu fassen, beide Begriffe möglichst zusammen- zuschmelzen. Diesem Gedankengange kann das Gute als etwas von der Tätigkeit Abgelöstes, dem Menschen Gegenüberliegendes erscheinen. Die moderne Denkweise verficht dagegen, daß von einem Guten nur in Beziehung auf ein lebendtätiges Wesen die Rede sein kann, und daß nur nach der Bedeutung für dieses sich die Schätzung näher bestimmen läßt; damit empfahl es sich, statt von Gütern von Werten zu Dieser Grundgedanke kann aber sehr verschiedene Fassungen reden. annehmen und hat das in Wahrheit getan. Gilt als der Träger des Lebens lediglich das empfindende und fühlende Einzelsubjekt, und demnach der Wert der Geschehnisse nach der Leistung und Behagen bemessen, wird Lust und Unlust zum entscheidenden Maßstab gemacht, so sieht man nicht, wie davon wird sich* für dessen Befinden Bewegungen oder gar Erhöhungen des Lebens ausgehen könnten. Denn die Lust schmiedet den Menschen an die eigne dunkle fruchtbare Zuständlichkeit, sie macht das Leben innerlich eng bei aller Weite Erhöhung des Wesens, alle unmittelbare Freude an Menschen und Dingen, alles Aufnehmen des nach außen, sie verwehrt Gegenstandes in den Lebensprozeß. lich alle derjenige schwer nehmen, innere Diesen Mißstand wird nament- der in dem seelischen Stande des Menschen große Aufgaben und Verwicklungen erkennt Denn eine solche Lage bringt die Forderung eines kräftigen Emporklimmens, ja einer inneren Umwälzung mit sich ; diese aber sind ausgeschlossen, wenn das Leben starr an den bloßen Zustand gebannt bleibt. Unvergleichlich kritisch-idealistische versetzt und bestrebt ist ^ gewinnt. bildeten. sie viel höher steht eine andere Art, welche die Denkweise Kants auf den Boden der Gegenwart den Erfahrungen der Zeit gemäß weiterzuführen Hier bildet die Tatsache den Ausgangspunkt, daß unser So zuerst bei den Stoikern, die auch einen Terminus (a^ia) für ihn Nikolaus von Kues, der erste moderne Deiiker, nennt Gott den Wert der Werte (valor valorum). ^ Einen besonders klaren und bedeutenden Ausdruck findet diese Be- wegung in den „Präludien" Windelbands, namentlich in den Abschnitten „Was ist Philosophie?", „Normen und Naturgesetze", „Kritische oder genetische Methode". Zum Grundbegriff des Geisteslebens. 24 Leben und Tun nicht in eine bloße und blinde Tatsächlichkeit des Geschehens aufgeht, sondern daß unsere uns zu geistige Art steter Beurteilung zwingt; diese Beurteilung aber erfolgt nach bestimmten Normen, die Willkür überlegen und auch von der Verwirk- aller lichung beim Menschen unabhängig sind. Werte ersichtlich, Lust und Unlust und vollziehen die jenseit liegen, für sich die eine In diesen Normen werden alles bloßen Nutzens, eine innere Erhöhung des Lebens jenseit aller Unbedingtheit in Anspruch nehmen dürfen. ^ So erkennen wir hier ein bedeutendes Streben, dem Leben des Menschen von innen her einen Halt und einen Inhalt zu geben, ihn durch machen, zu ohne ihn in der Mensch die Fährnisse zugleich verwickeln, Aufgabe abzustecken. anderen Wege dem Selbstbesinnung kritische als Naturgetriebe überlegen zu einer spekulativen Metaphysik auch der Philosophie eine eigentümliche In der Tat ist nicht zu ersehen, wie auf einem dem der Selbstbesinnung und der Selbstvertiefung durch die Verfeindung von Subjekt und Objekt die drohende Spaltung des Lebens überwinden könne. Was uns Bedenken erregt, gewiesenen hier Stelle ist selbst mit innerer Notwendigkeit ob ob man an der nicht die über sie hinaustreibe. Fragen mögen sich dabei erheben. Erlebnisse die lediglich dieses, abschließen könne, Bewegung Verschiedene Können die Werte als einzelne genügende Sicherheit erlangen, wird nicht ihre Ur- sprünglichkeit anfechtbar sein, so lange sie ein bloßes Nebeneinander bilden, sich nicht zur Einheit eines Ganzen zusammenfassen ? 2 ferner die in den Werten eröffnete höhere Stufe des Wird Lebens gegen die bestrickende Macht der natürlichen und sozialen Selbsterhaltung aufkommen und sich durchsetzen können, wenn so faltet behauptet? sie uns nicht ein den Werten wie ent- Dieses aber wird schwerlich ohne eine Um- neues geistiges Selbst verschafft, das sich kehrung der vorgefundenen Lage möglich in sein, und das führt doch der Werte vertritt namentlich Münsterberg in Werte" (1908) mit großer Kraft und Wärme. " Die Notwendigkeit einer solchen Verbindung hebt auch Münsterberg nachdrücklich hervor; er sagt im Vorwort zu seiner „Philosophie der Werte": „Die Gesamtheit der Werte muß grundsätzlich geprüft und aus einer Grund^ Diese Überlegenheit seiner „Philosophie der tat einheitlich abgeleitet werden. fehlt, ist ein in sich Das, was unserem Philosophieren heute geschlossenes System der reinen Werte; erst dann kann die Philosophie auch wieder aufs neue zur wirklichen Lebensmacht werden, wie es zu lange ausschließlich die Naturwissenschaft gewesen ist" (VI). Subjektiv — Objektiv. 25 mag sie von wieder zu irgendwelcher Metaphysik, der alten noch so verschieden sein. So erblicken wir in der Lehre von den Werten weniger einen Abschluß Bewegung. Über die Bewegung wenig hinaus, und die Menschheit verbleibt in dem peinlichen Hin- und Herschwanken zwischen Arbeit und Seele, zwischen der Absorbierung des Subjekts fertigen Philosophie als kommt eine aussichtsreiche einstweilen diese durch das übermächtige Objekt und der Verflüchtigung des Objekts durch die Selbstherrlichkeit des Subjekts. Diese Lage mit ihrer Verwicklung treibt die Frage hervor, ob ganze Scheidung von Subjekt und Objekt, ob nicht nicht die Anerkennung inneren eines neben Bereiches alle Außenwelt von der Haus aus verfehlt sei, ob nicht bei solcher Fassung das Wahrheitsstreben den unlösbaren Widerspruch enthalte, zugleich scheiden und Von verbinden, auseinanderhalten und zusammenführen zu wollen. entgegengesetzten Ausgangspunkten her sind neuerdings Avenarius und Mach zu dem gleichen Ergebnis gekommen, jene Scheidung als unnütze und irreleitende Verdoppelung aufzugeben. Die Ver- eine setzung der Empfindungen in ein Inneres, die Introjektion, erschien ebenso verfehlt, wie die Herausstellung von Bewußtseinsvorgängen nach außen, die Projektion. eine einzige, und es verbot Erfahrung zu mittelbare Statt sich zweier Welten ergab sich damit jedes Hinausgehen Dingen. ^ jenseitigen Wendung des Problems, deren Streben über die un- Diese scharfsinnige nach Vereinfachung einen sichtlichen Eindruck auf die Zeit macht, nach ihrer technischen Seite zu prüfen, ^ liegt außerhalb Aufgabe; unserer daß sie 2. Aufl. Siehe Mach, Die Analyse der Empfindungen. gibt keine Kluft zwischen Psychischem und Physischem, keine Empfindung, der ein äußeres, von ihr Draußen, auf ihrem S. 206: «Es und verschiedenes Ding kein Drinnen Es gibt nur einerlei Elemente, aus welchen sich das vermeintliche Drinnen und Draußen zusammensetzt, die eben nur, je nach der temporären entspräche. Betrachtung, drinnen oder draußen sind. — Die sinnliche Welt gehört dem physischen und psychischen Gebiet zugleich an." S. 33: »Ich sehe keinen Gegensatz von Psychischem und Physischem, sondern einfache Identität bezug auf diese Elemente." Siehe auch Wlassak Nr. 18, S. 202): »Kein naiver Mensch findet einen findung in seinem Bewußtsein, (in in der »Zukunft" 1902, Baum irgendwie als Emp- sondern immer nur als Bestandteil seiner auch dann, wenn der Baum nicht gesehen, sondern nur erinnert wird; auch das blasse Gedankenbild steht in keinem anderen Umgebung. Dies gilt Verhältnis zum Beschauer als der gesehene Baum." Zum Grundbegriff des Geisteslebens. 26 nächsten Gebiet der Wahrnehmungs- physiologisch-psychologischen lehre scheinbar abgeschlossene Fragen wieder in Fluß bringt und den problematischen Charakter des landläufigen wissenschaftlichen Naturbildes aufdeckt, ist sicherlich ein Verdienst. Der Grundbehauptung aber beizutreten verbietet uns schon die Erwägung, in Wirklichkeit daß schon unser Erkennen daß namentlich aber jenseit Innenleben entwickelt, durch Wechsel allen erweist.^ daß unser Ich mehr ist als ein Strom von sinnlichen Empfindungen, ein selbständiges aller intellektuellen gegenüber das Verarbeiten enthält, Vorgänge sich ein Mannigfaltigkeit aller und Wandel hindurch eine und beharrende Art Für solche Selbständigkeit des Inneren spricht auch das Ganze der geschichtlichen Bewegung; denn durch alle Arbeit und Verwicklung hindurch hat sich der Mensch immer weiter von der ein immer mehr das äußere Begegnis in inneres Erlebnis verwandelt, immer mehr Gegenwirkung gegen die zuströmende Fülle geübt. bloßen Sinnlichkeit entfernt, Wenn Mach ^ Das alles ist kein bloß das Ich als etwas Unbeständiges intellektuelles und Unselbständiges behandelt, so liegt das zum guten Teil an einer Vermengung von Bewußtsein des Ich und lebendigem Ich selbst. So heißt es z. B. a. a. O. S. 3: »Die scheinbare Beständigkeit des Ich besteht vorzüglich nur in der Kontinuität, in der langsamen Änderung. Die vielen Gedanken und Pläne von gestern, welche heute fortgesetzt werden, an welche die Umgebung im Wachen fort- während erinnert (daher das Ich im Traume sehr verschwommen, verdoppelt sein oder ganz fehlen kann), die kleinen Gewohnheiten, die sich unbewußt und unwillkürlich längere Zeit erhalten, machen den Grundstock des Ich aus. Größere Verschiedenheiten im Ich verschiedener Menschen, als im Laufe der Jahre in einem Menschen eintreten, kann es kaym geben. Wenn ich mich heute meiner frühen Jugend erinnere, so müßte ich den Knaben (einzelne wenige Punkte abgerechnet) für einen anderen halten, wenn nicht die Kette S. 17: „Man wird dann auf das Ich, welches schon der Erinnerung vorläge." während des individuellen Lebens vielfach variiert, ja im Schlaf und bei Versunkenheit in eine Anschauung, in einen Gedanken, gerade in den glück- ganz fehlen kann, nicht mehr den hohen Aber besteht denn nicht mit lebendiger Kraft eine Einheit geistiger Art gegenüber allen Wandlungen und Verdunklungen des Bewußtseins, und wirkt nicht aus solcher Einheit geistiger Individualität alles vordringende wissenschaftliche und künstlerische Schaffen, entspringt aus ihr nicht alle durchgreifende Leistung auch auf praktischem und technischem Gebiete? lichsten Augenblicken, teilweise oder Wert legen." Diese Erfahrungen des geistigen Lebens bestätigen gegenüber jener Ver- flüchtigung des Ich vielmehr die Überzeugung Goethes: „Und keine Zeit und keine Macht zerstückelt Geprägte Form, die lebend sich entwickelt." — Objektiv. Subjektiv Phänomen, kein bloßer Erklärungsversuch, reicher Tatsächlichkeit, der nächsten und 27 sondern eine Eröffnung sichersten, die wir kennen, die allein uns die sinnlichen Eindrücke denken und überdenken lehrt. So wenig erklären sich eine solche Tatsächlichkeit für eine bloße Illusion und das Rad der Weltgeschichte zurückdrehen läßt, so gewiß behauptet jene Scheidung zwischen Subjekt und zwischen Objekt, Innenwelt und Natur eine unabweisbare Notwendigkeit. c) Die positive Behauptung. a. In Einführung. welcher Richtung sollen wir nun weiter suchen? Ist die Scheidung nicht zurücknehmbar und führt kein Weg von der einen Seite zur anderen, so bleibt keine andere Möglichkeit, als den Gegensatz in den Lebensprozeß selbst aufzunehmen, diesen von innen her so zu erweitern, daß er sich nicht erst nachträglich auf eine neben ihm befindliche Welt bezieht, sondern daß er selbst eine Welt enthält; innerhalb des Menschen selbst muß Ganze das Welt zur einer Wirkung kommen, die dem Gegensatz überlegen ist, und diese Welt muß nicht durch die Besonderheit des Punktes hindurch, unmittelbar uns zugänglich sein; sondern dann und nur dann kann es für den Menschen irgendwelche Wahrheit geben. Diese in Wendung mag beim Wahrheit die fehlt es ihr nicht Anblick auffallend dünken, ersten an geschichtlichen Anknüpfungen, um nur einer Zusammenfassung bedürfen, in dem scheinbar Neuen Altes entdecken zu lassen. Wie kam denn die Menschheit dazu, die Begriffe des Wahren und des Guten zu bilden und sie von der bloßen Tatsächlichkeit und Nützlichkeit abzuheben, wie konnte sie überhaupt über die Meinungen und Neigungen des bloßen Menschen irgend hinausstreben? Ein merkwürdiges Phänomen ist Denn mag noch hier nicht zu sein, was als wahr urtd als gut zu gelten habe, vor aller Ungewißheit verkennen. so der Antwort steht die Tatsache der Frage, und diese etwas Großes Denn und Folgenreiches. brechung des bloßpunktuellen Seins, des Wesens, die in sie sie Streit viel ist enthält darüber unmittelbar eine Durch- bezeugt eine innere Weite dem scheinbar Fremden etwas Eigenes sieht und sucht Sicherlich nämlich kann sich der Mensch kümmern und bekümmern, was nicht um nichts ernstlich irgendwie zu seinem Leben Zum Grundbegriff des Geisteslebens. 28 und Wesen Beziehung in vöUiges Jenseits kann ihn schHeßlich zu ihm gehört; steht, ja nicht im mindesten erregen. beim Wahren und Guten das Streben auf eine Welt, des nächsten Lebenskreises nicht die einem an auch aus sich Bewegung viel muß Kreise teilhaben, Welt umspannen, wenn uns üben, so die jenseit müssen wir dann nicht von Haus liegt; weiteren ein Nun geht unser Leben Anziehung auf ihr Inhalt so viel uns erzeugen soll? in Freilich muß dann der Begriff unseres Selbst verändern, aber die Begriffe haben den Tatsachen und nicht die Tatsachen den Begriffen zu dienen, warum sollten wir uns also gegen eine solche Veränderung sträuben? Eine schwere Frage und Aufgabe bleibt es danken einer Weltnatur des Menschen eine jenem Ge- freilich, präzisere Fassung zu geben, aber auch dafür haben die letzten Jahrhunderte auf der Höhe Leistungen ihrer Hauptstück der deutlich genug einen Weg gezeigt. Es ist ein Größe Kants, von der bloß psychologischen Er- klärung die Erforschung der Möglichkeit geistiger Inhalte abzuheben, z. von der Frage, wie der einzelne Mensch zum Erkennen, zur B. Moral u. s. w. kommt, die zu unterscheiden, welche inneren Be- dingungen das Bestehen von Wissenschaft und Moral sowohl die logische als die ethische über der psychologischen. hat. So wird Betrachtung selbständig gegen- Das mag zunächst bloß als eine neue hinfällig ohne ein Methode erscheinen, aber diese Methode wäre Leben neues Leben, ein Daseins, Leben aus dem Ganzen, charakter. ein jenseit der Einzelvorgänge des seelischen ein Leben mit einem Welt- Was sich aber von- solchem Leben nach einzelnen Rich- tungen entwickelt, das hat keinen festen Grund und Halt, wenn es sich nicht zu einem Ganzen zusammenfaßt und als Erweisung einer neuen Stufe anerkannt wird, die nicht unter, Gegensatz von Subjekt und Objekt sondern über dem steht. In anderer Art und doch zu verwandtem Ziele wirkt die moderne Kunst auf der Höhe ihres Schaffens. Die Objektivität eines Goethe wird gefeiert, er selbst begrüßte es mit freudigem Dank, sein Denken ein gegenständliches nannte. lichkeit bedeutet aber keineswegs eine Aufsaugung des Subjekts durch als Heinroth Eine solche Gegenständ- Unterdrückung und möglichste das Objekt, ein bloßes Wieder- geben des äußeren Eindrucks der Dinge, sondern eine Begegnung und gegenseitige Durchdringung von Objektivem und Subjektivem auf dem gemeinsamen Boden des Innenlebens; die Dinge empfangen dabei selbst eine Seele und vermögen ihre eigene Art getreulich mit- Subjektiv das zuteilen; menschliche Leben zu einem Inhalt geführt. Leere — Objektiv. 29 aber wird von der anfänglichen Hier wird nicht eine subjektive Stimmung den Dingen aufgedrängt, sondern ihnen ihr eigenes Leben und Weben abgelauscht oder abgerungen; der Dichter „erscheint damit wie ein Zauberer, der die sonst stummen Wesen zum Sprechen bringt, dem öffnet, der ganze Unermeßlichkeit der Welt seelisch die sich alle ihrer eigenen Mannigfaltigkeit er- Natur zuführt und zugleich das Lebendige, Wesentliche, Wirksame aus den Dingen heraussieht" Lebensanschauungen der großen Denker, (s. 7. Aufl., 446). Goethe nennt das eine Synthese von Geist und Welt, „die von der ewigen Harmonie des Wahrheit diese Synthese erfolgt sondern Außenwelt, Daseins die seligste Versicherung gibt«; innerhalb nicht zwischen der Seele der So bestehen nicht bloß zwei, sondern drei Arten künstlerischen Schaffens: satz einer objektivistischen eine sich stellt und überlegene Art und der zu einer Innenwelt erweiterten zwischen Seiten und Polen ihres Lebens. Seele, in einer subjektivistischen entgegen, die dem GegenBehandlung wir eine souveräne Erst diese souveräne Behandlung dringt sowohl über die nannten. ^ seelenlose Gegenständlichkeit, als über die formlose Zuständlichkeit zu einer Eigenständlichkeit vor, bei welcher der Lebensprozeß eine Welt nicht nachträglich sucht, sondern damit gewinnt er einen erst sie aus sich selbst Inhalt, nicht als entfaltet; Abbildung eines vor- handenen Daseins, sondern durch schöpferische Synthese einer neuen Welt. hat, Sollle, was so in der Kunst eine unbestreitbare Wirklichkeit nicht auch für das Ganze des Geisteslebens gelten, könnte über- haupt die Kunst sich darum bemühen, wenn nicht hinter ihr ein Ganzes So sollten wir getrost den hier gewiesenen und ihn mutig zu Ende gehen, mag er uns noch des Geisteslebens stünde? Weg verfolgen so weit von daran ist dem üblichen Welt- und Lebensbilde entfernen. nicht zu zweifeln, Denn daß nur im Widerspruch damit sich eine Welt von innen her aufbauen und das Leben und Wirken eigentümlich gestalten des begriff läßt. Erwägen wir also, wie Geisteslebens, 2. das Verhältnis der Grund- sich 1. des Menschen zur und zugleich der Anblick des geschichtlichen Lebens, 3. das Problem der Wahrheit ausnimmt, wenn wir jenen Weg be- Geistigkeit treten und damit die Voraussetzungen dessen entwickeln, Ergebnis jeder irgendwie festhalten muß. ^ S. „Wahrheitsgehalt der Religion". 2. Aufl., S. 95. was im Zum Grundbegriff des Geisteslebens. 30 ß. Der Grundbegriff des Geisteslebens. Ein Leben geistiger Art es als das, gilt was den Menschen aus- So muß und über die Stufe des bloßen Tieres hinaushebt zeichnet wohl mehr sein als das natürliche, uns mit den Tieren gemein- same Seelenleben; Wahrheit zeigt schon eine flüchtige Betrachtung in Das Seelenleben der niederen mehr als eine Begleiterscheinung und eine Hülfe des Naturprozesses; alle Entwicklung von Intelligenz und Geschicklichkeit bleibt hier ein bloßes Werkzeug zur Erhaltung des Individuums oder der Gattung; als solches Werkzeug gelangt es nicht zu einem inneren Zusammenhange, einem sicheren Beisichselbstsein, einem alsbald einen wesentlichen Unterschied. Stufe nicht ist eigentümlichen Dies Inhalt. aber es, ist was die Wendung zur Hier erscheint ein neuer Lebens- geistigen Stufe des Lebens bringt. prozeß: das Innere, bisher eine bescheidene Zutat, ein Anhang einer fremden Welt, will auf jetzt sich und selber stehen eigne eine Zu einem Ganzen verbunden wäre demnach Wirklichkeit bilden. geistiges Leben selbständig gewordene und zu einem Inhalt gelangte Innerlichkeit; die sonst in eine unermeßliche Vielheit zerstreute in lauter Abhängigkeitsverhältnisse verstrickte Wirklichkeit und erreicht Zusammenhang und ein Leben, das allererst ein hier einen inneren Selbstleben heißen kann. Wird Aber aus solcher Behauptung erwächst sofort eine Frage. mit jenem Selbstleben ein Sonderreich lichkeit der Dinge liegt, zufrieden erstrebt, und das neben der Wirk- oder sicher bei sich selbst, verbleibt auch in jener Wendung ein Zusammenhang mit der großen Nur dies letztere entspricht dem Befunde des Lebens. Welt? im Streben zu sich selbst bleibt großen Welt befaßt, es sich zu ziehen, es kann nicht ruhen und rasten, und überwunden in Denn das Geistesleben zugleich mit der kann sich selbst nicht finden, ohne diese an sich bis es sie vollauf Darum aufgenommen hat ist all sein Gehalt zugleich eine Behauptung, die Behauptung, das Letzte, Ganze, Allumfassende, der Kern der gesamten Wirklichkeit zu sein. aber kann es Drngen durch nur sein, wenn die Weiterbildung, die es an Dies den die Aneignung bewirkt, diese zur Höhe ihres eigenen Wfesens führt, wenn der Gehalt des Geisteslebens die eigene Wahrheit der Dinge bedeutet unerträglicher Das Geistesleben wird Widerspruch, wenn es neben in sich selbst ein und gegenüber der Subjektiv nicht innerhalb ihrer steht, Welt, ihm — Objektiv. wenn 31 Wendung zu nicht in der sich die Wirklichkeit selbst vollendet. Die Anerkennung dessen verwandelt sie in ein in Fluß und Reich von aufsteigender Bewegung. Die An- unsere Welt versetzt fangsstufe bildet die Natur, aus ihr quillt das natürliche Seelenleben Aber auf. Leben dies durchgängigen einen bildet indem es eine gewisse Innerlichkeit entwickelt und die völlige Bindung an Selbstlebens wieder aufhebt sie zugleich Diesen Widerspruch und Stärke des Lebensaffektes gehalt- durch die Versagung alles durch Äußeres, ein Widerspruch, das stellt aller in sinnlose Getriebe der Tierwelt jedem denkenden Beobachter eindringlich, ja erschütternd vor Augen. Erst im Geistesleben beginnt eine Lösung des Widerspruches, indem sich nun das Leben gegen sich selber mehr bloß nach außen hin, sondern auch nicht kehrt. Als eine solche Stufe des Alls kann das Geistesleben nicht eine und bloße Eigenschaft einzelner Punkte sein erst nachträglich aus einzelnen Betätigungen zu einem Ganzen zusammenschießen, es muß vielmehr von Haus aus ein Ganzes, ein selbständiges und sich selbst angehöriges Leben sein. Mannigfaltigkeit aller so Zu einem solchen Ganzen gehört eine wie auch dem Gegensatz von Subjekt und Objekt überlegene Einheit. Dies Ganze entwickelt sich mittels des Gegensatzes von Subjekt und Objekt, aber es bleibt ihm überlegen und von Kraft und Gegenstand, hält beide Seiten auch in der Scheidung zusammen, auf geistigem Boden kann jede einzelne sich nur zusammen mit der anderen entfalten und ihre eigene Höhe So sind hier nicht sowohl die beiden Seiten einander ent- finden. gegengesetzt, als vielmehr der Stand ihrer Einigung, der Stand der Volltätigkeit dem der Spaltung, dem des halbseitigen und zugleich Das bloße Subjekt ist vom Geistesleben aus ange- leeren Lebens. sehen ebenso etwas äußerliches wie das Objekt; nicht die Beziehung der einen auf Seite die andere, sondern nur die schöpferische Synthese erzeugt eine Innerlichkeit und zugleich eine selbst befindliche Wirklichkeit; eine solche volle, bei sich kann nie von draußen dargeboten werden. Es hat aber solche Überwindung des Gegensatzes von Subjekt Bedingung und Voraussetzung: die Betrachtung darf ihren Ausgangspunkt nicht in einem gegebenen Geschieht Sein, sie muß ihn im Lebensprozeß selber nehmen. und Objekt nämlich eine jenes, so unerläßliche wird entweder die Welt oder das Subjekt als Zum Grundbegriff des Geisteslebens. 32 etwas für sich bestehendes und dann aber läßt sich bei sich abgeschlossenes festgelegt; vom einen zum anderen nie gelangen, immer unter der Macht des Gegensatzes. verbleiben wir dann Im Lebens- prozeß dagegen kann von vornherein das eine auf das andere angewiesen und sein, die Beschaffenheit jeder Seite sich nach dem bemessen, was im Ganzen geschieht und erreicht wird; dann verschwindet die Starrheit des Gegensatzes, dann kann der Scheidung ein Zusammenhang überlegen bleiben. Endlich diene zur Aufhellung und Abgrenzung dieser Fassung des Geisteslebens noch eine Bemerkung historischer Art. klärung dem Mechanismus neben kannte Die Auf- der Natur keine andere Wirklichkeit als das Nebeneinander einzelner Seelen, sie sprach nicht von einer nur von sondern Geisteswelt, Kant brachte die einer Geisterwelt. Erst Wendung in Fluß, welche die geistige Arbeit des 19. Jahrhunderts beherrscht, Abhebung eines GeistesDenn bei ihm erscheint jenseit die deutliche lebens vom bloßseelischen Getriebe. des Unterschiedes der Individuen eine gemeinsame geistige Struktur, ein Grundgewebe, das Aber staltet. als sich die alle Sache geistige Betätigung beherrscht blieb weder das Neue fest bei ihm zugleich behandelten sie unbedenklich das menschliche Geistesleben als absolutes Geistesleben zeuger Wirklichkeit. aller besonderes Tun, Die speku- erhoben das Geistesleben zur vollen Selbständig- lativen Nachfolger aber ge- genug bei sich selbst zusammenschloß, noch eine deutliche Abgrenzung des Geistigen erfolgte. keit, und insofern noch unvollendet, und machten jenes zum Er- Das konnten nicht sie und mehr und mehr ward dies wohl ohne ein das Denken, für das Ganze des Geisteslebens einzusetzen; daraus entstand nicht nur ein viel zu knappes, auch zu anthropomorphes Bild der Welt, es drohte sich zugleich alle Wirklichkeit in einen Dementgegen verflüchtigen. das menschliche Dasein sei hinausgehoben: das Geistesleben, sondern er gewinnt mit an einer höheren Stufe das Geistesleben nicht rastlosen teil der Mensch erzeugt nicht am Geistesleben und da- der Wirklichkeit. als eine Prozeß zu entschieden das Geistesleben über Zugleich erscheint besondere Betätigung, nicht als eine besondere Seite des Lebens, sondern als bei sich selbst befindliches, wirklichkeitbildendes Leben, das unser menschliches Tun keineswegs ganz erfüllt, sondern als ein hohes Ziel zu ihm wirkt. — Objektiv. Subjektiv y. 33 Das Verhältnis des Menschen zum Geistesleben. Dieses Selbständigwerden des Geisteslebens mit seiner Abhebung vom bloßen Menschen verwandelt dessen Verhältnis zum Geistes- leben aus einer scheinbar selbstverständlichen Tatsache in ein schweres Problem. Wie kann der Mensch, verschwindender Punkt, befindlichen Welt teilhaben, Er kann es leben darstellt? für die nächste Betrachtung ein am Ganzen einer Welt, einer bei sich selbst welche sich nunmehr das Geistes- als wenn von vornherein Wesen angelegt, er ihm Das Geistesleben darf ihm sicherlich nur, das Geistesleben als Möglichkeit in seinem irgendwie verbunden unmittelbar ist. nicht durch die Vermittlung seiner besonderen Natur zugeführt dabei all sich selbst entfremdet werden, es muß ihm seiner Unendlichkeit irgend gegenwärtig sein, es als und Ganzes mit muß ihn damit von innen her, wenn auch zunächst nur der Möglichkeit nach, zu Ohne ein solches Inne- einem Weltleben und Weltwesen erweitern. wohnen der Geistigkeit gibt es für den des Weiterkommens; würde er in Menschen keine Hoffnung dem Geistesleben nicht sein echtes Selbst ergreifen, so könnte es nie eine Macht für ihn werden; böte und hielte es nicht mit richtender Kraft allem menschlichen Unternehmen Ziele und Maße vor, so wären wir dem Wechsel und Wandel der Erscheinungen wehrlos preisgegeben, so entfiele für uns alle Möglichkeit einer Wahrheit jenes nicht einen unwandelbaren Pol Nur das Geistesleben, nicht der bloße Mensch, kann eine absolute Festigkeit gewähren. Solches Teilhaben des Menschen Gesamtanblick seines Wesens. am Geistesleben verändert den Jenes ist nur möglich jenseit des unmittelbaren Daseins, so gewinnt sein Leben einen tieferen geistigen Grund; zugleich Betrachtung, scheidet sich von der empirisch-psychologischen welche mit den unmittelbaren Vorgängen des Seelen- lebens zu tun hat, eine noologische, welche sich jene geistige Grundlage mit ihrer Selbsttätigkeit Bei solchem zum Vorwurf macht. zwiefachen Anblick erscheint der Mensch in sich Das Geistesleben ist und eine Aufgabe, unerschütterliche Ruhe und nie befriedigtes Streben, innerster Kern und fernes Ziem- selbst bei als ein Gegensatz und ein Problem. ihm zugleich eine Tatsache er selbst aber erscheint zugleich klein im Abstände, sein Leben als groß wird ein der Verbindung, als unablässiges Suchen des in eigenen Wesens und wird damit erst einer wahrhaftigen Geschichte 3 Eucken, Grundbegriffe. 4. Aufl. Zum Grundbegriff des Geisteslebens. 34 Denn wie fähig. i<önnte wo entstehen, das Streben von außen abhängig wäre, nicht von innen her durch lediglich und Ziel gelenkt festes solche eine gerichtet ein würde? Auf dem Boden der menschlichen Geschichte überwindet das Geistesleben langsam die anfängliche Zerstreutheit und Ohnmacht; es geschieht das, indem unter besonders günstigen Umständen eine Kernbildung innerhalb des Lebensprozesses indem Tätigkeits- komplexe indem suchen, Art geistiger erfolgt, zusammenschießen und schließlich ein durchzusetzen sich Lebenssystem, charakteristisches Aufbau einer geistigen Wirklichkeit, unternommen wird. licheres Beispiel gibt es davon als das griechische Schaffen, eine wie es Wie eine solche mit ausgeprägter Art Leben und Welt umspannt. Synthese der Kein deut- Wahrheit des von ihr dargebotenen ausschließliche Lebensinhaltes behauptet, so zerlegt sie das Dasein in ein Für und ein kann nichts dulden, was ihr fremd oder feindlich entgegen- Wider, sie steht. So erwächst Bewegung und Kampf, diese erschließen Erfahr- ungen und treiben das Leben dadurch weiter; so mögen sich neue mag Konzentrationen vorbereiten, die ähnliche Schicksale haben, so durch Werden und Vergehen der einzelnen Phasen der Wahrheitsgehalt des Ganzen wachsen. von einem wird, während begründenden sich Das freilich nur, wenn alle Bewegung und richtenden Geistesleben umspannt ohne das gegenüber den starren Widerständen und den schweren Hemmungen der menschlichen Lage nun und nimmer irgendwelche Wahrheit durchsetzen könnte. der geschichtliche Prozeß als eine fortschreitende nicht subjektiver, sondern substantieller Art; es Damit erscheint Verinnerlichung muß sich damit eine immer größere Entfernung von der unmittelbaren Lage vollziehen, die unter dem Gegensatz steht und daher einer vollen Innerlichkeit wie einer echten Wirklichkeit entbehrt. Innerhalb dieser Bewegung findet sich auch ein Platz für den Gegensatz, den die Ausdrücke „subjektiv" und „objektiv" recht unzulänglich bezeichnen. Das Geistesleben ist Selbstleben und Welt- leben zugleich, ein Selbst entfaltet sich hier zur Welt und die Welt gewinnt ein Selbst, das eine gehört sammengehörigkeit verhindert wegung das nicht, zum anderen. Aber solche Zudaß in der geschichtlichen Be- Leben bald mehr zur Konzentration, Expansion geht; tiefung in sich dort das Streben selbst, hier bald mehr zur nach Verinnerlichung und Ver- das nach Weite und Sachlichkeit, dort die Gefahr eines Einströmens bloß menschlicher Elemente, hier die Subjektiv — Objektiv. 35 des Vordringens einer seelenlosen Welt; vielleicht besteht ein Rhyth- mus des Geschehens, der gibt. Aber durch Wandlungen hindurch alle bald bald jenem diesem, die Führung beharrt das Streben des Geisteslebens nach einer den Gegensätzen überlegenen Einheit. Auch bleibt von jenem subjektiven oder objektiven Zuge innerhalb des Geisteslebens grundverschieden ein Subjektivismus oder Objektivismus gegenüber dem Geistesleben, Subjektivismus, ein der aus den Zuständen des bloßen Subjekts eine Welt hervorspinnen möchte, ein Objektivismus, der eine Wahrheit glaubt von den bloßen Dingen Das eine her durch Austreibung des Geistes erreichen zu können. muß rasch ins Leere sinken, wenn sie nicht versteckt aus eben dem überlegenen Geistesleben schöpfen, dessen Anwie das andere erkennung sie ablehnen. Ergebnisse für den Wahrheitsbegriff. S. Was an Wandlungen ersichtlich wurde, das muß sich auch auf ' den Begriff der Wahrheit erstrecken und ihn eigentümlich gestalten. Wahrheit bedeutet jetzt nicht mehr die Übereinstimmung mit einem draußen befindlichen Gegenstande, sondern ein Aufsteigen zu einem Leben, das aller menschlichen Willkür überlegen Gegensatz von Subjekt und Objekt durch die umspannt. um eine Verwandlung des Daseins in Selbst- Es handelt sich hier tätigkeit, und das den ist, tätiges Schaffen mit ihrem umbildenden Wirken von aller bloßen Be- tätigung innerhalb eines gegebenen Daseins wesentlich verschieden ist. Dies Wahrheitsstreben hat nichts mit einem ruhenden Sein zu tun, das unabhängig von allem Leben bestünde, vielmehr und sie ist Aber das Leben, das hier in Frage Wahrheit innerhalb des Lebens, das Leben. Sache des bloßen Menschen, sondern Wirklichkeit Inhalten zu nur erreichbar durch steht, ist nicht eine ihm gelangt das Ganze der in und zugleich einem Beisichselbstsein allererst und Werten, die Wahrheit aber ist nicht ein bloßes zur Erhöhung dieses Lebens, sondern Wahrheit Alle intellektuelle einer gesamtgeistigen Wahrheitserkenntnis der liegt die auf einer aller gehört zu seinem Wesen. ruht schließlich auf wesentliche Fortschritt der prinzipieller Wahrheit, Gewinn der Wahrheit sie Art Weiterbildung nicht Sache zu Mittel eines des Lebens. So ist einzigen Augenblicks, sondern nur durch den Lauf der weltgeschichtlichen Arbeit mit ihren Versuchen, Erfahrungen, Wandlungen dringt der Mensch allmählich 3* 36 Zum Grundbegriff des Geisteslebens. zu ihr vor, es gibt kaum etwas so törichtes als der Anspruch philosophischer Systeme, in einem gegebenen Zeitpunkt die ganze der Wahrheit erschöpfen und alle wir so im Suchen verbleiben und Rätsel lösen zu wollen. Fülle Daß zugleich unvermeidlich im Irren, das kann uns in keiner Weise erschrecken, wenn wir der Über- zeugung sind, daß alles menschliche Streben eine Welt des Geisteslebens hinter sich hat, die sich nur durch Freiheit aneignen die aber nicht auf unsere Willkür gestellt ist. läßt, Theoretisch - praktisch. (Intellektualismus a) I — Voluntarismus.) Geschichtliches. |ie Frage des Intellektualismus und Voluntarismus steht mit der *-^ eben behandelten in engstef Verbindung, es wird bei ihr nur direkter ins Seelische gewandt, was uns dort als Weltanblick beschäftigte. Auch hier ein Gegensatz von Lebenstypen, auch hier eine Bewegung der Jahrtausende. Nur der Unterschied, daß sich hier unsere Zeit ihres eigenen Zieles weit sicherer fühlt. Denn entschieden überwiegt hier das Streben, den Schwerpunkt des Lebens in das Wollen zu verlegen als in dasjenige, was allein ihm Wärme, Kraft und Festigkeit Wie mag es kommen, daß eine alte Streitfrage uns geben könne. plötzlich Sehen so einig findet? wir, ob die Geschichte zur Auf- klärung dessen einiges beitragen kann. Die Ausdrücke Intellektualismus und Voluntarismus sind mo- dernster Prägung; jenes taucht in den philosophischen Kämpfen zu Beginn des in 19. Jahrhunderts auf, es findet sich z. B. Schellings Bruno (s. Werke IV, 309), als Gegensatz zu Materialismus; Voluntarismus ist die erst in den Ausdrücke griechischen letzten Jahrzehnten aufgekommen.'^ theoretisch Philosophie — praktisch zurück; der Dagegen führen der in die Blütezeit Gegensatz der theoretischen bis und der praktischen Vernunft erscheint zuerst bei Aristoteles (voG? und 7rpaxTi>cd?). Jene hat die Aufgabe, die große Welt ^ewpTjTixd; ^ Gebildet ist das Wort von Tönnies, der darüber in der Wiener „Zeit" vom 23. März 1901 folgendes berichtet: „Diese Termini (d. h. Voluntarismus und voluntaristisch) sind zuerst vom Verfasser dieses Memoire gebraucht worden in seiner Abhandlung »Zur Entwicklungsgeschichte Spinozas", Viertel- Von Paulsen, der sie bald angenommen und durch seine Autorität in Umlauf Der Begriff der „voluntaristischen" Psychologie ist mehr und mehr jahrsschrift für wissenschaftliche Philosophie 1883. adoptierte, hat Wundt sie gebracht. zu allgemeiner Geltung gelangt". Zum Grundbegriff des Geisteslebens. 38 mit ihren ewigen Ordnungen zu erkennen, die praktische Vernunft dagegen befaßt und Wandel mit den menschlichen Dingen, die dem Wechsel sich unterliegen. Sie nicht bloß Einzelerkenntnis (An- ist wendung der allgemeinen Sätze auf den einzelnen Fall), sondern sie hat auch eigentümliche Prinzipien, aber ihre Gesamtleistung wird der theoretischen Vernunft entschieden untergeordnet. So auch in der Gedankenwelt und dem Sprachgebrauch der Scholastik; wenn Thomas von Aquino von cognitio practica redet, so versteht er darunter einfach eine Erkenntnis, die sich auf das Handeln bezieht. praktischer Philosophie Für die Neuzeit von theoretischer und hat namentlich Ch. Wolff die Unterscheidung Umlauf gebracht und dabei jener den in unbedingten Vorrang gegeben. ^ Wie in der Einteilung der Philosophie so folgt ihm auch im Sprachgebrauch Kant, zieht er die bekannte Umwälzung dahin, daß »was durch Fi-eiheit möglich als das, der Sache aber voll- in nunmehr das Praktische das Übergewicht gewinnt ist", und zugleich einen selbständigen Gedankenkreis hervorbringt. «Die ihm in das Gebiet des Theoretischen praktische Vernunft greift bei zurück, indem sie Postulate erzeugt, also theoretische Voraussetzungen, welche der Kritik der reinen Vernunft zweifelhaft waren" (s. TrendelenLogische Untersuchungen, burg, 3. Aufl. 457). II, Da nach ihm nur auf diesem Gebiet die Vernunft eine volle Selbsttätigkeit erreicht, so ergeben sich von hier die tiefsten Einblicke in die Wirklichkeit, und eröffnet es sich absolute Wahrheit.^ nirgend anders als hier dem Menschen eine Von Kant ist nur ein Schritt zu Fichtes Lehre: praktische Vernunft »die einmal das Praktische aber die ist als bloße Wurzel aller als Quell neuer Wahrheiten übergeordnet. Es spiegelt sich aber So z. B. Logica § 92 ' versam ex Metaphysica : der Geschichte in Palam principia Vernunft". So wird Anwendung untergeordnet, sodann igitur est, petere der Ausdrücke ein philosophiam practicam uni- debere. § 93: Metaphysica philo- sophiam practicam praecedere debet. Die Art, wie Kant aus der praktischen Vernunft Überzeugungen ab- " leitet, z. B. nicht ohne Bedenken und fand manchen Widerspruch. So sagt Harms (Gesch. der Philosophie seit Kant S. 247): »Kant nennt die ist Ideen Postulate der praktischen Vernunft, sie sind aber gar keine Postulate der praktischen, sondern sie sind Postulate der theoretischen Vernunft in der Erkenntnis der praktischen, der handelnden Vernunft im sittlichen Leben des Das Wort praktische Vernunft ist bei Kant selbst zweideutig; denn wird darunter verstanden die handelnde Vernunft, dann aber auch die Erkenntnis von der praktischen Vernunft." Geistes. einerseits — praktisch. Theoretisch 39 fundamentaler Gegensatz, die Frage, ob der Welterkenntnis oder dem — denn vornehmlich wird unter Führung unseres Lebens und Vernunft verstanden — moralischen Handeln, dies , unserer die Überzeugungen gebühre. Die Antwort darauf entscheidet zugleich über unsere Stellung zur Wirklichkeit und damit über das Es entstehen zwei widerstreitende Lebens- Bild der Wirklichkeit selbst. typen, der eine vornehmlich auf Weite Wärme und Kraft bedacht, und Klarheit, der andere auf der eine mehr um Gesetzlichkeit, der um Freiheit bemüht. andere mehr Die griechischen Denker Intellekt, praktischer die Leitung erteilen einmütig den Vorzug dem oder maßvollere es unterscheidet sie lediglich die schroffere Ausführung des Grundgedankens. Diese Schätzung des war der naturgemäße Ausdruck der hier Intellekts vorhandenen Überzeugung, daß der Mensch einer unwandelbaren Weltordnung angehöre, die ihn mit sicherem Dasein und herrlicher Pracht umfängt.. Nichts Größeres gab es hier zu erstreben Anschauung eines solchen Kosmos als die mit .ihrer Befreiung von aller Kleinheit des Alltages und allem Wirrwarr So verficht Aristoteles, dieser reinste Ausdruck der griechischen Kultur, die unbedingte Überlegenheit des der menschlichen Verhältnisse. über Lebens forschenden veränderlichen Dingen das befasse handelnde, das den Menschen mit und ihn von seiner Umgebung ab- Nur soweit die Forschung reicht, soweit reicht nach Glückseligkeit. Auch die Wendung zur Moral, welche hängig mache. ihm echte die Stoa brachte, vom Denken, eine bedeutet nicht sowohl eine Ablösung des Lebens Aufnehmen tätiger Energie Steigerung des Denkens zur Denkhandlung. ein als in das Denken, Und das letzte Aufleuchten des griechischen Geistes in Plotin bringt eine Erhebung des Denkens zu voller Souveränität und weltschaffender Größe; im Untergange selbst Geisteskraft, der steigert seine das Griechentum das Bekenntnis zu der Kulturarbeit eine unermeßliche Weite und eine wunderbare Klarheit verdankt. Das Christentum mußte nach Schätzung brechen. in Denn wo das Verhältnis des Erscheinen sich seiner innersten Natur mit jener das Hauptproblem des Lebens Menschen zu Gott verlegt, und wo mit dem neuer Tiefen auch schwere Verwicklungen, ja dunkle Abgründe im Menschenwesen zur Empfindung kommen, wo es demnach einen Aufstieg und eine Erneuerung gilt, da muß sich das Streben des Menschen von der Erkenntnis der Welt auf den Stand seiner Seele und weiterhin auf den Aufbau eines neuen Zusammen- Zum Grundbegriff des Geisteslebens. 40 hanges der Menschheit richten. Der Intellektualismus wird damit in Aber diese innere Wandlung ist in der Ge- der Wurzel gebrochen. staltung und den allgemeinen Verhältnissen wenig zum Ausdruck gekommen was die Herzen erfüllte, fand nicht die Kraft zur Schöpfung ; einer entsprechenden Gedankenwelt Lediglich bei Augustin erscheinen bemerkenswerte Ansätze dazu, wie er denn alle Wirklichkeit auf Wollen (nihil aliud quam voluntates) und in seiner Psychologie dem Willen als der die Seele einigenden Kraft die leitende Stellung zurückführt Aber ein durchgebildetes Lebenssystem und eine entsprechende gibt. Gedankenwelt hat auch Augustin daraus nicht entwickelt; so ist die Gestaltung des Christentums unter einem starken Einfluß derselben Denkweise verblieben, die es überwinden wollte, so leidet das Christen- tum bis zum heutigen Tage an einem Zwiespalt innerster Gesinnung und greifbarer Ordnung. Der griechische Intellektualismus beherrscht auch das christliche Dogma; mögen an die Stelle der Lehren von der Welt Lehren von Gott getreten sein, es daß die bleibt dabei, Erkenntnis über die Wahrheit und den Wert des Lebens richtige Auf der Höhe der Scholastik wächst noch der Einfluß entscheidet. des griechischen Intellektualismus, ein logisches Räsonnement dringt bis die in fehlt nicht Scotus,2 Tiefen der christlichen letzten Gedankenwelt vor.^ dem Nominalismus, der praktisch gerichteten Mystik; Reformation bringt diesen Zug zum siegreichen Durchbruch. bemüht des Es an Gegenwirkungen zu gunsten des Willens, so bei Duns mit höchstem sich griechischen platonischer Eifer, Intellektualismus Prägung zu befreien, die Luther das Christentum von der Macht sowohl der, aristotelischer als neu- wie er meint, seinen echten Tatbestand verdunkelt oder verflüchtigt hat; Melanchthon aber nennt «das Herz mit seinen Affekten« «den wichtigsten teil Aber des Menschen". und den Haupt- bei aller Verstärkung des Willens hat auch der Protestantismus nicht die Kraft gefunden, den innersten Lebenstrieb in ein Lebenssystem zu verwandeln, auch er hat schließlich wieder der Macht des Intellektualismus gehuldigt. ^ Denn mochte die Nur äußerlich angesehen dient im Mittelalter die Philosophie als Magd der Theologie, innerlich hat damals weit mehr die Philosophie der Theologie ihren Stempel aufgedrückt, " Er sagt z. B. (s. est habitus speculativus, Stöckl, Philos. d. Mittelalt. credere est visio speculativa, sed practica. fruitioni. II, S. 788): Fides non nee credere est actus speculativus, nee visio sequens Nata est enim ista visio conformis Theoretisch für Spekulation die — praktisch. Dauer abgelehnt werden, 41 ein Wissen anderer Wissen von geschichtlichen Daten, aber immerhin ein Wissen Art, ein erschien als unerläßlich zur Rettung der Seele; auch der Begriff des Glaubens erhielt eine Färbung, intellektualistische stark die neue Kirche aber wurde vornehmlich eine Gemeinschaft der Lehre, eine Schule des Es entstand eine neue Orthodoxie, an reinen Wortes. Selbstgerechtigkeit und Unduldsamkeit der griechischen voll gewachsen. Die Neuzeit beginnt mit voller und freudiger Ergreifung der Denkarbeit; das Denken ist es, von dem sie eine Befreiung vom Druck der geschichtlichen Überlieferung hofft, das Denken, das eine Klärung des unerträglich gewordenen Chaos verspricht, das Denken endlich, welches das Gewebe kleinmenschlicher Interessen zerreißt und die Unendlichkeit des Weltalls eröffnet. Art ja ist Gegenüber der griechischen das Denken aus ruhigem Anschauen weit mehr rastlose Arbeit, stürmisches Vordringen, aus Aneignung einer gegebenen Welt zum Aufbau einer neuen geworden. vom, Großen Eine solche beherrscht die Aufklärung bis ins Kleine, sie beherrscht nicht nur ihre spekulative Richtung mit der Kühnheit ihrer Weltentwürfe, sondern auch die empiristische mit ihrer Richtung auf das Menschenleben. hier wird von def klaren und deutlichen Erkenntnis Denn auch alles Heil er- eine andere Art des Erkennens, aber es ist ein Erkennen, wartet, es ist das den Kern der Arbeit trug freilich auch Wie bildet.^ die Aufklärung in jede große Bewegung, selbst einen sich so Rückschlag: und Überspannung des Erkennens erzeugte mit Notwendigkeit einen Zweifel an seinem Vermögen gegenüber der Welt die Steigerung und seiner Herrschaft über den Menschen.^ Aber ein bloßer Rück- schlag hat noch nie die Herrschaft über die Geister gewonnen, es bedurfte positiver Wandlungen, um die Menschheit in eine andere Bahn zu lenken. Solche Wandlungen erfolgten auf philosophischem Boden in Kant; sowohl sein Nein als sein Ja hatten bei diesem Problem eine unver^ S. z. B. Locke zu Beginn seines Essay: Our business here is not to know all things, but those which concem our conduct. ^ So z. B. bei Pascal, so mehr noch bei Bayle, dem bedeutendsten SkepEr sagt z. B. oeuv. div. 1727, III, 89 b: Ce ne sont tiker der Aufklärung. pas les opinions generales de l'esprit, qui nous determinent ä agir mais passions presentes du coeur. les Wie bei Bayle die Moral allein dem Leben eine Festigkeit verleiht, so sagt auch Friedrich II., sein getreuer Schüler: Les sciences doivent etre considerees cite comme des moyens qui nous donnent plus de capa- pour remplir nos devoirs (s. Zeller, Fr. d. Gr. als Philosoph S. 183). Zum Grundbegriff des Geisteslebens. 42 gleichlich größere je Wucht als eine andere wissenschaftliche Leistung und gründlicher als je wird das Vermögen des Schärfer zuvor. bloßen Erkennens geprüft und werden die Bedingungen seines Gelingens ermittelt; das Ergebnis diese Erschütterung wird ist eine schwere Erschütterung, aber mehr als ausgeglichen durch die Steigerung des moralischen Tuns zu einer moralischen Welt und die Anerkennung dieser Welt als des Kernes aller Wirklichkeit. Erst mit solcher Um- wälzung erhielt der Intellektualismus eine ebenbürtige Gegenwirkung, nun gelangte erst ein seit Jahrtausenden vorhandenes Streben zu Wir wissen, daß trotzdem der Intellektualismus im Panlogismus Hegels noch einmal sein wissenschaftlicher Klärung und Durchbildung. Haupt erhob, so kühn erhob wie nur je, aber wir wissen auch, daß nur ein rasches Hinwegeilen bald ein über Kant dies möglich machte, und daß um so stärkerer Rückschlag kam. zug der Zeit gegen den Intellektualismus. Einfluß Schopenhauers der Neigung praktische und seiner Willenslehre, wir gewahren der Religion und Theologie, die ihren es in Schwerpunkt in Aufgaben und Forderungen zu verlegen, wir gewahren es in dem Zuge des gesamten Lebens, das hinter Seitdem geht der Haupt- Wir gewahren das in dem praktisch -sozialen Grübeln über Weltprobleme Fragen zurückzustellen, die sich mit immer größerer Dringlichkeit erheben. Auf dem eigenen Gebiet der Wissenschaft aber wirkt zu gunsten der neuen Überzeugung namentlich die Psychologie, und Interessen eigne indem sie die beherrschende Macht der Triebe auch über das Vorstellungsleben aufdeckt, ja seine Bewegung als vom Willen geleitet aufweisen möchte. Solcher Schätzung des Willens entspricht die Neigung, dem Überwiegen des Verstandes alle möglichen Schäden der gegenwärtigen Die Grundlage unserer geistigen Existenz und Lage aufzubürden. die Hauptrichtung unseres Strebens sind unsicher geworden: das verschuldet, so heißt es, der Intellekt, der alles beweisen, d. h. auf Um- wegen ermitteln möchte und damit alles unmittelbare Leben mit seiner Gewißheit verscheucht; der Meinungen uns umfängt eine unsägliche Zersplitterung und Schätzungen: das kommt, so sagt man, vom Vorwalten der Verstandesarbeit, welche die Individuen auf ihre eigene Reflexion stellt und sie damit unvermeidlich auseinandertreibt; beklagt wird ein Mangel an Ehrfurcht vor göttlichen und heiligen Dingen: das liegt, so hören wir, an einer Überhebung des menschlichen Selbst- bewußtseins, und die Haupttriebkraft dabei ist der Intellekt mit seinem Kraftgefühl und seinem Wissensdünkel. Trägt demnach der Intellekt Theoretisch — praktisch. 43 die Hauptschuld aller Irrung, so läßt die Befreiung von seiner Herrschaft eine Gesundung des gesamten Lebens erwarten. Hat der heutige Voluntarismus die Kraft zu solcher Befreiung? Der Voluntarismus. b) Der Voluntarismus Erscheinung keine ist einfacher Art, die Hauptepochen der Geschichte haben ihn ihrer Hauptrichtung gemäß verschieden In der gestaltet, religiösen jede besonderen Voluntarismus. ihren hat Gedankenwelt ging die — daß Behauptung dahin, sowohl die Offenbarung Gottes als ihre Annahme durch den Menschen Sache eine des Willens sei, der nicht sich Darin lag eine Betonung der Selbständigkeit, weiter ableiten lasse. Ursprünglichkeit und reinen Tatsächlichkeit des religiösen Geschehens, die Abweisung aller Versuche, es aus weiteren Zusammenhängen verständlich zu machen. Zum Ausdruck den bringt Gegensatz bekannte Formel, die nach, Thomas von Aquino Gott das Gute gebietet, nach Duns Scotus das Gute gut Spezifische der gleichlichkeit gleich Religion, ihre ist, weil Gott es weil Unabhängigkeit es gut ihre ist, Das gebietet. und daß Unver- kam bei dieser Denkweise voll zur Geltung, aber zu- entstand die Gefahr einer Ablösung vom übrigen Leben, Zusammenhängen; wie es hier nicht 2u einer vollen geistigen Durchdringung und Aneignung des Wahrheitsgehaltes kam, so konnte leicht die Tatsächlichkeit in starre und eines Herausfallens aus allen äußerliche Positivität und daß, die Ursprünglichkeit Man möchte Willkür umschlagen. wer über die Vernunft hier hinausstrebt, und Freiheit in blinde an Plotins Wort denken, leicht in Gefahr kommt, aus ihr herauszufallen. Auf dem Gebiete der Philosophie stellt der Voluntarismus anders dar: hier gilt es eine Verlegung des sich Zentrums des Lebens dem Erkennen ins Wollen, namentlich in das moralische Wollen. Antrieb dazu gab vornehmlich ein Irrewerden am Vermögen des Erkennens; wie es unfähig dünkte, zum letzten Grunde der Dinge durchzudringen, so schien es auch dem Leben keinen sicheren aus Den Halt gewähren zu können. Sollte also nicht auf alle Wahrheit in vollem Sinne verzichtet werden, so war eine andere Quelle zu suchen, und eine solche schien nach Erschütterung des religiösen nichts anderes dem großen zu bieten Sinne, als des Menschen wie Kant dies versteht, sittliches Glaubens Handeln. In wird dies zur Offen- Zum Grundbegriff des Geisteslebens. 44 barung einer neuen Welt, welche die Tiefe letzte Diese Welt aber läßt sich ebenso wie das bildet. der Wirklichkeit Handeln sittliche selbst nicht jedem theoretisch demonstrieren oder als ein Vorhandenes sondern aufweisen, der das sie besitzt eine Überzeugungskraft nur für den, Grundgeschehen anerkennt und auf sittliche nimmt; sich so geht eine Tat dem Erkennen voran, und was aus ihr an leitenden Überzeugungen hervorgeht, das hat nicht den Charakter theoretischer Erkenntnisse, sondern den praktischer Postulate. Die gewaltige Er- regung und eingreifende Wandlung, die das bewirkt hat, stehen uns vor Augen. allen deutlich richtige Würdigung dieser Wendung Die schwer, und notwendige weil ist namentlich deshalb Wahrheiten fruchtbarste hier mit problematischen Fassungen aufs engste verquickt sind. Ein solche Wahrheit daß liegt vor allem letzten Überzeugungen Welt, sondern durch der in deutlichen Einsicht, unsere nicht durch den Befund der uns umgebenden was im Bereich des bestimmt werden, das Innenlebens vorgeht und an Wirklichkeit ersichtlich wird; das macht Versuchen ein Ende, durch Spekulation allen in ein inneres Wesen der Dinge einzudringen und von ihm her die Wirklichkeit zu verstehen. In andere, daß schlossen enger Verbindung mit dieser Wahrheit steht aber die der Bestand des allen geweckt und geschieht, vor Augen entfaltet sein dem Einzelnen Innenlebens nicht mühelos und ge- liegt, sondern daß dieses Leben erst will, und daß nach dem Maße, wie das auch das Bild Die Entzweiung der Menschen im Streit der Welt um sich gestaltet. die Wahrheit, sowie der persönliche Faktor im Wahrheitsstreben können hier ihre volle Würdigung finden. Aber sobald wir vom Allgemeinen des Gedankens zur n;iheren Ausführung fortschreiten, erwachen Zweifel über Zweifeln. etwas anderes, die Grundtatsachen des Innenlebens in den Es ist Mittel- punkt der Erkenntnisarbeit zu stellen und in ihnen die entscheidenden Daten zu finden, etwas anderes, jene direkt zu einer eignen Erkenntnisquelle zu machen. ist dieses. Ebenso notwendig wie jenes, ebenso unmöglich Denn der Befund des Innenlebens läßt sich nicht un- mittelbar, wie er vorliegt, als sicherer Ausgangspunkt ergreifen, sondern er ist erst durch die Erkenntnisarbeit zu klären und zu durchleuchten; was dabei aber in ihm als Grundtatsache und als Wahrheit befunden wird, das hat eine Allgemeingültigkeit und zugleich einen Zwang, das läßt sich inneren unmöglich auf eine persönliche Zustimmung Theoretisch 45 Es gibt mathematische Wahrheiten von solcher Schwierig- stellen. keit, — praktisch. daß nur ein kleiner Teil der Menschen sie klar zu durchschauen vermag, das ihrer Allgemeingültigkeit auch nur den mindesten tut Wenn daher auch die Lebenswahrheiten ihre volle Über- Abbruch? zeugungskraft erst bei Entwicklung des entsprechenden Lebens langen können, ja des Menschen in sich so werden sie dadurch in keiner Weise trägt, zu bloßen Möglichkeiten, sondern werfen könnte, er- wenn die Wendung zu ihnen eine Entscheidung der eine annehmen, der andere ver- die sie behalten und die volle Notwendigkeit Allgemeingültigkeit; die Subjektivität liegt nicht in der Sache, sondern nur im Verhältnis des Menschen zu besitzen, das mit innerlich einem ihr, nichts kann volle Wahrheit subjektiven Faktor behaftet ist. Von solchen Erwägungen aus müssen wir den Begriff der praktischen Vernunft als schief und irreleitend grundsätzlich ablehnen, es gibt nicht und eine praktische Vernunft nebeneinander, son- eine theoretische dern es gibt nur eine einzige Vernunft, die mit zu t)an hat; dem ganzen Leben den Begriff dieser Vernunft aber ist als wesentliches in Und es schwebt die Merkmal das der Selbsttätigkeit aufzunehmen. Vernunft nicht der Luft, sondern in frei sie ist die Vertreterin eines bei sich selbst befindlichen Lebens, eines Beisichselbstseins der Wirk- Ohne ein solches gibt es keinerlei Wahrheit. lichkeit Dazu behauptet der Begriff der praktischen Vernunft meist nicht die Höhe, die er Hier vollzieht er eine Kant einnahm. bei wälzung des ganzen Lebens, bewirkt er eine Versetzung Um- in ein ur- sprüngliches Schaffen, und gibt er dem Leben in besonderer Richtung wenn irgend wenn auch eine andere Metaphysik als die der ontologischen Spekulation. Je mehr sich aber dieser metaphysische Charakter verwischt, desto mehr wird das Reich der einen Weltcharakter etwas, so ist mit strenger Allgemeingültigkeit; dies Metaphysik, praktischen Vernunft aus der Tiefe der ganzen Wirklichkeit zu etwas besonderem neben anderem, desto weniger allgemeingültigen die daraus hervorgeht, wird läßt sich hier zu einer Die Lebensgestaltung aber, Wahrheit gelangen. leicht ein Gebiet des praktischen und moralischen Lebens gegen die Kulturarbeit isolieren, damit aber jenes und auf zu subjektiv und empfindungsmäßig, dieses die bloße Leistung gerichtet gestalten, den Gesamtstand des Lebens zu aber durch solche Spaltung stark herabdrücken. die Kulturarbeit und auseinanderfallen; je äußerlich In Wahrheit dürfen die letzten Überzeugungen des Menschen mehr es tun, sie desto nicht mehr muß unserem Zum Grundbegriff des Geisteslebens. 46 Leben durchwaltende eine Größe Seele und sich der Voluntarismus im Leben der zugleich alle ent- weichen. Wieder anders gestaltet Hier erscheint er als wissenschaftliche Theorie zunächst Gegenwart. dem auf psychologischem Gebiet in von Vorstellungslebens den auch gesamten den Streben, Trieben die Abhängigkeit des und Neigungen aufzuweisen, des Vorstellungslebens Verlauf als durch ein Willensmoment bedingt zu zeigen, wie das vornehmlich in ist neue und fruchtbare Erkenntnis Viel Apperzeptionslehre geschieht. Wundts auf diesem Wege gewonnen, ja der Gesamtanblick der Sache ver- Nur in der Richtung mögen tieft. entstehen, und Wille als zwischen zentraler der durch den ganzen liegt, hier noch Fragen und Zweifel ob dabei nicht oft weniger ein Gegensatz zwischen Intellekt und peripherer Seelentätigkeit vor- Umfang des Lebens geht. gestaltet sich auf modernem Boden Für das Ganze des Lebens der Voluntarismus und das Überwiegen der praktischen Aufgaben und zwar dahin, daß in weitem Zuge das Befinden der Menschheit, Daseins, der Menschheit des unmittelbaren für das Ziel der Ziele und auch das Erkennen zu einem bloßen erklärt macht wird; es erscheint als eine zur Förderung des Wohlseins der Menschen wirkt. Richtung des modernen Lebens entspricht, die Geschichte ersehen. Eine Mittel dafür ge- unnütze Grübelei, wenn es nicht ließ Daß dies der schon der Blick auf Ermüdung an dem Kampf um die Weltprobleme ist eingetreten, und die Fragen der inneren Bildung, der Bildung des ganzen Menschen zu einer weltumspannenden Persön- werden lichkeit, durch die wirtschaftlichen, technischen unablässig anschwellenden politischen, Probleme weit zurückgedrängt, im be- nimmt der Kampf um die ökonomische Selbsterhaltung immer mehr alle Kräfte in Anspruch und stellt immer mehr das Leben und Handeln unter den Gesichtspunkt der Zweckmäßigkeit; wie sollte in solchen Zusammenhängen noch irgendwelcher Platz sondern für einen Selbstwert des Wissens bleiben? Ein Versuch, von jener praktischen Haupttendenz aus eine eigentümliche Theorie des Wissens zu entwickeln, ist namentlich im Pragmatismus gemacht, Amerika und England aus immer mehr die Kulturwelt so sei er etwas genauer betrachtet. der von beschäftigt; Theoretisch 47 Der Pragmatismus. c) Der Pragmatismus daß — praktisch. ist in Deutschland noch so wenig bekannt, es vor aller Erörterung einer kurzen Orientierung bedarf; halten uns dabei namentlich an die Vorlesungen wir von William James, Zweck einer Aufklärung über den Pragmatismus verDer Ausdruck und der Begriff Pragmatismus im hier ge- die eben den folgen.^ meinten Sinne stammt von Charles Pierce, der ihn zuerst amerikanischen Zeitschrift „Populär Science" wandte. Zwanzig Jahre und glänzender Weise weitergebildet. in später hat James in der vom Jahre 1878 ver- Sache aufgenommen die Als weitere Führer sind Dewey (New York) und Schiller (Oxford) zu nennen; von letzterem stammt der Ausdruck «Humanismus." Es nicht ist daß hier wohl zuerst geschichtliches Interesse, in ohne ein kultur- einer philosoph- ischen Bewegung Amerika vorangeht, wie denn auch dort vornehmlich der Pragmatismus eine weitverbreitete Strömung geworden Was, Europa anbelangt, so hat er außer England namentlich in ist. in Einfluß gewonnen. Italien Über das Verhältnis des Pragmatismus zu anderen. Denkrichtäußert James sich folgendermaßen: »Der Pragmatismus ungen repräsentiert sophie, eine uns durchaus vertraute nämlich die empirische Richtung, in einer radikaleren Richtung allein der Philo- in er repräsentiert sie und zugleich einwandfreieren Form als die war, die sie bisher angenommen hatte" (S. 31 a. a. O.), ferner, »er stimmt dem Nominalismus darin überein, daß er sich überall an das Einzelne hält, mit dem Utilitarismus darin, daß er überall den praktischen Standpunkt betont, mit dem Positivismus in der Vermit achtung, die er den bloß sprachlichen Problemlösungen, überflüssigen Fragestellungen O. S. 33). (a. a. und metaphysischen Abstraktionen entgegenbringt" Der Pragmatismus sondern eine Methode zu sein, legt diese Wert darauf, kein System, Methode aber besteht in der Beziehung alles Erkenntnisstrebens auf das menschliche Dasein und was hier sich bewährt, Das Wahre wird damit ein Teil Förderung; seine gelten. nur kann des Guten, als Wahrheit „wahr heißt „Der Pragmatismus". Ein neuer Name für alte Denkmethoden. VolksAns dem Englischen übersetzt von Wilhelm Jerusalem, 1908. — Beachtenswert ist auch der Aufsatz von Jerusalem in der Deutschen Literaturzeitung vom 25. Januar 1908: „Der Pragmatismus. Eine neue philosophische Methode." * tümliche philosophische Vorlesungen. Zum Grundbegriff des Geisteslebens. 48 was alles, dem auf sich Überzeugung Gebiete der intellektuellen aus bestimmt angebbaren Gründen als gut erweist" «Alle 48). (S. Theorien sind nur Werkzeuge, nur Anpassungen der Gedanken an und keineswegs Offenbarungen oder intellektualistische die Tatsachen, Lösungen eines göttlichen Welträtsels" 122). (S. In solchem Ge- dankengange gelten dem «Humanismus" die Wahrheiten als Erzeug«Die Wahrheit macht keinen anders- nisse des Menschengeschlechts. gearteten Anspruch und heit und Reichtum, Alle diese Ansprüche sind bedingter Art« (S. 146). legt Fassung gibt Solche keine andere Pflicht auf als Gesund- Forschung insofern der durchaus eine eigentümliche Richtung, als es nunmehr nicht sowohl die Begründung von als Prinzipien nicht jetzt Menschen Sache in sondern betrachtet, Konsequenzen gilt, Natur gegenüber dem auf den Menschen gerichtet und Entwicklung die als die alles ihre in eignen einer nach der Leistung für ihn bemessen wird. Was ehesten Der das besagt und welche deutlich Streit durch die Wandlung Beispiele, des Materialismus wird bewirkt, es welche James und des Spiritualismus tritt am bringt. selbst in eine neue Beleuchtung und wird zugleich einer Entscheidung zu- völlig geführt, wenn nicht das Recht der Prinzipien erörtert, sondern die Wohl der Menschheit erwogen wird. Materialismus bedeutet in diesem Zusammenhange die Denkweise, welche* die höheren Phänomene durch die niederen erLeistung eines jeden der Gegner für das klärt und blinden Kräfte bestimmen Leitung und ihre die Geschicke der Welt durch ihre blinden Teile läßt, Spiritualismus diejenige, welche die dem höheren Elemente zuweist und damit den Geist nicht zu einem bloßen Zeugen und Berichterstatter des Weltlaufs macht, sondern ihn Frage, denselben eingreifen tätig in welche der beiden läßt. so kann die Antwort nicht zweifelhaft sein. tischen Ergebnisse Spiritualismus mit des Stellen wir nun die Beleuchtungen mehr das Leben fördert, Denn die letzten prak- Materialismus sind trostlos, während der Bejahung einer sittlichen Weltordnung gibt. «Der Glaube an geistige seiner unseren Hoffnungen freien Spielraum Wesen in all seinen Formen hat es immer mit einer Welt der Vertun, während die Sonne des Materialismus Meer der Enttäuschung untergeht" (a. a. O. S. 67). Dem heißung zu die Erörterung des religiösen Prinzipien her wird es Problems: statt von in einem entspricht spekulativen vom Bedürfnis des Menschen aus behandelt. »Nach pragmatischen Grundsätzen ist die Hypothese von Gott wahr, — praktisch. Theoretisch wenn im weitesten Sinne des Wortes befriedigend sie nun immer noch besteht den andern O. (a. a. so daß sie wirkt, in und das Problem daß auszubauen, Wahrheiten wirkenden Einklang mit sich sie bringen läßt" S. 192). Wer sich weite zeigt, Hypothese die darin, Was wirkt. dieser Hypothesen Schwierigkeiten restlichen die mögen, die Erfahrung sein 49 in Ganze einlebt, kann wohl das der Zeit Kreise gewinnen zu verstehen, daß Indem etwas, vermochte. was sonst nur nebenbei und an einzelnen Stellen es es beachtet wurde, zur Hauptsache macht, wird eine Beleuchtung der Dinge gewonnen, den Vorzug der Einfachheit und der die hat. Eine große Fragen, die keine Vereinfachung teien diese gestattet; als alle unnütz Beziehung aber scheint einen Maßstab in würdigen und damit die Sache dem zu entziehen indem augenscheinlich, ist zu gewähren, der die verschiedenen Behauptungen fangenheit zu Verständlichkeit Beziehung zur Lebenshaltung haben, müssen; ausscheiden leichten voller Streit Unbe- der Par- die Wahrheit erhält hier mehr Unmittel- mehr Beweglichkeit und Flüssigkeit, indem sie so in die Bewegung des Lebens hineingestellt wird und zu ihrer barkeit und Fruchtbarkeit, Förderung mitzuwirken hat; gerade unsere Zeit mit ihrer Zersplitter- ung der Überzeugungen scheint eine derartige Lösung zu empfehlen.^ Das Positive der Leistung wird aber durch eine scharfe Kritik des überkommenen Wahrheitsbegriffes wesentlich unterstützt. Aber so viel Anregung das alles, vertreten von bedeutenden und geistvollen Männern, bringen mag, als Letztes und Ganzes angesehen druck muß des es uns doch als ein Pragmatismus stammt Irrtum gelten. Der starke Ein- namentlich daher, daß hier die gewöhnliche Betrachtungsweise umgekehrt wird; wie aber, wenn dabei der Begriff der Wahrheit selbst auf den So aber geschieht es in Wahrheit des Wahrheitsbegriffes, und das ist Kopf zu stehen kommt? Das eben die ist das Wesentliche bewegende Seele des Wahr- daß der Mensch dabei etwas erreicht, was jenseits aller Meinungen und Neigungen liegt, und was völlig unabhängig von seiner, ja von aller Menschen Zustimmung gilt. Damit scheint heitsstrebens, seiner ' James (a. a. O. S. 122) bemerkt in dieser Hinsicht: „Die tatsächliche Unruhe, die jetzt in der theoretischen Philosophie herrscht, der Wert, den jeder Denktypus für gewisse Zwecke besitzt, die Unfähigkeit eines jeden dieser Denktypen, alle anderen zu verdrängen, alles das legt die pragmatische An- schauung nahe." Eucken, Grundbegriffe. 4. Aufl. 4 Zum Grundbegriff des Geisteslebens. 50 dem Menschen neues Leben zu winken, wesentlich ein und Großwerden des Wesens, eine Befreiung Wirklichkeit, von ein innere Gemeinschaft eine bloßmenschlichen aller Weit- mit der Art. Wo dagegen das Wohl des Menschen und der Menschheit zum höchsten und zum richtenden Maß gemacht wird, da sinkt die Wahrheit Meinung herab, und das ist eine innere Ziel zu einer bloßnützlichen sie in Was immer sie an Überzeugungskraft hätte, das müßte dem Augenblick verlieren, wo sie als ein bloßes Mittel durch- schaut würde. Zerstörung. Wahrheit »instrumentale" Wahrheit nur ist ist möglich, Selbstzweck als eine keine Wahrheit. Das heißt nicht behaupten, daß die Wirkung der verschiedenen Lehren auf den Zustand des Menschen ein unwichtiger Gegenstand kann sicherlich sei; Aufklärung und Anregung davon ausgehen, viel wenn jene Wirkung mehr beachtet und ihren Gründen nachgeforscht wird. Aber zunächst haben wir es dabei mit einem bloßen Phänomen zu tun; was in ihm Kern und Schale, Recht und Unrecht sei, das ist erst herauszustellen. Dem Pragmatismus Wahrheiten es so und auf, daß sicher, ander geraten? lauter in er selbst sieht darin einen Vorteil. diese Wahrheiten gegenseitig vertragen, Wahrheit die sich löst daß nicht sie sich friedlich einzelne Aber ist und freundlich schwere Konflikte mit ein- in Wer sollte wohl bei solchen Konflikten der Schieds- richter sein? Endlich ist das betrachtet, was das, Gelingen allem Zweifel enthoben. Kulturleben, das als nimmt; um dieses wie der Pragmatismus als höchstes und Gedeihen des Lebens, Denn es das sich Leben in ist hier Ziel keineswegs nichts anderes der Breite des Daseins aus- Leben aber als ein sicheres Gut zu betrachten, muß man von dem Kulturenthusiasmus und der optimistischen Stimmung beherrscht sein, die früheren Zeiten näher als der unsrigen lagen. Denn ist dies Leben, wenn es für sich selbst als das Letzte genommen wird, all der Mühe und Arbeit, all der Sorgen und Aufregungen, all der Schmerzen und Entsagungen wert, die es kostet? Ist dies Leben, genauer betrachtet, mit und all seinem prunkenden Schein seiner inneren Leere, mit seiner durchgängigen Unlauterkeit und Unwahrhaftigkeit nicht ein schwerer Widerspruch? Und ein Mittel zur Aufrechterhaltung dieses problematischen Lebens sollte das Streben nach Wahrheit sein? Uns will von allen Arten des Glaubens keiner gewagter als erscheinen ein derartiger unfundierter Lebensglaube. Theoretisch — praktisch. 51 Die eigne Behauptung: der Aktivismus. d) Jerusalem hebt im Vorwort zur Übersetzung von James' »Pragma- meine Annäherung an die neue Richtung hervor und be- tismus" »Euckens merkt dabei: Aktivismus ruht auf bestimmten meta- physischen Voraussetzungen, während der Pragmatismus rein empirisch Wahrheit begrüße In (S. VII). ist" ich sympathisch die Wahrheit in eine engere Beziehung mit sie nicht als eine Sache des bloßen Intellektes minder Wahrheit Sein da ich teile als die das Streben, dem Leben zu bringen, zu verstehen, nicht Ablehnung eines Wahrheitsbegriffes, dem Übereinstimmung mit einem die neben uns befindlichen Es fragt sich nur, was unter Leben verstanden wird, und gilt. zwischen dürfte was dem, in jenen Worten „empirisch" und Dort be- „metaphysisch" genannt wird, eine weite Kluft bestehen. sagt Leben die menschliche Zuständlichkeit, das menschliche Befinden — ob des Einzelnen oder der Gattung, kommt hinaus — wir aber denken beim Suchen selbe , schließlich auf das- einer engeren Ver- bindung der Wahrheit mit dem Leben an das Geistesleben Beisichselbstsein des Lebens, aller menschlichen Zuständlichkeit bildet, ja eine völlige Daher bedeutet auch gegenüber etwas wesentlich Neues Umkehrung die als ein das mit seinen Inhalten und Werten des nächsten Standes besagt. Verbindung der Wahrheit mit dem Leben dem Pragmatismus und dem Aktivismus etwas sehr verschiedenes: dort wird die Wahrheit ein bloßes Mittel für ein höheres Ziel, das gilt uns als eine innere licher Bestandteil des Lebens Zerstörung, selbst hier wird sie und darf daher und ein wesent- nie ein bloßes Mittel werden. So wenn hat es auch hier die verschiedenen und dort einen wesentlich anderen Sinn, Leistungen im Kampf um die Wahrheit nach ihrer Fruchtbarkeit für die Lebensentfaltung bemessen werden. Dort bildet das Maß der Nutzen des Menschen mit all seiner Wandelbarkeit, hier wird es das Bestehen und der lühalt des Geisteslebens, und die verschiedenen Behauptungen werden daraufhin zu prüfen sein, was sie dafür an sachlicher Vertiefung und Erweiterung bringen. Eine Denk- Beides kann bis zu vollem Gegensatz auseinandergehen. weise kann dem Menschen als Menschen harte Opfer — auferlegen von allem und ihm das Leben eher schwerer als leichter machen wahrhaft Großen läßt sich dieses behaupten -, aber zugleich kann es das geistige Leben gehaltvoller und größer machen, während um- Zum Grundbegriff des Geisteslebens. 52 was dem Menschen gekehrt, arg herabdrücken und Genuß geistig recht behagliches Dasein gewährt, ein Geistesleben leer kann. Daß sein können, Zeiten zeigt voller das Leistung Gegenwart die deutlich genug. ^ Mit Lebens dem Pragmatismus in Tätigkeit, wollen auch wir eine Versetzung des aber diese Versetzung dünkt uns nicht möglich vom gegebenen Dasein mit seinen festen Verkettungen aus, sondern nur durch eine Umkehrung dieses Daseins, durch die Ergreifung eines neuen Ausgangspunktes und die Entfaltung eines neuen Lebens. Das ist eine Art von Metaphysik: wir leugnen es nicht, vielmehr Um- verlangen wir aufs entschiedenste eine Metaphysik, da nur in kehrung der ersten Lage möglich und wird, irgendwelche ein ursprüngliches daher keine es Metaphysik gibt und geistige selbsttätiges Leben Selbsterhaltung ohne Wiederum kommen wir damit auf die Notwendigkeit eines selbständigen Geisteslebens als einer neuen Stufe der Wirklichkeit, als der Entfaltung ihrer eignen Tiefe. Nach dem allen scheint uns der Gegensatz des Intellektualismus und des Voluntarismus genügt nicht, nicht den Kern der Sache zu Es treffen. den Schwerpunkt des Lebens von der einen seelischen Tätigkeit in eine andere zu verlegen, da dies keine wesentliche Ver- änderung und Erhöhung bewirkt und den abgesteckten Kreis nicht überschreiten läßt. Vielmehr handelt es sich um den Gegensatz und eines bei aller Emsigkeit innerlich geDamit aber tritt die ganze Frage unter einen eines selbsttätigen, freien bundenen Lebens. wesentlich neuen Anblick. Intellekt e) und Intellektualismus. Die Eigentümlichkeit der aktivistischen Behauptung dürfte ehesten erhellen durch eine Betrachtung ihrer Stellung sowohl Intellektualismus herabzusetzen, als zur intellektuellen Arbeit. dazu hat sie nicht am zum Diese Arbeit irgend den mindesten Anlaß, ihr kann dem Hauptleben Befindliches und ganz wohl Entbehrliches gelten. Denn die geforderte Umkehrung des Lebens, die Wendung zur Selbsttätigkeit, wird sich ohne energische sie nicht als etwas neben intellektuelle Arbeit nun * Für eine weitere und nimmer vollziehen und behaupten lassen. Erörterung des Wahrheitsbegriffs sei „Grundlinien einer neuen Lebensanschauung" (1907) verwiesen. auf meine Theoretisch Hierfür daß sie — praktisch, 53 auch das Zeugnis der Geschichte beachtet, welche sei überall zeigt, wo von der Erkenntnisarbeit groß gedacht wurde, da, nicht als eine bloße Begleitung des Lebens oder als eine nach- trägliche Deutung eines sondern vielmehr Entfaltung Plato, wesentliches Stück des Lebens, ohne dessen Höhe eigene seine es „gegebenen" fertigen Tatbestandes erschien, als ein Spinoza und Leibniz; überall schien gehalt des Lebens bei bei den Geistes- ganzen Tiefe nach zu eröffnen und ihn seiner dem Menschen zu vollem Besitze Unrecht das Wissen als Mochte dabei zu machen. zu das Ganze des Lebens erscheinen, jedenfalls war es mehr als ein bloßes Abbild, sondern in dem Leben. jedenfalls stand es nicht neben, Aber so entschieden wir es demnach ab- lehnen müssen, den Intellekt was an der Gegenwart uns liches Geistesleben verlangt zum Sündenbock für alles zu machen, wer ein bei sich selbst befindund nur von ihm einen wesenhaften Inmißfällt: des menschlichen Lebens erwartet, der halt • und Origenes, so das Wissen erst So könne. erreichen nicht wie Clemens so bei Kirchenvätern, ist gegen Hinneig- alle ung zu einer intellektualistischen Lebensgestaltung gesichert, der wird vielmehr die Überflutung namentlich der Neuzeit und Gegenwart von intellektualistischen wird aber ihn Überflutung in Strömungen besonders stark empfinden. anderer Weise betrachten und beurteilen lassen als So sei zunächst auf diese Machtentwicklung des den Voluntaristen. Intellektualismus das Auge gerichtet; dann erst läßt sich ermessen, ob die erstrebte Gegenwirkung ihr in Wahrheit gewachsen a. Es Sorge für das Ganze des Geisteslebens diese die ist. Die Überflutung des modernen Lebens durch den Intellektualismus. Der Intellektualismus wirkt auf uns zunächst von der Ge- schichte her in mannigfacher Gestalt: er wirkt aus dem Altertum, das Geist und Intellekt dem christlich- kirchlichen Leben, dem wirkung immer zu behandeln gleichbedeutend als wieder eine pflegte, aus der Glaube trotz aller Gegen- überwiegend intellektuelle Tätigkeit wurde, aus der Neuzeit, welche die von ihr erstrebte Lebenserhöh- ung namentlich von der Tätigkeit des Intellektes erwartete. Sie veraber solche Schätzung bis in die Gegenwart hinein nicht nur ficht in der Richtung, welche Innenleben ausging, sie als Aufklärung, tut es fast erkennen beherrschten Denkweise. Spekulation noch stärker in der u. s. w. vom vom Natur- Denn noch immer pflegen die Zum Grundbegriff des Geisteslebens. 54 Naturforscher Geist und Bewußtsein einander gleichzusetzen und das Geistesleben als eine bloße Abspiegelung einer draußen befindlichen Welt zu verstehen. Dem entspricht es, alles Heil, auch Er- alle höhung des moralischen Standes, vornehmlich von einer Berichtigung der Begriffe zu erwarten.^ aber Nichts zeigen oft Macht des Intellektualismus deutlicher die dürfte daß Tatsache, die als die selbst wieder zu ihm zurückgekehrt wollte einen neuen Inhalt, aber ist Gegenwirkung gegen ihn So zieht es sich durch unterlag damit alsbald der feindlichen Macht. die ganze Geschichte des Christentums, Der 19. Jahrhundert hinein. den setzung aller Kraft positive und irrationale das Man und ihn nur verstärkt hat. man gab ihn in der alten Form und so reicht spätere Schelling eingewurzelten z. auch es das in B. sucht mit Ein- Rationalismus Denkweise zu verdrängen. durch eine Aber er bringt Neue als eine bloße Lehre; die Annahme dieser Lehre, das Bezu ihr, scheint das ganze Leben ins Wahre zu stellen. kenntnis Was ist leicht macht das anders als Rationalismus es und Intellektualismus? Viel- mancher heutige Gegner des Intellektualismus ähnlich wie Schelling. Gefährlicher noch, weil tiefer eindringend und Wirkungen des Intellektualismus durch sind die neuen Gewöhnungen und alten Erkenntnisarbeit gaU von alters des her Denkens. die mehr versteckt, sein Festwurzeln in Als der Kern der Heraushebung allgemeiner Größen aus der grenzenlosen Vielheit der Erscheinungen, im Altertum in vollem Einklang mit der Gesamtauffassung der Wirklichkeit, indem einfache und unwandelbare Formen ihr Grundgerüst zu bilden schienen; seit dem Verlassen solcher Überzeugung mit entschiedenem Unrecht, indem das Heraussehen der großen Züge aus der Erfahrung und die Verbindung der Mannigfaltigkeit zu einem Ganzen weit ^ So zeigt es System des mit besonderer Anschaulichkeit 19. Jahrhunderts, das System Comtes. das gi'ößte realistische Es seien nur einige be- zeichnende Stellen aus dem cours de philosophie positive (4. Aufl. 1877) angeführt. opinions. I, 40/41 heißt es: le mecanisme social repose finalement sur des Nach IV, 113 trägt die Hauptschuld an der unerquicklichen Lage der Gegenwart die anarchie intellectuelle; so eine philosophie convenable. ist das dringendste Bedürfnis Als der tiefste Grund der politischen Korruption erscheint l'impuissance et le discredit des idees generales. Überhaupt ent- Comte die Epochen der Geschichte den Stufen des Erkennens. Auch der heutige Monismus glaubt durch eine Berichtigung der Begriffe das sprechen bei ganze Leben heben zu können. — praktisch. 55 verlangt als ein bloßes Abstrahieren Theoretisch mehr besagt und mehr weit gleicher Züge.^ Der intellektualistischen Überschätzung des Strebens zum All- gemeinen entspricht ein merkwürdiger Kultus abstrakter Begriffe, der im Jahrhundert 19. besondere eine Höhe erreicht hat. Wie viel Macht üben jetzt Begriffe wie Vernunft, Kultur, Gesetz, Wert, FortHumanität schritt, u. s. w., Unbestimmtheit scheint sie Gerade ihre Begriffe höchst vager Art! zu empfehlen, liebsamen Entscheidung enthebt; sie indem sie uns einer un- sind oft wie leere Anweisungen, Dabei wird Hegel getadelt, die jeder nach seinem Belieben ausfüllt. dessen Begriffen doch eine zusammenhängende Gedankenwelt einen bestimmten Inhalt gab! Den Einfluß intellektualistischer Denkweise verrät auch die landläufige Neigung, unser Handeln nach Art eines logischen Schlusses als eine Subsumtion eines einzelnen Falles unter ein allgemeines Gesetz zu verstehen. In Wahrheit würde die wissenschaftliche Arbeit selbst nicht viel vermögen, logischen Formen erfüllenden im besonderen nichts neues erreichen, wären die nicht das bloße Denkprozesses. Jenseit Gefäß eines sie belebenden und der Wissenschaft aber wird die Verkehrung noch augenscheinlicher, wenn z. B. das politische Leben, ja alle menschliche Handlung nach jenem Schema der Anwendung allgemeiner Sätze auf den besonderen Fall die richterliche Tätigkeit, ^ Auch der Terminus Abstraktion bekundet jene Wandlung; ihr ent- sprechend hat er zwei Hauptphasen durchlaufen, eine logisch-metaphysische und eine psychologische, Abstrakt zurückgeht. (s? von denen jene auf äipatpEaew? Xeyo'tAeva) Aristoteles, diese auf Locke heißen bei Aristoteles die von der Materie abgesonderten Formen, namentlich die mathematischen Größen; so hält es auch das Mittelalter fest (abstrahere formam a materia intellectu). moderne Auffassung sieht im Abstrahieren ein allmähliches Herausheben gemeinsamer Eigenschaften aus der Breite der Erscheinungen. Die alte Bedeutung überdauert die Herrschaft der antiken Formenlehre; so heißt Erst die es z. def. B. in Baumeisters definitiones philosophicae ex systemate Wolfii collectae, DCCXXXV: absü-ahere ea dicimur, si ea, quae in percepüone distingu- untur, tanquam a re percepta sejuncta intuemur. Kant versteht in seiner Logik (s. VIII, 92, Hartenst.) unter Absü-aktion „die Absonderung alles Übrigen, worin die gegebenen Vorstellungen sich unterscheiden." Er will daher nicht sagen „etwas abstrahieren" (abstrahere aliquid), sondern „von etwas abstrahieren" und meint „abstrakte (conceptus abstrahentes) gebrauches erklärt sich Bedeutungen. Begriffe sollte nennen." man eigentlich abstrahierende Das Schwanken des heutigen Sprach- zum guten Teil aus dem Durcheinanderlaufen beider Zum Grundbegriff des Geisteslebens. 56 verstanden Denn das wird. heißt eine in sie starre Schablone und sowohl der Ursprünglichkeit als der Individualität berauben. Das ist auch eine Wurzel des viel bestrittenen und inpressen mitten aller Bestreitung unablässig anschwellenden Bureaukratismus. Schließlich sei noch daran daß der Intellektualismus erinnert, mit seiner Neigung, Denken und Geist einander gleichzusetzen und die Welt hauptsächlich handeln, eingesickert die in tief als einen Vorwurf der Betrachtung zu be- Sprache, namentlich in die der Wissenschaft, Die Ausdrücke aber, so unverbindlich ist. sie scheinen, führen leicht unter die Macht der Sache. So umfängt uns der Intellektualismus von allen Seiten mit einem dichten und feingewobenen Netz; subjektive aller Affekt vermag daraus nicht zu befreien, selbst die Behauptung des direkten Gegenteiles wie wir sahen, lenkt, zurück. Wendung Eine auf Umwegen leicht in die alte Bahn könnte nur aus der Anerkennung dessen hervorgehen, daß die eigene Arbeit des Intellektes einen positiven und produktiven Charakter erst erlangt, wenn sie sich einem Ganzen des Geisteslebens empfangend und fördernd einfügt, wenn sie von gesamtgeistigen Synthesen geleitet und von solchen Energien getrieben Daß wird. als aber es Wahrheit so in steht, läßt sich sowohl direkt indirekt erweisen: alle echte Leistung intellektueller Art stand in engem Zusammenhange mit Gesamtbewegungen des Geisteslebens; wo immer hingegen die Arbeit solchen Zusammenhang fallen ließ, da ist sie rasch zu leerem Formalismus oder schwankender Reflexion gesunken. Ein solches Verfechten der Abhängigkeit des Intellektes ist mit der Anerkennung einer Größe Ganzen vom Ganzen und Bedeutung innerhalb des aufs beste vereinbar. ß. Die Begründung des Erkennens im Lebensprozesse. Wer vom Erkennen ein Registrieren seine nähere Gestaltung Geisteslebens gering denkt, wer in ihm nicht mehr bloßer Erscheinungen keinerlei sieht, als der braucht sich über und über sein Verhältnis zum Ganzen des zu machen. Wer aber in ihm eine Sorge Durchleuchtung und innere Aneignung der Wirklichkeit sucht, dem wird jenes zu einem schweren Probleme. Wirklichkeit unterwerfen und aneignen, irgendwelches Vermögen an die Widerstände sie Wie läßt sich eine fremde wenn die Arbeit nicht heranbringt, nicht eine Kraft gegen der Dinge einzusetzen hat, wie kann eine Erfahr- ung uns wertvoll werden, wenn sie nicht eine von innen kommende Theoretisch — praktisch. 57 Bewegung aufnimmt und weiterführt, wie kann sie eine Antwort erteilen, wenn ihr nicht zuvor eine Frage gestellt ist? Woher aber soll das Erkennen die Kraft zu jener Leistung finden, wenn nicht der gesamte Lebensprozeß eine innere Konzentration vollzieht, seine zu einem Ganzen verbindet und aus ihm Tätigkeiten den Kampf Umgebung aufnimmt? Eine derartige Bewegung würde aller Betätigung, wie so auch dem Erkennen eine eigentümliche Art gegen die und Richtung Ein solches Zusammenschießen des Lebens erteilen. zu einem charakterhaften Ganzen steckt einen eigentümlichen seinsraum ab und eigentümlich wie die Erfahrung, gestaltet Da- so das Grundverhältnis des Menschen zur Wirklichkeit und seine Arbeits- Daraus hat auch welt. das Erkennen Wie könnte empfangen. Großen und Unterscheidenden die die seine und Wege zu Ziele einer die griechische Philosophie in dem ihrer Art verstehen, der sie nicht als Wendung derjenigen Lebenssynthese zur Wissenschaft verstünde, dem Ganzen der griechischen Kultur zugrunde liegt? Diese Synthese erfolgte unabhängig von der nicht intellektuellen Arbeit, war nicht sie bedurfte vielmehr ihrer unablässigen Hülfe; ein Werk des bloß auf seine eigene Kraft gestellten Erkennens. So überhaupt nur ein Erkennen, besitzt gegründet ist aber sie das in einer Lebenssynthese und aus ihrer Tiefe schöpft, sichere Richtungen und zwingende Notwendigkeiten, nur ein solches vermag den Gegenstand zu erfassen und zu durchdringen, nur ein solches kann der Wirk- Zusammenhang geben. Warum macht die lichkeit einen lebendigen Scholastik bei allem Eindruck, warum hat sie, trotz breitester Wirkung im menschlichen Fleiß und allem Geschick einen so dürftigen Kreise, geistig nichts Erhebliches- gefördert? Weil ihr die gestaltende Kraft eines charakteristischen Lebens fehlte, weil sie daher ihren Be- keine griffen innere zu geben, vermochte. Nähe und keine zwingende Überzeugungskraft Schon deshalb mußte sie der neueren Philo- sophie erliegen, weil in dieser und durch sie ein neues Leben emporstieg. Eben dieses ist Leibniz und Kant, von es auch, tüchtigen was schöpferische Schulgelehrten, Denker, wie wie Wolff und Herbart, unterscheidet, daß jene neue Lebenssynthesen zum Durch- bruch bringen und ihrer Arbeit eine vollziehen. So sind in siö Erhöhung der Wirklichkeit Mehrer im Reich des Geistes, nicht bloße Bearbeiter und Zergliederer eines gegebenen Befundes, sie reflektieren nicht bloß über die Wirklichkeit, sie erweitern unsere Wirklichkeit. Es bekräftigt aber solchen Zusammenhang des Erkennens mit Zum Grundbegriff des Geisteslebens. 58 dem Ganzen des Geisteslebens nichts nachdrüci<licher als die eigenen Erfahrungen der Logik, die enthoben glaubt. leicht aller Die Unverbrüchlichkeit jener Gesetze ist klar und Aber unbestritten. und Bindung und Beziehung sich mit der Allgemeingültigkeit Umwandelbarkeit ihrer Gesetze alle und Formen ergeben keineswegs Gesetze schon ein lebendiges Denken; das wirkliche Denken des Menschen ist keineswegs bloß eine gleichförmige Anwendung jener Denkgesetze, es hat darüber hinaus eine Eigentümlichkeit, die alle Mannigfaltigkeit beherrscht und durchdringt, und die Lebensprozesses hervorgehen kann. nur aus dem Ganzen eines Demgemäß das Denken in ist seiner feineren Struktur verschieden nach der Art des Lebensganzen, dem es angehört. Daß das künstlerisch gerichtete Griechentum das Denken enger mit der Anschauung zusammenschloß, daß es rasch und unmittelbar zu einer Synthese strebte und alles Grenzenlose mied, daß es die Elemente des Lebens als gegeben und unveränder- hinnahm, lich das auch seine Erkenntnisarbeit gestaltet einzelnen logischen Operationen ferner die Denkweise des alters den Einfluß eines eigentümlich. Wie ausgehenden Altertums bis in die sehr bekundet und des Mittel- neuen, von der Religion beherrschten Lebens! Indem dort alles sichtbare Dasein zum bloßen Gleichnis einer unsichtbaren die Ordnung Behauptungen ahnt wird, verlieren die Begriffe ihre Ausschließlichkeit. und schaut durch den ihre Sprödigkeit, Die allegorische Deutung sinnfälligen Tatbestand hindurch eine höhere Welt, ohne dabei jenen zu einer gleichgültigen Erscheinung So ist derselbe Gegenstand Bild und Sache, Sinnund Geistiges in Einem; daß darin zugleich eine Bindung herabzusetzen. liches und eine Befreiung, eine Bejahung und eine Verneinung, damit aber ein unhaltbarer Widerspruch liegt, das empfindet jene von Stimmung und Ahnung beherrschte, beinahe traumhafte Denkweise dieser Denkweise aber ruht der Sakramentenlehre u. s. w. nicht. mittelalterliche Kirchenbegriff, Auf die Die Höhe der Scholastik wird klarer und nüchterner, aber da es ihr bei aller Tüchtigkeit syllogistischen Ver- fahrens an einer selbständigen Synthese und an einer entsprechenden Energie des Denkens fehlt, so fehlt ihr auch die Kraft eines disjunktiven Verfahrens; grundverschiedene Welten, wie der weltfrohe Aristotelismus und das weltfremde alte Christentum, ferner innerhalb des Christentums die kirchliche Ordnung und die alle Gestalt überfliegende Mystik, vertragen hier sich miteinander friedlich und freundlich; sie scheinen zu völliger Harmonie gebracht, wenn eine geschickte An- Theoretisch — praktisch. 59 Ordnung und Abstufung einen direkten Zusammenstoß verhütet. Man als auch, wo einem kräftigen Denken denkt im Schema eines Sowohl Entweder sich alsbald ein — — oder ergeben hätte. Auch die logische Methode der neuen Wissenschaft mit ihrer energischeren Disjunktion und ihrer schärferen Analyse, ihrem Flüssigmachen auch der Elemente und ihrem Streben ins Unbegrenzte, sie zeigt deutlich genug einen Zusammenhang engen dem modernen mit. Lebensideal der Kraft und Bewegung. wer So bejaht eine besondere Art des Geisteslebens, dieser Art der Forschung den Typus aller Forschung sieht. in Wie aber jede ausgeprägte Zeit so ihre besondere Art der Logik hat, auch jeder selbständige Denker; ohne eigene Logik kann hat sie keine eigene Denkweise, keine eigene Lebensgestaltung bestehen. kräftiger diese Gestaltung, desto tiefer wird ihr Einfluß bis Je in die einfachsten Elemente und Tätigkeiten des Denkens sich hineinerstrecken. So muß die Verkettung mit dem Lebensganzen die Denkarbeit konkreter, individueller, reicher gestalten. neue -Fragen und Aufgaben. dem Lebensganzen Es gilt Zugleich entstehen damit zu zeigen, was das Erkennen näher aufzuweisen, wie es in ihm zur vom zur Verkettung der leistet, Wesentlichen, Scheidung des Zufälligen Mannigfaltigkeit, zur Herausbildung einer Allgemeingültigkeit wirkt. Auf den mag gerade die Allgemeingültigkeit als bedroht ersten Blick wenn das Erkennen in so nahe Beziehung zu besonderen Lebenssystemen tritt. Wird sich nicht damit die Wahrheit in eine Vielheit von Wahrheiten auflösen und ein zerstörender Relativismus erscheinen, gänzlich Das würde doch nur der Fall das Feld gewinnen? sein, wenn alle Lebenssynthesen gleichwertig nebeneinander stünden, nicht ihrer aller Arbeit einer einzigen umfassenden Synthese diente, an der sich messen hätte. Aber könnte nicht eine solche Synthese der Bewegung als Endziel vorschweben und zugleich von Anfang an zur alles zu und Richtung des Lebens und mit ihm des Denkens Gestaltung wirken? Daß Behauptung neue Probleme hervorruft, eine gegen keineswegs sie; sind Grundanschauung nicht sowohl Y- Daß stätigt es jede spricht echte Probleme, so werden sie die es belasten als verstärken. Die bewegende Kraft im Wahrheitsstreben. sich in der Tat Erwägung der um echte Probleme handelt, das bewas im Kampf um die Wahrheit Frage, Macht hat und die Entscheidung bringt. Daß es nicht die bloßen Gründe und Beweise sind, das läßt uns jede Disputation zwischen Zum Grundbegriff des Geisteslebens. 60 abweichenden Überzeugungen Gedankenarbeit, anders sollte es auf dem beim Zusammenstoß der Geister in größeren Felde der Disputation, der ersehen; deutlich stehen? Jeder überträgt die Gründe des anderen in die eigene Sprache und Denkweise und verändert sie damit völlig; so steht gewöhnlich Monolog neben Monolog, zu einem wirklichen Dialoge kommt es selten. Überzeugungskraft ihre nicht ihr In Wahrheit gibt den Gründen logisches und dialektisches Ver- mögen, sondern der Gehalt und die Gewalt des Geisteslebens, der geistigen Konzentrationen, der Lebensenergien, aus denen sie schöpfen. So verteidigt in aller Erörterung prinzipieller Fragen im Grunde ein jeder sich selbst und seine eigene Art; erst aus solcher geistigen Selbst- erhaltung strömt Kraft, Glut und Leidenschaft Eine fruchtbare Aussprache und die Möglichkeit einer Bewegung. Verständigung entsteht nur Art in die intellektuelle gemeinsamen einen da, wo die Verwandtschaft der geistigen Boden würden bereitet; Aristoteles und Thomas und Voltaire, sie alle treffliche Logiker, sich wohl gegenseitig überzeugt haben, wenn sie noch so lange miteinander gestritten hätten? Mur einen flachen und haltlosen Menschen können bloße Gründe aus seiner geistigen Art hinauswerfen; auf bloß intellektuelle Erwägung gestellt, könnte der Mensch seines eigenen Wesens nimmer sicher und froh werden; denn er müßte in steter Furcht sein, daß nicht ein stärkerer Dialektiker komme, ihn überwinde und zum Gegenteil zwinge. Augustin, So sind es auch im geschichtlichen Leben Gedankenbilder, geistigen die Energien, freischwebenden die nicht die abgelösten sondern es sind die Ideen, Lebenskonzentrationen, welche die Geister beherrschen und die Leidenschaften entzünden. bewußte Anhänger der Hegeischen die mit Ideen hervortreiben, weitertreibt als überwältigender und daß Notwendigkeit nichts stärker aufrüttelt, logischer Widerspruch, ein Bewußte und un- Denkweise sagen uns oft, daß ihre Konsequenzen nichts zwingender Gewiß, Konsequenzen und Widersprüche können eine unwiderstehliche Gewalt über den Menschen erlangen. Aber sie tun das nicht von der bloßen Logik aus. Konsequenzen nicht gezogen, doch nicht können sehr nahe empfunden. Es kommt und werden doch hier alles darauf an, daß die aufgenommen werden, daß Lebensenergien entfalten und ein geistiger Probleme in die geistige Selbsterhaltung durch hindurch sich sie liegen Widersprüche mögen handgreiflich sein und werden Existenzkreis gestaltet; nur eine solche Aneignung, eine solche Auf- — praktisch. Theoretisch 61 macht die Konsequenzen unabweisbar und die Widersprüche unerträglich; es nähme ist der Arbeit intellektuellen Eigenleben das in der Einigung, die Kraft der Zusammen- im besonderen der Grad welche der Logik die Macht fassung des Lebens, die erst verleiht, Vermögen zu üben wähnt. Das ruhige Ertragen widersprechender Gedanken massen verrät immer eine geringere Konzentration des Lebens; wie jenes für das kindliche Denken charakteristisch ist, so ist es das auch für naivere Zeiten und für sie oft aus eigenem den Durchschnittsstand der Menschheit gegenüber den Forderungen selbständiger Geistigkeit. Der Mangel an Logik verschuldet nicht, er bekundet nur jenen Mangel. So licher und der geistigen Lage der Gegenwart nichts verdrieß- in ist hemmender, nichts die Unempfindlichkeit als Widerspruch von Gedanken massen; an Lebensenergie Selbsttätigkeit inmitten sie verrät einen starken den Mangel wahrhaftigem Eigenleben und an zentraler Art, für rührigster Betätigung. Der Lebensbestand ist Kompromisse an der Oberfläche sollen sie beschwichtigen, sie scheinen ausgeglichen, wenn die Schroffheit des direkten Zusammenstoßes einigermaßen vermindert heute voll fundamentaler Gegensätze; ist. Oder auch Widerspruch vermengt. alten Gedankenmassen werden die unbedenklich ineinander bei allem sachlichen geschoben und miteinander Wie oft müssen sich die grundverschiedenen Welten des und der modernen KulturentwickNicht minder laufen solche Behandlung gefallen lassen. ethisch-religiösen Idealismus lung eine bei allermodernsten Schriftstellern So ungen durcheinander. z. grundverschiedene Lebensstimm- B. bei Nietzsche antike und moderne, klassische und romantische, künstlerische und dynamische Denkweise; wer für solche Dinge ein Ohr der muß darin grelle Disso- Die Masse der sog. Gebildeten aber, wie nanzen empfinden. alles hat, kräftigen Eigenlebens entbehrt, sie empfindet nicht den mindesten Schmerz an geistigen Dissonanzen, eher sieht sie darin ein buntes und unterhaltendes Spiel; je mehr Widersprüche, desto «eigenartiger«, desto «interessanter"! Nichts zeigt die Abhängigkeit des Denkens von der Energie Lebens greifbarer des geistigen Alle eingreifenden der jeweilige als die Bewegungen der Religion. Wendungen kamen hier dadurch in Befund unerträgliche Widersprüche Fluß, daß empfinden ließ, daß im besonderen, was der Lauf der Zeit und die Anpassung an die menschliche Lage an äußeren Einrichtungen, Übungen, Formeln Zum Grundbegriff des Geisteslebens. 62 hervorgebracht mit den Forderungen einer gesteigerten Inner- hatte, Aber wie wenig war die zusammenstieß. unversöhnlich hchkeit Empfindung, Durchlebung, Überwindung solcher Widersprüche das Werk bloßlogischer Betrachtung! lag der Kontrast zwischen und dem Verlangen vor aller Im Zeitalter der Reformation z. B. der Veräußerlichung des Kirchenwesens ernster Seelen nach Verinnerlichung deutlich Augen; der größte Gelehrte jener Zeit, Erasmus, sah ihn, Warum ist nach Ausweis seiner Schriften, ebenso klar wie Luther. nun Luther der Reformator geworden und nicht Erasmus? Sicherlich nicht, weil jener der größere Logiker war, denn das war entschieden Erasmus. Sondern weil ihm jene Lage mit ihren Widersprüchen und geistreicher Reflexion blieb, nicht eine Sache kühler Betrachtung sondern weil sie ihm zu einer persönlichen Angelegenheit und zu- gleich zu einem heftigen Schmerz, einer unerträglichen Notlage wurde. Bei solcher völligen Aneignung wurde ihm eine Lösung des Konfliktes zur zwingenden Notwendigkeit, zur Seele seines Lebens, geistigen Selbsterhaltung, die alle andere zu einer Erwägung mit elementarer Die Gewalt einer solchen Selbsterhaltung gab Wucht verdrängte. dem schlichten Manne das Vermögen und auch das Recht, eine überkommene, in der Überzeugung der Menschheit geheiligte Ordnung anzugreifen aus und den Aufbau ursprünglicher Helden, an geistiger einer das Wirken neuen zu wagen; Notwendigkeit machte ihn zu einem dem gemessen Erasmus mit allem überlegenen Wissen, Geschmack und Scharfsinn als klein erscheint Entscheiden so in den geistigen Kämpfen nicht freischwebende intellektuelle Erwägungen, sondern die begründenden Lebensprozesse und der Gehalt der von ihnen umspannten geistigen Wirklichkeit, so sind auf sie die Gedankenmasseh zurückzuführen, so hängt aller wesentliche Fortschritt an einer Weitererschließung jener Wirklichkeit. So vollzog und vollzieht sich die Überwindung alternder Gedanken- massen nicht durch ein plötzliches Erscheinen sondern dadurch, daß die Schranken des in überlegener Gründe, jenen Gedankenmassen verkörperten Lebens bemerklich werden, neue Konzentrationen oder doch Bewegungen aufstreben, durch das in scheinbar sicherem wird; mag es äußerlich Besitz Befindliche veraltet sicher zu herrschen glaubt. ist Leben und entwertet den früheren Glanz behaupten, die Herrschaft über die Seelen; es Ideen in die Energien, ein frisch sich regendes geschlagen, auch es verliert wo es noch Daß sich so die Entscheidung von den von den intellektuellen Erwägungen in die Theoretisch schöpferischen — praktisch. 53 das muß zur Vertiefung Lebensentfaltungen verlegt, der Arbeit und zur Befestigung des Strebens wirken. Unvergleichlich größer wird das Bild des geschichtlichen Lebens, wenn sowohl Lehren gegen Lehren wird das Problem durchgängig stehen; hier nicht Lebensmächte gegen Lebensmächte als zurückverlegt, beträchtlich wenn es die Wurzeln der Lehren erst zu ermitteln, die letzten Triebkräfte erst aufzudecken, stoßes den entscheidenden Punkt des Zusammen- herauszustellen erst Aber gilt. alle Mühe der Arbeit wird dann von der Überzeugung getragen und beseelt, daß im Menschenleben ursprünglichere Kräfte, tiefer gegründete Notwendigkeiten walten, Gedankenarbeit aus eigenem Vermögen als die 8. sie Konsequenzen für die Erkenntnisarbeit. Solche Verbindung der Erkenntnisarbeit mit und dem Aufbau Geisteslebens aufbringen kann. dem Ganzen des einer geistigen Wirklichkeit muß für ihre eigene Gestaltung eingreifende Folgen haben; bei ' der dringend gebotenen Beschränkung nur insofern betrachtet, als mit der Lösung wichtige Aufgaben lösbar oder doch angreifbar diese seien hier werden, die sich sonst einer erfolgreichen Behandlung entziehen. Immer noch waltet darüber viel Unsicherheit, wie die Philosophie eine selbständige Aufgabe finden könne. jener zur Einigung Die Einheit oft verbinden, entweder ist gegenüber den einzelnen Wissenschaften erteilte eine Antwort, kann bloße sie nicht solle die genügen. Zusammenstellung, Ergebnisse Denn jene dann muß man sehr freigebig mit der Bezeichnung Wissenschaft sein, um eine solche Enzyklopädie als Wissenschaft anzuerkennen, oder sie bedeutet eine Weiterbildung Prinzipes, Prinzip bloß aus kann und Umwandlung, dann bedarf es eines neuen dem eine solche hervorgehen könnte. Das gesuchte nun weder von außen gegeben werden, noch aus intellektuellen Punkt, der uns zugänglich ungen es muß im Ganzen kommen wir auf den letzten Bewegungen entspringen, des Lebensprozesses liegen. Hier ist; erst nach seiner Art und seinen Erfahr- muß sich das Grundverhältnis des Menschen zur Wirklichkeit, sowie die Bedeutung seines Lebens und Seins bestimmen; erst von hier aus kann eine Verbindung, eine Abschätzung, eine Weiterbildung der Ergebnisse der einzelnen Wissenschaften erfolgen. prozeß erst tritt Dieser Grund- uns nicht im unmittelbaren Eindruck entgegen, herauszuarbeiten; das aber ist die er ist Aufgabe der Zentraldisziplin der Philosophie, die von alters her Metaphysik heißt; die anderen Zum Grundbegriff des Geisteslebens. 64 haben dann die neue Beleuchtung den einzelnen Ge- Disziplinen zuzuführen. bieten Solche Fassung engen Zu- den auch erklärt sammenhang der Philosophie mit der Persönlichkeit des Menschen, ohne jene zu einer bloßen Ausstrahlung der individuellen Art herab- Denn zusetzen. ohne Erweisung einer erfolgen nicht Lebens; Durchdringen zu jenem Grundprozesse kann das insofern letzthin ist Weite und Tiefe des Kraft, Maß das Maß des Lebens auch das des Denkens. Wendung dem Eine wesentliche Förderung bringt ferner jene Daß Probleme der Wahrheit. wenn wäre, sie auf Wahrheit endgültig zu verzichten Übereinstimmung unseres Denkens mit einer eine draußen befindlichen Welt bedeutete, darüber Um so zweifelhafter Mit könnte. Anknüpfung der heute kein Zweifel. Nein entgegentreten Lebensprozeß das erhält es gibt keine intellektuelle Wahr- ohne eine gesamtgeistige Wahrheit, diese aber bedeutet nichts anderes als eine Verwandlung der Welt in Eigenleben, eine innere Bewältigung der Wirklichkeit. den den an Problem eine neue Beleuchtung; heit ist das solchem das Ja, ist Menschen hinaus das Das hat zur Voraussetzung, den Geistesleben den Menschen aber Wirklichkeit bildet; für aufhörliches Streben und Weiterstreben, ist daß über Grund der die Sache ein un- letzten ein Vordringen und Empor- klimmen, ein wachsendes Ringen mit den Widerständen ungeistiger und Innerhalb dieses Strebens gibt halbgeistiger Art. sahen, fruchtbares keinerlei Erkennen des sächlichkeit zunächst nicht mehr als Versuche, der Welt drinnen und draußen können es, wie wir Begründung eine Aber diese Synthesen sind Synthesen Lebens. ohne erst bei aller in Tat- im Kampf mit Vermögen erweisen; sie ihr das Erkennen aber hat bei solcher Auseinandersetzung eine leitende Stellung, es ist unentbehrlich zur Klärung und Prüfung, unentbehrlich zur Erringung der Allgemeingültigkeit, zur Austreibung alles Klein- menschlichen und zur Herausbildung des Weltcharakters des GeistesEin lebens. solches kritisches Wirken kann es nicht üben ohne ein gewisses Heraustreten aus der Besonderheit jener Synthese, aber die Kritik kann nicht weiterführen, wenn sie nicht einer neu auf- steigenden Synthese dient. Als festen weiteres Problem gehört Zusammenhang des Menschen ging, hierher die Ausgangspunkt der Erkenntnisarbeit. ist mit Frage Seit der der sinnlichen jene Frage unabweisbar geworden. nach einem unmittelbare Welt verloren Aber vergeblich hat Theoretisch Erkennen das einen — praktisch. Punkt festen bei sich 55 und Letzte erreichen Nicht anders Sicherste auftrat. als gesucht; selbst wieder erschienen unerwiesene Voraussetzungen ist in immer dem, was als das jener feste Punkt zu durch eine Zusammenfassung des ganzen Lebens zur Einheit und durch eine gleichzeitige Verwandlung in eigene Tat; nur damit kann eine axiomatische Gewißheit entstehen und auch Erkennen zugehen. bedeutet diese Einheit im dem Für den stets eine Aufgabe; eine volle Tatsache könnte Streben befindlichen Menschen Weges bringen, was in unabsehbarer Ferne liegt. Aber das Streben selbst wäre unmöglich, wenn nicht das, was für uns Menschen eine unermeßliche Aufgabe ist, im Geistesleben selbst erst das Ende des die begründende Tatsache wäre. Es ein alter Einwand ist gegen die Philosophie, daß sie lediglich Meinungen neben Meinungen stelle und diese tausende bis zur Unübersehbarkeit anhäufe, im Lauf der Jahr- ohne die spätere Leist- ung der früheren sicher überlegen zu machen. Gewiß verbleibt in der Philosophie ein Element der Freiheit und der Entscheidung, sie teilt mit der Religion, der Moral, der Kunst und allen edlen Dingen die Eigenschaft, immerfort der eigenen Tat zu bedürfen und sich nie- mandem aufzwingen zu lassen. Aber deshalb wird sie noch kein bloßes Neben- und Nacheinander menschlicher Meinungen: davor behütet sie sicher die Erkenntnis ihres engen Zusammenhanges mit dem Das bringt Bewegung in enge Verkettung mit der Evolution Lebens in der Menschheit, und wie die Wendepunkte Streben des Menschen nach einer geistigen Wirklichkeit. ihre geschichtliche des geistigen dieser Evolution fundamentale Tatsachen erschließen, so treiben auch die philosophische Arbeit in neue Bahnen. sie Unser Verhältnis zu den großen Problemen kann nicht mehr das der alten Griechen sein, seit prozeß das Christentum so durchgreifende Wandlungen im Lebens- vollzogen, so schwere Konflikte, Tiefen in ihm aufgedeckt hat; es wachsen, seit hat. solche fruchtbare dem Mittelalter entdem Menschen und der und das Innenleben zu größerer Zeigt so aber auch die Neuzeit schärfer zwischen Welt geschieden geweckt ist aber auch Selbständigkeit Erfahrung nicht den Denker in enger Verbindung mit der Geschichte und dem Ganzen der Menschheit? Denn immer nur Mög- Seine Selbständigkeit braucht er darüber nicht einzubüßen. was die lichkeiten Umgebung dem Menschen zuführt, sind und Anregungen; eine Wirklichkeit und greifbare eine Gestalt erwächst daraus nur durch eine vordringende Tat, die immer Eucken, Grundbegriffe. 4. Aufl. 5 Zum Grundbegriff des Geisteslebens. 66 So wird das eine auf das andere eine Sache des Einzelnen bleibt. angewiesen, das Ganze aber, das beides umspannt, gewinnt unver- kennbar an Größe. £. Konsequenzen für die Behandlung der Geschichte der Philosophie. Die Anerkennung solches Zusammenhanges der Philosophie mit dem Ganzen muß auch auf die Behandlung der Ge- des Lebens schichte der Philosophie einen starken Einfluß üben. kann genügen, es einzelnen die Systeme in mehr Nicht ihrem unmittelbaren Bestände darzustellen und aneinanderzureihen, sondern zur Aufgabe wird, begründenden die Lebensinhalte und herauszuarbeiten die Leistung der Denker dadurch in größere Zusammenhänge zu stellen. Nicht sowohl was jene sagten, als und Aussprache welche Art geistige ihre wie dazu kamen, es zu sagen, sie zeigte, wird jetzt Hauptproblem. Das geschichtlichen und menschlichen Umgebung aufzuklären, zum das Verhältnis des Denkers zu seiner zwingt, freilich nicht in der Art eines landläufigen kulturgeschichtlichen Verfahrens, das Dinge auf den die Kopf indem stellt, es das Innere vom Äußeren, das Große aus der Summierung des Kleinen, das Ewige vom Zeitlichen herleitet. aber wird sich nunmehr danach bemessen, was sie für die Eröff- nung neuer Tiefen, In taten. diesem für Die Bedeutung der einzelnen Leistungen die Erweiterung Sinne alles ist der geistigen Wirklichkeit große Denken ein Vordringen, Neubilden und Schaffen. Macht solche Zurückbeziehung des philosophischen Strebens die Behandlung seiner Geschichte in gewisser Richtung verwickelt, Denn nach jenem Maß- so wirkt sie in anderer zur Vereinfachung. stabe gemessen, heit als dürfen nur einige wenige Erscheinungen in Wahr- schöpferisch, als Weiterbildner der Substanz des Lebens gelten; auch heben sich dann aus der scheinbar chaotischen Fülle einige wenige Typen hervor, wie der Begriffe sich schärfer meiste oder ein die bei aller subtile lebens. Erörterung, der Menschen, Wir So mag Kern von der bloßen Umhüllung scheiden. zeigt, ist bloße Das> Um- Gelehrsamkeit verschiedener Art, mehr minder geistreiches Räsonnement, schäftigung Veränderung der Lagen der Hauptsache immer wiederkehren. was der unmittelbare Anblick aber, gebung: in untauglich alles das tauglich zur Be- zur Erhöhung des Geistes- sind ärmer und reicher, als wir gewöhnlich meinen, ärmer im Umfang, reicher im Inhalt unseres Besitzes. — praktisch. Theoretisch bloßen Form des Systems entgegenwirken, die von der Hauptsei keines- Die Verkettung zum System drängt die einzelnen unterschätzt. Sätze enger leicht Die Bedeutung der systematischen Form sache ablenkt. wegs 57 mag jene Zurückverlegung auch der Überschätzung der Endlich zusammen, sie Widersprüche schärfer heraus, treibt die zur gleichmäßigen Durchbildung, zur Organisation der Ge- sie wirkt Aber das dankenwelt. alles tut nur unter Voraussetzung eines sie lebendigen und belebenden Inhaltes, den nur Synthesen und Energien des gesamten Lebens erzeugen. Fehlt solcher Inhalt, so kann alle und Geschicklichkeit in Aufbau und Anordnung das System nicht vor einem Sinken zu einem leeren Gehäuse bewahren. logische Kraft Wie ist viel durchgearbeiteter ist Wolffs System die schroffen Ausbau Gedanken seiner gewirkt verhindert, der Geisteswelt wie wenig andere. und doch auch auf als gerichtet, der bloßen die hat er durch seine Gedankenarbeit so Auf jenes Wesentliche, auf schaffende Kraft und den treibenden Kern, Augenmerk das von Leibniz, Widersprüche seines Wesens an einem systematischen Weiterbildungen stark als Augustin wurde schon durch jener damit der größere Philosoph? sei Form aber die also vornehmlich das nicht mehr gegeben ihr gebührt. Doch genug der Erörterungen; auch würden sie bloße Ausschnitte aus einem bei weiterer Ausführung größeren Gedankenkreise bleiben. Wir verweilten hier et\('as länger, weil der Nachweis wichtig schien, daß das eigene Interesse der Erkenntnisarbeit über die bloße Erkenntnisarbeit hinaustreibt. Zugleich aber wurde ersichtlich, daß es nach einer anderen Richtung treibt als Vielleicht manche der Männer, stehen pflegen, dem könnten wir nur freudig begrüßen. nach der des Voluntarismus. die Voluntaristen nahe, was wir versuchen. Persönlichkeiten stehen zu heißen Solche Übereinstimmung Aber wie immer es mit den mag, der Unterschied einer Verschiebung innerhalb des Seelenlebens und einer Erhebung über alles empirische Seelenleben sei in keiner Weise verdunkelt. Idealismus a) Die Ausdrücke. |ie Ausdrücke Idealismus I und Realismus sind durch übermäßigen *-^ Gebrauch so verschliffen Wissenschaft treten sie — Realismus. und sind. für die ver- einen alten und bleibenden Gegensatz, bewegende Frage Gegenwart der daß Aber immerhin abgegriffen, unbrauchbar geworden fast ist. Wegen sie der zugleich eine solcher Beziehung mögen zunächst die Termini kurz erläutert sein. Das Wort Idealist taucht in der Philosophie zuerst gegen das Ende des 17. Jahrhunderts auf;^ wenn Leibniz das Wort im Gegensatz zu Materialist (s. 186a Erdm.) verwendet wie sonst Formalist, so denkt er dabei an Philosophen, welche wie Plato in der Form das Wesen der Dinge sehen. und Aristoteles Alsbald aber übte die moderne Bedeutung des Wortes Idee eine Wirkung auch hierher. Aus einer urbildlichen Form wurde Idee, zuerst in der französischen Sprache, zur bloßen Vorstellung, zu einem subjektiven Gedankenbilde; durch Descartes und Locke drang — nicht ohne Widerspruch Neuerung auch System, das die Realität alle in Lehre wöhnlich in Idealismus bedeutete dann ein Wirklichkeit jenseit der Vorstellungen einer Berkeley's die Philosophie; Außenwelt mit jenem tadelndem Sinn leugnet. Im und damit besonderen Ausdruck bezeichnet. als - die wurde Und zwar ge- eine Verflüchtigung der Wirklich- So rechnet Wolff die Idealisten samt den Materialisten und zu den «drei schlimmen Sekten" (s. Wolff von seinen keit Skeptikern Schriften S. 583); durchgängig war man bis gegen ^ Ende des 18. Jahr- Näheres darüber s. Vaihinger in den „Straßburger Abhandlungen zur In der Kunsttheorie scheint der Ausdruck noch weiter Philosophie" S. 94 ff. zurückzureichen. Wenigstens ward mir von befreundeter Seite die mir hier mitgeteilt, daß schon in Pacheco's arte de la nicht kontrollierbare Notiz Pintura (Sevilla 1649) Idealist zur Bezeichnung einer Kunstrichtung dient. Idealismus — Realismus. hunderts ebenso eifrig darauf bedacht, zu verwahren, als später, sich sich 69 gegen den Idealismus zu ihm zu bekennen.^ stück zu Idealismus in diesem Sinne bedeutete dem Als Gegen- 18. Jahrhundert Realismus die Behauptung einer außerhalb des Denkens befindlichen Durch Herbart und Welt. 2 seine Schule hat sich diese Bedeutung der Ausdrücke durch das 19. Jahrhundert hindurch bis zur Gegenwart erhalten. Dann aber bewirkte, wie bei vielen Ausdrücken, so auch hier die Kantische Philosophie eine wesentliche Verschiebung. ^ noch der herkömmlichen Terminologie folgt zunächst daher (z. nunft) den Idealismus mit dem Skeptizismus zusammen. Kant selbst und stellt B. in der Vorrede zur 2. Aufl. der Kritik der reinen Ver- Die Präg- ung des Ausdruckes transzendentaler (auch formaler oder kritischer) Idealismus erfolgt im Hinblick nicht auf Plato, sondern auf Berkeley; seinem »empirischen", er einen Dingen jenseit stellt neuen „psychologischen« Idealismus „materiellen", Idealismus entgegen, der die Existenz von der Vorstellung keineswegs leugnet oder bezweifelt, der aber die Formen der Anschauung und des Denkens für bloßsubjektiv erklärt; damit werden Gegenstände einer uns mög- alle lichen Erfahrung zu bloßen Erscheinungen, danken keine an „die außer unseren Ge- Diese Verschieb- sich gegründete Existenz haben." ung enthält insofern einen fruchtbaren Keim zur Weiterbildung, als den Träger der Formen, das Subjekt der Erkenntnis, dabei nicht sowohl der Einzelmensch in seiner Besonderheit als die gemeinsame Struktur unseres Wesens, Indem bildet. sich so die geistige das Problem Organisation des Menschen aus der Psychologie in die Geisteslehre verlegte, konnten bald in weiterem Sinne Idealisten alle welche die Überlegenheit der geistigen Tätigkeit gegen die heißen, Macht der Außenwelt verfechten. So schreibt W. von Humboldt (Briefw. S. 485): „Am Ende Idealisten ^ z. Schiller B. sind an wir ja beide und würden uns schämen, uns nachsagen zu lassen, daß Wolff (de differentia nexus rerum sapientis et fatalis necessitatis S. 75) will in keiner Weise Plato zu den Idealisten gerechnet wissen; wohl nenne den er die Körperwelt Erscheinung, aber er verstehe darunter keineswegs mit Idealisten eine bloße Vorstellung. Im Mittelalter bildete bekanntlich Realismus den Gegensatz zu Nominalismus; seine Anhänger hießen gewöhnlich reales; realista erwähnt Prantl (Geschichte der Logik IV, 221) zuerst bei Petrus Nigri (um 1475). * ^ II, Näheres darüber s. Trendelenburg, Logische Untersuchungen, 512 ff. 3. Aufl. 70 Zum Grundbegriff des Geisteslebens. die Dinge uns formten und nicht wir die Dinge".^ Zur Durch- setzung dieser Bedeutung hat niemand kräftiger gewirkt als Fichte. In deutlicher Verwandtschaft damit, aber zugleich in eigentüm- licher Färbung, verwendet den Ausdruck der deutsche Neuhumanis- mus, diese neueste Phase der So Renaissance. mit der F. A. Wolf dankenreiche Abhandlung, erklärt das ge- die „Museum der Altertumswissenschaft" (1807) eröffnet, als die „erste Bedingung aller höheren Ausbildung" Richtung des Geistes"; »ideale die ver- er gemäß seinem Lieblingsspruch, daß, „überall das Nützliche zu suchen ganz und gar nicht für großgesinnte und steht aber freie Menschen paßt" einer harmonischen Entfaltung nicht irgendwelcher Folgen darunter Polit. (Aristot., aller wegen, die Richtung des Lebens auf das Nützliche, sondern das Schöne. mus läßt Realisten die nicht Diese Fassung des Idealis- durch Persönlichkeit und Lebenswerk niemand mehr ge- hat fördert als Goethe, mit so einen 1338 b2), das Streben nach Geisteskräfte allein ihrer selbst, gutem Rechte er in anderer Hinsicht sich Der Sprachgebrauch des nannte. philosophische und die 19. künstlerische Fassung Jahrhunderts in eins zu- sammenfließen; indem so der Idealismus ein Bekenntnis zur Selbsttätigkeit und zum Selbstwert des Geisteslebens wird, des erkenntnistheoretischen Schulproblems des Stelle 1 8. tritt an die Jahrhunderts und bleibende Frage der Menschheit. eine alte b) Zum Kampf der Lebensgestaltu,ngen. Die Formulierung des Gegensatzes von Idealismus und Realismus läßt verschiedene Fassungen zu, die hinauskommen. selbe Ist in der Sache aber alle auf das- der Hauptstandort des Lebens die sichtbare oder eine unsichtbare Welt, und sind die Hauptziele des Lebens hier Besonders eingehend hat sich Schiller mit den Ausdrücken in der Abhandhmg ,,Über naive und sentimentalische Dichtung" beschäftigt. Als ^ ihm hier, wer sich durch die Notwendigkeit der Natur bestimmen läßt, als Idealist, wer sich durch die Notwendigkeit der Vernunft bestimmt. Die Schulgelehrten empfanden die Wandlung wohl und widerstrebten ihr. So sagt Plattner (Phil. Aphorismen I, 412): „Man fängt itzt an, den Begriff des Idealismus gar zu weit auszudehnen. Der zeither gewöhnlich gewesenen Bestimmung nach ist es dasjenige System, welches das Dasein alles dessen leugnet, was nicht Geist ist." — „So wie man itzt den Idealismus versteht, Realist gilt wären alle die, welche die Sinnenwelt als eine Erscheinung betrachten, mit alle Philosophen ohne Ausnahme Idealister." anderen Worten — Realismus. Idealismus sich nicht ist sondern nur der Natur, als eine neue und höhere Stufe der Wirklichkeit verstehen wesentlich oder Fortführung eine als 71 Entwickelt sich im Menschen ein Leben, das oder dort zu suchen? alle läßt, Betätigung nur eine Begleiterscheinung oder geistige Werkzeug eines seinem Kerne nach naturhaften Lebens? Hat kein anderes Ziel als die Erhaltung und Pflege des ein der Mensch menschlichen Kreises, wie er im unmittelbaren Dasein vorliegt, oder gewinnt das Menschenleben einen Sinn und Wert nur durch das Teilhaben an Ordnung? niedere zu bloßmenschlichen allem einer Ist, wo gewöhnliche die und höhere Stufen und erklären damit Befinden Ansicht scheidet, das die Höhere vom Niedern her ihm zurückzuziehen, zu Höhere den Schlüssel zum Verständnis des Niedern? durch der alle vom Größten Gehalt Fassungen diese bis zum und Wert bis hindurchscheint, einzelnen Gebiete alle oder bildet das Der Gegensatz, muß das Leben im Denken wie im Handeln, Kleinsten, in überlegenen Wirklichkeit in in hinein grundver- Das wird auch für den Begriff schieden, ja entgegengesetzt gestalten. der Wirklichkeit selber gelten, und es wird sich der Idealist mit Fug und dagegen Recht dem nach sträuben, Realismus gemessen zu werden. des Wirklichkeitsbegriff Das aber geschieht, wenn die Welt des Idealismus zu einer gegebenen und gesicherten Welt nur hinzu- kommen scheint, wenn sie wie ein bloßer Zusatz und Schmuck behandelt wird; demgegenüber wird der Idealist darauf bestehen, daß seine Gedankenwelt allererst Wirklichkeit möglich mache, den Begriff einer Welt und einer und daß die sinnliche Welt einen Halt wie einen Wert nur aus jener gewinne. Dem Idealismus ergeht es hier oft ähnlich wie der Religion. So lange diese das Leben beherrschte, galt ihre Welt als die allernächste und unbestreitbare, ein Augustin Idee eines höchsten Wesens aus, überwand alle Zweifel von der und ein Thomas von Aquino nannte Erst nachdem die überirdische Welt schlechtweg das Vaterland (patria). die Stellung der Religion erschüttert und ihr Inhalt verblaßt war, konnte bei ihr der Gedanke der Jenseitigkeit und der Transzendenz in ist, den Vordergrund treten. Wie sie in Wahrheit schon aufgegeben wo sie in erster Linie als transzendent erscheint, so ist auch die Sache des Idealismus schon verloren gegeben, wenn seine Welt eine ferne schließende als und fremde, durch mühsame Gedankengänge erst zu ergilt. Den Gegensatz aber in solcher Schärfe denken, das heißt alle Zum Grundbegriff des Geisteslebens. 72 Vermittlung zur Unmöglichkeit machen, auch den sog. Realidealismus, falls er eine solche bedeuten will. Wohl mag, ja muß der Idealist anzueignen streben, auf die sich der Realist stützt, sich die Tatsachen und dieser wird das in umgekehrter Richtung tun. Beide aber tun es, indem sie den Tatbestand von ihrer Überzeugung aus neu beleuchten ein und umgestalten, das aber ist weniger ein Ausgleichen als Vertiefen des Gegensatzes. Der Realismus des 19. Jahrhunderts. a. Zu einer Untersuchung des Problems drängt zwingend die Tatsache, daß der Verlauf des neue Phase gebracht hat. Aufgaben vornehmlich tat es die neuere in überkommene Kultur hatte 19. Jahrhunderts , alten Streit in eine der Richtung des Idealismus; Lebensführung, religiöse dahin bis den Bis dahin suchte das Kulturleben seine die Lebensarbeit vor allem aber auch die vornehmlich von innen her geführt und den Forderungen des Denkens die äußeren Verhältnisse zu unterwerfen gesucht. Es fehlte dabei nie an einer Gegen- wirkung realistischer Art, aber sie bot weniger eine charakteristische Gestaltung des Ganzen als sie ein zäher Widerstand der Individuen war, die von den Freuden und Leiden der sinnlichen Welt viel zu wurden, um sich zur geforderten Höhe des Lebens stark festgehalten aufschwingen und auf ihr halten zu können. wirkung von sie Eine solche Gegen- Mochte bemessene Grenzen. lauter Kleinkräften hatte unablässig zur Abbröcklung und Herabdrückung wirken, keines- wegs war sie im stände, den Idealismus durch Entgegenhaltung eines neuen Lebenssystems bis zum Grunde zu erschüttern. es, was der Realismus des 19. Jahrhunderts Das aber ist unternommen hat; die seine Meinung, kann alle Ziele der Menschheit nächste Welt, so ist aufnehmen und alle Wünsche erfüllen, ohne sie kömmliche Fassung des Idealismus herabzustimmen. gegen die her- Solches Unter- nehmen ist mehr als eine andere Deutung, eine neue Zurechtlegung des überkommenen Tatbestandes, es schöpft seine Kraft vornehmlich aus der Tatsache, Menschheit Realismus er das daß die mehr geworden Welt des unmittelbaren ist als je zuvor. Nur Daseins der weil der neue dem Idealismus eine neue Wirklichkeit entgegenhält, kann Denken und Streben der Menschheit zu gewinnen So stoßen hier nicht eine Bestätigung der sowohl Lehren als Wirklichkeiten hoffen. zusammen, Behauptung des vorigen Abschnittes, daß der Idealismus — Realismus. 73 Kampf der Geister nicht sowohl auf die Deutung als auf die Ge- staltung des Tatbestandes geht. Zur Steigerung der nächsten Wirklichkeit verbinden sich im mannigfachsten Bewegungen. Weit tiefer hat uns 19. Jahrhundert die die Natur in ihr Gewebe einblicken lassen, weit mehr beschäftigt sie das Tun und beherrscht sie das Denken; den Gewinn des Wissens aber verwandelt das technische Geschick alsbald in einen Gewinn für das Leben und bringt diesem die erfreulichste Bereicherung, Beschleunigung und Kräftigung; ein staunenswertes Wachstum menschlichen Vermögens treibt mehr und mehr das starre Schicksal aus der Welt und nimmt selbst den Widerständen ihre Bitterkeit, indem Über- es sie in einen Antrieb zur Tätigkeit, eine Aufforderung zur windung Zugleich eröffnet das menschliche Zusammen- verwandelt. sein größere und größere Aufgaben. uns, wie viel die Gestaltung des Immer mehr überzeugen wir gemeinsamen Lebens bedeutet, und dem vorgefundenen Stande eine erhebliche Steigerung möglich, eine Hebung der Wohlfahrt und ein allgemeineres Glück erreichbar ist. Wie innerhalb der Staaten die einzelnen Kräfte wie gegenüber hier zu einer freieren und volleren Betätigung gelangen, so findet zugleich und Unterscheidende der Völker eine bereitwillige läßt Gesinnungen und das Eigentümlich Anerkennung, die Ausbildung nationaler Art Auf wirtschaftlichem Gebiet trifft das Streben nach Kräfte erstarken. gleichmäßigerer Verteilung der Güter mit schweren Verwicklungen der aus technischen Gestaltung der Arbeit unermeßliche Leidenschaften; die Macht zusammen und erzeugt der materiellen Lebens- bedingungen gelangt nun zuerst zu deutlicher Anschauung und voller Würdigung; scheint an steigert sich auch die innere Lage Probleme zu diesem wie hängen. das Glück Das alles des Lebens ergänzt gegenseitig, die Erfolge wie die Probleme dieses und neuen Lebens schmieden den Menschen immer fester an die unmittelbare Welt. In solchen Leistungen wächst auch der Träger der Arbeit, die Menschheit, und zwar die Menschheit wie wie eine Gedankenwelt in neuem Bilde verklärte. sie sie leibt und lebt, nicht Geschichte und Gesellschaft wirken dahin zusammen. Im Nebeneinander wie im Nacheinander rücken die Kräfte einander näher, verbinden sich zu gemeinsamem Werk und gewinnen das Bewußtsein einer durch- gängigen heit, wie Solidarität. So steht vor uns in großen Zügen die Mensch- sie sonst zerstreute Zusammenhängen einfügt, Kräfte verbindet, den Einzelnen festen das Vermögen des Ganzen unermeßlich Zum Grundbegriff des Geisteslebens. 74 So kann steigert. zum Gegenstand sie der Verehrung und des Glaubens werden, so kann sie alle praktische und ethische Betätigung des Menschen an sich zu ziehen suchen. muß alle einzelnen Gebiete, Diese neue Denkweise und Wissenschaft, eigentümlich Kunst aller Tätigkeit gestalten, Berührung mit den Dingen Ablösung von ihnen, Gewebe, alles wie scheint die und idealistischen Nur die zu führen, während ein Sicheinspinnen der Seele in ihr eigenes Streben schattenhaft, matt und unwahr macht. treibt; solchen in B. aber menschlichen Kräfte von Wandlungen scheinen Lebensgestaltungen So ist Bewegung es das Verlangen nach echter Wirklichkeit, das hier alle trägt z. gebietet den engsten Anschluß an die Welt um uns. blasser Möglichkeit zu lebendiger Wirklichkeit die sie älteren, alle alle wie Nebelgebilde der siegreichen Klarheit eines neuen Tages zu weichen. ß. Ist Die Schranken des neuen Realismus. das eigene Schicksal nicht alle Schatten und die Wendung Daß die Sache nicht ganz einfach liegt, zeigt der realistischen Lebenswelle. Gewiß hat sie das Licht dieses Tages ohne ohne allen Zweifel? nur die menschliche Meinung überwältigend fortgerissen, sie hat auch die Arbeit ein gewaltiges Stück gefördert, sie hat in unser Dasein einen rascheren Fluß, ein mannhafteres Ringen mit den Widerständen, mehr siegreiches Vordringen gegen die Unvernunft gebracht. erzeugt, und Aber zugleich hat das Anschwellen der Bewegung Probleme die den vom Realismus abgesteckten Kreis überschreiten die Selbständigkeit dieses Kreises gefährden. Zur ausschließ- lichen Wirklichkeit des Menschen konnte das realistische System nur werden, wenn sich Verwicklungen durch den eignen Fortgang alle wenn alle selbständige Innerlichkeit mehr mehr verschwunden, und der Mensch ganz und gar in ein Werkzeug der Arbeit verwandelt wäre. Statt dessen hat jener Fort- der Arbeit gelöst hätten, und gang deutlich gezeigt, Arbeit aufgeht. daß der Mensch keineswegs in die Zunächst hat er die Arbeit immer mehr in bloße einen harten Kampf ums Dasein verwandelt, in einen Kampf der Individuen, Stände und Völker; die Gegensätze sind immer schroffer, die Schlachtlinien immer breiter geworden. verraten deutlich genug, Die Leidenschaften dieses Kampfes daß hinter der Arbeit empfindende und glücksdurstige Wesen stehen, die von der Arbeit etwas begehren und verlangen, sei es selbst auf Kosten der Arbeit. Läßt sich den daraus Idealismus — Realismus. 75 erwachsenden Gefahren begegnen, ohne die Gesinnung anzurufen, d. h. eine Größe, für die strenger Realismus keinen Platz hat? ein Es reichen aber die Verwicklungen über den Zusammenstoß der dem Wesen der immer nur einen Teil der menschlichen Kräfte, sie entwickelt einen immer kleineren Teil, je feiner und verzweigter sie wird; immer geringer wird der Bruchteil des Ganzen, den das Individuum zu umspannen arbeitenden vermag. Leben wirkliche ist folgerichtig Menschen an nicht in nimmt aber sie jenen Umgebung zuerkennen Verzicht als einen Verlust mehr in ihm, als darf. da ihm der besteht; nicht gleichgültig und Schmerz. Also der Realismus ihm zuerkennt Weiter die Arbeit bindet den die Leistung, ihr gilt alle Kraft als verloren, die sich Leistung nach außen, kann eigenen entwickelt gleichgültig sein, Berührung mit der der in Mensch augenscheinlich und Die Arbeit müßte dem Realismus sondern er empfindet ihn hin, scheinen sie verbunden. Solches Liegenlassen von Kräften, solcher Verzicht auf den ganzen Menschen alles hinaus, Kräfte untrennbar Arbeit umsetzt. macht einen solchen Stand Streben nach Leistung, Damit aber richtet sie Sinnen alles gleichgültig gegen den Stand der Seele, sie Erfolg den Menschen aufsaugen und überhaupt nicht gelten lassen. Das und Anerkennung muß immer mehr alles selbständige Seelenleben unter- Aber freuen drücken, es hat es in Wirklichkeit sehr zurückgedrängt. können wir uns solcher Zurückdrängung wir eine peinliche Leere; ja nicht, vielmehr empfinden wo aber eine solche Empfindung erwacht, da verfliegt alsbald die Befriedigung an der Arbeit, und uns mit allen ihren Erfolgen in eine seelische Ferne. sie rückt Beim Ganzen der Menschheit entspricht jener völligen Verwandlung des Daseins in Arbeit ein Verblassen eines geistigen Lebensgehaltes; mehr gemeinsame Ideen und Überzeugungen lich wo nicht die Menschheit inner- zusammenhalten, da entschwindet immer mehr eine gemeinsame Gedankenwelt. Darauf aber scheinen wir nicht so leicht und nicht ohne schweren Schaden verzichten zu können, denn damit hängt alles zusammen, was unserem Leben einen und eine Seele gibt. Das sind nicht bloße Erwägungen Selbstwert, eine Größe grüblerischer Reflexion, es sind unbestreitbare Erlebnisse und Erfahrungen der modernen Menschheit Oder kann jemand leugnen, daß alle glänzenden Triumphe der Arbeit ein Aufkommen und Vordringen einer tiefen Unzufriedenheit, einer pessimistischen Lebensstimmung nicht verhindert haben? Zum Grundbegriff des Geisteslebens. 76 Das 19. Jahrhundert hat den Anblick der Welt und die Lage des Menschen so gefördert, wie kein anderes Jahrhundert; so ließ sein Schluß ein stolzes und freudiges Kraftgefühl erwarten. Wenn der wirkliche Anblick der Dinge ein völlig anderer ist, so enthält gewiß die Rechnung einen die realistische Lebensbewegung die Seele nicht eliminieren läßt; sich Fehler. die Seele eliminieren wollte, und der Versuch der Verneinung wieder stärker hervorgetrieben. selbst hat die Seele überkommenen Formen des Idealismus. Kritik der y. Dieser aber dürfte darin bestehen, daß Solche Erfahrung zwingt, die ganze Frage zu revidieren und Recht und Unrecht bei den streitenden Parteien möglichst zu scheiden. Das Verlangen nach Lebens voller Wirklichkeit des soviel Macht erlangt, kundet, wenn nicht die überkommenen idealistischen eine solche Wirklichkeit hätten vermissen lassen. in Wahrheit: jene Arten eine religiöse, be- sie Lebensformen So aber stand des Idealismus wurzelten im eignen Wesen des Menschen. nehmlich zwei: hätte schwerlich wie das Vordringen des Realismus nicht es mehr fest Es waren aber dieser Arten vor- die zu uns vom Christentum durch seine verschiedenen Gestaltungen wirkt, und eine künstlerische, vom oft unterdrückten, so doch Griechentum her einen wenn auch die nie ganz versiegten Lebensstrom bildet. Die religiöse Lebensgestaltung mit ihrer Begründung des menschlichen Daseins auf eine überweltliche Ordnung, ihrer Erhebung von der Zeit zur Ewigkeit, von allem Außenleben zu einer reinen Innerlichkeit behauptet Macht; auch trotz aller Schwächung immer noch eine große wo sie abgelehnt wird, wirkt sie im Verborgenen fort. Nähe aber und ihre sichere Überzeugungskraft hat Menschen verloren. Sie hat sie verloren schon deshalb, weil zwischen der überkommenen Gestalt der Religion und der modernen Gedankenwelt eine tiefe Kluft entstand; selbst wer diese Kluft überbrücken zu können hofft, der hat nicht mehr Ihre sie seelische für den modernen die Unmittelbarkeit und die volle Gewißheit des alten Glaubens. Wenn aber die Religion nicht das Allergewisseste leicht zum ist, so wird sie Allerungewissesten. Mehr noch hat die Religion dadurch an Macht verloren, daß dem Menschen der Neuzeit nicht mehr in derselben Weise aus eigenen Erfahrungen hervorquillt wie dem Christen der alten Zeit. Dieser Zeit entsprang die Wendung zur Religion aus stärkster sie Idealismus — Realismus. Empfindung menschlicher Ohnmacht, aus und windlicher Schranken nur Lebens; ort des eine Rettung ließ und sicherste Welt, der schlechthin feste Standals ein Abglanz oder ein Symbol jener Welt Die Neuzeit hingegen ver- behielt das nächste Dasein einen Wert. Aufkommen wie ihre Eigentümlichkeit einem jugendlichen dankt ihr einem starken Lebenstriebe des Menschen; von da aus Kraftgefühl, verwandelt sich ihm die Welt in eine unermeßliche Aufgabe, Bearbeitung fallen nur die des geistigen Selbst er- wurde daher tieferen Gemütern, wie einem warten, diese Überwelt Augustin, die nächste So Widersprüche. starrer Wendung zu einer Überwelt 77 einer Erfahrung unüber- er Schranken eigene Entwicklung scheint deren innerlich wächst; hier endgültigen Verzichte, ihre und auch weitet sich selbst starren alle in und alle Welt zu höchster Vollendung die hier Vielleicht liegt die Sache nicht zu führen. Anhänger moderner Denkart meint, es ganz so einfach, wie der vielleicht entfaltung selbst schließlich unsere Grenzen, zur ^Empfindung bringen. der Stärke, und es wird die Kraft- unser Unvermögen ja Aber einstweilen herrscht das Bewußtsein fehlt zugleich ein eigener, ein unmittelbarer, ein Damit aber verliert sie überwältigender Antrieb zur Religion. ihre zwingende Kraft und sichere Wahrheit. Größer noch ist die Gefahr eines Unwahrwerdens beim künstlerischen Idealismus. legenen Standort Er suchte die Welt nicht von einem übersondern her, durch ein ihr selbst angehöriges Wirken zu vollenden: die Gestaltung, die in Begegnung von Innerem und Äußerem, von gebung alle Seele und Welt erfolgt, schien mit ihrer Form- Mannigfaltigkeit des Lebens zusammenzufügen, einzelnen Glieder gegeneinander abzugrenzen Ebenmaß zu verbinden. das Geistige aber aus Alle bloße Naturkraft wurde damit veredelt, dem dunklen Wirklichkeit tagesheller Schacht Mit gefördert. künstlerische Lebensform erzeugt, hat das menschliche Dasein sie der Seele verfeinert, ihre und zu harmonischem sie ein hat ebenso sich Durchbildung des Lebens gezeigt. solcher tätiges als der Möglichkeit Leistung hat zu die wie vornehmes Leben gehoben und das Gewebe unentbehrlich zur geistigen Aber ist sie stark und gehaltvoll genug, um es ganz ausfüllen zu können? Gehört nicht eine be- sondere Naturbegabung, Vermögen dazu, hier schöpferisches um den Schwerpunkt des Lebens zu finden, erwächst daraus nicht ein Aristokratismus, sich ein auch solcher der nicht nur die anderen ausschließt, sondern Ausschließung freut? Muß ferner nicht ein Zum Grundbegriff des Geisteslebens. 78 Mensch, ein Volk, eine Zeit schon um im Gestalten Großes zu müssen nicht Gestaltung fiihren zu jenes künstlerische leicht zu endlich und Großes zu Wo solche Tiefe fehlt, da bleibt können? Leben erreichen, um sie zur eine Tiefe der Seele irgend besitzen, sie an die Oberfläche gebannt, da sinkt es zu Und wenn Tändelei, einer der Fülle des Lebens stehen, in erfahren leerem Scheine herab. schweren Verwicklungen und harten Widersprüche, die unheimlichen Abgründe menschlichen Daseins des ja anerkannt voll — und dahin drängt eben die Erfahrung des Jahrhunderts, — kann da wohl die Kunst den Anspruch behaupten, werden, 19. von sich Leid in sie leicht aus Schwere zu heben, alle Kann Freude zu verwandeln? dahin neigen, jene alles sie des Licht, alles aber nicht, so mag in Unvernunft abzuschwächen und das Das aber weckt bald den Dasein möglichst ins Schöne zu malen. Widerspruch Trübe es und Wahrheitssinnes, dessen als Vertreter darf sich der Realismus fühlen. Noch augenscheinlicher ist sein Recht gegen den landläufigen inmitten aller Erschütterung und Zerreibung der Formen des Idealismus das Allgemeine der Richtung ohne es irgend näher zu bestimmen und zu begründen. der Idealismus, besonderen festhält, Ein solcher wissen, „Schöne", geben. Idealismus ohne über ihren Vollauf verständlich idealistischen Lebensformen nicht genügen; friedigt, schwärmt was dieses »Höhere« ob wie er ihn freilich für „Höheres", sei,^ irgendwelche demnach, daß dem neu das die erwachten der Realismus eifrig vertritt, ohne zu er preist das „Gute", „Wahre", Inhalt ist etwas ist ihn Rechenschaft zu überkommenen Wahrheitsdrange ebenso völlig be- eine andere Frage. * „Höher" ist als Lieblingsausdruck für eine neue, vermeintlich vornehmere Denkweise wohl namentlich in der Sturm- und Drangzeit der deutschen Literatur aufgekommen. Dann suchte mit Vorliebe die Romantik dadurch die eigenen Ziele und Begriffe von denen des Durchschnitts zu scheiden, so verwenden auch Schleiermachers Jugendschriften das Wort sehr oft. Man spricht von „höherem" Leben, „höheren" Gefühlen, „höherer" Bildung, „höherer" Sittlichkeit u. s. w., bis der Ausdruck endlich dem Spott verfiel („höherer Blödsinn"). Der soliden und klaren Denkweise Kants widerstrebte der Ausdruck gründlich. Als Feder ihm einen „höheren" Idealismus zugeschrieben hatte, bemerkt er dagegen (IV, 121 Hart.): „Bei Leibe nicht der höhere. Hohe Türme und die ihnen ähnlichen metaphysisch -großen Männer, um welche beide gemeiniglich viel Wind ist, sind nicht für mich. Mein Platz ist das fruchtbare Bathos der Erfahrung." Idealismus 79 Erörterung des Wirklichkeitsproblemes. 5. des Lebens scheint Eine Wirklichkeit durch reichbar — Realismus. ob er- Tuns mit der des Verkettung fortwährende eine sichtbaren Umgebung; dem Realismus nur Wahrheit durch eine solche Verkettung in Wirklichkeit für den Menschen entsteht, das läßt sich sehr bezweifeln. Denn nur um kann keit des Tuns mit in keiner Weise Umgebung der Hülle und Fülle, nicht aber ergibt lebnis doch erlebbare Wirklich- erlebte oder handeln, alle andere Wirklichkeit liegt außerhalb und kann ihn seines Kreises kettung ihm eine von sich es wird die Leistung sie indem erst, kümmern. ergibt Jene Ver- nun Leistungen in zum Er- damit Erlebnisse; auf eine Einheit zurück- sie des Seelenlebens umspannt wird. bezogen und von einem Ganzen Ein solches Seelenleben aber kann der Realismus mit seinen Mitteln unmöglich erklären, für sich und doch bedarf Denn selbst. er seiner aufs dringendste ihm eigentümliche Welt entwickelt die er eigenem Vermögen, er würde, eigenen Mittel angewiesen, wie alle inneren Zusammenhänge, so alle nicht aus und Lebenssysteme So zugleich sich bildet seine stillschweigende selbst auf ausschließlich als ein Ganzes seine zerstören. Voraussetzung ein Seelenleben, das und auch den Gegensatz von Subjekt und Objekt die Mannigfaltigkeit Grund solcher Voraussetzung läßt sich dartun, daß die Weltumgebung für den Menschen weit mehr bedeutet, daß er aus ihr weit mehr zu gewinnen vermag, als der DurchschnittsDann aber wird in Wahrheit der Realismus idealismus zugestand. umfaßt. von auf Erst einer' Gedankenwelt des Idealismus Lebenssystem Wird aber er ist zugleich umsäumt, ja umspannt, als überhaupt nicht möglich ohne den Idealismus. der Seele jede Selbständigkeit versagt und sie möglichst von draußen abgeleitet, so entsteht ein schreiender Wider- spruch; mag er sich leidlich verstecken schleichungen in den Begriffen und Betrachten Realismus. Es lassen, unablässige Er- Lehren verraten ihn bald. wir das System Comtes, des größten Denkers des ist Grundlegung bei der alles zu entfernen, was irgend aus den Begriffen stammt. Aber sobald es eifrigst darauf vom bedacht, Idealismus vom Entwurf zur Ausführung, von der Kritik zum Aufbau vordringt, verschiebt sich der Anblick der Sache. Je mehr nämlich jenes geschieht, desto mehr sehen wir jene an- und einer idealistischen Fassung nähern, namentlich wird nur mit Hülfe einer solchen Verwandlung fänglichen Größen sich verändern Zum Grundbegriff des Geisteslebens. 80 Überganges vom Erkennen zum Handeln der kritische Punkt des und der physische Zwang überwunden ein in moralisches Sollen So ergibt sich schließlich ein Lebensganzes, aber es ergibt verwandelt. sich nur mit ständiger Hülfe desselben Gegners, an dessen Vernichtung die Wahrheit des Lebens zu liegen schien. nun wohl eine derartige zwiespältige Welt die Bedürfnisse Sollte im besonderen das ethische Verlangen befriedigen des Geisteslebens, Wiederum mag Comte herangezogen sein, wiederum voll- können? Umschlag, nur tut er es hier in umgekehrter Richtung. zieht er einen Der Ausgangspunkt nämlich der Abschluß idealistischer, ist realist- ischer Art. Die tiefangelegte Natur des Mannes empfindet die Schäden der Zeit durchaus im Sinne des Idealismus, er nimmt sie so schwer, daß ohne ein Aufbieten ursprünglichen Schaffens, ohne eine MögErneuerung durchgängiger lichkeit Gegenwirkung alle verloren Was er aber vom Realismus her als Heilmittel bringt, scheint. genug, dürftig es Aufklärungen über die Natur, sind ist sowie Ver- der gesellschaftlichen Organisation, von denen jene änderungen in Umwälzung, jener Sieg des Das kann Guten erwartet wird. nicht geschehen ohne einen krassen Optimismus gegenüber dem Menschen, sonst würde der Widerspruch zwischen der Größe der Aufgabe und Das aber ist der Kleinheit der Mittel allzu handgreiflich werden. typisch für den Realismus: entweder er nimmt das Lebensproblem sehr flach, oder er verwickelt sich in Widersprüche, die, konsequent durchdacht, ihn zerstören. selbst Befriedigt nun wohl ein Lebens- system das Verlangen nach Wahrheit und den Durst nach Wirklichkeit, das um so widerspruchsvoller wird, je mehr es dem Gesamt- befunde des menschlichen Lebens gerecht werden will? Dieser begrifflichen war. ihrer Atmosphäre, Denn mögen hat in vom welche gänzlich dessen Gestaltungen Behauptung noch so der Kultur entspricht die Erfahrung der Die Bewegung zum Realismus erfolgte innerhalb einer Menschheit geistigen Erörterung erschüttert sein, Idealismus in dem gesättigt Besonderen die Gesamtentwicklung jahrtausendlanger Arbeit ein Allgemeineres der Denkweise, der Empfindung und Schätzung von jener Besonderheit abgelöst und es tief in das Ganze des Lebens, auch in das Innere der Seele einsickern lassen; das umfängt auch die realistische Lebens- ordnung und dient ihr zu unablässiger Ergänzung, Milderung, Berichtigung. Je mehr sich aber der Realismus zur Selbständigkeit erhebt und seiner Eigenart bewußt wird, desto gründlicher muß er — Realismus. Idealismus jene idealistischen Elemente vertreiben. 81 Aber zugleich wird eine innere Niederlage. vollzieht er dem äußeren Sieg eine Selbstverengung und Selbstzerstörung, aus Bei solcher weltgeschichtlicher Dialektik wäre der Verlauf der Sache mit voller Ruhe zu betrachten, und es könnte das gewaltige Wechselspiel der Gedankenmassen reine Freude um ein Schauspiel handelte. Aber es handelt sich um das Geschick des Menschen, um Vernunft oder Unvernunft seines Daseins, um den Gewinn oder Verlust einer Seele. wenn bereiten, es sich nur Und das läßt sich nicht so ruhig betrachten. Die Notwendigkeit eines neuen Idealismus. s. So wenig der Realismus mit digen kann, viel zu viel seiner Daseinskultur daß eine einfache Rückkehr zum als uns befrie- im Bestände des Lebens verändert, hat sich alten Idealismus möglich wäre. draußen und drinnen weit mehr Unvernunft zutage Nicht bloß ist getreten als jenem Idealismus vor Augen stand, auch die weite Aus- delyiung starrer Tatsächlichkeit und blinder Gleichgültigkeit des Weltlaufs gegen die Zwecke des Geisteslebens hat für uns eine viel zu Nähe und Gegenwart, als daß wir darüber so rasch hinwegkommen könnten als jener. Sind aber die Verwicklungen und die Widerstände so sehr gewachsen, so muß sich auch die Gegenwirkung verstärken, so muß sich der Idealismus zu größerer eindringliche Tiefe zurückverlegen und eine festere Grundlage suchen. nur das aber ihm nicht die faltung des können, wenn zur Anerkennung Er wird gelangt, daß bei Aufbringung besonderer Leistungen, nicht eine Ent- Lebens nach besonderen Richtungen, sondern die Er- reichung eines wesen- und wahrhaften Lebens schlechthin in Frage und damit überhaupt kein echtes Leben ohne eine das Dasein begründende Tiefe und ihre Aneig- steht, daß kein es Selbstleben nung gibt Zugleich aber muß eine schärfere Abhebung des Geisteslebens vom Stande des bloßen Menschen erfolgen. Der Welt der Natur und des sichtbaren Daseins, die uns mit überlegener Macht umfängt, kann die geistige Betätigung gewachsen nur werden, wenn sie eine neue Stufe der Wirklichkeit, Gesamtleben der Geistes- ein welt vertritt und aus seinen Kräften schöpfen darf. Sonst fehlt dem Idealismus ein und nur wenn eine Halt fester ein gutes bei sich selbst befindliche Geisteswelt in Recht; uns wirkt und uns zu er- füllen vermag, wird die dem Idealismus wesentliche Forderung ver- und daß die Größen und Güter der neuen Welt ständlich erfüllbar, Eucken, Grundbegriffe. 4. Aufl. 140095 6 Zum Grundbegriff des Geisteslebens. 82 Zwecken der Menschen unvergleichlich überlegen und als allen als unabhängig von allem menschlichen Mögen und Meinen, von allem menschlichen Tun und Treiben behandelt werden; nur dann wird es möglich, daß nicht daß hat. Die Menschheit nach sich Wahrheit ihre sie dern vom Menschen erhalten, son- bemißt, wie ihnen zum Maß des viel das heißt diese innerlich zerstören; wie aber heit Wahrheit sein Leben Wahren und Guten machen, über die Mensch- ist hinauszukommen, wenn das unmittelbare Dasein Wirklichkeit giltpi Durch Sache wird immer wieder die eine Frage Entscheidung fordern, ob Dasein zu dienen hat, oder ob in der in dem mensch- einem gegebenen Wendung zum Geistesleben und zugleich ein Reich von echten Gütern eine neue Art Wirklichkeit Gewinnt das Geistesleben hervorbricht. die ganze hervorbrechen und eine unser Streben lediglich all Wohlsein, der menschlichen Wohlfahrt lichen als Verdunklung und Abschwächung der alle nicht eine Überlegenheit gegen das menschliche Getriebe, so muß aller Idealismus fallen, aber mit ihm fällt läuft in völlige auch menschlichen gefährden, sicher und weit überlegen. lebens in Ideen gelangen, heit es ver- so kann alle Unzu- jene Überlegenheit anerkannt, des Weise die und Wert unseres Lebens, und Leere. Wird aber länglichkeit aller Sinn so bleibt Standes die das Geistesleben Grundtatsache aller in keiner Verwicklung Mag sich an alle Entfaltung des Geistes- unserem Bereich Kleinmenschliches anhaften und mögen nur mit Hilfe der Interessen zur Wirkung gewöhnlich mag ferner das Geistesleben auf dem Boden der Mensch- von verschwindenden Anfängen her geworden und sich sehr langsam, auch unter mannigfachen Rückfällen, fortbewegt haben, das alles gefährdet nach jener Scheidurig nicht im mindesten die Grund- des Geisteslebens; tatsache unwillige Anerkennung der ja der Widerstand der Menschen, die auch der geistigen Notwendigkeiten, der Geistigkeit, womit das menschliche Tun und Treiben zu umkleiden das alles kann nur die Überzeugung ver- stärken, daß mehr Schein sich liebt, in der Menschheit vorgeht, als aus dem Menschen des unmittelbaren Daseins stammt. Wenn daher heute auf der Höhe der geistigen Arbeit die Wage Es sei dabei das Wort Kants gegenwärtig (III, 260 Hart.): »In Ansehung der sittlichen Gesetze ist Erfahrung (leider!) die Mutter des Scheines, '- und es ist höchst verwerflich, die Gesetze über das, was ich tun soll, von demjenigen herzunehmen oder dadurch einschränken zu wollen, was getan wird." Idealismus wieder mehr sich — Realismus. 83 zum Idealismus neigt, so ist nur zu wünschen, daß solche Bewegung bleibe, sondern daß das Entweder - oder mit voller Schärfe hervor- nicht abschwächenden Vermittlungen stecken in gekehrt und die unerläßliche Umkehrung mit vollem Nachdruck gefordert werde. muß auch auch positiver Der Idealismus darf vordringender, Art sein. er darf nicht nicht Denn nur so bloß abwehrender, kann es er er muß gelingen, der bloß kritischer, wachsenden Veräußerlichung, Verflachung und Scheinhaftigkeit einer bloßen Menschenkultur eine echte Geisteskultur entgegenzusetzen und der Überwältigung und Unterdrückung siegreichen Widerstand zu leisten, womit jetzt Natur, Geschichte und Gesellschaft den Menschen bedrohen. Ohne einen Glauben an die Größe und den Wert der Menschheit feste kommen wir nicht weiter, ein solcher Glaube aber muß Grundlagen haben. 6* Zum Erkenntnisproblem. B. Denken und Erfahrung. 1. (Metaphysik.) Geschichtliches. a) I—« inige Notizen zur Terminologie seien vorangeschickt. Der Aus*~^ druck Erfahrung ist im Lauf der Zeit immer vieldeutiger geworden und zeigt auch bei den einzelnen Denkern so viele Schwank- daß ungen, er kaum einen Terminus festen Abgrenzung der eine sprachliche vorwissenschaftlichen Erfahrung von der wissenschaftlichen Erfahrung hervorgegangen. — Schon Erfahrung schaftlichen Nicht einmal bildet. alltäglichen, ist aus aller Arbeit die Stoiker bildeten den Begriff der wissen- (ejATrsipia Die jjtsö-oSixTj). Erfahrungsphilo- sophen der Neuzeit waren geneigt, durch Herabdrückung der griechischen Ausdrücke Empirie, empirisch, Empiriker zur Bezeichnung der niederen Stufe eine Scheidung herbeizuführen, auch die deutsche Schulphilosophie des 18. Jahrhunderts suchte eine »empirische oder gemeine" und eine »gelehrte" Erfahrung auseinanderzuhalten. Kant verwendet fahrungsphilosoph energisch ist empirisch in jenem Sinne, oft gegen des den 19. Comte, Jahrhunderts, „empirisme". Zu Auch und der größte Erverwahrt diese Unterscheidung nicht gelangt; lediglich die Sonderung »Empiriker« für niedere und die die aus der kantischen Philosophie gedrungen von „Empirist« für die höhere Stufe, stammen dürfte, kann als durch- gelten. Bedeutender a priori sich allgemeiner Geltung aber ist die Geschichte der hierher gehörigen Ausdrücke und a posteriori; ihre Wandlungen spiegeln die Hauptphasen des Erkenntnisstrebens und erstrecken Wirkungen bis in die Gegenwart. — Entsprungen fahren, sind die Ausdrücke dem aristotelischen Ver- das Allgemeine als das (begrifflich) Frühere, das Einzelne Denken und Erfahrung. als einen festen Sprachgebrauch bildete das Spätere zu bezeichnen; Höhe des Mittelalters. Ex prioribus beweisen, einem Albert dem Großen von den Gründen her, ex daraus aber das 85 heißt erst die posterioribus von den Folgen her beweisen; a priori und a posteriori in Bedeutung erwähnt Prantl gleicher Abendlande IV, 78) zuerst bei Albert von Sachsen, einem Gelehrten Diese des 14. Jahrhunderts. bis die Neuzeit,^ in dem Ende im (Geschichte der Logik sie ist Bedeutung behielten die Ausdrücke auch heute noch nicht erloschen. Mit gemäß der stärkeren des 17. Jahrhunderts aber beginnt, Hervorkehrung der Frage nach dem Ursprünge der Erkenntnis, eine Verschiebung von der Methodenlehre zur Erkenntnislehre. A priori So vor- heißt nun, was aus der Vernunft, a posteriori, was aus der Erfahrung stammt. Diese Unterscheidung nehmlich bei Leibniz. aber sich ließ relativ und flacher absolut, und tiefer verstehen. A priori erkennen bedeutete zunächst nicht mehr als ein Erkennen aus gewonnenen Einsichten vor näherer Befassung mit der schon besonderen Sache, also durch bloße Schlußfolgerung; ^ woher das Erkennen letzthin Aber schon stamme, blieb dabei unerörtert. bei Leibniz selbst und dann bei seinen Nachfolgern bezeichnet a priori auch, was von aller Erfahrung unabhängig ist und lediglich der VerBei Kant erreicht Erfahrung selbst ihm Bewegung nunft angehört.^ indem die griffen und Sätzen a priori möglich zu werden er gebraucht nicht selten diese Höhe, ihre durch ein Gefüge von Be- erst den Ausdruck in scheint. Aber auch dem laxeren Sinne. Um diese Zeit dringen die Wörter über die Schule hinaus in den allge- meinen Sprachgebrauch, zugleich gewinnt a priori eine feste deutsche ^ penser) So heißt es z. B. : seit a priori, in der sogenannten Logik von Port Royal en prouvant les effets par seit les causes, ce en demontrant au contraire les (l'art de qui s'appelle demontrer causes par les effets, ce qui s'appelle prouver a posteriori. ' quod experiundo quod vero ratiocinando nobis §435: quicquid ex iis coUigimus, So sagt z. B. Wolff (psychologia empirica § 434) : addiscimus, a posteriori cognoscere dicimur: innotescit, a priori cognoscere dicimur. quae nobis jam innotuere, cum ante ignotum esset, innotescit, adeoque idem a priori cognoscimus. ' Leibniz stellt id ratiocinando nobis der philosophie experimentale qui procede a posteriori la pure raison ou a priori entgegen (s, 778 b. Erdm.). Lambert sagt im „Neuen Organon" §639: „Wir wollen es demnach gelten lassen, daß man absolute und im strengsten Verstände nur das a priori die Erkenntnis durch heißen könne, wobei wir der Erfahrung nichts zu danken haben." Zum Erkenntnisproblem. 86 Der Bezeichnung.! Sprachgebrauch laxere moderne Empirismus, namentlich mit wenn der vor, liegt der Entwicklungslehre, Hilfe auch das a priori aus der Erfahrung abzuleiten sucht. A priori ist was nicht das einzelne Individuum sondern was dann das, Niederschlag der Erfahrung des gesamten Menschengeschlechts als ein kommt und seinem Denken bestimmte durch Vererbung an jenes Bahnen So schöpft vorschreibt. nicht der Einzelne, anderes Problem gröbliches erst Kants, ein völlig und es ist wenn man glaubt, Kant und Spencer zu denken schärfer das des absoluten a priori Mißverständnis der Sache, Darwin durch als wohl aber die Das ist jedoch Menschheit lediglich aus der Erfahrung. ein erwirbt, lernen, widerlegen ehe man Man können. sich m.it solchen sollte Problemen befaßt. Wenn schon solcher Wandel und solche Unsicherheit im Ausdruck verwickelte Probleme der Sache vermuten so stellt die Geschichte der Philosophie diese klar und deutlich vor Augen, sie jahrtausendlangen einen zeigt ^ Kampf, Dieser Kampf war aber bei schwillt. der läßt, immer gewaltiger an- aller seiner Leidenschaft wenig Als Übersetzung von a priori diente in früheren Jahrhunderten «von vornen her", das schon Luthers Tischreden vorkommt in (s. Ausgabe von Förstemann IV, 399) und sich bis ins 18. Jahrhundert erhält. Als Quelle für „von vornherein" bezeichnet Campe Lessings Ernst und erste Falk, auch ich kann den Ausdruck nicht weiter zurückverfolgen. Er bezeichnet aber den Begriff nur in dem laxeren, bloß relativen Sinne, Absolut verstanden, begegnet sich a priori mit „rein", das auch eine lange Geschichte Anaxagoras voüc xaO^apd; hat. Seit yoCiv cpif)aiv «utov (d. h. (s. Aristoteles de anima 405 a 16: (xo'vov tov vouv) tüJv ovtwv octcXoGv Eivat y.ai «[Aty^ T£ xa"i xaO-apov) dem älteren Sprachgebrauch das Einfache, Lautere, Ungemischte zum Gemisch der sinnlichen Welt. Die Neu- bedeutet es des Geistigen im Gegensatz platoniker und ihrem Gefolge das Mittelalter übertragen den Begriff auf in das Erkennen und nennen rein eine von allen sinnlichen Bildern freie Erkenntnis (s. z. B. Scotus Erigena, pura erklärt die intellectio reas versatur. stand", ihr si in de div. nat. 657 D, 658 B). Auch Descartes eine solche quae circa nullas imagines corpo- In diesem Sinne während nimmt die Wolffsche Schule „reinen Ver- den Gegensatz zu Erfahrung bildet ratio pura entspricht (s. Wolff psych, emp. § 495 „reine Vernunft" und damit dem a priori est, als : non admittimus nisi definitiones ac propositiones Auch Gottsched folgt diesem Sprachgebrauch, s. z. B. ratiocinando a priori cognitas). = Erste Gründe der gesamten Weltweisheit (1739), S. 485 reiner Verstand ohne sinnliche Vorstellungen, S. 486 reine Vernunft, == wenn sich in unsere Vernunftschlüsse keine Erfahrungssätze mit einmengen. So entspricht Kants Verwendung von reiner Vernunft dem gelehrten Sprachgebrauch. — Denken und Erfahrung. weil fruchtbar, er das Problem Stelle wo aufnahm, Man stritt darüber, woher das Erkennen Entscheidung liegt seine 87 an der nicht stamme, ob aus der Mitteilung der Dinge oder der Selbsttätigkeit Das wäre aber des Denkens. direkt zu entscheiden nur, wenn das Was des Erkennens, unser Erkenntnisbesitz, allem Zweifel enthoben wäre, nicht die Frage nach dem Woher immer wieder in die nach dem Was zurückgriffe. Dieses aber geschieht in Wahrheit. Wir sind keineswegs einig über den Tatbestand des Erkennens, sondern die grundverschiedene Bilder ein und beweisen setzen Streitenden von ihnen aus, sie beweisen damit immer nur für sich selbst, nicht für die anderen; die geschichtliche Bewegung wird Monologen, welche die Gegner nicht einen fruchtbaren Austausch in die eigene Behauptung nicht zu ermitteln Das Was des Erkennens aber hineintreibt. ohne in sondern jeden nur immer weiter bringt, ein Zurückgreifen eine Folge von ist auf die letzten Fragen, im besonderen auf dem unsere Untersuchung überall begegnet, das eine Grundproblem, das Problem, ob das Leben und Streben des Menschen lediglich die Bewegung der Natur fortsetzt, oder ob es eine neue Stufe der Wirklichkeit einführt. Im eigenen Gebiet des Erkennens aber erzeugt der über seinen Ursprung immer wieder die Frage, Streit ob neben den Einzelwissenschaften auch eine selbständige Philosophie möglich und nötig ist, so daß den in Streit auch dieses Problem mit hin- einspielt. So gewiß die Frage des Ursprunges der Erkenntnis die Arbeit der Philosophie seit Plato begleitet, der Neuzeit erlangt. erst in zuerst das Seelenleben eine leitende hat sie Das aber aus dem Grunde, weil nun und die Weltumgebung deutlich auseinander- zugleich aber die einzelnen Seiten ihr traten, zulegen Stellung und gegeneinander abzugrenzen Vermögen näher dar- hatten. Sie traten aber weiter auseinander, nicht weil ein gesteigerter Scharfsinn auf diesen Gedanken kam, sondern weil das Leben in seinem Grundbestande nach entgegengesetzten Richtungen auseinanderging. wann die durch lange Arbeit Einerseits ge- und mannigfache Erfahrungen selbst vertiefte Innerlichkeit ein so starkes Selbstgefühl, in sich um sich für den Kern der Welt erklären und den Versuch eines Aufbaues der ganzen Wirklichkeit aus selbständiger Gedankenarbeit wagen zu können; andererseits erhob sich die sinnliche Welt, unter Abwerf ung der mittelalterlichen Verschleierung, so mächtig gegenüber dem Zum Erkenntnisproblem. 88 Menschen und eine zeigte ermeßlichkeit des Lebens, daß Baues, Festigkeit des solche Un- wie aus sicherer Überlegenheit auch sie dem menschlichen Dasein und damit dem Erkennen seinen Inhalt zuzuführen schien. Ein so schroffer Gegensatz verbot alle friedliche auf einer der Seiten mußte der Schwerpunkt Vereinbarung, und liegen, je nach der Entscheidung das Erkenntnisbild sich grundverschieden gestalten. So entstanden die Systeme des Rationalismus und des Empirismus mit entgegengesetzten ihren Durchblicken der Wirklichkeit. Der Empirismus nimmt seinen Standort beim Bewußtsein des Einzelnen, mit einleuchtender Klarheit vermag er zu zeigen, wie dies Bewußt- sondern ihn langsam, von und unter Leitung der Umgebung ge- seinen Inhalt nicht fertig mitbringt, sein Eindrücken einzelnen her Die Philosophie winnt. hatte hier Bewußtsein zurückzubeziehen, nur sie nur die Erkenntnisse auf das als empirische sich in diesen Zusammenhängen halten. schließlich ' eine bloße Assoziation Psychologie konnte Das Erkennen wird hier von Empfindungen und Vorstell- ungen ohne allen inneren Zusammenhang, auf eine Durchleuchtung der Wirklichkeit wird ganz und gar verzichtet. heißen ob kann, ja hinauskommen und ein gemeinsamer bleibt bestreitbar schaft winnen das läßt, Gründen worden. bestritten dabei sich Ob das noch Wissen- über die bloßen Individuen Besitz und ist der Menschheit mit alsbald ge- triftigen Völlig anders der Rationalismus. Sein Ausgangspunkt ist die Tatsache der Wissenschaft; ihre Eigentümlichkeit präzis daß erfassen, das scheint nicht sie ihm die Überzeugung zu begründen, von außen her dem Menschen sondern nur zufallen, aus der Werkstätte selbsttätigen Denkens hervorgehen kann. lich sind nisse, anders es welche Nament- formale Eigenschaften der wissenschaftlichen Erkenntalle Ableitung von außen zu verbieten scheinen. Was kann die Quelle der ewigen und allgemeingültigen Wahr- heiten sein, die das Gebäude der Wissenschaft tragen, als die eigene Natur des Geistes? Es wird aber damit das Erkennen vornehmlich ein volles Herausarbeiten dessen, was dem Vernunftwesen von Haus aus innewohnt; das analytische Verfahren bildet den Kern der wissenschaftlichen, namentlich der philosophischen gestaltet sich die Arbeit; einem Leibniz Philosophie zu einer universalen Mathematik, welche die Voraussetzungen des Erkennens immer weiter zurückschiebt und Gleichungen mehr und mehr die umsetzen möchte. Aber wenn damit ein systematisches Gefüge der ganze Wirklichkeit in rationale Denken und Erfahrung. 89 Wissenschaft erreicht wird, so verwandelt sich zugleich die Welt mehr und mehr in von bloßen Formen und Beziehungen, die immer blasser und ärmer zu werden. So mußte ein Reich Wirklichkeit droht der Empirismus dem unermeßlichen beherrschende keine Stoffe Form, der Rationalismus aber den Formen keinen genügenden Inhalt zu geben. Kant strebte Kraft nach ganzer mit Überwindung des einer Gegensatzes, dessen beide Seiten in seiner eigenen Natur zusammen- Er gehört insofern auf die trafen. Erkennen energisch hinaushebt und über ihm aus einen macht. Aber dieser Rationalismus schlag dadurch, Selbsttätigkeit daß das Denken erzeugt, sondern gebunden Stoffes ist; rationalistische Seite, als er das bloße Assoziation der Vorstellungen die bei einen empiristischen Ein- die Erkenntnis ihr an nicht aus reiner Darbietung die eines so kann es nicht eine Welt der Dinge, sondern nur ein Reich der Erscheinungen erreichen. wandt ist auch Zusammenhang systematischen erhält Dem Empirismus ver- ein starker Tatsachensinn, der überall auf eine präzise Erfassung des Eigentümlichen und Unterscheidenden dringt, während der Rationalismus dies zu Gunsten einfacher Gedankenreihen abzu- Ebenso entschieden wie Leibniz vorwiegend schleifen geneigt war. denkt Kant quantitativ, denkt dieser in vorwiegend qualitativ, wie jener in Stufen, Das hier unternommene schiedsrichter- Gegensätzen. liche Verfahren hat nicht nur den Vorzug, das Problem systematischer zu behandeln, als je zuvor geschehen war, es hat auch das dem Menschen eigentümliche Erkenntnisvermögen mit besonderer Schärfe zu begrenzen versucht. eine neue Aber bei aller Größe der Behandlung, die Epoche für das gesamte Problem beginnt, ruft die neue Kann das Denken zugleich an eine fremde Welt gebunden werden und eine Selbstständigkeit bewahren? Verrät nicht schon der Umstand die große Antwort sofort neue Fragen und Zweifel hervor. Verwicklung der Sache, daß Kants Untersuchung nirgends umständ- wo es gilt, die Denkfunktionen und die Sinneseindrücke zusammenzubringen? Auch kann das Er- licher und gebnis des künstlicher ist als Schiedsspruches Rationalisten nicht, da, keine der Parteien befriedigen. Den weil Kants gewaltige Steigerung der Denkarbeit unvermeidlich über die Bindung an ein Ding an sich und die Be- schränkung auf ein Reich von Erscheinungen hinaustreibt, den Empiristen nicht, weil er die Frage aufwerfen kann, ja aufwerfen muß, ob jenes Flechtwerk von Formen, das nach Kant die Erfahrung erst Zum Erkenntnisproblem. 90 möglich macht, nicht umgekehrt aus ihr selbst allmählich erwachsen sei; damit aber würde auch sein Sinn ein wesentlich anderer werden. Die unsichere Lage, die in das Erkennen solcher Streit würde weit mehr empfunden werden, brächte versetzt, nicht die praktische Philosophie der Gedankenwelt eine Ergänzung wie eine Befestigung. Aber auch ihre Begründung ist nicht allem Zweifel enthoben. So kann es nicht befremden, daß die Bewegung der Philosophie über die kantische Lösung des Erkenntnisproblemes hinaustrieb, daß der Auch nun Gegensatz erst Wandlung, hierher wirkte die geistige Charakter des 19. Jahrhunderts schichtlich-gesellschaftlichen noch fernlag. ^ Ansicht war. Spannung die am ja erreichte. den meisten das Aufsteigen einer ge- bestimmt, der Wirklichkeit, wie Kant sie Dieser neuen Denkweise konnten sich beide Parteien bemächtigen und mit ihrer Hilfe zu gelungen höchste seine Die Geschichte leisten erhielt suchen, was bisher nicht dabei hier und dort ein grundverschiedenes Ansehen: dort ward sie eine einzige, von innerer Notwendigkeit getriebene Bewegung, hier ein Sichaufschichten einer Wendung endlosen Mannigfaltigkeit. Mit der wuchs der zur spekulativen Rationalismus zu jener Geschichte Konstruktion, den die Denkprozeß -durch seinen eignen Fortgang die ganze Wirklichkeit erzeugen ließ und Tatbestand allen ein Die Erfahrung bloße der Vernunft zu verwandeln suchte. fahrung sollte hier ganz Werk mehr und mehr in verschwinden. Das als 'Er- analytische Verfahren des älteren Rationalismus wich damit einem synthetischen, die Philo- sophie gestaltete sich zu einer weltumspannenden, Geschichte durchwaltenden Logik, sie zog alles namentlich die echte Erkennen an sich und ließ den Einzelwissenschaften keinerlei Selbständigkeit. Völlig Ihm bot namentlich die Entwicklungslehre naturwissenschaftlicher Art Mittel und Handhaben zu umgekehrt verfuhr der Empirismus. dem Unternehmen, allen vermeintlichen Eigenbesitz des Geistes aus der Erfahrung abzuleiten. Das Erkennen wurde hier eine wachsende Wie Kant, in Einklang mit dem älteren Rationalismus, der PrinzipienBewegung versagt, zeigt u. a. folgende Stelle aus der Vorrede zur Kritik der reinen Vernunft (III, 11 Hart.): »Nun ist Meta* lehre eine geschichtliche physik nach den Begriffen, aller Wissenschaften, die wir hier davon geben werden, die sich eine solche Vollendung, die einzige und zwar in kurzer und mit nur weniger, aber vereinigter Bemühung versprechen darf, so daß nichts für die Nachkommenschaft übrig bleibt, als in der didaktischen Manier alles nach ihren Absichten einzurichten, ohne darum den Inhalt im Zeit, mindesten vermehren zu können." Denken und Erfahrung. 91 die Umgebung, der Kampf ums Dasein sollte diese immer zweckdienlicher, immer ökonomischer gestalten; daraus sollte alles entstanden sein, was unser Denken an durchgehenden Rich- Anpassung an tungen und Formen aufweist, und was sich vom bloßen Individuum her als ein a priori ausnimmt. und Alles innere logische Gefüge der Erkenntnis weicht dabei der bloßen Tatsächlichkeit, alles Erklären dem bloßen Schildern. Philosophie, alles Hier ist kein Platz eine selbständige für Erkennen echter Art wird zur Naturwissenschaft. Nur als ein Herausheben und Zusammenstellen ihrer Hauptergebnisse kann die Philosophie sich behaupten. Die wirkliche Arbeit des 1 9. Jahrhunderts ging zwischen diesen Gegensätzen ihren Weg; begünstigte in seinen ersten Jahrzehnten das hochgespannte Selbstgefühl des Menschen und die vorwaltende Beschäftigung mit den Fragen der inneren Kultur den Rationalismus, so unterstützte die Wendung des Lebens zum sinnlichen Dasein und der unermeßliche Zustrom von Tatsächlichkeit aus Natur, Geschichte, politisch-praktischem Leben den Empirismus. Fühlte sich vorher der Mensch im Mittelpunkt der Wirklichkeit und durfte er glauben, bei sich ihre Fäden zusammenfassen und aus seiner geistigen Tätigkeit ihr anfängliches Dunkel gänzlich klären zu können, so übermannt ihn jetzt das Bewußtsein seiner verschwindenden Kleinheit; aus Zentrum in die die Wirklichkeit Peripherie verwiesen, von darf nicht er sich aus hervorzubringen, dem mehr hoffen, muß er vielmehr be- scheiden und unterwürfig ihre Eröffnung erwarten. Aber nicht nur solche Notwendigkeit, auch ein inneres Verlangen treibt den Menschen zur Hingebung an die Erfahrung. Unmittelbarkeit, als sie die der Welt in mehr Es Tatsächlichkeit, ist die Sehnsucht nach mehr mehr Reichtum des Lebens, Gedankenwelt des Rationalismus mit ihrem Einfangen ein Netz freischwebender Begriffe und Formen bot. Dies Verfahren beginnt als eine Verarmung und Verflüchtigung des Lebens empfunden zu werden; dem gegenüber wird »ein unersättliches Verlangen nach Realität die gewaltige Seele der gegenwärtigen Wissenschaft" (Dilthey).i ^ Mit Recht sagt James (Pragmat. S. 9): „Seit hundertundfünfzig Jahren scheint der Fortschritt der Wissenschaft nichts anderes zu bedeuten als eine Vergrößerung der materiellen Welt und eine stete Verminderung der Bedeutung des Menschen. Das Resultat ist die Zunahme der naturalistischen stete und der positivistischen Fühlweise." S. auch S. 8: «Niemals hat es so viele Menschen von entschieden empirischer Geistesrichtung gegeben als heutzutage." Zum Erkenntnisproblem. 92 Natürlich fehlte es nicht an Versuchen, zu vermitteln und aus- Eine Wiederaufnahme kantischer Denkweise lieferte den zugleichen. Nachweis, daß die Erfahrung, so wertvoll mag, aus eignem sie sein Vermögen nun und nimmer ein wissenschaftliches Erkennen erzeugt, daß es dazu einer fortwährenden Hilfe des Denkens bedarf. In ähnlicher Gesinnung ließ schaften Voraussetzungen fertigen vermögen, solche sich daß die einzelnen Wissen- dartun, enthalten, die sie selbst nicht zu die vielmehr über sie hinausweisen. Gegenbewegung war mehr recht- Aber eine negativer als positiver Natur, sie mochte ungelöste Rätsel jenseit der Erfahrungswelt zeigen, nicht aber eröffnete sie ihr gegenüber einen neuen Lebens- und Gedankenkreis, nicht trieb sie zu einer eigentümlichen philosophischen Betrachtungs- weise und in diesen die Philosophie einer selbständigen Philosophie; wurde Zusammenhängen lediglich eine kritische und reflektierende Umsäumung der Einzelwissenschaften, dem die Fachgelehrten die anziehendste Beschäftigung bieten mag, die aber zur Erhöhung des Geisteslebens kaum etwas beiträgt, die zugleich, beim Mangel eines beherrschenden Prinzips, zu die Subjektivität überwinden vermag. punkte nicht Ideen- und Überzeugungswelt, tausenden besessen hatte. wie die bloß individueller Stand- So entfiel eine gemeinsame Menschheit sie seit Jahr- Und das Ungeheure dieses Verlustes, die damit drohende Zersplitterung und innere Verarmung der Mensch- kam gegenüber der Freude an der massenhaft zuströmenden kaum zur Empfindung. Das kann jedoch nicht lange Denn das Verlangen nach einer zusammenhängenden so bleiben. Gedankenwelt und einer inneren Einheit des Lebens wurzelt zu tief, heit Tatsächlichkeit um sich auf die Dauer unterdrücken zu lassen; schon heute ist der Beginn einer Gegenbewegung deutlich genug. die Einzelwissenschaften selbst, indem ihr Mehr Einheit fordern eigner Ausbau sie zu einer gründlicheren Beschäftigung mit ihren Prinzipien und Voraussetzungen führt, diese aber zur Aufdeckung der Zusammenhänge mit anderen Gebieten treibt. Von den und zu einem Streben nach einem Ganzen Seiten erschallt wieder der Ruf verschiedensten nach einer Synthese. Die Synthese ist aber nicht echt, so lange die Verbindung eine bloße Zusammenstellung bleibt; zur Wurzel durchgreifen kann sie nur bei kräftiger Herausarbeitung gemeinsamer Ideen und Überzeugungen, das aber fordert einen dem Nebenein- ander überlegenen Standort, es fordert eine selbständige Philosophie. Stärker noch drängt dahin das gemeinsame Leben. Die Kehr- Denken und Erfahrung. Seite 93 der völligen Hingebung an die unmittelbare Welt, der völligen Verwandlung des Lebens in Arbeit immer weniger über- läßt sich sehen: der Mangel einer Zurückbeziehung auf eine überlegene Eindie heit, das Leben verwandelt, Besitz allererst die und damit Selbstleben in in äußeren Eindrücke, die Ungewißheit über das Ganze bei festigung im Einzelnen. Alles geistige eignen überströmender Fülle der geistige Leere bei aller und zugleich Leben Bealler Sinn und Wert unseres Daseins gerät damit in Zweifel, der Boden entschwindet dem Menschen unter den Füßen; so treibt es zwingend, wieder die auf letzten und einen Kampf Grundlagen seiner Existenz zurückzugehen um die Erhaltung einer Seele zu führen. Solche Probleme verbieten einen Abschluß bei der bloßen Erfahrung, treiben uns, neue Möglichkeiten zu suchen sie und unser Verhältnis zur Wirklichkeit gründlich zu revidieren. Zugleich aber tritt die Philosophie wieder auf den Plan, die Philosophie nicht als zur Bearbeitung der Erfahrungswelt, sondern eine bloße Gehilfin als die Trägerin eines eigenen Gedankenreiches, als eine Kraft des Schaffens und Weiterbildens. b) Das Recht einer selbständigen Philosophie. Die Frage nach der Selbständigkeit der Philosophie bilde den Beginn der Untersuchung, da ihre Beantwortung über den Gesamtanblick des Erkennens entscheidet. Aber wie ist hier zu einer Antwort Daß sich beim Erkenntnisproblem nicht unmittelbar an zu gelangen ? die Vergangenheit anknüpfen, sich nicht aufnehmen und fortführen läßt, von dort ein Faden einfach das zeigte der Überblick der welt- geschichtlichen Bewegung, und das fand sich bestätigt durch die eigen- tümliche Lage der Gegenwart; wir empfinden heute mehr die Ab- weichung als die Übereinstimmung mit früheren Leistungen, wir sehen uns von ihnen eher Wege versperrt des geistigen Lebens trieb als gewiesen. Natur und Seele weiter auseinander, sie verwehrt damit auch Die Entfaltung innerlich weiter und dem Erkennen ein un- mittelbares Zusammenfassen beider, sie zwingt es zu einer Entscheidung für das eine oder das andere. So geschah es, grundverschiedene Weltbilder erschienen und rissen die Wahrheit an sich. war stark genug, trieb es die Aber keins um das Feld gänzlich einzunehmen, immer wieder Forschung vom einen zum andern zurück. Solche Er- fahrung empfahl eine friedliche Verständigung, eine schiedsrichterliche Abgrenzung; eine solche schien am ehesten erreichbar durch Zum Erkenntnisproblem. 94 ein Anerkennen verschiedener Faktoren im Erkennen und die Zu- Das geschah durch weisung des einen hierher, des anderen dahin. von Unterscheidung kantische die Form und Lösung scheitert an der Schwierigkeit, durchaus sondern nur verschiedenen, zusammenzubringen, das von eine beiden die nicht Faktoren logische Tätigkeit So scheinen wir uns weder entscheiden, noch beides miteinander Zu solcher Erfahrung aus dem Ganzen der zu können. festhalten Aber diese verschiedenartigen Empfindung und sinnliche zu gemeinsamer Wirkung zu verknüpfen. für Stoff. UnmögUchkeit, ja Geschichte gesellen sich widerstreitende Eindrücke und Antriebe der Gegenwart. bloß mit zugleich Macht. Wir empfinden mehr und n^ehr die innere Leere eines befaßten Lebens und Denkens, der Erfahrungswelt aber umklammert uns die Erfahrung mit immer wachsender Wir wollen mehr Selbständigkeit des Denkens, aber die Ab- neigung gegen die spekulativen Systeme läßt uns bei jedem Schritte vorwärts zaudern und macht uns mißtrauisch gegen alle Metaphysik. Eine so verworrene Lage zwingt, das Problem direkt ins Auge zu fassen und in eigner Weise zu behandeln. Was ist es, von was den Menschen über die Erfahrungsund solchem Streben eine Macht verleiht? Ist es das Denken selbst, dessen Natur auf diesen Weg führt und auch die Mittel zu seiner Verfolgung gewährt? So hieß es von dieser Frage sei begonnen, welt hinausstreben alters her, es, läßt so hören wir vielfach auch heute. Das Denken, so scheint enthält Forderungen, welche die Erfahrungswelt nicht befriedigt, auf deren Befriedigung aber eine innere Notwendigkeit seines eignen Wesens ihr es bestehen heißt. So muß es jene Welt verwandeln, ja gegenüber eine neue entwerfen, da doch die eigne, innere Not- wendigkeit ihm mehr als alle Eindrucke der Umgebung gelten muß. Das wäre einfach und überzeugend, wenn nur nicht die vom Denken behauptete Notwendigkeit über das Denken hinaus gelten wollte und die von ihm entworfene Welt den Anspruch erhöbe, die eigne Wahr- heit der Dinge zu sein. Das aber geschieht, und indem es geschieht, überschreitet das Denken den eignen Bereich; das Recht dazu kann nur durch künstliche Annahmen stützen, deren Verfolgung in immer weitere Schwierigkeiten verwickelt. Wie ließe sich hier dem es Vorwurf entrinnen, daß das Denken nur menschliche Bilder in die weite Welt hineinsieht? Auch ist es ein eignes Ding mit jener vermeintlichen Denknotwendigkeit. Da sie selbst jede weitere Begründung ablehnt und ablehnen muß, so käme die Entscheidung an das Ge- Denken und Erfahrung. das Gefühl an fühl, einer unwiderstehlichen Zwanges. und fühlen, alle in schlechterdings unvermeidlich nicht Subjektive ins als Hegel denknotwendig: läßt Wirklichkeit in Bewegung umsetzen, Herbart möchte Bewegung aus der alle Wahrheit ein solcher Forderungen widerstreitende das Denken eines läßt sich In Wahrheit verfechten hervorragende Philosophen und Individuelle? direkt Unabweisbarkeit, Aber das Gefühl führt Q5 entfernen; Wirklichkeit jener feiert den Widerspruch als die treibende und erhöhende Kraft des Weltprozesses, Wessen Denknotwendigkeit dieser will ihn in keiner Weise dulden. ist die echte, welche soll uns anderen binden? nun Eine Weltaufhellung vom bloßen Denken her wäre nur unter Einer Bedingung erreichbar: das Denken müßte die ganze Wirklichkeit in sich tragen oder durch seine Bewegung erzeugen; dann wäre das Selbsterkennen des Denkens zugleich und der Welterkennen, ein Lebensprozeß hätte seine Wahrheit bei sich selbst, er brauchte sie nicht So trieb die Konsequenz der Sache bestätigt zu haben. von außen von Plotin die 'Philosophie und ihn ließ immer wieder auf diesen Weg bis Hegel mögliche Überwindung des Spaltes einzig die als Denken und Sein begrüßen. zwischen Aber wie der Absor- viel bierung der ganzen Wirklichkeit durch das Denken widersteht, und wie sich dabei die Welt in ein zu verwandeln droht, das griffe ungen des Hegeischen Systems bloßes Schattenreich formaler Be- der Neuzeit durch die Erfahr- ist viel zu deutlich geworden, um sich bald vergessen zu lassen. Wenn aber weder das Denken mit dem Sein zusammenfällt, vDn ihm ein draußen befindliches Sein erreichen läßt, so ist Denken aus überhaupt kein Erkennen möglich, freischwebenden vom im besondern kein Aufbau eines selbständigen Gedankenreiches neben noch sich der Erfahrungswelt. darauf, Alle daß das Denken Aussicht eines Gelingens in weitere Zusammenhänge beruht tritt also und damit ein anderes Verhältnis zur Wirklichkeit gewinnt; das aber tut es in von vornherein den ganzen intellektuellen Kreis des Menschen, sondern dieser wird zunächst von den Assoziationen der einzelnen Vorstellungen mit ihrem mecha- Wahrheit. nischen auf Das Denken Getriebe nicht eingenommen; das Denken mit das Gegenständliche, tischen bildet seinen inneren Gesetzen, seiner Richtung seinem synop- Umspannen der Mannigfaltigkeit gegenüber dem sukzessiven Verlaufen der Vorstellungsketten hat sich dagegen erst aufzuringen und durchzusetzen. Das aber kann es nur, und es ist überhaupt Zum Erkenntnisproblem. 96 lebendige eine neuen kommen nur Kraft Lebensstufe, die vom bloßen Damit aber wie wir ihn im des Geisteslebens, lernten. Im geistigen Wendung der gesamten Wirklichkeit, eine Tiefe entwickelt sie Ausdruck einer ein aufsteigt Seelenleben fassen Leben erkannten wir eine der Menschen im wir auf den Begriff Unterschied Stück und ein als erst und sich zugleich zu bei einem Weltleben am Geistesleben teilgewinnen heißt daher zugleich an zusammenfaßt; einem Weltleben teilgewinnen; es sind nicht Erfahrungen des bloßen Punktes, es sind Erfahrungen vom Ganzen, ungen und Wandlungen hervorgehen. die aus seinen Dies neue Leben zeigte sich auch dem Gegensatz von Subjekt und Objekt überlegen, es halbes Sein, das zu seiner Ergänzung eines dern es ist trägt in es kein ist Fremden bedürfte, son- Leben über jenen Gegensatz hinausgehoben, als volltätiges sich Beweg- den Umriß einer selbständigen Wirklichkeit und Bewegung zur vollen Durchbildung dieser Wirklichnun ist der Träger des Denkens und alles Erkenntnisstrebens, nicht der bloße Mensch und das einzelne Individuum; das Erkennen aber erscheint in neuem Lichte, wenn es strebt in seiner — keit. Dies Geistesleben weder auf sich selbst noch auf ein draußen gelegenes Sein, sondern erstwesentlich auf das Geistesleben geht, von dem es selbst umspannt wird; Welterkennen kann es nur werden, wenn das Geistesleben den Kern der Wirklichkeit bildet. Eine solche Begründung im Geistesleben und zugleich ein Weltcharakter kommt allem Wissen zu, aber es ist leicht zu ersehen, wie hier die Philosophie eine besondere Aufgabe findet. Alles Erkenntnis- streben beruht auf einem Verhältnis von Ganzem zu Ganzem. Verhältnis dies kann als eine stillschweigende Aber Voraussetzung im Hintergrunde bleiben, und die Arbeit kann sich auf einzelne Gebiete oder einzelne Beziehungen richten; es bedarf einer eignen Wissenwelche die Sache schaft, gründende Tatsache zu wie halt ihre Stellung als Ganzes behandelt, vor allem die be- voller Klarheit herausarbeitet und ihren Ge- zur umgebenden Welt zu ermitteln sucht; ist die Philosophie. So gewiß das GeistesZusammensetzung aus einzelnen Punkten, sondern ein diese Wissenschaft aber leben keine inneres Ganzes ist, so zuversichtlich läßt sich erwarten, daß sich mit der Philosophie ein neuer Weltanblick eröffnet, daß sie bei allem, was sie von den einzelnen Wissenschaften empfangen muß, ihnen auch eine selbständige Leistung entgegenzusetzen vermag und von hier aus alle dargebrachten Tatsachen von neuem in Probleme verwandelt. Denken und Erfahrung. So 97 der Angelpunkt aller philosophischen ist Betrachtung und das Axiom der Axiome die Tatsache eines weltumspannenden Geistes- Schon daß überhaupt eine neue Stufe der Wirklichkeit über lebens. die Natur hinaus anerkannt wird, verändert den Anblick der Welt und auch zeigt Geistesleben es ist es den Anspruch erheben, an sich zu Beleuchtung. Aber das der Wirklichkeit zu ihrer eignen es das Beisichselbstsein des Lebens bedeutet, gewiß Stufe sein wollen; als solche aber muß und abschließende die letzte anderer in es die Wendung muß, so gewiß Innerlichkeit, so Natur die nicht nur irgendwelches Mehr gegenüber der Natur, alles von sich aus zu verstehen Dieser Anspruch messen. aber führt und alles notwendig zur Frage, wie weit das im Menschen gegenwärtige Geistesleben solcher Aufgabe gewachsen die Widerstände wollen erwogen, die einer lichkeit zusammen sei, Überwindung geprüft ergibt sich eine Mög- aus Größe und Schranke sein, dem Menschen eigentümliche Art. zusammen stellt der Philosophie eine besondere Aufgabe Alles und eröffnet wird damit ihr einen selbständigen charakteristische Züge Weltdurchblick, empfangen, von ihre Arbeit denen hier namentlich drei herausgehoben sein mögen. Wenn die Philosophie vom Ganzen des Geisteslebens zum 1. Ganzen der Wirklichkeit strebt, eines gegebenen stellen, sie so liegt ihre Arbeit nicht innerhalb Raumes, sondern findet nicht ihre Welt, das Ganze, das sie sucht, sie hat diesen sondern sie Raum erst herzu- hat sie erst zu bilden; tritt ihr nie von außen her entgegen, es will von innen her entworfen sein, es verlangt eine Synthese schöpferischer Art. Zur Selbständigkeit wird dieses Weltbild der Philosophie namentlich dadurch getrieben, daß das von ihrer Synthese umspannte Dasein ohne Umwandlung was ist es bietet, sammenzufügen. viel nicht in sie einzugehen vermag. Denn um sich ohne weiteres zu- zu verschiedenartig, Namentlich das Zusammentreffen von Natur und Innenwelt in Einer Wirklichkeit treibt zwingend zur Umwandlung des ersten Anblicks. Schon dadurch ist namentlich der modernen Ge- dankenarbeit ein Trieb zur Metaphysik unzerstörbar eingepflanzt, daß die Neuzeit den Gegensatz gebracht hat, und daß von Natur und Seele zur vollen Klarheit dieser Gegensatz sich beim Versuch einer unmittelbaren Zusammenfassung notwendig zu einem unerträglichen Widerspruch sophischen steigert. Tätigkeit Zugleich selbst, wie ist auch das eine Sache der philo- viel von dem Umfange unseres Lebens- und Gedankenkreises in jene Synthese eingeht und zu ihrer Eucken, Grundbegriffe. 4. Aufl. 7 Zum Erkenntnisproblem. 98 Denn Gestaltung mitwirkt. nicht alles uns irgend Bekannte ist bei Auch der beherrschende Mittelpunkt, der alles um sich gruppiert und das Ganze eigentümlich gestaltet, will immer erst gewonnen sein und läßt sich an verschie- jener philosophischen Synthese gegenwärtig. denen suchen. Stellen Nachdem die Die mittelalterliche Gestaltung Umkreis der Religion ganzen gehen Zeiten weit hier Geisteslebens des unterworfen auseinander. seinen erhob die Auf- hatte, klärung das Verlangen nach einer größeren Weite der Wirklichkeit; sie fand aber eine solche in dem Nebeneinander von Natur und einzelnen Seelen, einem Nebeneinander, das sich nicht ohne Gewalt- beherrschenden samkeit einer Einheit unterwerfen Die ließ. von Kant anhebende Bewegung erzeugt den Begriff eines selbständigen Geisteslebens und macht es mit seiner geschichtlich-gesellschaftlichen zum Kern des Ganzen. Entfaltung Aber da sie das Geistesleben mehr und mehr in das bloße Denken setzt, so wird die davon um- spannte Wirklichkeit zu eng, ein Rückschlag wird unvermeidlich, und dem Gesichts- dieser Rückschlag droht das Geistesleben wieder aus kreis der Philosophie zu rücken und damit in das Wirklichkeitsbild der Aufklärung zurückzufallen; zugleich wird der Mangel eines be- herrschenden Mittelpunktes peinlich empfunden, den in Wahrheit nur ein selbständiges Geistesleben zu Daß so immer von Wirklichkeit in springende die gewähren vermag. zum Probleme neuem Punkt der Synthese liegt, Freiheit der philosophischen Arbeit; den einzelnen Wissenschaften wird deutlich zeigt bei allem sie wie wird, philosophische Synthese eingeht, viel und wo der größere die Zusammenhange mit durch die Forderung kühnen Vordringens und mutigen Vorausentwerfens zur Spekulation getrieben. Die Hilfe intellektueller Phantasie dabei unentbehrlich; was aber an Gestalten entwirft, das wird diese Phantasie sie dem Menschen machen können, ohne eben der Erfahrungswelt eindringlich nicht ist Bilder zu entlehnen, über welche die Philosophie hinausführt. Das alles ist Gefahren, voller großes Unternehmen, und sein in Freiheit Welt in eine Gefahren der aber ohne solche gibt es kein wenn die Philosophie unser ganzes Da- verwandeln möchte und uns aus einer gegebenen eigne, selbstgebildete Freiheit tragen. versetzt, Immerhin so muß sie auch die erscheint das Wagnis der Philosophie bei unserer Fassung in ganz anderer Beleuchtung als in den Systemen freischwebender Begriffskonstruktion. Denn bei uns geht das Streben vor allem auf eine Tatsache, die das Denken Denken und Erfahrung. begründet, auf die Tatsache selbst lebens; ist was ihm in das liegt, gg weltumfassenden eines als eine Tatsache ist Geistes- zu ermitteln, das aufzuweisen, nicht abzuleiten; wie es zur umgebenden Welt steht, welchen Widerstand es windung ihr in der Tatsächlichkeit, aber und wie findet, weiterzubilden selber sich das hat, es zu seiner Über- eine Frage alles ist einer Tatsächlichkeit, die nicht freilich von Zusammenfassung des Lebens, außen her zufallen kann, sondern in im Aufklimmen zu und Messen vom Ganzen zum einem Sehen Ganzen immer neu zu erringen ist. Darin steckt eine freie Tat, die sich keiner Zeit und keinem Individuum aufzwingen läßt, die damit aber keineswegs eine Sache und Ge- individuellen Beliebens schmackes wird. 2. Erst die Philosophie rechtfertigt ein Streben über ein bloßes Kennen der Dinge hinaus zu einem Erkennen. nichts anderes als ein selbstfinden, ein Hineinziehen Selbsterkennen. Erfahrungswelt mit ihrem nahme der Dinge Denn Erkennen ist das eigene Leben, in Das bietet nun und nimmer die Nebeneinander, aber auch das subjektive Seelenleben, in liche Fürsichsein, wird es nicht erreicht. sein trägt ein Sich- mit der Auf- das bloßmensch- Denn ein solches Fürsich- bloß die eigne Zuständlichkeit in die Welt hinein, es ver- menschlicht sie und daher auch ist in Vollendung seiner höchsten dem Grade nach von der kindlichen Personifizierung der Um- nur gebung verschieden, welche die Anfänge kennzeichnet. Eine Innerlichkeit, die den Dingen nicht von draußen aufgedrängt wird, sondern eignes Sein ihr ihnen erschließt, sucht selbst in Kraft die Widerstände Kampf mit phie aber zum erst im Geistesleben, das findet, das mit seiner in innere ist es, sie in sich umspannenden verwandelt und den Die Philo- gestaltet. welche diese Bewegung zur inneren Durchleuchtung, der Wirklichkeit auf sich nimmt. dem Menschen Vergeistigung der Welt wie weit Hemmungen ihnen zu einem inneren Erlebnis Verstehen solche erscheint und einer gegebenen Lage gelingt, Wie gelingen das weit eine kann, und eine andere ist Frage, aber schon die Aufwerfung des Erkenntnisproblems bekundet eine völlige bloßen Wendung und Kennen der Dinge. zerstört Alle lassen die Tatsache unangetastet, alle Befriedigung Hemmungen und bei alle einem Zweifel daß beim Menschen eine Aufhellung der Wirklichkeit beginnt; wie aber könnte er über das Ganze der Welt irgend denken, dächte er nicht aus dem Ganzen der Welt? So treibt die Bewegung zwingend über alles bloße Anordnen und Zum Erkenntnisproblem. 100 Aufschichten der Erscheinungen hinaus wäre das Erringen einer Seele; ihnen nicht irgendwie Die berufene Vertreterin dieses Verlangens nach Seele Philosophie; die ist Leben und Denken menschliche überlegen. aber zum könnten nicht als Schranken empfunden werden, selbst die Schranken sie kann Aufgabe jene der Verinner- lichung der Wirklichkeit mit besonderem Nachdruck angreifen, wo das Geistesleben mit voller Klarheit als der Träger jenes Strebens anerkannt und die ganze Weite des Daseins zu ihm in Beziehung gesetzt wird. Endlich 3. ist es die des Erkenntnisstrebens mit Philosophie, welche den Zusammenhang dem Ganzen des Geisteslebens deutlicher herausstellt, als es irgend sonst geschieht, und damit jenem Streben mehr verleiht. Sicherheit, Kraft und Bedeutung Die Philosophie bedarf jenes Lebens, weil nur sein Gehalt und seine Kraft den Stand Suchen einer fruchtlosen Reflexion in ein sicheres Schaffen erhebt bringt; das sie über und aus tastendem Leben bedarf der Philo- sophie, weil es nur durch sie seine volle Durchleuchtung, Einigung und Ursprünglichkeit erreicht. Wie die Philosophie aus dem Gesamtleben hervorgeht und sich nach seiner eigentümlichen Lage verschieden jede Vergleichung verschiedener Zeiten gestaltet, das zeigt uns und verschiedener Kulturen. Wie grundverschieden ist z. B. die Art und das Streben der Philoim indischen und im vorderasiatisch -europäischen Kulturgemäß dem Typus des Lebens hier und dort! Dort weniger eine Durchdringung und Überwindung der Welt als eine Ablösung und Befreiung von ihr, nicht eine Steigerung des Lebens, um es sophie kreise auch gegen die härtesten Widerstände durchzusetzen, sondern eine Herabstimmung, Verschwinden, dagegen ein ein eine Auflösen tiefsinnige, aller Härte, ein aber tatenlose kräftiger Lebenstrieb, Verschwimmen und Kontemplation; hier ein zähes Festhalten des Daseins gegenüber allen Widerständen, ein Zurückkehren zur Selbstbejahung aus aller durch Erschütterung und scheinbaren Vernichtung, ein Vordringen alle stellung Hemmungen zur Entwerfung neuer Welten und zur Her- neuer Lebensformen. Zugleich wird auch die Philosophie mehr Eindringen in die Welt, mehr Ringen mit ihren Widerständen, mehr Fortschreiten durch das Überwinden dieser Widerstände. Doch wir brauchen nicht in die Ferne zu schweifen, um den engen Zusammenhang des philosophischen Strebens mit dem Gesamtstande — des Geisteslebens zu gewahren; die eigne Erfahrung des 19. Jahr- Denken und Erfahrung. mit ihn zeigt Systeme freischwebender Spekulation während fortreißen, die einer zeugungskraft gewinnen? zugleich ändert hat. so schien Verlangen. Heute winzigen der statt heute die seitdem sich Über- rechte Gesamtlage und die Tiefe der Welt zu letzte der Vollbesitz dagegen des Kleinheit und Dinge, Wiederbelebung keine Weil alle Wirk- Vernunft zu verwandeln schien, so durften seine Begriffe mutigem Vordringen Welt, unwiderstehlich im Mittelpunkte der Welt; wie dieses Schaffen lichkeit in hoffen, die Grundstimmung des Lebens aufs wesentlichste verDamals fühlte sich der Mensch mit seinem geistigen die Schaffen bei unsere Väter konnten uns ganz fremdartig anmuten und auch durch sie Versuche eifrigsten Warum Klarheit. völliger Hunderts 101 beherrscht Menschen erschließen Wahrheit kein uns Empfindung der die gegenüber der kühnes zu unermeßlichen im Zentrum fühlen wir uns an der Peripherie der faßt sich das geistige Leben nicht zu einheitlichem Schaffen zusammen, heute bedrängen zugleich uns schwere Verwick- lungen im eignen Kreise des Menschen. überhaupt ein Lage wieder Kraft dafür sammeln sichtig vom Saume Behauptet sich bei solcher philosophisches Streben, Dinge der so müssen wir erst und scheinen nur langsam und vorher einigermaßen zu vordringen können. Wie aber wirkt sie auch trägt sich in Philosophie aus ein Aufstreben bloß Erzeugnis die dahin zurück. Jede dem Gesamtleben des ganzen Geisteslebens, intellektueller Geschicklichkeit, und eine Bekräftigung der gesamten schöpft, so große philosophische Leistung sondern sie ist ein kein Werk geistigen Art, auch eine Selbst- erhaltung weltumspannender Persönlichkeit. Das eben kennzeichnet wahrhaft große philosophische Leistungen, daß sich in ihnen nicht bloß eine Klärung der Begriffe, eine Erweiterung des intellektuellen Horizontes vollzieht, sondern durch ihre Arbeit eine Weiterbildung des Lebensprozesses selbst, Die Philosophie erfolgt. Dinge, sie sondern nach ihrer sie ein Wachstum der geistigen Wirklichkeit liefert keineswegs bloße Abdrücke fertiger nimmt selbst am Bilden und Bauen innersten Natur teil; so ist keineswegs eine kühle Betrachtung, Nur ein solcher Zusammenhang mit dem Gesamtleben erklärt die Stellung und Bedeutung sondern eine Sache kräftigen Lebensaffektes. der Philosophie im menschlichen Dasein, die sonst einen rätselhaften Widerspruch enthält sophie als ein Denn äußerlich angesehen, erscheint die Philo- Durcheinander von Systemen, die sich gegenseitig Zum Erkenntnisproblem. 102 und widersprechen sich ihrer in Ablehnung als Leben geistige Wirkung und ganzen scheinen, die dazu im großen aufzuheben einander mehr bei der Menschheit Zustimmung fanden. verarmen und verkümmern, wo es alle Beziehung Aber zugleich sehen wir das So zur Philosophie aufgibt. es ist am wie dürftig wird Religion; wie eng, entsagt! Der Widerspruch löst sich augenscheinlichsten der bei sie, wo sie aller Philosophie bei Anerkennung jener Ver- kettung der Philosophie mit dem Ganzen des Lebens. Denn damit wird zu ihrer Hauptleistung nicht eine Ablieferung fertiger Lehren, sondern die innere Erhöhung des Lebensprozesses, der Gewinn an und Ursprünglichkeit, die Befähigung, mehr Ganzes, mehr Wesenhaftes in den Dingen zu sehen. Diese Verbindung der Philosophie mit dem Leben ist auch Selbständigkeit mehr Inneres, geeignet, eine Auseinandergehen ihr Richtungen verschiedene in ver- machen, ohne diese einander gleichzusetzen und damit ständlich zu allgemeingültige Wahrheit aufzugeben. Das nämlich die Entscheidung auch über die Art der Philosophie, bringt was als Mittel- punkt des Lebens erklärt wird, und wie die Gestaltung, die daraus hervorgeht, in Frage, zum Ganzen der Wirklichkeit steht kommt Zunächst ob überhaupt eine Zusammenfassung zum Ganzen voll- zogen wird, oder das Leben bei einem bloßen Nebeneinander verbleibt; in diesem Falle entsteht überhaupt keine Philosophie. Versuch einer Zusammenfassung aber wird die Hauptfrage oder gehört, sichselbstseins ob wozu auch das Durchschnittsleben der Ge- das natürliche Dasein, sellschaft Beim die, ob ein ihm überlegenes Reich des Bei- Lebens mit geistigen Inhalten und Werten den des Hauptstandort des Denkens dort bilde; entsteht der Naturalismus mit seinem Empirismus, hier der Idealismus mit seiner Verfechtung eines a priori. Eine Entscheidung weitere entsteht innerhalb des Idealismus bei Beantwortung der Frage, wie das aufstrebende geistige Leben sich zu entgegensetzt. eignen Kraft den Widerständen verhält, welche die Weltlage ihm Glaubt allen der Idealismus Widerstand durch unmittelbar volle Entfaltung überwinden der und auch das scheinbar Feindliche sich ganz und gar assimilieren zu können, so entsteht ein reiner Idealismus; dieser aber wird zu spekulativer Konstruktion und zur Geringschätzung der Erfahrung neigen. hingegen der Widerstand geistige Kraft entfalten und als überwinden zu in zu groß lassen, erscheint, um sich Wo durch da wird sich der Pessimismus der Erkenntnislehre einen Skeptizismus erzeugen; Denken und Erfahrung. Denkweise diese so erklärt, im Sinne des Idealismus und gehört steckt das Ziel daher auf seine Seite, aber da sie das Leben beläßt 103 es für schlechthin sie unerreichbar unter der peinlichen Macht eines Wo aber der Widerstand wohl in fundamentalen Widerspruches. seiner Schwere anerkannt wird, aber eine Weiterbildung des Lebens trotzdem möglich scheint, die wenigstens seinen innersten Kern von Lähmung befreit, da wird sich ein Idealismus entwickeln, den man einen positiven nennen könnte; er drängt zu einer Metaphysik, jener die von schieden einer bloßen Begriffskonstruktion bleibt. Von aus würde der Wirklichkeit eindringt darf der als friedlich sich ma^cht solche unterals den Tatbestand in schwer genug mit ihren gleichwertige Möglichkeiten als miteinander Ausdruck volle genug tief nicht es können nicht damit entstehen, und und deutlichste reiner Idealismus Die Haupttypen des philosophischen Denkens, Widerständen nimmt. gelten der nicht erscheinen, ein abstrakter die hier aufs jener vertragen, der Wahrheit sondern nur eine gelten; aber zugleich Verbindung mit dem Leben augenscheinlich, daß die Entscheidung des Menschen wesentlich durch seine eigne Lage und Erfahrung wie auch durch die Arbeit und weiligen Zeit bestimmt sein wird, die Stimmung der je- und daß wir daher, so gewiß nur eine einzige Wahrheit besteht, uns schwerlich je einträchtig bei ihr zusammenfinden werden. Wir brauchen dabei nicht zu fürchten, daß solche engere Verknüpfung der Philosophie mit dem Ganzen des Lebens sie dem Wechsel und Wandel geschichtlicher Lagen preisgebe und einem zerstörenden Relativismus überliefere. Denn das würde nur geschehen, wenn das Leben geistige gesellschaftlichen Menschen wäre. lediglich Erzeugnis des geschichtlich- ein Daseins und zugleich eine Erscheinung In Wahrheit ist am bloßen geschichtlich-gesellschaftliche alle Geistigkeit nur die Entfaltung eines zeitlosen, allem bloßmenschlichen Dasein überlegenen Geisteslebens; und nur echter Art, ist teilhat. die Kultur soweit sie an nur eine Seele Es wirkt etwas Überzeitliches in jeder großen geschichtlichen Erscheinung, etwas Übermenschliches des Menschen. in jedem geistigen Aufschwung Dies Überzeitliche und Übermenschliche, mit Einem Worte das Absolute, herauszuarbeiten, dazu sonders hat einem solchen Geistesleben berufen. Gesichtskreises, sie Denn nicht kann kraft nur hat sie ist die Philosophie be- die größte Weite des der Freiheit des Denkens zu den ursprünglichen Tatsachen und am ehesten zugleich zu einer Betrachtung Zum Erkenntnisproblem. 104 sub specie aeterni vordringen; sie kann einer durchgreifenden in Umkehrung unser Leben dem bloßen Strom der Dinge entreißen und es bei sich selbst befestigen, sie Leistungen liegenden indem üben, kann eine Kritik an sie dieselben allen vor- auf den Grund- prozeß und die inneren Notwendigkeiten zurückführt und daran mißt, sowie ihnen von da aus neue Aufgaben vorhält. Mit solcher Um- kehrung der ersten Lage bringt die Philosophie nur eine durchgängige Notwendigkeit des Geisteslebens zum Ausdruck, und dient sie seiner Erhebung zur Schon in vollen Selbständigkeit und Ursprünglichkeit. dem Streben danach liegt eine Wendung des Lebens und eine Befreiung, es verändert den Anblick des Lebens wie der ganzen Wendung bedeutend Wirklichkeit. Das allein machen, daß ihre Vorhaltung absoluter Forderungen die Gering- genügt, zum Bewußtsein fügigkeit unseres Besitzes Tiefen ahnen läßt. c) diese bringt zu und uns weitere ^ Die Wendung zur Metaphysik. Die Philosophie, so sahen wir, gewinnt eine eigentümliche Auf- Erhebung über die Erfahrungswelt, gabe nur in nicht erst nachträglich, ihre Arbeit sich aber ^ sie enthält sie sie von vornherein. kommt dazu Daher hat von Haus aus eine beträchtliche Spannung; diese steigert zu einem schroffen Widerspruch durch die besonderen Er- Es sei hiezü eine Stelle des tiefsinnigen Steffensen angeführt, obschon Gedankengang sich nicht völlig mit dem unsrigen deckt. Er sagt (siehe Gesammelte Vorträge und Aufsätze S. 6): »Nicht aus sich selber, aus ihren Werken oder der besonderen Macht oder Reinheit ihrer Leidenschaft schöpft sie (d. h. die Philosophie) ihren Ruhm, sondern aus der lichten Höhe, in welcher der Gegenstand schwebt, dem sie sich ergeben hat und dessen Mitteilungen sie empfängt. Darum mag sie ohne Gefahr ihre eigene Ohnmacht bekennen und dann und wann auf eine Weile verstummen und sehr unscheinbar einhergehen; ihr altehrwürdiges Dasein bezeugt doch den Menschen das Hereinleuchten einer vollkommenen Erkenntnis in den veränderlichen Schein dieser Welt und unserer alltäglichen Denkart. Wie die irdischen sein Entfernungen zu der Tiefe des Fixsternhimmels, so verhalten sich die Begriffe und Maße der empirischen Wissenschaft zu dem Erkennen, nach welchem die gewaltigsten Überzeugungen der gemeinen Ansicht, wenn sie dieselben mit der Gewißheit vergleicht, von deren Ahnung sie ausgeht, erscheinen ihr nur als schwankende Meinungen des die Philosophie emporstrebt, Augenblicks. auftut, und Ein Standpunkt, vor dem ein so unermeßlicher Horizont sich wird seine Selbständigkeit und Unabhängigkeit schon zu behaupten wissen." Denken und Erfahrung. Die Art, wie hier das Geistes- fahrungen des menschlichen Kreises. leben durchaus widerspricht vorliegt, Widerspruch deutlich erkennt, ausweichen, entweder das Welt zu eignen der Entscheidung aufzugeben Geistesleben oder im es und zum Träger behaupten Das Geistesleben kann machen. nicht die Wirk- beherrschen und an sich ziehen ohne eine volle Selb- lichkeit nicht ständigkeit, Wesen; wer diesen seinem kann der Gegensatz zur vorliegenden Welt zu einer 105 in unserem Kreise aber bloße Begleiterscheinung, bildet es von der Natur aus eine vom geschichtlichen Dasein aus ein nachZusammenseins; das Geistes- trägliches Erzeugnis des menschlichen vom Ganzen zum Einzelnen, im nächsten Dasein ist alle Verbindung eine Zusammensetzung aus einzelnen Elementen; zum leben geht und Ursprünglichkeit, das Dasein Geistesleben gehört Selbsttätigkeit Verkettung und damit eine Gebundenheit durchgängige zeigt eine alles Wirkens; das Geistesleben gibt seine Wahrheit als zeitüberlegen, das menschliche Leben verläuft in der Zeit und Nun folgen. kann das Geistesleben Welt- als ohne auch einen eigentümlichen Weltanblick zu wirken, kraft muß ihrem Wandel uns unmöglich bei er- zeugen; müssen wir also auf einem solchen bestehen und begegnen wir dabei einem durchgängigen Widerstände der nächsten Welt, so ist Sache im die Gegensatz Steigerung der Spekulation zur Metaphysik, istischen stellt, zu durchzuführen. ihr zur Weltüberlegenheit Wie wird Gegensätzlichkeit überhaupt diese die charakter- die Züge der Philosophie kräftiger ausprägt und klarer heraus- so wird sie namentlich die Umkehrung des Weltbildes verstärken, welche in jener liegt; sie wird zugleich erkennen lassen, gegebene Welt sich nicht völlig Seins verwandeln läßt, sie dagegen Widerstand leistet; und harte aber muß Kämpfe erzeugen. Ja in das Gesamtbild unserer Welt schwere ein geschichtliches Element; nichts physik als ein zu lassen. In solches, wenn auch ist nicht die kommt damit charakteristischer für die Meta- so doch ahnen dem allen wachsen die Probleme, es wächst der Ab- das Unternehmen müßte des Lebens. Verwicklungen nicht erkennen, stand zwischen den Zielen der Sache stünde daß die eine Entfaltung eines geistigen in sondern daß Widerstand dieser das Mit solcher hinter In als ein und den Mitteln des Menschen, vermessenes Wagnis erscheinen, der Metaphysik des Denkens eine Wahrheit trägt alles Leben das Metaphysik zur deutlichen Aussprache bringt. echte Geistesleben entwickelt sich bei der Metaphysik Problem in sich, Denn alles Menschheit nicht nur in Zum Erkenntnisproblem. 106 sondern einer Überlegenheit, Welt; die Moral z. Selbsterhaltung, sondern ernem B. Weltgetriebe in Reich Das sie möglich das uns läßt muß sie im direkten Gegensatz zu sich und Interessen bereitet, sie diesen sich kleinlicher eigner ein Zu ohne Zweifel, Weltanblick. aber nicht wir Un- Verwicklungen durch einen Rückzug auf Phänomen und etwa die entfliehen Denn nicht moralische Persönlichkeit als ein sicherer Halt ergreifen. nur Zwecke sind uns zugefallen und auferlegt. moralische unmittelbare auch gehört aber Verwicklungen, schwere ergibt zur nächsten hartem Kampf dagegen ihr Reich erbauen. behaupten und haben einem Widerspruch in nicht nur etwas anderes als die natürliche selbstischer einem solchen ist der ihr notwendigen Einheit des steht solche Persönlichkeit mit Lebens und Ursprünglichkeit des Handelns in schroffem Widerspruch zu dem Nebeneinander und sie enthält unmittelbar der Gebundenheit der nächsten Welt, Weltbehauptung, eine sie hat damit selbst Dieser Weltcharakter aber wird einen Weltcharakter. dem Menschen nur mit Hülfe eines Bildes der Wirk- zur eindringlichen Gegenwart lichkeit; Behauptung von die der Gegenwart einer neuen Ordnung der Dinge, so treibt eben die Selbsterhaltung der Persönlichkeit zu einer Demnach wird Metaphysik. um die Aufrecht- der Metaphysik in erhaltung einer selbständigen Philosophie gekämpft, die Philosophie zusammen ohne den Fortgang zu jener. Die Leugnung der Metaphysik bekundet entweder, daß die Bewegung zur Philosophie bricht nicht Kraft um gegenüber den Widerständen der nächsten Weg zu verfolgen, oder daß ein flacher Optimismus genug hat, Welt ihren die Widerstände unterschätzen läßt. Mit der Aufgabe wachsen immer der Aufbau zu überwinden Aufschwung Charakter, die das wird sich hier bekommt Festhalten lassen, überschreitet, Aber wenn damit auf sondern die in allem, noch jetzt sich Hemmungen einen heroischen hier nie rein das in was den andeutenden sein. noch weiteren Spielraum gewinnt, Ganze ein bloßes Bild; durch alle Unzuläng- der Darstellung können Notwendigkeiten wirken, angesehen, Der steigern. Hülfe von Bildern angewiesen die Phantasie nie wird deshalb das lichkeit das Gedankenforderungen werden Begriffe umsetzen Umriss auch die Widerstände; was einer selbständigen Philosophie an hatte, wie freilich Ursprünglichste und Gewisseste die, unseres geistig ganzen Lebens sind; gerade jene Unzulänglichkeit der Darstellung kann die Gewißheit der Grundtatsache nur noch stärker empfinden lassen. Denken und Erfahrung. des Alle Tiefe was in Lebens hat einen symbolischen Charakter; geistigen und von ihr ursprünglich aufsteigt hier aus die ganze Wirk- Formen nur geht in die menschlichen und seelischen lichkeit trägt, unvollkommen die Leistung dieser hat nur in der Zurückbezieh- ein; ung auf jenen Grund und Wahrheit; 107 sie der Durchleuchtung von daher eine in der Unwahrheit, sobald verfällt sie davon ablöst sich So geschieht es vor allem und mehr als ein bloßes Mittel sein will. der Religion, die zur bloßen Mythologie zu sinken droht, in ihre Formen und Begriffe den auf unablässig nicht wenn geistigen Orundprozeß zurückbezogen und von da aus beseelt werden. Auch auf der Höhe der Kunst waltete oft die Empfindung, daß das Schaffen durch alles das wohl anregen und sich drücken lasse. ;,lch hindurch Darstellen habe etwas erweise, Tieferes angemessen aus- nicht aber beleben, mein Wirken und Leisten immer nur all symbolisch angesehen, und es ist mir im Grunde ziemlich gleichgiltig gewesen, ob ich Töpfe machte oder Schüsseln", so bekennt es von Überall der Wider- sich, ein Goethe (Gespräche mit Eckermann). spruch, daß der Lebensprozeß im innersten Kern über das Bloß- menschliche hinaus zu selbständiger Geistigkeit und absoluter Wahrheit gehoben wird, menschlichen Art Aufforderung, durch alle führung zu behaupten. für allen Zweifel klug wie der überall damit die Unzulänglichkeit hindurch das Notwendige Aber gegen hier Verwicklung der Aus- alle bequemer Angriffspunkt ein ist und Kleinglauben, nirgends mehr als hier werden lange die Sache von draußen her kühl sich die Geister scheiden: so und Entfaltung die Schranken seine überwinden vermag, zu Grundtatsache die festzuhalten, und daß nicht etwas fremdes hat der Zweifel ge- betrachtet wird, Überwindung wird nur möglich, wo die Aufgabe als der Kern des eignen Lebens ergriffen und als eine Sache wonnenes Spiel; seine Eine Vermittlung gibt der geistigen Selbsterhaltung betrieben wird. es zwischen solchem Entweder — Oder nicht. Wenn demnach die Metaphysik nur das Geschick alles Geisteslebens teilt, Aufgabe das selbständig sein darin, hervorzukehren, gültigkeit will, den Widerspruch damit aufzurütteln trieb einzupflanzen. hat sie eine Klarheit aller aber das Leben und ihm einen Denn indem so mit voller aus zwingenden besondere und Schärfe trägen ihm gegenüber und eine dann aber Fort- das zur geistigen Selbsterhaltung Notwendige dem Durchschnitt entwunden, durchgebildet, Gleich- inneren ihm als nicht befestigt abzulehnende Zum Erkenntnisproblem. 108 Aufgabe entgegengehalten wird, kommt eine Unzufriedenheit, eine Bewegung Unruhe, eine treibt es zu einer Emporarbeitung und weist zugleich durch innere dem Streben bestimmte Bahnen. Ziele sammenhange unserer Betrachtung was gelten, in Leben; das in die dem Zu- Metaphysik nicht als sie den Bewegungen dieser Arbeit aufs engste verflochten bedeutende Kultur hat ihre eigne Metaphysik, und Wesen aus, etwas den Bestrebungen und Erfahr- farbloser Gestalt über Wollen jene Dabei kann nach ungen der weltgeschichtlichen Arbeit schwebt, sondern innerstes Zerlegung jene jede sie spricht darin sich will sie wird mit sein, selbst hier ihr einen wesenhaften Charakter und zugleich eine lebendige Seele erringen, wird in greifen, seits ihr sich selbst zum Ideal. Die Metaphysik muß einerseits er- was als vorwaltende Kraft eine Kultur durchdringt, anderer- hebt sie das Ergriffene über Standes hinaus zu öffnet sie von sie hier Bloßmenschliche, alle Schranken des vorgefundenen und absoluter Giltigkeit, vollendeter Gestalt aus Kampf gegen einen alles Unzulängliche, Niedere der üblichen Lebensführung, eine energische Scheidung in ein Für oder Wider. er- bewirkt sie So vollzog die platonische Ideenlehre eine Erhebung der künstlerischen Weltanschau- ung des Griechentums ins Metaphysische, wobei wandelbaren Ewigkeit voransteht, so Aufklärung eine erhielt und die Gedankenwelt der im System Leibnizens mit metaphysische Gestalt seiner Unendlichkeit des Kleinen die Idee einer un- seiner Verwandlung der Philo- sophie in eine universale Mathematik. Überall ein Bestreben, vom Gipfelpunkt menschlicher Leistung ins Absolute vorzudringen und in Umkehrung der nächsten Lage solche sie ist ein Herausarbeiten dessen, was von den be- sonderen Zeitlagen aus an zeitloser Wahrheit erreichbar war; sich dabei ergab, das versank nicht mit der Zeit, wenigstens Aus als Möglichkeit und Aufforderung, solcher Fassung Schon der Name konnte was sondern es bleibt, stets gegenwärtig. der Metaphysik läßt sich ganz wohl den Angriffen begegnen, mit denen * eine Die Metaphysik wird durch Beziehung zur Geschichte keine Hingebung an die bloße sondern Zeit, Denken und Sein unserem Selbständigkeit geistiger Art zu erringen. sie von alters leicht Vorurteile her zu kämpfen hatte. erwecken,^ aber auch in Der Ausdruck Metaphysik entstand dadurch, daß Andronikus Rhodius, in seiner Anordnung der aristotelischen Schriften ein Zeitgenosse Ciceros, die Untersuchungen über die „erste Philosophie" (rptÖTT) cp(Xoao9(a) hinter die Physik stellte: [jL£Ta xa tpuatzä (näheres s. Bonitz, Kommentar zur aristotelischen Denken und Erfahrung. der Sache wird die Metaphysik einen anderen jetzt müssen, als ihn frühere Zeiten versuchten. Bruch mit Weg einschlagen Es gilt einen entschiedenen freischwebenden Spekulation, jener 109 welche aus bloßem Denken glaubte, eine neue Welt erzeugen zu können. welche im Wissen jener älteren Denkweise, gehalt des Lebens glaubte und ermitteln Das entspricht den gesamten Geistesihn von hier aus den übrigen Gebieten zuführen zu können, während wir jetzt das Wissen ein in tiefer gegründetes Geistesleben hineinstellen und es vereint um die Entfaltung dieses Lebens und zu- mit den übrigen Gebieten um Wahrheit kämpfen lassen. Besonders aber bildet die neue Metaphysik den schärfsten Gegensatz zum ontologischen und damit gleich und dogmatischen Charakter der Meta- zugleich abstrakten physik. Die Art, wie Aristoteles die Aufgabe der »ersten Philosophie" dahin bestimmt 6v), hat, älteren das Seiende als Seiendes zu betrachten (to ov Eigenschaften die allgemeinsten des Seins zu ermitteln, hat •jl von vornherein die Sache in eine erschienen damit der Dinge und wurden zum Hauptgerüst, als eine gewisse Bahn schiefe formale gelenkt. Beschaffenheiten als Denn es das Wesen in das alle Besonderheit bloße Ausführung einzutragen war; die Metaphysik wurde Metaphysik, S. 3 ff.). Daraus wurde schon eine Bezeichnung der Disziplin selbst (xä -paYp.aT£ta). im [ista ersten Jahrhundert n. Chr. Ta tpnaixä, tj puta xa «puaixi Die Singularform metaphysica gehört der Scholastik an, sie dürfte aus der Übersetzung des Averroes stammen. Der Name war insofern nicht von Anfang an den Begriff mit der Vorstellung behaftete, als habe die Metaphysik mit etwas Femliegendem, Jenseitigem, zur nächsten Wirklichkeit erst Hinzugedachtem zu tun. So heißt es schon bei dem Neuplatoniker Herennius (s. Brandis, Abh. der Berl. Akad. 1831, p. 80): {j-et« xa glücklich, cpuatxa als er XEyovTat, ä~tp cpuasoj; Ü7:£pf,pTai xai jjTup atriav za\ Xdyov slaiv. Auch den Scholastikern, z. B. Thomas, gilt die metaphysica soviel als transphysica. Kant aber sagt (VIII, 576 Hart.): „Der alte Name dieser Wissenschaft [xera Ta »uitxa gibt schon eine Anzeige auf die Gattung von Erkenntnis, worauf die Absicht mit derselben gerichtet war. Man will vermittelst ihrer über alle Gegenstände möglicher Erfahrung (trans physicam) hinausgehen, um womöglich das zu erkennen, was schlechterdings kein Gegenstand derselben sein kann." Die Freunde der Metaphysik strebten dem gegenüber nach neuen Bezeichnungen. Clauberg, der bedeutendste Cartesianer Deutschlands, emp- Ontosophie oder Ontologie, aber bald übertrug sich die Ungunst auf das neue Wort; schon Wolff klagte (s. Philos. prima sive ontologia 1): vix aliud hodie contemtius est nomen quam Ontologiae. Auch würde Ontologie fahl nur die ältere Art der Metaphysik bezeichnet haben, die uns heute unmöglich dünkt. Ist es übrigens nicht bemerkenswert, daß nie ein Denker ersten Ranges eine „Metaphysik" unter diesem Namen geschrieben hat? Zum Erkenntnisproblem. 110 Das ergab eine Verschiebung der Ge- damit zur bloßen Ontologie. dankenwelt ins Abstrakte und Formale, eine Zurückstellung des eigentümlichen Inhalts des Menschenlebens. Zugleich aber ergab es einen Dogmatismus, indem jene formalen Eigenschaften vor aller näheren Erfahrung und unabhängig von erkennbar dünkten und daher von der Metaphysik den allemal für Bewegung ein- aller geschichtlichen übrigen Wissensgebieten wurden. Ein solches als Wahrheiten unantastbare dogmatisches Verfahren nahm zugeführt zugleich der Metaphysik eine innere Bewegung und den anderen Wissenschaften ihre Selbständigkeit. Kein Wunder, daß jene ontologische und dog- matische Metaphysik von den verschiedensten Seiten her Widerstand Entwicklung der modernen Forschung die fand; ist möglich nur geworden unter Abschüttelung der alten Metaphysik. Aber ist die Abweisung ein Verzicht nicht einer auf alle besonderen Metaphysik der Art und jede Metaphysik, vielmehr wird Kant wohl Recht behalten mit der Überzeugung »irgend eme Metaphysik immer in der Welt gewesen und wird auch wohl ferner - ist anzutreffen darin sein" (Hart. 111, 25). wird Jedenfalls Meta- die physik, welche sich aus den Zusammenhängen unserer Betrachtung ergibt, nicht von den Vorwürfen getroffen, welche die Zerstörung wo das Erkennen selbst eine Entfaltung des Lebens in sich trägt, und wo es vor allem auf der alten Metaphysik bewirkten. die Denn hier, Durchleuchtung und Vertiefung dieses Lebens geht, Metaphysii^ das Denken und Leben nicht ins wird die Abstrakte locken, sondern es seiner eignen Tatsächlichkeit und Bestimmtheit zuführen, sie wird mit ihrem Zusammenschluß aller Mannigfaltigkeit die einzig- artige Individualität Klarheit unseres Seins und unserer Welt erst mit voller herausstellen. Alle einzelnen gesamte Gebiete wie Religion, Kunst, Größen und Aufgaben, auch Moral, werden die klägliche Farblosigkeit der üblichen Fassung erst überwinden, wenn sie inner- halb eines umfassenden Lebenszusammenhanges einen und erhalten, ein bestimmtes Ziel auch wird nur der die Wirklichkeit in jener Verdichtung festen Platz Inhalt, den zum Ganzen erschließt, über das Recht und die Bedeutung der Formen des Seins entscheiden. So ist es nicht eine Lust an allgemeinen Formeln, sondern das Ver- langen nach mehr Charakter, nach ursprünglicher Tatsächlichkeit, nach energischer Durchbildung unseres Lebenskreises, was die Forschung zur Metaphysik treibt. Ebenso sicher ist eine Metaphysik, welche den Zusammenhang Denken und Erfahrung. Hl des Erkenntnisstrebens mit einem begründenden und umfassenden dem Vorwurf eines erstarrenden Dogmatis- vor Geistesleben wahrt, Eine solche Metaphysik wird mit der weltgeschichtlichen Be- mus. wegung nehmen und zugleich selbst eine Ge- engste Fühlung die ohne damit zur bloßen Zeitgeschichte zu sinken. schichte gewinnen, Wir haben heute keine Metaphysik, und es gibt nicht wenige, die das für einen Gewinn erachten. nur, wenn stande sich befände, Aber ein Recht darauf hätten sie unsere Gedankenwelt einem vortrefflichen Zu- in wenn auch ohne Metaphysik unser Leben und Streben beherrschten, hielten und vom kleinmenschlichen Getriebe heit eine grenzenlose Zersplitterung, in ist allem Überzeugungen In befreiten. Wahr- eine klägliche Unsicherheit Überzeugungen, der Prinzipiellen feste hohe Ziele uns zusammen- Ohnmacht gegen eine das Kleinmenschliche, eine Seelenlosigkeit in überströmender äußerer Wer das ruhig zu ertragen vermag, den Fülle nicht zu verkennen. werden theoretischen Erörterungen nicht zur Metaphysik führen; alle wer 'aber eine zwingende Aufgabe darin Kulturleben und sich zu erkennt, daß auch unser einem charaktervollen Ganzen zusammenfasse gewinne zu engerer Verbindung wie eine innere Selbständigkeit zu schärferer Scheidung der Geister, der wird mit uns an der Metaphysik festhalten und für die d) alte Aufgabe neue Bahnen suchen. Der Gesamtanblick der menschlichen Erkenntnisarbeit. Die bisherigen Untersuchungen enthalten prinzipielle Überzeug- um ungen vom Erkennen, die nur entwickelt zu werden brauchen, ein eigentümliches Gesamtbild zu die Fassung des verfochtene hier schichtlich Geistesleben gilt uns zugleich unmittelbaren Stufen welche ist den es ge- befreien und strebens vollauf steigern, was neue Stufe der Wirklichkeit als eine Von ursprüngliche Tiefe gegenüber eine als Seelenleben, zusammentreffen. dem in hier Entfaltungen aller die Bedingtheit unseres anzuerkennen; ja es beiden der aus wird es möglich, zugleich Abhängigkeit nach außen menschlichen mag nun sich sonst feindselig gegenüberstand wendig Abbruch Das besonderen Geisteslebens, den Kern der Erkenntnisarbeit von zu Im überkommenen Gegensatz zu überwinden verspricht. Das gegenüber der Natur, und dem ergeben. sich Erkenntnisgegenseitig und einander not- tat. Geistesleben galt uns als volltätiges Leben, das nicht Zum Erkenntnisproblem. 112 zwischen Subjekt und Objekt verläuft, sondern den Gegensatz von Haus aus umspannt; die Aufgabe kann Seins, sondern nur in muß dann in sich selbst verschiedene Stufen der Aus- eines jenseitigen Es liegen. hier nicht in der Abbildung der eigenen Durchbildung prägung enthalten, die Bewegung zur höheren Stufe aber durch eine Notwendigkeit Ganzen gefordert werden; was des das wird irgend schon angehört, der Verwandlung So steht es erst mit beim Er- auch Bewegung liegt innerhalb des gesamten Lebens. kennen, auch seine Denn auch Tätigkeit zu seinem vollen Besitz Selbsttätigkeit. in seiner der Vorwurf, dem es zu tun hat, muß sich inner- mit halb, nicht jenseit des Geisteslebens befinden; ein gänzlich draußen Gelegenes könnte nichts erregen und nichts bewegen, es würde das Denken gar nicht berühren und könnte ihm nun und nimmer auch nur zum Probleme werden. wenn Dies geschieht nur dann, ein Gegenstand der Gedankenwelt irgend schon gegenwärtig ist, die Art wie er aber, entspricht, ja zwingender gegenwärtig Antrieb zur der Natur ist, widerspricht; dieser Weiterbildung. behauptung, die das Geistesleben Vordringen zur höheren Stufe in des Geisteslebens Widerspruch So ist eine es nicht dann wird ein Selbst- der Erkenntnisarbeit mit ihrem vollzieht. kann zum Erkenntnisproblem nur dem Lebensprozeß irgend schon einverleibt ist, so muß eine innere Erweiterung des Lebens vorangehen, wo das Erkennen in Fluß kommen soll. Die weltgeschichtliche wie die alltägliche Steht die Sache derart, so werden, was Erfahrung bestätigt diese Behauptung mit überzeugender Klarheit. Denn sie zeigt, daß auch solches, was den Menschen mit eindringlicher Nähe umfängt und mit den stärksten sinnlichen Wirkungen berührt, ihm innerlich völlig fremd bleiben kann und seinem Er- kennen nicht zum Probleme wird. der an keiten, sie Fragen stellt; Nur dem antworten die Dinge, nur dem erschließen sich Wirklich- der ihnen Möglichkeiten entgegenbringt; auch der härteste Widerstand übt eine seelische Wirkung nur nach Verwandlung innere eine Hemmung; Völker und Zeiten treffen, welcher Gegenwehr mitgebracht fertig verfechten ^ zu schwerste können Mißstände in Individuen, ohne sie stark zu erregen und zu irgendtreiben. werden, übereinstimmend Daß sondern große die geistigen erst zu Künstler Organe bilden und große sind, nicht das Erzieher. ^ Bekannt ist Herbarts Auslassung über den neunzigjährigen Dorfschul- meister (Werke X, 8): „Wollten wir nur sämtlich bedenken, daß jeder Denken und Erfahrung. Auch ja 113 die weltgeschichtliche Betrachtung zeigt, wie spät ganz Nahes, äußerlich schon Angehöriges zu eignem Leben geworden das eigne Streben bewegt hat; und Wie langsam dünkt. deckung der Natur, des Sehens zu erfolgte erschloß spät die Kunst verfeinern, künstlerische die sich z. der Gegenwart, um dann Ent- der die Gestalt B. zuerst den Dingen mehr in und neue Seiten an ihnen zu eröffnen! Auch sich entdecken das Menschsein und was daraus an Gemeinschaft, des Lebens selbst, und Empfindens fand wie wie müht sich auch Landschaft, die Art zu erkennen, was später leicht selbst- die Vorbereitungen dessen verständlich und ist läßt zugleich die Voraussetzungen sie es nicht fließt, vor, fertig der Mensch erst entdecken müssen, hat innerer Art Fortbildungen errang er Die er Bewegungen und durch es Erziehungslehre spricht von der stellungskreis Aufnahme neuer Eindrücke in den Vordes Individuums; nun wohl, es gibt auch eine welt- geschichtliche Apperzeption, auch das Ganze der Menschheit Apperzeption als einer mag, nichts aufzunehmen, dem innere es nicht eine ver- Bewegung ent- gegenbringt. Was so im Einzelnen bereitwillig anerkannt wird, das muß, ins Ganze gehoben, das Erkenntnisproblem rücken. Denn damit wird klar, Menschen liegt, daß und in alle eine neue Beleuchtung Erkenntnis innerhalb der daß Arbeitswelt des Fortschritte des Erkennens gibt ohne ein V/achstum dieser Arbeits- Wahrhaft große welt. solche, die nicht den Lebenskreis Leistungen auch des es keine wesentlichen Erkennens sind einem vorgefundenen Kreise angehören, selbst verändern. nur sondern Die moderne Wissenschaft wäre unmöglich gewesen ohne den modernen Menschen mit seiner kühnen Erhebung über die Welt und seiner Befestigung in der eignen Seele. Solche Zurückverlegung gestattet erst, den Erkenntnisprozeß als ein immanentes Vorgehen zu fassen und jenes Dilemma zu vermeiden, daß das Denken entweder mit einem fremden Sein zu tun habe oder aus sich selbst Aber eben alles Sein diese hervorspinnen müsse. Anerkennung der Selbständigkeit des Geistes- nur erfährt, was er versucht! Ein neunzigjähriger Dorfschulmeister hat die Erfahrung seines neunzigjährigen Schlendrians; er hat das Gefühl seiner langen Mühe; aber hat er auch die Kritik seiner Leistungen und seiner Methode?" Fröbel aber meint, daß der Mensch, ,,um die Natur zu verstehen, sie durch eine ihm eigentümliche Kunstweise gleichsam von Neuem in und aus sich schaffen muß." Eucken, Grundbegriffe. 4. Aufl. 8 Zum Erkenntnisproblem. 114 und der Immanenz ^ des Erkenntnisprozesses ist geeignet, das Eigentümliche der menschlichen Lage und zugleich die Bedeutung lebens der Erfahrung zur vollen Geltung zu bringen. und Je selbständiger und mit ihm das Erkennen überlegener nämlich das Geistesleben gefaßt wird, desto größer wird der Abstand der nächsten Lage, desto deutlicher erhellt, daß nur unter gewissen Bedingungen und durch harte Arbeit der Mensch daran teilzunehmen vermag, daß auch das Geistesleben ihm nur durch irgendwelche Erfahrung zugänglich wird. Der Mensch wird zunächst von der untergeistigen eingenommen, lichkeit mit mechanischen ihren könnte diese Stufe höhere die Höhere es hat erfolgt Stufe der Wirk- die intellektuell in der sinnlichen Vorstellung Verkettungen keiner Weise in zum Ausdruck kommt; Aber dieses wirksam. irgendwie er wäre nicht auch überschreiten, in seinem hat für den Lebensprozeß keine volle Gegenwart, sondern eine solche Bereiche zu erst gewinnen; selbst die Anregung dazu gewöhnlich nur unter besonderen Bedingungen, bei Verwick- lungen und Widersprüchen der niederen Stufe; die Geschichte zeigt wie mühevoll und langsam das Erkenntnisstreben deutlich, in Fluß Und eben im Vordringen mußte es auch eine eigentümliche kam. Lage beim Menschen anerkennen, die sondern nur als eine sich Tatsache hinnehmen nicht begrifflich ableiten, läßt. Insofern trägt das menschliche Erkennen einen Erfahrungscharakter. Aber das anerkennen heißt keineswegs dem Empirismus huldigen. Denn jener Erfahrungs- charakter wäre nicht zu erkennen selbst gegen die bloße Erfahrung; ohne eine Überlegenheit die Schranken des Menschen gelangen zur Einsicht nur, ständigen und überlegenen Geistesleben seine Lage zu und die Gebundenheit sofern er an einem selbteil hat und von hier aus würdigen vermag. Es hat aber die Erfahrung für das Erkennen eine zwiefache Bedeutung: sie im Innern. Jenes ist Begrenzung nach außen und sie ist Determination ist Bedingungen gebunden tätigkeit sie, wenn bleibt die geistige Tätigkeit an jenseitige und sich daher nicht zur vollen Selbst- zu erheben vermag; dieses, wenn sie die volle Bestimmtheit ihrer eignen Art erst im Zusammenstoß mit Widerständen erreicht, erst durch Versuchen und Erfahren hindurch ihrer selber inne wird und zu reiner Selbsttätigkeit gelangt. In beiderlei Bedeutung ist das Wir nehmen hier Immanenz in dem alten und ursprünglichen Sinne, wonach es ein Geschehen bezeichnet, das innerhalb des Lebensprozesses ver* bleibt, nicht über ihn hinausgeht; siehe den Artikel Immanenz-Transzendenz. Denken und Erfahrung. 115 Erkennen des Menschen auf Erfahrung angewiesen; entbehrHch sowohl gebung als für fang für seinen Inhalt. als für eigne Beschaffenheit, sowohl für seinen seine un- sie ist hier das Verhältnis des Geisteslebens zur UmUm- Was sich beim Menschen an Erkennen entwickelt, das befindet zunächst gegenüber der Unermeßlichkeit einer fremden Welt, sich und das kommt nur weiter in aus der Welt lediglich des Empfangenen arbeitung Berührung mit dieser Welt, das kann empfangen scheinen. zu kann namentlich die sinnliche Natur, sich es nie Auch für weite in der Be- wie Gebiete, davon ablösen; was daraus die Gedankenwelt des Menschen eingeht, ist nicht rein in in Gedanken- größen umzusetzen, es behält eine Bindung an etwas Jenseitiges und zugleich eine Undurchsichtigkeit. mit den Dingen Aber mag und eine Beziehung auf die hier eine Berührung Dinge noch so not- wendig sein, diese Berührung und Beziehung erzeugt nicht das Er- kennen; es entwickelt sich unter Bedingungen und Beschränkungen, aber es bleibt auch dabei vornehmlich ein Werk des Geisteslebens; es entwickelt sich nicht aus der Erfahrung, sondern nur an der Er- fahrung, wie Eindrücke nicht in die Gedankenwelt eingehen können, ohne eine wesentliche Umwandlung zu erleiden. Wie grundver- nimmt sich dasselbe Naturphänomen in der unmittelbaren Empfindung des naiven Menschen und in der Gedankenwelt des schieden Mit Recht sagt Hegel. Naturforschers aus! »Die Natur des Geistes — nicht ein anderes Ursprüngliches in sich aufzunehmen, oder es ist eine Ursache sich in ihn kontinuieren zu lassen, nicht sondern sie abzubrechen und zu verwandeln" (Wke. IV, 229). Aber nicht Geisteslebens ist Ausdehnung, nur die für uns Menschen eine Frage und eine Aufgabe. auch die innere Art des Weder erfüllt das Geistesleben in fester und klarer Gestah unmittel- noch zieht es uns bar unser eignes Leben, schritt hinein, in einen sicheren Fort- wie der intellektuelle Optimismus der konstruktiven Philosophie es annahm, sondern wir müssen von kleinen, nicht ein- mal unbestreitbaren Anfängen allmählich vordringen, und wir finden in solchem Streben die mannigfachsten Hemmungen und Gefahren, wir unternehmen vieles in froher Zuversicht, was sich im Verlauf als unmöglich erweist, hergeworfen. wir nicht wir werden scheinbar oft im Zickzack hin- und Was wir aber mit aller Mühsal erringen, das gewinnen durch kluge Überlegung, sondern durch ein zu Ende Gehen eingeschlagener Richtungen; unseres Vermögens wie unserer Zum Erkenntnisproblem. 116 Schranken werden wir inne erst durch Lebensentwicklung und Lebens- Im besonderen erfahrung. erreicht unser Leben nur durch Kampf seine volle Tiefe, erst der Widerstand treibt es zur Aufbietung seiner ganzen Kraft und Wachstum der zu voller Ursprünglichkeit. Dabei bedeutet das reinen Sieg über das Feindliche keinen Geistigkeit und bringt keine volle Aufhellung. Vielmehr mag die innere Steigerung Probleme und Widerstände hervorrufen, und neue Ansprüche, sich damit der Anblick der Wirklichkeit immer positiver, immer irrationaler muß aus dem Erkennen etwas Eine solche Tatsächlichkeit gestalten. wesentlich anderes machen, als es der Rationalismus wollte; Punkt für Punkt wird es hier auf die gewiesen. Einem glatten Erfahrungen des gesamten Lebens hin- Abschluß nahe glaubte man den ersten Anfängen, aus wachsender Einsicht sondern klarer, rätselhafter geworden. in immer mehr An- ist erkennung ungelöster Probleme hervorgegangen, die Welt nicht nur sich So ist ist uns eben auf der Höhe des modernen Lebens der Gesamtanblick des Erkennens alles Die Wirklichkeit erhebt sich vor unseren Augen eher als einfach. als ein Stufenreich mit einem Fortgang vom Unorganischen zum Organischen, vom Unlebendigen zum Lebendigen und zur Seele, von der naturgebundenen zur geisterfüllten Seele. Von jeder Stufe aus ergibt sich ein eigentümlicher Durchblick der Wirklichkeit; der Kampf wird nicht enden, ob die unterste oder die oberste Stufe den Ausgangspunkt der Erklärung zu bilden habe. Die Philosophie kann nicht umhin, die Wirklichkeiten, welche sich auf der höchsten Lebens- Offenbarungen zu behandeln und von stufe eröffnen, als die tiefsten ihnen sie, aus das Aber nun des Ganzen zu entwerfen. erfährt daß die von dort entwickelten Größen für die Welt unter uns daß diese ihnen eine starre Eigenart entgegensetzt, auch nicht passen, erfährt sie, daß diese Welt durch höhere Stufe mit Bild seinen als Begriffen ihr ganzes Wirken und Sein jene gleichgültige Nebensache behandelt. Weder noch scheint unserer eine mit seinen Kräften in Welt sich durchsetzen zu können, was wir für den Kern aller Wirklichkeit zu erachten nicht lassen können. daß an den Menschen seine geistige Überall der Widerspruch, Natur Anforderungen stellt, denen seine bloßmenschliche Art nicht zu entsprechen vermag; die geistige sein Selbsterhaltung zwingt ihn, intellektuelles Vermögen Wahrheiten zu bejahen, denen nicht voll gewachsen ist, ihren Grund- gedanken energisch zu behaupten, ohne ihm eine angemessene Ausführung geben zu können. So bewirkt es notwendig eine geistige Denken und Erfahrung. 117 Verarmung, wenn das intellektuelle Vermögen über den Gesamtinhalt des Lebens entscheiden e) soll. Würdigung des Rationalismus und des Empirismus. Nach diesen Erörterungen läßt sich eine schiedsrichterliche Würdigung der streitenden Gegner unternehmen; sie wird zeigen, daß jeder bedeutende Wahrheitsmomente gegen daß den anderen kehrt, vertritt er aber ins und sie siegreich Unrecht gerät und die eigne Stellung nicht behaupten kann, sobald er bei sich selbst einen Abschluß letzten sucht. Der Rationalismus des Selbständigkeit alle Umgebung, in hat seine Geisteslebens Stärke und der Verfechtung in der Überlegenheit gegen seiner der Verfechtung der Überzeugung, daß das Leben daß erstwesentlich nicht von außen nach innen geht, sich, um mit Plato zu reden, nicht einem Blinden von außen her Augen einsetzen' Ohne diese Überzeugung gibt es überhaupt keine Wahrheit. lassen. Denn der die Auslieferung unserer Erkenntnis an die Eindrücke volle Umgebung würde ihr alle Festigkeit, allen Zusammenhang, alle innere Durchleuchtung rauben, Individuen preisgeben. würde sie der Zufälligkeit der bloßen Es ist eine axiomatische Notwendigkeit, wenn der Rationalismus dagegen ein a priori Nur sei dabei das verficht. a priori nicht als eine in der Seele jedes Einzelnen fertig vorhandene Größe, sondern es sei als der Mensch standen, das a priori enthält ein Grundgesetz des Geisteslebens ver- sich erst die Behauptung, aneignen muß. Ein solches daß das Geistesleben Normen in sich trägt, die das Streben aus aller Irrung immer wieder zur Wahrheit lenken; es enthält auch die Behauptung, daß das Geistesleben seinem Wesen nach übergeschichtlicher Geschichte bildet. Art ist, kein bloßes Produkt der Ohne solche Übergeschichtlichkeit könnte es nie die geschichtlichen Bildungen einer überlegenen Kritik unterwerfen, sondern würde es ganz und gar ihrem Wechsel und Wandel ausgeliefert. Mit der Verfechtung so unerläßlicher Wahrheit hat der Rationalismus ein überlegenes Recht gegenüber gerät ins Unrecht, reichen zu oder doch indem er indem können, leicht dem Empirismus. er jene Wahrheiten indem er das hohe Ziel als eine gegenwärtige zugängliche Tatsache behandelt. Dies ohne weiteres das menschliche Geistesleben leben an sich, als Aber er unmittelbar glaubt er- absolutes Geistesleben behandelt geschieht, als Geistes- und damit den Zum Erkenntnisproblem. 118 auch für die Schranken Sinn für das Charakteristischmenschliche, des Menschen abstumpft; es geschieht, indem er Leistungen, die das Denken nur im Zusammenhange mit dem Ganzen eines selbständigen ihm vermag, Geisteslebens aufzubringen den Gedankengrößen selber zuweist nimmt; ihre belebende Tiefe und damit es geschieht, in- dem er unser Geistesleben ohne weiteres auf sicherem Wege glaubt und innere Verwicklungen anerkennt. keinerlei Alles zusammen gibt dem Rationalismus die Neigung, das Dunkle und Feindliche unserer Weltlage abzuschwächen und wegzuerklären, das dem Individuelle — So der Inhalt Form zu opfern. zu glatte, dünne, blutlose Wirklichkeit, das viel Denken wie das Leben hafte. den Allgemeinen, Daraus entsteht eine Formale und Schatten- gerät ins Abstrakte, zeigt es besonders anschaulich das Bild der Geschichte, Wendung zur spekulativen Begriffs- das der Rationalismus in konstruktion Die Bewegung scheint hier von vornherein erzeugt. der im Element der Vernunft befindlich, da hat; dünken hier Gegensätze und Kämpfe nur ein Mittel zur alle Steigerung der Vernunft, und in eine große nicht bloß und Mehr der Vernunft jedes auch die Unvernunft zu steigern hältnissen eine alles Irrationale scheint sich schließlich Harmonie aufzulösen, während in Wahrheit der Kampf innerhalb der Vernunft, sondern wird, führt Wahrheit den Ver- in sie und immer von neuem zu bestätigen nunftcharakter erst zu erringen mehr noch um sie gemenschlichen Ver- in pflegt; hier scheint die Epoche auf der anderen sicher fortzubauen und, was an welt- geschichtlicher Erfahrung erwächst, dauernd gesichert zu sein, da in Wahrheit der Kampf immer wieder greift, eine feste in Grundlage immer neu zu erringen der Mensch als ein reines ist, zum Probleme wird; Erfahrung geistiger Art immer neu scheint die letzten Elemente zurück- Werkzeug der und alle hier er- geistigen Arbeit, da vielmehr seine Neigung überwiegend dahin geht, das Geistesleben der natürlichen und sozialen Selbsterhaltung unterzuordnen, es damit arg zu verkehren und seinen eignen Zwecken zu entfremden. Bei Verkennung des Dunklen und Feindlichen solcher Geschichte listische ihre Kraft und Tiefe; je ausschließlicher geschichtliche Leben daß immerfort des Ganzen um nicht in desto mehr entleert und klar, daß das sicherem Fortgang aufbaut, das Ganze gekämpft, vollzogen werden die rationa- Wird dagegen Behandlung durchgeführt wird, verflüchtigt sie die Wirklichkeit. verliert jene muß, so sich sondern immerfort eine Bejahung tritt vor den Prozeß die Denken und Erfahrung. und Tat, freie es entfällt alle Möglichkeit \\g einer Kon- rationalen struktion. muß das Sichausleben des Rationalismus So in der Richtung des Empirismus mit seinem lichkeit einen Rückschlag Durst nach Tatsäch- und seiner willigen Anerkennung der Schranken des Menschen erzeugen, wie denn auch auf geschichtlichem Boden der Empirismus namentlich da zu Macht und Ansehen gelangt wo die Mängel ist, überkommenen Rationalismus augenscheinlich wurden. eines den Hintergrund des neuesten Empirismus Auch der Widerwille bildet gegen die spekulative Begriffskonstruktion. Aber der Empirismus bringt den Erfahrungscharakter unserer Gedankenwelt keineswegs zu angemessenem Ausdruck, er Prozeß der Erfahrung keit, er faßt ohne die es doch kein wissenschaftliches Erkennen gibt. alles Geistesleben selbständige Menschen aus eine den einem schroffen Gegensatz zur Selbsttätig- in Geistigkeit leugnet, muß und zugleich eine Indem vom bloßen er Erkenntnis zu Dies aber, was in Wahrheit unmöglich ist,^ kann entwickeln suchen. einen leidlichen Schein des Gelingens nur erreichen, indem es versteckterweise eine geistige Welt voraussetzt und ihr entlehnte Größen verwendet. Damit aber ergibt sich schiefer Anblick der Wirklichkeit. bis ins Einzelne hinein ein Der Empirismus richtet beim Er- kenntnisprozeß das Augenmerk gänzlich auf die Leistung und übersieht die geistige Tätigkeit, und vergißt, daß er für die darin wirkt; er haftet am Gegenstand uns etwas nur durch unser Aneignen wird; er sieht die Determination des Erkennens durch die Erfahrung, aber er sieht nicht, daß diese Determination innerhalb eines umfassenden Gedankenraumes und durch die eigne Bewegung des Geistes, durch ^ eine Mitteilung außen von erfolgt; - er ist so nicht ausschließ- Die Unmöglichkeit mit den Mitteln des Empirismus zu einer Wissen- schaft zu gelangen, ist eben neuerdings von hervorragenden Forschern nach- drücklich hervorgehoben. es einen Windelband (Präludien, 2. Aufl., S. 303) nennt „hoffnungslosen Versuch, durch eine empirische Theorie dasjenige zu begründen, was selbst die Voraussetzung jeder Theorie bildet", und Husserl L Bd S. HO) bemerkt zum gleichen Gegenstande: „Der schwerste Vorwurf, den man gegen eine Theorie der Logik erheben kann, besteht darin, daß sie gegen die evidenten Bedingungen der Möglich- (Logische Untersuchungen keit einer , Theorie überhaupt verstoße." ist der Sprachgebrauch, indem er Erfahrung und Denken einander entgegensetzt, als vermöge die Erfahrung etwas ohne das Denken zu leisten. Mit Recht bemerkt dagegen schon Robert Boyle (the - Nicht ohne Schuld daran Zum Erkenntnisproblem. 120 von der Fülle des Einzelnen eingenommen, daß lieh Zu- er ihren sammenhang wie selbstverständlich erachtet und über den Bäumen den Wald nicht sieht. Dem Empirismus scheint aus den Dingen entgegenzukommen, gelegt wie hat, was Wahrheit die Tätigkeit in in sie hinein- der Begriff der Erfahrungswelt alles eher B. z. als Daß es ein Gesamtproblem der Erfahrung gibt, d. h. daß der Boden, auf dem Erfahrung entein Erzeugnis der bloßen steht, zu gewinnen erst um einzelne Daten, nur Erfahrung ist.^ ist, auch daß wir bei der Wahrheit nicht sondern um Gesamtgestaltungen und Ge- samtüberzeugungen kämpfen, verdunkelt werden. Es kann sich aber das sollte nach Kant nicht so leicht dem Empirismus nur ver- dunkeln, weil ihm nur besondere Seiten der Wirklichkeit gegenwärtig sind, die ihre Tiefe Es und ihren Umfang in keiner Weise erschöpfen. sowohl das geschieht nach Seite des des Objekts, wie es in Kürze heißen mag. Leben sich zunächst in Subjekts als nach Seite Da unser Denken und Bewußtseinsvorgängen abspielt, so bleibt der Empirismus dabei stehen und verkennt, daß der Inhalt des Bewußtseins selbst nicht verständlich sie schon ist, in namentlich alle es einander ein tiefer gegründetes Selbstleben des Geistes ohne eine Umkehrung der ersten Ansicht, wie der Bildung eines einheitlichen Ich aber gibt ohne nicht erfolgt, innere Synthese erst möglich macht; keine einzelner Wissenschaft. Das Seelenleben Bewußtseinsvorgänge auflösen, wie sie aber ohne eine solche in das ein Neben- heißt allen Zusammenhang preisgeben und damit auch die Möglichkeit einer Wissenschaft von Grund aus zerstören. Innern Christian virtuose gegen Schluß): 'tis When we say, experience corrects reason, an improper way of speaking, since tis reason formation of experience corrects the judgment it itself, that upon the In- had made before. Merkwürdig ist es überhaupt, wie oft man sich heute auf eine Erfahrung ohne zuvor ihre Bedingungen irgend zu untersuchen und ihre Möglichkeit zu sichern. Nirgends dürfte dies heute mehr geschehen als auf pädagogischem Gebiet. Man richtet neue Schulgattungen ein und erklärt bald, die Erfahrung habe sie als vortrefflich erwiesen; man neigt dahin, Einrichtungen fremder Völker einzuführen und beruft sich dafür auf die Bewährung durch die Erfahrung bei jenen. Aber ist gesagt, daß was dem einen Volke frommt, für das andere, vielleicht unter wesentlich anderen Lebensbedingungen befindliche, ebenfalls passe? Und wenn eine Einrichtung hie und da vielleicht unter besonders günstigen Bedingungen gute Erfolge hat, ist damit ein durchgängiger Vorzug erwiesen? Die Erfahrung hefert ein Zeugnis nur, wo wesentlich gleiche Umstände vorliegen; ob aber dies der Fall, darum pflegt man sich wenig zu kümmern. * beruft, Denken und Erfahrung. Nach 121 Seite des Objekts aber haftet der Empirismus viel zu aus- schheßlich an der äußeren Natur und verkennt die EigentümHchkeit Was von seinen Lehren ein gewisses Recht gegenüber der Natur hat, das wird zum Unrecht in der Ausdehnung über die ganze Welt. Was uns an sinnlicher Wirkung der anderen Daseinsgebiete. zugeht, das nie sich läßt voll in umsetzen und geistige Tätigkeit von innen aus entwickeln; so verbleibt hier immer eine Fremdheit und Gebundenheit, und es nicht hinauszukommen. Aber schon beim ersten Anblick des mensch- ist über ein Registrieren und Beschreiben und Strebens stellt sich lichen Lebens die Sache anders. Auch hier werden zunächst uns einzelne Vorgänge zugeführt, aber es verbleibt beim bloßen Eindruck, jene nicht der Betrachtende sich diesen in lassen sich auf den erzeugenden und mit einander verbinden; indem Lebensprozeß zurückverfolgen kann Prozeß zu versetzen vermag, das Fremde in eignes Leben verwandeln. Kann aber so der Mensch mit dem Menschen leben und fühlen, ihn nicht bloß wie ein fremdes Ding von außen betrachten, so ergibt sich über das bloße er Noch weiter aber gelangen wir, wenn innerhalb des menschlichen Kreises ein Geistesleben anerkannt, Beschreiben hinaus ein Erkennen. der Standpunkt der Erkenntnisarbeit gewählt, die Gesamtheit hier des geschichtlich-gesellschaftlichen Lebens sowohl als der Erfahrungen am einzelnen Punkt darauf bezogen, von dort durchleuchtet und Nun und nimmer kann zusammengefaßt wird. sich die Be- hier handlung eine bloße Feststellung der dargebotenen Erscheinungen muß genügen lassen, Messen und Umwandeln sie eine innere Aneignung, mit ihr aber ein vollziehen. Denn was im menschlichen Kreise an Entfaltung des Geisteslebens vorliegt, das ist seinem ersten Befunde nach mit behaftet, daß keine Klärung ohne vielem so Menschlichen, ZufäUigen Zeitlichen, eine energische Scheidung und eine Zurückführung zur eignen Art erfolgen kann, zugleich hier, aus jenes Leben besonderen Beziehungen und Richtungen, den vorliegt herauszuheben, sie und aufstrebt, in gilt umfassendes Ganzes ein es denen erst von ihm her jene Mannigfaltigkeit aufzuhellen und zu einem Zusammenhange zu verbinden. Höhe dessen, was sich dem Menschen an In Wahrheit liegt die Erkenntnis erschließt, an dieser Stelle: in den charakteristischen Entfaltungen des Geisteslebens und dem Aufbau einer geistigen Welt; hier liegt daher auch die Entscheidung über das Ganze der Weltanschauung, von hier aus ist der Gesamttypus unserer Weltanschauung zu entwerfen, von hier Zum Erkenntnisproblem. 122 muß irgendwelche Würdigung auch das finden, was das mensch- aus ist Das alles Dasein an Schranken und Widersprüchen enthält. liche Erfahrungen, voller voller Bewegungen und Vertiefungen, nun und nimmer aus bloßen Begriffen hervorgehen können, damit durchaus jenseit liegt aber ebenso gewiß Beiden bloßen Sphäre des der die es liegt Rationalismus, es über allem Vermögen des bloßen Empirismus. deutliche Abhebung des Geisteslebens vom mensch- fehlt die das treibt den Rationalismus zur Überspannung des lichen Dasein: Menschen, den Empirismus zur Verleugnung des Geisteslebens; jener vermag dem Erkennen keinen lebendigen Inhalt, keinen wissenschaftlichen Charakter zu geben. dieser vermag Beiden ihm auch der ist Fehler gemeinsam, das Erkennen nicht einem größeren Ganzen des Geisteslebens einzufügen kenntnisproblem und Zusammenhang damit im behandeln; zu solcher bei Isolierung Erkennen entweder zu niedrig oder zu hoch bewertet. treten dabei dem Erkennen Ursprünglichkeit, Er- das Beide ver- unentbehrliche Momente: der eine die andere der das wird die Tatsächlichkeit. Aber es bedarf eines neuen Standortes, um diese Wahrheitsmomente zu einem Ganzen zu verbinden und weder einseitig an der Größe noch an der Schranke des menschlichen Erkennens zu haften, sondern Schranke und Größe mit einander anzuerkennen. Wenn der Empirismus trotz aller augen- scheinlichen Schwächen immer von neuem aufsteigt und die Menschen überwältigend fortreißt, so liegt das weniger an seiner eignen Leistung als an den Mängeln des Wahrheitsbegriffes, der im Rationalismus zu überwiegen Daß er die Wahrheit über die Meinung und pflegt. Spaltung der Menschen emporhebt, ihr eine völlige Unabhängigkeit vom Menschen gibt, das bleibt sein Verdienst und sein Recht; wo dies Recht irgend unsicher wird, da ist eine Vernichtung der Wissenschaft nicht zu verhüten. Aber solange der Abstand nicht irgend- welche Überwindung findet und die Wahrheit nicht irgendwie zu unserer eignen Sache gemacht wird, behält sie etwas Kaltes und Totes; unerfindlich bleibt, wie sie uns mit überwältigender Kraft zu bewegen, und wie sie das Ganze des Lebens zu erhöhen vermag. So sehr wir es ablehnen müssen, die Wahrheit mit dem Pragmatis- mus nach der Brauchbarkeit für das Leben zu messen, haupt nach einem draußenliegenden Ziele zu messen, so sie über- muß doch das Erfassen der Wahrheit als Entwicklung eines neuen Lebens ver- standen und die Sache als nicht jenseit, sondern innerhalb des Lebens befindlich verstanden werden. Es handelt sich schließlich nicht Denken und Erfahrung. die jenseit des Lebens darum, eine Wirklichkeit zu erfassen, sondern Leben ein gewinnen, zu läßt sich ein innigeres Ver- ohne daß wir dem Empirismus ver- hältnis zur Wahrheit erreichen, der zu keiner Wahrheit gelangen würde, fallen, Glauben an sie liegt, das eine Wirklichkeit aus sich Verfolgung dieser Bahn In entwickelt. 123 wenn er nicht den zu seiner Arbeit schon mitbrächte. dem Empirismus und dem Rationalismus wirken, wie wir sahen, entgegengesetzte geistige Strömungen; von der Art und Lage In der Zeiten wird es abhängen, ob diese oder jene jeweilig das Über- Wo der Gedankenkreis als im wesentlichen ge- gewicht gewinnt. und wohl übersehbar gilt, wie im Altertum und im Mittel- schlossen alter, auch bei der deutschen schätzung Erfahrung der Philosophie, da wird konstruktiven man sich zu einer Unter- die innere Leistung voranstehen, da wird neigen. Wo hingegen die Empfindung der Enge des bisherigen Gesichtskreises vorwaltet und ein Verlangen nach Erweiterung aufkommt, da wird von der Erfahrung alles Heil umwandelnde Tätigkeit erwartet und weiterbildende, die leicht So geschah geistiger Art übersehen. im 19. Jahrhundert und geschieht ja land 19. Jahrhundert vollzog, wirken, weil ihr hier ein Die un- auch heute. es vielfach ermeßliche Erweiterung des Gesichtskreises die das Bacon, so geschah es es bei in Natur und Geschichte, mußte besonders stark in Deutschenergischer Rückschlag gegen die zu straffe Zusammenfassung der konstruktiven Systeme zur Seite ging. Aber je mehr eine solche empiristische Bewegung sich ausdehnt und je einnimmt, das Feld ausschließlicher sie desto notwendiger Wir sahen den Empirismus zu einem leidlichen Abschlüsse nur kommen, weil er innerhalb einer vorge- wird eine Gegenwirkung. fundenen, seinen eignen Begriffen überlegenen, Gedankenwelt wirkt; erschüttert und diese aufgelöst Gedankenwelt aber werden, samer jene Richtung wird. je ja widersprechenden muß um so mehr und selbständiger So untergräbt sie Fortgang die ihr unentbehrlichen Ergänzungen und äußeren Siege innerlich wird sonnenklar, sobald Eine solche auch ihre aus eignen Mitteln in jetzt sich unduld- muß daher im Unzulänglichkeit alles Wendung sehen wir trotz aller Gunst, Empirismus noch erfreut, zusammenbrechen; sie je durch ihren eignen bestreiten will. deren sich der lebensfremden exaktwissenschaftlichen Kreisen vorbereiten. Immer deutlicher wird, daß alle Aufschichtung und Anordnung von Kenntnissen noch keinerlei Er- Zum Erkenntnisproblem. 124 kenntnis, keinerlei Ideen, keinerlei Überzeugungen gewährt, daß aber ohne solche der Mensch nicht auskommen kann, bleiben, nicht zu einer Kulturmaschine herabsinken ein Seelenwesen Es soll. eine Notwendigkeit des geistigen ist Lebens, es ist im be- der gegenwärtigen Kultur, welche sondern die eigentümliche Lage das wenn anders er Denken zwingend über den Empirismus Ohne hinaustreibt. eine Selbständigkeif und Ursprünglichkeit des Denkens kann keine Aber jene Selbständigkeit Kultur bestehen. übersehen und läßt sich Bahnen fort- vergessen, so lange das Leben in vermeintlich sicheren läuft, lange es so nicht von schweren Verwicklungen und WiderHeute aber stehen wir ganz und gar unter sprüchen bedroht wird. dem Eindruck schwerer Verwicklungen und Widersprüche, wir er- kennen die Notwendigkeit einer gründlichen Revision des gesamten Kulturbesitzes, alles welk die Notwendigkeit energischen einer unwahr Gewordenen, und einer fassung und Steigerung aller Wahrheitselemente; Erschütterung, daß die Ungewißheit greift und Wie sollten um das Ganze des in Ausscheidung kräftigen ja so Zusammengeht die tief die letzten Elemente zurück- Geisteslebens kämpfen zu zwingt. solchen Aufgaben irgend weiter gelangen wir nun bei und ursprünglicher Tätigkeit, ohne eine Selbstbesinnung und Selbsterweckung des Geisteslebens, ohne ohne ein Vermögen selbständiger Erhöhung und Erneuerung, welche neue Möglichkeiten und neue Tatbestände erschließt? Das alles aber liegt dem Empirismus fern. So notwendig daher die Zeit einer Innern Wandlung bedarf, so notwendig muß sie ihn hinter sich lassen. So sehr wir nun die Wendung der philosophischen Forschung der Gegeneine geistige entwirft wart dabei zum Idealismus begrüßen, und so sehr wir die Scheu verstehen, der alten Metaphysik wiederaufzunehmen: die Art wir einer gründlichen Erneuerung und so gewiß durchgreifenden Kräftigung des Lebens bedürfen, ebenso notwendig bedürfen wir eines Idealismus aufrüttelnder und vordringender Art, nicht bloß ein kritischer, er bedeutend der kritische ein solcher Idealismus aber darf muß auch ein positiver sein. Idealismus, der philosophischen Forschung vorherrscht, und Empirismus seine daß sie nur mit heute darin auf der ist, Denn so Höhe der dem Realismus Grenze zu weisen, im besondern darzutun, Hülfe des Gegners ein Ganzes des versteckter Lebens und Wissens erreichen, so entschieden er ferner in einzelnen Hauptrichtungen des Lebens das Wirken Ordnung der Dinge zeigt, und Walten einer neuen er faßt diese Hauptrichtungen nicht ge- Denken und Erfahrung. nügend in ein Ganzes zusammen; ein 125 Ganzes aber ist unentbehrlich, Bewegung sein geistiges Selbst ergreifen, den Schwerpunkt seines Lebens dahin verlegen und zugleich eine Umkehrung seines Leben vollziehen soll. Ohne eine solche, ohne wenn der Mensch in jener eine Versetzung in ein ursprüngliches Leben von der anderen Seite her kann aber das neue Leben schwerlich die Kraft gewinnen, einer andersartigen sich gegen die ungeheuren nächste Weltlage intellektuellen um Ordnung gegenüber selbständig zu werden und bietet. Hemmungen durchzusetzen, Demnach treibt welche die uns nicht eine Lust an Abenteuern zur Metaphysik, sondern die unerläßliche Forderung einer Selbsterhaltung des Geisteslebens. 2. — organisch. Mechanisch (Teleologie.) I und Probleme Mechanisch |ie Begriffe *-^ eine besonders reiche Geschichte. und Organisch haben Sie zeigt nicht nur große Gegensätze der Weltanschauung und Methodenlehre und einen harten Kampf um den Charakter der wissenschaftlichen Arbeit, sie ist auch Nuancen und Schwingungen und ergibt damit einen eigentümlichen Durchblick der Gesamtbewegung. Die Gegen- feinerer voll leiserer der Jahrtausende aber erstrecken sätze So sei Gegenwart. bei diesem sich bis in Arbeit der die Problem unser Augenmerk vornehm- der Geschichte zugewandt. lich a) Zur Geschichte der Ausdrücke und Begriffe. Wie Ausdrücke mechanisch und organisch, so sind auch die die Begriffe alt, aber erst spät haben sie einander gefunden. erscheint, als technische Bezeichnung der Herstellung von Aristoteles spätere Schrift Jahrtausende Maschinen, (A7];^avixrj, (i^ rä [A7]x^avix,a. i und dient wie ein eingebürgerter Ausdruck (XTj^^avtxa), In es Mechanisch Kunst der Erfindungen, der dessen Namen trägt bei eine diesem Sinne durchlief das Wort die seit Descartes zur Bezeichnung einer Theorie, welche die Bildung der Natur, nach Art der menschlichen Werke, nicht aus einer Innern Triebkraft des Ganzen, sondern aus der Zusammenfügung kleiner von Haus aus bewegter Teilchen des Stoffes erklärt; die Werke der Natur scheinen von denen des Menschen nur durch ihre größere Feinheit, also quantitativ, nicht ^ (pUatv rr,i In dieser ::pa^at, -ziy(yri<i Siot Schrift wird der Ausdruck so erklärt: "OTav xo j^aXsröv änopiav riapiyji xai oelrai to Ttpö; ta? xotaux«? ar.oploic, ßor]0-ouv 8io xe'yvy)?. [lipoc, |jL7])(^av7Jv qualitativ oeV, Tt Tiapa xai xaXoü(jL£v (Arist. 847 a, 16). Die Kunst erscheint hier als eine Art Überlistung der Natur. Mechanisch — organisch. Die technischen Mittel der Erklärung verschieden.^ Mechanik theoretische 127 als Bewegungslehre. 2 In liefert dabei die Umlauf dürfte den Terminus mechanisch namentlich der Chemiker und Philosoph Robert liebte und gern auf dem Werke verwandte. Selbst am Ausdruck „Natur" nahm Anstoß und wollte ihn durch mechanismus universalis ersetzt Boyle gebracht haben, der ihn besonders seiner Titel er wissen. Die Naturwissenschaft der folgenden Zeiten faßte den Terminus bald bald laxer, strenger; Erörterungen darüber waren gang und Aber gewöhnlich wurde unter mechanischer Erklärung eine gäbe. Erklärung körperlicher Beschaffenheiten aus der Figur und Bewegung verstanden. Übertragung auf seelische Vorgänge Eine mechanisch und materiell gelten zunächst ganz fern, dabei lag oft als gleich- Eine mechanische Erklärung seelischer Vorgänge heißt bedeutend. ^ daher zunächst eine Ableitung aus bloß körperlichen Ursachen. In der Sache aber unternimmt schon Spinoza, den Bestand des Seelenlebens aus ,dem Zusammenwirken einzelner Vorstellungen zu erklären, wie Descartes ^ ipsa (sc. facile percipi § 203): »Nullum corpora naturalia discrimen agnosco, et fadorum operationes sensu (principia philos. IV, sagt: arte facta) ut aliud nisi inter quod arte plurimum peraguntur instrumentis adeo magnis, ut hoc enim requiritur, ut ab hominibus fabricari possint: queant. Contra autem naturales effectus fere semper dependent ab aliquibus organis adeo minutis, ut omnem sensum effugiant. Danach bringt jede Verfeinerung der Maschinen die Kunst der Natur ein Stück näher. ^ Descartes princ. phil. IV, §200: Figuras et motus et magnitudines corporum consideravi atque secundum leges Mechanicae, certis et quotidianis experimentis continuatas, quidnam ex istorum corporum mutuo concursu sequi debeat, examinavi. § 203 Et sane nullae sunt in Mechanica rationes, quae non etiam ad Physicam, cujus pars vel species est, pertineant, nee minus — naturale est horologio ex bis vel arbori ex hoc vel illo : illis quam Quamobrem rotis composito, ut horas indicat, semine ortae, ut tales fructus producat. ii qui in considerandis automatis sunt exercitati, cum alicujus machinae usum sciunt et nonnullas ejus partes aspiciunt, facile ex istis, quo modo aliae quas non vident sint factae, conjiciunt; ita ex sensilibus effectibus et partibus corporum naturalium, quales sint eorum causae et particulae insensiles, investi- ut gare conatus sum. ' So stellt Descartes selbst dem Un körperlichen das mechanicum et corporeum entgegen (Briefe I, 67), und dieselbe Bedeutung liegt vor, wenn Wolff (psych, rat. § 395) behauptet, daß die Einsichten der anschauenden Erkenntnis nihil — mechanice quoque in cerebro absolvi qua quid in universal! repraesentatur, quod non aeque (cognitio symbolica) inesse notioni, mechanice repraesentatur in corpore. Zum Erkenntnisproblem. 128 er denn die Seele eine Maschine nennt (automaton geistige spiritu- Leibniz verfeinert das trotz seiner Betonung der Einheit der ale). und Wolff Seele. ^ einer Übertragung des Wortes, dann Psychologen französischen die hunderts bilden es näher durch. Schließlich des kommt 18. Jahr- auch zu und „mechanisch" wird erst bildlich, das Innere der Seele verwandt.- lehrhaft für es Kant verallge- meinert den Ausdruck, indem er ihn überträgt auf »alle Notwendig- Begebenheiten der keit Kausalität, ob man der in gleich ihm unterworfen sind, wirklich In nach Zeit dem Naturgesetze der daß Dinge, die darunter nicht versteht, Maschinen sein müßten." materielle der Naturphilosophie aber entwickelt er klar und scharf den Gegen- satz einer mechanischen und einer dynamischen Erklärung.^ Auch organisch begegnet uns zuerst bei Aristoteles, dem großen Aber es hat dort einen anderen Sinn als jetzt. Entsprechend dem Stammwort opyavov, Werkzeug, bedeutet organisch Bildner der Sprache. „werkzeuglich", es wird gebraucht zweckmäßig angelegten Körpers, teilen, nämlich welche solchen, zusammengesetzt vom Ganzen des lebendigen, öfter aber von einzelnen aus ungleichartigen Körper- Bestandteilen So gewiß der Begriff nur Lebewesen zu- sind. kommt, so enthält er selbst nicht das Merkmal inneren Lebens, er ist daher auch nicht über das besondere Gebiet hinaus zur Bezeichnung ^ qu'elle S. z. B. est, Erdm. 153: II faut considerer aussi que l'äme, toute simple a toujours un sentiment compose de plusieurs perceptions ä fois;'Ce qui opere autant la pour notre but, que si eile etait composee de pieces comme une machine. - man die Übertragung noch im Werden, er sagt im „Wenn diese Veränderung durch innere Triebfedern, (mich Bei Lessing sieht 7. Literaturbriefe: plump auszudrücken) durch den eigenen Mechanismus des seiner Seele erfolgt Mit besonderer Energie hat Herbart die Vorstellung von einer Mechanik ist." Innenlebens durchgeführt; er erklärt Organismus der Vernunft aufzulösen stellungsreihen, erklärt in es (III, 255) als seine einfachen Aufgabe, Fibern, „den die Vor- deren Entstehung nur aus der Mechanik des Geistes konnte werden." ^ S. IV, 427 (Hart.): „Die Erklärungsart der spezifischen Verschiedenheit der Materien durch die Beschaffenheit und Zusammensetzung ihrer kleinsten Teile, als Maschinen, ist die mechanische Naturphilosophie; welche aus Materien, nicht als Maschinen, bewegender Kräfte, d. i. diejenige aber, bloßen Werkzeugen äußerer sondern ihnen ursprünglich eigenen bewegenden Kräften der Anziehung und Zurückstoßung die spezifische Verschiedenheit der Materie ableitet, kann die dynamische Naturphilosophie genannt werden." Mechanisch — organisch. 129 eines lebendigen Ganzen, etwa in der politischen Theorie, verwandt; wo ^pyavijcd; bei Aristoteles Stellen, es gibt mit mechanisch übersetzen läßt. ^ kaum anders als Diesen Sinn behielt das Wort unsich verändert durch Mittelalter und Neuzeit hindurch bis in das 18. Jahr- Jenen Begriff des Werkzeuglichen konnte sich auch die hundert. - neue mechanische Theorie aneignen, unbedenkHch wurden im 18. Jahr- dem Be- hundert organische (natürliche) und künstliche Maschinen der Maschine untergeordnet; griff von organischen Maschinen sprechen hatte damals gar nichts Befremdliches. zu ^ Erst das Aufsteigen der deutschen Blütezeit mit ihrem Verlangen und eignen Bewegung der Natur hat dem Ausdruck organisch das Merkmal des Lebendigen hinzugefügt und nach einer Beseelung Besonders hat dahin Kant mit seinen es zur Hauptsache gemacht. und Unterscheidungen gewirkt, aber auch Herder, präzisen Begriffen Jacobi u. Von den natürlichen Lebewesen seien nicht vergessen.^ a. übertrug sich die neue Bedeutung zunächst auf Staat und Gesellschaft, ^ ' dann auf Recht, Geschichte Pol. 1259 b, yevEJEw; xa\ (pO-opä? rapl S. Z. B. Tot; atüjiaat, Bi a; yEvvtiJsi, 23: u. s. w. Organisch wird nament- 336a, 2: xal xa? Suvatisi? aTioStSdaai Xiav 6pyavtxt3;, oupaipoüvTe; aroprJoEtev av Ti;, Tco'xEpov ettiv xaxa to eiSo; atttav. ttiv ap£Tr' xt; SouXou napa xa; opyavixa; xat Staxovtxas oXXrj TijJUWTEpa xouxtov. * Der letzte bedeutende Ausläufer der Scholastik, Suarez (1548—1617), anima I, 2, 6): Dicitur corpus organicum, quod ex partibus dissi- sagt (de milaribus componitur. Noch für den Sprachgebrauch der Wolffischen Schule bezeugt Baumeister: Corpus dicitur organicum, quod ad peculiarem quandam actionem aptum ^ nannte Saint-Simon machine organisee (s. noch Paul Barth, vi compositions suae est. um 1813 die Gesellschaft Vierteljahrsschr. f. eine wissenschaftl. veritable Philos. XXIV, I, S. 72). Kant definiert (V, 388 Hart.): „Ein organisiertes Produkt der Natur S. 386 in welchem alles Zweck und wechselseitig auch Mittel ist." heißt es: „Ein organisiertes Wesen ist also nicht bloß Maschine; denn die hat lediglich bewegende Kraft; sondern es besitzt in sich bildende Kraft, und zwar eine solche, die es den Materien mitteilt, welche sie nicht haben (sie * ist das, organisiert)". Jacobi (Hume 172): „Um die Möglichkeit eines organischen Wesens zu denken, wird es notwendig sein, dasjenige, was seine Einheit ausmacht, zuerst: das Ganze vor den Teilen zu denken." Sachlich wurden damit nur aristotelische Gedanken wieder aufgenommen und genauer formuliert. Kant spricht auch von einem „wahren Gliederbau" der reinen spekulativen Vernunft, „worin alles Organ ist, nämlich alles um eines willen und ein jedes Einzelne um aller willen" (III, 28 Hart.) ' Die Übertragung des Ausdrucks Organisation auf das politische Gebiet dürfte zuerst in den Bewegungen der französischen Revolution erfolgt Eucken, Grundbegriffe. 4. Aufl. 9 Zum Erkenntnisprablem. 130 ein Lieblingswort der Romantik, lieh einzelne Schulen gebrauch ein. es über dringt aber zugleich und Richtungen hinaus in den allgemeinen SprachMechanisch und organisch, am Ursprung ziemlich gleicher Bedeutung, bilden also schließlich den schroffsten Gegen- und bezeichnen jetzt Hauptgegensätze der Weltanschauung.^ satz b) Zur Geschichte des Problems. Der sachliche Gegensatz, den nunmehr weit reicht zurück, Demokrit und hat er Aristoteles. im Altertum Hauptvertreter wie sie in Kürze heißen mag, Die Vorstellung den Ausdruck — des Organismus eingebürgert, von dem auch die Formel stammt, daß organischen Wesen das Ganze den Teilen vorangehe. ^ sein, sich die das Ganze vor die Teile, das Lebendige vor das Leblose und erklärt von dort nach hier. stellung erstreckt in Die künstlerische und synthetische Denk- entschiedene Oberhand. stellt Worte bezeichnen, Auf der Höhe des klassischen Altertums gewinnt die organische Lehre, weise jener Zeit die seine sofort hat — nicht namentlich Aristoteles bei dafür einem Die Vor- über ihr nächstes Gebiet hinaus- auf den innerlichen Sinn aber haben dem Worte erst deutsche Denker und Kant sagt (V, 387 Hart.): „Genau zu reden, hat die Or- Dichter gegeben. ganisation der Natur nichts Analogisches mit irgend einer Kausalität, dem die Anmerkung hinzu: »Man kann umgekehrt einer gewissen Verbindung, die aber auch mehr in der Idee als in wir kennen" und fügt in einer der Wirklichkeit angetroffen wird, durch eine Analogie mit den genannten So hat man sich, bei einer neulich unternommenen gänzlichen Umbildung eines großen Volkes zu einem Staat, des Worts Organisation häufig für Einrichtung der Magistraturen u. s. w. und selbst des ganzen Staatskörpers sehr schicklich bedient. Denn jedes Glied soll freilich in einem solchen Ganzen nicht bloß Mittel, sondern zugleich auch Zweck, und, indem es zu der Möglichkeit des Ganzen mitwirkt, durch die Idee des Ganzen wiederum seiner Stelle und Funktion nach beunmittelbaren Naturzwecken Licht geben. stimmt sein." In derselben Schrift (der K. d. Urteilskraft) sagt er S. 364 »So wird ein monarchischer Staat durch einen beseelten Körper, wenn er nach inneren Volksgesetzen, durch eine bloße Maschine aber (wie etwa eine Handmühle), wenn er durch einen einzelnen absoluten Willen beherrscht wird, in beiden Fällen aber nur symbolisch vorgestellt." ^ So z. B. bei Trendelenburg, s. Log. Untersuchungen (3. Aufl.) II, 142ff. * Pol. 1253a, 20 heißt es: xo oXov JupoTspov ava^xaiov etvai xou [xtpou?. xi? yap xoü oXou oux eaxat noui ouSk X^V^ ^^ Kl' op-wvufxw; tforcEp £i liycn xT^v Xi'd-ivTiv. 8ta<p^S-ap£toa yap Eoxat xoiauxr). Danach ist der Staat früher als das Individuum. avatpou(xE'vou Mechanisch — organisch. den — freilich erst nach und auf das Weltall, bald auch und am meisten bei den späteren Stoikern — auf das Staat Aristoteles Vom Altertum fließt sie in das Christentum Ganze der Menschheit. und gewinnt hier durch die ein Innigkeit. 131 Färbung eine besondere religiöse Sie befestigt sich später zu der Idee der Kirche als des ^ mystischen Leibes (corpus mysticum) Christi. im Sie erhält Mittel- und Sinn- mit seiner untrennbaren Verkettung von Geistigem alter lichem eine greifbare Gestalt und beherrscht mit solcher die mittelalterUche Gesellschaftslehre; ^ sie eigentümlichen Ordnungssystems, ist ein Hauptstück dem das des jener Zeit Einzelnen Geistig- alle von einem Ganzen, und zwar einem sichtbaren Ganzen her zu- keit gehen läßt. Jene organische Lehre wirkte stark sowohl auf das praktische Gebiet als Dort gebot auf das wissenschaftliche Verfahren. eine sie unbedingte Unterordnung des Einzelnen unter das Ganze, das ihm zur Entwicklung seiner Vernunft erst sie 'dem Einzelnen das Bewußtsein, und an verhelfe, aber innerhalb des Ganzen gab zugleich etwas zu bedeuten. Mit besonderer Freude verweilt das spätere Altertum bei dem Ge- Eigentümliches seiner Stelle Unersetzliches (i^ipo?), sondern ein ein Glied des Ganzen tröstet einen Marc Aurel danken, daß der Einzelne, nicht bloß ein Teil Glied (pi>.o;) des Weltalls der Vernunftwesen«, in diese sei. »Ich bin Überzeugung den Gefahren und Dunkelheiten des Lebens. aber entwickelt namentlich die Vorstellung, daß Die alle alte Kirche Christen als Glieder des gottgeweihten Gemeinwesens in Schicksal und Tat auf einander angewiesen, einander solidarisch verbunden seien. Nicht minder folgenreich ist jene Denkweise für die wissenschaft- Denn hier quillt aus ihr die teleologische Betrachtung, vom Altertum mit mächtiger Wirkung bis in die Gegen- liche Arbeit. die sich wart erstreckt. War das Ganze das Ursprüngliche und Überlegene, so bot es den Schlüssel * zur Erklärung der einzelnen Glieder und Bezeichnend für den griechischen Ursprung der Vorstellungsweise ist die Tatsache, daß unter den Evangelien nur das Johannesevangelium sie bietet (Weinstock und Reben), das stark unter griechischen und philosophischen Einflüssen steht. ^ Dem entspricht es, daß die Analogie zwischen dem Staat und einem lebendigen Körper nicht beim allgemeinen Gedanken verbleibt, sondern gern bis ins Einzelne ausgeführt wird. So hat z. B. Johann von Salisbury für jeden Teil des Staates ein entsprechendes Körperglied aufzuweisen versucht (s. Gierke, Das deutsche Genossenschaftsrecht III, 549). 9* Zum Erkenntnisproblem. 132 Das Ganze Leistungen. war aber nach platonisch-aristotelischer Vorstellung eine unwanddbare Form, ein bei sich selbst befindliches und befriedigtes Leben; so setzte es aller und Schlußpunkt, Das in sich selbst einen festen Ziel- i reicht des Lebendigen hinaus in das gesamte All. ein lebendiges, fest Bewegungen sich gliedartig einfügt; die über das Gebiet Die Welt dem ineinandergefügtes Ganzes, vollendetes sich in gilt hier als alles Einzelne laufen nicht wirr durchein- um dort in ein ander, sondern eine jede strebt zu einem Endpunkt, beharrendes, Bewegung Wirken überzugehen. (evepysia) Aber besonders fruchtbar ist jene Betrachtungsweise innerhalb Heimatsgebietes: bei den Lebewesen. ihres Nicht nur werden hier bei den einzelnen Tierarten alle Organe und Funktionen auf ein allumspannen- des Leben bezogen und daraus verstanden, Mannigfaltigkeit organischer Bildung als Normaltypus, der durch alle Diesen Normaltypus zeigt Stufen wirkt. alle rein der Mensch, so läßt sich erscheint auch es Entfaltung eines einzigen von ihm aus das weite Reich durch- leuchten und der unermeßliche Stoff unter durchgehende Gedanken bringen. Es entstand damit eine Art von vergleichender Anatomie und Physiologie, sowie eine Entwicklungsgeschichte; auch das Seelenleben der Tiere wird vom Menschen her aufzuhellen gesucht. So wenig uns jetzt jene Betrachtungsweise genügen kann, eine gewisse Zusammenfassung und Anordnung des Stoffes hat sie ihrer Zeit und langen Jahrhunderten geboten. Auch im Altertum aber Widerständen nicht an es fehlte dagegen, gelangten nicht aus bloßer Gegenwirkung zur Leitung der sie Das geschah Arbeit. in erst organische und Klarheit wurde. wo der Kampf gegen des Strebens nach Freiheit der Neuzeit, Lehre ein Hauptstück jene Die Befreiung erfolgt zuerst im Gesamtzuge des Lebens, indem das moderne Individuum die Bindung an eine Organisation und greifbare durch als eine unerträgliche * S. Aristot. TT? oüot); £<jti ?V£xa. S. femer lQ9a, 8'auTT), Tou teXou; S'?vexa raXXa, teles 30: im\ des Geisteslebens da- Bedrückung empfindet und Physik 194 a, 28: xtvr-'<j£(jj? Vermittlung abweist, zum All erstrebt und daraus eine sichere ein unmittelbares Verhältnis ouve^^ous die r, ti i] Sk <puais (pu'at? auxr) Sirrr, av arj f| ou te'Xo? xa\ {ikv r ahla. a; f, ^vexa. av yap touto Eo^^atov xai to ou teXo? tt^ xtv/jaew?, uXt) r S'w; jJioptpr', xeXoi; Nach Aristo- -ou ?v£xa. könnte der Zufall wohl einzelne zweckmäßige Bildungen hervorbringen, nun und nimmer aber die durchgehende Zweckmäßigkeit; zweite Buch seiner Physik. s. darüber das Mechanisch — organisch. Überlegenheit gegen sichtbare alle Ordnung 133 So zuerst erringt. in der Renaissance und in der Reformation, so auch in der politischen und wirtschaftlichen Befreiung, wie sie namentlich von England ausging. Mit solcher direkten Begründung des Lebens auf das Indi- viduum scheint es unermeßlich an Kraft, Vernunft und Wahrheit zu Alle Zusammenhänge erscheinen hier als das Werk der gewinnen. Individuen, haben nur sie Nach Leibniz gewährt soviel Recht, Unendlichkeit des Alls Und entwickelt sie wie fem hier jene organische Lehre! liegt Gleichzeitig erfolgt das Individuum als Individuum das trägt auch eine sich in die ihnen ganze aus sich selbst heraus; Umwälzung Wissenschaft der Die überkommene Erklärung der Natur aus dem Ganzen und Innern wird unerträglich, Deutung, nicht sind die als sondern Bild, Werke Lehre, der scholastischen Kräften erscheint sie als ein rantiae, s. B. als mit ihren ist, weil es Erklärung gibt vollwichtige jener Zeit voller Klagen über lichkeit z. das als eine durchaus subjektive das energisch abzuweisen bloßes Bild, als ein sich man empfindet die So versteckte Bild- inneren Formen und «Asyl der Unwissenheit« (asylum igno- Oldenburg an Spinoza). Demgegenüber wird zur Grundbedingung einer echten Erkenntnis, daß alles Innere aus der Natur vertrieben und alle Zusammenhänge in kleinste Elemente aufgelöst werden; die Ermittlung und Verfolgung dieser Elemente ver- spricht zugleich ein Durchsichtigmachen der bis dahin verworrenen Wirklichkeit und Denn vom Kleinen her lassen sie sich bewegen eine Macht über die sonst unzugänglichen Dinge. und umgestalten. Für das Große der künstlerischen Art der Alten fehlt hier aller Sinn; So sie war ja auch durch die Scholastik aufs stärkste verkümmert. die mechanische Naturerklärung der Neuzeit; in direktem und be- wußtem Gegensatz zur Denkweise macht älteren zur Hauptsache, unternimmt sie sie die Elemente von ihnen her allen Aufbau, zerlegt überkommene Kontinuum bei Raum, Zeit und Bewegung in diskrete Größen und gibt sie damit allererst eine exakte Begreifung Mit der Leugnung aller inneren Zusammenhänge der Phänomene. sie fällt das natürlich auch die teleologische Betrachtung; die verschiedensten Erwägungen verbinden anthropomorph, als sich unklar, zu ihrer Verwerfung: als unfruchtbar. sie erscheint als Für die Einheit der Natur haben jetzt statt der Zwecke die Gesetze zu sorgen, die überall gleichmäßig wirken und als einfache Grundformen keit beherrschen. Das alles ergreift die Geister alle Mannigfaltig- mit elementarer Zum Erkenntnisproblem. 134 echtes Wissen Gewalt, mit der neuen Denkweise scheint zuerst ein und zugleich ein Zeitalter der Wissenschaft aufzusteigen, alle bisherige Arbeit konnte dafür nur als eine Vorstufe gelten. Daß neue Denkweise die Probleme hervorrief, Fragen viele offen ließ und neue Der konnte tieferen Geistern nicht entgehen. bedeutendste Denker der Aufklärungszeit, behandelt Descartes, die mechanische Theorie nur als ein Prinzip der exakten Naturbegreifung, nicht als metaphysische Lehre von den letzten Gründen eine der Dinge, er zieht zugleich eine scharfe Grenzlinie zwischen sich und Sein treuer Schüler Robert Boyle verficht eine Demokrit. ^ zweck- ein unentbehrliches Gegenstück zu mäßig wirkende Ursache als den mechanischen Ursachen. ^ Berkeley macht geltend, daß die mechanische Betrachtung nur die Gesetze und Formen (modes) nicht seine Ursache. erkläre, eigentümlichen Typus entwickelt einen tief des Geschehens Leibniz ein: er greift der Weltanschauung, indem Natur mit ihrem Mechanismus zur Erscheinung einer er die ganze Wirklichkeit geistigen Besonders macht, die letzten Einheiten, die für die mechanische Betrachtung einen bloßen Grenzbegriff bilden, zur Haupt- und erhebt sache als sie Monaden mit innerem Leben Innerhalb der Natur will er alles Prinzipien mechanisch des Mechanismus aber scheinen erklärt selbst versieht. wissen, einer die Erklärung zu bedürfen und diese nur in einer zweckmäßig waltenden Weltvernunft finden zu können. ^ Es sieht aber Leibniz die Zweckmäßigkeit der Naturgesetze darin, daß sie alle dem Ziele dienen, ein möglichst großes Quantum von Kraft zur Existenz zu bringen. die er * criti kürzesten Wege eingeschlagen und die Überall findet einfachsten Mittel Die wichtigste Stelle dafür findet sich princ. philos. IV, § 202: (Demo- philosophandi ratio) rejecta est, primo quia illa corpuscula indivisibilia rejicio; deinde quia vacuum circa quod ego nullum dari posse demonstro, tertio quia gravitatem iisdem tribuebat, quam ego nullam in ulio corporum cum solum spectatur, sed tantum quatenus ab aliorum corporum situ et motu dependet atque supponebat, quo nomine etiam ego illam ipsa esse fingebat, ad illa ' refertur, intelligo S. z. B. primordia rerum (sive breviore ff. de ipsa natura sect. IV: infinita. sua sapientia Hamm autem partium motum sub ac potestate ita direxit, ut tandem tempore sive longiore, ratio definire nequit) in speciosam hanc ordinatamque mundi formam coaluerint. * Omnia in corporibus fieri mechanice, ipsa vero principia mechanismi generalia ex altiore fönte profluere (S. 161, Erdm.); s. auch 155a, Foucher II, 253. Mechanisch — organisch. Auch gewählt.^ die Schule hält daran Zusammengesetzte und alles während die Seele, scharfer Fassung damit ganze Körperwelt gehöre, ihm entzogen Einfaches, als daß dem Mechanismus fest, die I35 In sei.^ minder Wolff nach scholastischer Art eine Erklärung stellt aus den Wirkursachen und eine aus den Zweckursachen nebenein- ander und bildet dabei den Ausdruck Teleologie. ^ Natürlich wurde Lehre durch jenes die überlieferte organische und wegs mit einem Schlage gestürzt; dazu Stack und den Methoden der Schule. Begriffen teleologische Aufkommen der mechanischen Erklärung keinessie viel Auch zu fehlte tief den in es nicht an tüchtigen Männern, welche die Eigentümlichkeit des Lebendigen eifrig Aber sie fanden verfochten.* durfte es erst einer keit des steigen Ohr der Zeit. Dafür be- Das geschah namentlich mit dem Auf- suchen und sehen hieß. deutschen ein Verlangen nicht das neuen Lebenswoge, die anderes in der Wirklich- Humanismus; siegreich erhebt sich nach mehr Unmittelbarkeit des Lebens, innigeren Verhältnis zu Welt und Natur, in ihm nach einem nach einem Schauen der * S. 147b (Erdm.): semper scilicet est in rebus principium determinationis quod a maximo minimove petendum est, ut nempe maximus praestetur effectus minimo ut sie dicam sumptu. Den Einwand, ob nicht bloße Naturnotwendig- keit dasselbe Ergebnis hätte hervorbringen Cela serait vrai, si par exemple les loix können, beantwortet er dahiq (605b): du mouvement, et tout le reste, avait sa source dans une necessite geometrique de causes efficientes; mais il se trouve qiie dans la derniere analyse on est oblige de recourir ä quelque chose qui depend de causes finales ou de la convenance. So z. B. Baumgarten, Metaph. ed. VI, 1768, § 433: machina est com- positum stricte dictum secundum leges motus mobile. Ergo omne corpus in mundo est machina. — Machinae natura per leges motus determinata mechanismus est. At, quidquid non est compositum, non est machina, hinc nulla monas est machina. dari * S. philos. ration. sive logica cp. III, § 85: rerum naturalium duplices possunt rationes, quarum aliae petuntur a causa efficiente, aliae a fine. Quae a causa efficiente petuntur, in disciplinis hactenus definitis expenduntur. Datur itaque praeter eas alia adhuc philosophiae naturalis pars, quae fines nomine adhuc destituta, etsi amplissima sit et utilissima. Dici posset teleologia. Der Ausdruck causa finalis ist dagegen scholastisch; ich rerum explicat, finde ihn zuerst bei Abälard. * Natur, Obenan s. steht hier Cudworth mit seiner Annahme einer plastischen namentlich the true intellectual System of the universe (1678) I, 3, 19. Von den deutschen Gelehrten ist hier vornehmlich Rüdiger zu nennen, s. z. B. institutiones eruditionis seu philosophia synthetica pag. 109: physica vel mecha- nica est vel vitalis. Zum Erkenntnisproblem. 136 Dinge aus dem Ganzen; was dabei zunächst als Drang die Gemüter erregte, das künstlerischen Lebensgestaltung; von da ein ungestümer allmählich sich klärte zu einer aus fand sich leicht eine Rückkehr zu den Alten, die als das Muster lauterer und edler Natur erschienen. So war es kein Wunder, daß, Renaissance, auch die organische Denkweise eine Auferstehung Stück der jüngsten als ein daß sie mit wunderbarem Zauberklange die Gemüter lebte, er- ergriff und gewann. Eigentümlicherweise hat dieser neuen künstlerischen Denkweise wenig künstlerische wissenschaftlich zuerst der seiner Gemütsart nach Kant die Bahn gebrochen. mus zu Er hat es getan, indem er den Mechanis- bloßmenschlichen Denkweise einer herabsetzte und damit freien Platz für eine andersartige Betrachtung und Behandlung schuf; nur mußte für eine solche sich ein zwingender Anlaß finden. Einen solchen aber schien ihm das Reich der organischen Bildung zu bieten, indem unsere Begriffe nur mit Hilfe der Idee eines inneren Ganzen und es eines Zweckes eingehen kann. leitenden So hier die alte Lehre wieder aufgenommen, auch über das nächste ward dem Ganzen Gebiet hinaus das in alles der Welt zugeführt. in vorsichtiger Abgrenzung Betrachtung. flutete rasch gewann ein Bei Kant selbst und als eine Sache menschlicher Aber die vordringende künstlerische Lebenswoge überGrenzen und Dämme, die organische Denkweise alle stolzes Selbstbewußtsein und gab sich der Aufklärung gegenüber als ein Verstehen aus dem eigensten Leben und Wesen der Dinge heraus, während die mechanische Lehre nüchtern und Mit besonderer Energie schien. seelenlos Denkweise zum Ausdruck gebracht und Idee des hat alles Schelling die neue Naturleben unter die Organismus gestellt^ Begriff Gebrauch. und Ausdruck kommen dann Bei aller Festhaltung antiker rasch in den allgemeinen Elemente ist ein Einfluß modemer Denkweise dabei nicht zu verkennen. Die Idee des Organismus liefert hier weniger ein Bild vom Sein als vom Werden; die Wirklichkeit ein aus bildet Wandlung zunächst als hier weniger ein geschlossenes Kunstwerk eignem Vermögen fortschreitendes Lebewesen. für die Natur. weit fruchtbarer für das Reich So wird als die der Geschichte Einen bestrickenden Reiz gewinnt der Gedanke, * Den direkten Gegensatz zu mechanisch bildet ihm aber gewöhnlich dynamisch; dort scheint ihm die Welt als etwas Gegebenes, hier als etwas unablässig Werdendes verstanden zu werden. Mechanisch — organisch. daß alles geschichtliche Werden nicht stoßweise, sondern in ruhigem durch künstliche bewußtlosen Naturtrieb, nicht von sondern der eines Ganzen Fortgang, nicht aus Kraft I37 durch sondern Reflexion, einen dem bloßen Individuum erfolge. Indem her, diese sich Vorstellung auf Staat, Recht, Sprache u. s. w. überträgt, scheint durch- gängig eine reinere und reichere Tatsächlichkeit, ein größeres Bild vom Ganzen, ein innerlicheres und ruhigeres Verhältnis des Menschen Nun soll er sie nicht mehr von draußen zu den Dingen gewonnen. her meistern, sondern von innen nicht machen, nun kann satz die engste her miterleben, Lebens mit Stelle rationalen verbunden. das Recht B. z. Erzeugnis des Volksgeistes finden; und Wendung zu zur ein als er die Fülle des geschichtlichen Art anerkennen duellen ist sondern an seiner jedes aller So im Gegen- einer geschichtlichen Weltansicht, organischen Lehre der Aufklärung, jener Die Forschung historische indivi- würdigen. nahen Freundin die künstlerische Kontemplation; aufs aber hat es bezeichnend, ist zur hier daß^Schelling für den »dritten und absoluten Standpunkt der Historie" den der historischen Kunst erklärt. Aber auch die Einseitigkeit dieser historischen Betrachtungsweise und zugleich die Schranken der organischen Lehre ließen sich nicht Bedenken mußte schon das erwecken, daß jene lange verkennen. organische Lehre von der politischen und kirchlichen Reaktion, von Männern wie Adam Müller und de Maistre, dem Vater des modernen Ultramontanismus, mit besonderer alterlichen Sinne zur Individuen der als Wärme ergriffen und im mittel- Unterdrückung der Selbständigkeit sowohl der lebendigen Aber Gegenwart gewandt wurde. auch über diese besondere Gestalt hinaus kam das Problematische und Einseitige der organischen Lehre bald zur Empfindung. Jenen ruhigen Fluß des geschichtlichen Werdens hat sie weit mehr vorausgesetzt als erwiesen; was an Objektivität aus den Dingen gegenzukommen schien, das hat sie selbst in diese ist ihr Bild der Geschichte bilde brachte jene Augenmerk Dinge richtete, Naturkräfte Bewegung das auf von Leben starker auch kräftige Antriebe gab. Subjektivität. wertvolle Anregungen, und den inneren ent- hineingelegt; so Dem Naturindem sie das Zusammenhang der zum Suchen der Einheit der Aber wissenschaftlich fruchtbar sind diese An- regungen nur nach Verpflanzung auf den andersartigen Boden der modernen Naturforschung geworden; soweit jene organische Denkweise aus eigenem Vermögen einen Abschluß versuchte, hat sie sich in ge- Zum Erkenntnisproblem. 138 Dem Ganzen des Lebens wagte, oft wunderliche Gebilde verloren. aber brachte sie Gefahr, indem sie den Menschen kontemplativ zur Wirklichkeit verhalten ließ, sich überwiegend ihn weit mehr einlud, willfährig aufzunehmen und sich einzufügen als selbständig aufzutreten und eigne Wege zu bahnen; das Ganze war namentlich ungeeignet für eine Zeit, die voll großer Aufgaben war und schwere Verwicklungen in sich trug. So kam die Herrschaft wieder an die andere die Seite, nie ganz unterdrückt, sondern nur eingeschüchtert war, mit frischer Kraft neu hervor. Etwas anders gefärbt, aber im Grunde unverändert trat sie stieg die istische Aufklärung wieder auf, von ihr aus mochte jene human- Epoche mit ihrer organischen Lehre als eine bloße Episode erscheinen. Der Aufbau des Gesellschaftslebens von den Individuen her kam im modernen Liberalismus und in der modernen Freihandelslehre erst zur vollen Entwicklung, bis in die zweite Hälfte des neun- zehnten Jahrhunderts sehen wir A. Smiths scharf zugespitzte Theorie selbst von hervorragenden Gelehrten als eine und einen endgültigen Abschluß behandelt. ausgemachte Wahrheit Die Naturwissenschaft aber unternahm, unter schroffer Ablehnung der naturphilosophischen Spekulation, 'alle Reste vitalistischer Theorie aufs gründlichste aus- nun dahin gestellt, auch die organische Bildung mit ihrem Leben ganz und gar auf die physikalischen und zutreiben; die Aufgabe wurde chemischen Elementargesetze zurückzuführen. Unter den Philosophen hat namentlich Lotze eine solche universale Geltung des Mechanismus verfochten, Reiche freilich seelischen nicht ohne ihm, ähnlich wie Leibniz, Lebens einen jenes Übermechanische tieferen Grund zu in einem geben. Aber war Sache der Metaphysik, die Natur verblieb dem Mechanismus, und in der Zeit kam diese Bejahung mehr zur Wirkung richtig als jene verstanden Begrenzung. und — So umsichtig Lösung der großen Probleme; so blieb, das Prinzip erschien angewandt, viel der Mechanismus, als eine gesicherte der Ausführung zu tun ver- dünkte allem Zweifel enthoben. Da kam ein Widerstand, ein unerwarteter Widerstand, aus der eignen Bewegung des modernen Lebens, nicht aus Nachwirkungen älterer Zeiten, und weniger aus einer künstierischen Deutung der Wirklichkeit als aus wachsender Erfahrung, aus neuen Tatsachen und Die wirtschaftliche und industrielle Gestaltung des modernen Lebens treibt die Menschen enger zusammen und vervielfacht ihre Berührungen, sie differenziert und kompliziert die Arbeit neuen Aufgaben. Mechanisch — organisch. 13g und bindet damit den einen weit enger an den anderen, aber an Ganzes; ein vor der Gemeinschaft, die Einzelnen alle daraus erwächst, verschwindet die Isolierung, in welcher der Mechanismus die IndiHatte er alle Zusammenhänge von ihnen her abgeleitet, viduen sah. modernen Soziologie der Einzelne von vornherein so scheint der einem sozialen Zusammenhange angehörig, zum bloßen Produkt den Einzelnen dahin, vom Lehre die Milieu das Unsichtbare der Einflüsse zur Geltung und bringt auch seiner neigt Umgebung zu Zugleich wird die Wehrlosigkeit der Individuen gegenüber machen. den wirtschaftlichen Verwicklungen und Gegensätzen grell empfunden und mit ihr Notwendigkeit eines Gesamtwillens, die wie der ihn Staat verkörpert. Das alles drängt dahin, den Gedanken des Organismus wieder aufzunehmen, Philosophen hat namentlich Comte seine unter den und Politik von ihm aus gestaltet. Ethik Aber der Begriff wird bei ihm gegen die ältere Fassung beträchtlich verändert, er wird, wenigstens grundlegenden der in Ethischen ins aus Erörterung, der Histologie Fortschritte eine anschauliche Ausführung geben. ist auch die Gesellschaft ein Künstlerischen Es sind und namentlich die dem Grundgedanken Wie der lebendige Körper, überaus feines Gewebe aus lauter die so dem Naturwissenschaftliche versetzt. (Bichat), Elementen; so eng sind diese miteinander verschlungen, einzelnen daß das Tun und Lassen, das Wohl und Wehe des einen unmittelbar auch die anderen berührt. Die moderne Arbeitsteilung hat was von jeher galt, noch weiter gesteigert, die Anderen heit des Einzelnen an Damit scheint Augen, ein sie stellt die und an das Ganze leitendes Prinzip für Ethik gewonnen, das nur entwickelt zu werden braucht, dies, Gebundendeutlich vor und Politik um allem Handeln bestimmte Bahnen vorzuschreiben. In Wahrheit ist ein solches Prinzip nicht sowohl begründet als durch eine Vermengung antiker und moderner Art erschlichen; unvermerkt wird aus der Verwebung ein Ganzes innerer Art, aus dem Sein ein Sollen hervor. dem alten Boden, wenn das Ganze mit Forderungen an den Einzelnen kommt und sie ihm als Verpflichtung auferlegt. Das Dunkel, das dem Begriffe organisch von jeher anhaftete, wird durch solche Vermengung alter der Tatsache geht ein Wertbegriff, aus Schließlich befinden wir uns ganz auf und neuer Art bis zu unerträglicher Verworrenheit gesteigert. Aber man hält an dem Begriffe fest, weil es drängt, die Abhängigkeit des Zum Erkenntnisproblem. 140 Einzelnen vom Gesamtstande irgendwie zu bündigem Ausdruck zu bringen. So moderne Forscher der gerät unter widerstreitende Anregungen, und ein Auseinandergehen bis zu schroffem Gegensatz kann nicht Wunder nehmen. Gebiete trennen sich Nicht bloß die Individuen, auch die Am meisten Anklang hat die organische hier. Lehre bei den Soziologen, weit weniger bei den eigentlichen National- ökonomen gefunden; unter den Juristen sind ihr am ehesten hervorragende Germanisten gewogen. Mit dieser Bewegung auf sozialem Gebiet geht parallel eine Bewegung in der Naturwissenschaft; wie sie später einsetzte, so ist sie heute noch weit mehr mitten im Fluß und Kampf. Unverkennbar hat dazu an erster Stelle die moderne Entwicklungslehre gewirkt. Die darwinistische Form, gelangte, war in der in sie zuerst dem Eigentümlichen zu allgemeiner Geltung ihrer Art der Anerkennung des Organischen so abgeneigt wie nur möglich, sucht sie doch das ganze Gebiet des Lebendigen den Begriffen des Mechanismus zu Aber auch unterwerfen. massen oft ihrer in der Naturwissenschaft eignen Absicht entgegen. wirken Gedanken- Indem das Reich des Lebens weit mehr die Aufmerksamkeit und die Arbeit gewann, kam mehr zur Geltung, und es erwies sich die Zurückführung seiner Erscheinungen auf die physikalischen und seine Eigentümlichkeit weit chemischen Elementargesetze unvergleichlich schwieriger, Mitte des Jahrhunderts als um die Die Beobachtungen am der Entwicklungsmechanik, das angenommen war. die neuen Ausblicke Problem der Kontinuität des Lebens, die Mutationslehre mit ihrem Protoplasma, Aufweis sprunghaften Entstehens neuer Formen ander ergibt eine Scheidung der Geister. u. a., alles mitein- veränderte Lage. Dabei erfolgt eine Hoffen die einen von einer Verfeinerung wesentlich der mechanischen Begriffe eine intellektuelle Aneignung der neuen Tatsachen, so glauben die anderen auf ein neues Prinzip nicht verzichten zu können.* ^ In diesen Bewegungen erhebt sich von neuem S. u. a. Rindfleisch, Ärztliche Philosophie 1888 und Neovitalismus 1895. Roux, Einleitung zum Archiv für Entwickelungsmechanik der Organismen (1894), wendet sich dagegen, „die organische Form als Unerklärbares und bloß teleologisch Ableitbares zu bezeichnen" (3. 22), und bemerkt weiter: „Für den Forscher auf dem Gebiete der Entwickelungsmechanik gilt in hohem Maße das Wort »Incidit in scyllam, qui vult vitare charybdim." Die zu einfach mechanische und die metaphysische Auffassung repräsentieren Mechanisch — organisch. 141 auch das Problem der Teleologie, ihrer eignen Absicht nach weniger als ein Stück der Metaphysik, denn als ein Mittel naturwissenschaft- licher Erklärung, als „empirische" Teleologie,^ aber auch von anderen Rückfall in die Metaphysik bekämpft. als ein solche als Wird so die mechanische Lehre vom Gebiet des Lebendigen her, wenn nicht eingeschränkt, so doch über die herkömmliche Form, „die zu einfach mechanische Auffassung" (Roux), hinausgetrieben, Grundbegriffe mannigfach an- so werden weiter auch gegriffen. Schon die unermeßliche Verfeinerung, welche scheinbar ihre eignen elementare Vorgänge der unorganischen Natur enthüllten, lassen die Vorstellungen des älteren Mechanismus für das Unterlebendige selbst als viel zu roh erscheinen. Prinzipiell hat namentlich die energetische Naturlehre das mechanische Weltbild bekämpft, indem sie die Grund- vorstellung von der Materie als einem jenseits der Sinnesempfindungen befindlichen Sein, und als einem besonderen Träger der Naturerscheinungen alle zurückzuführen suchte. 2 hier den auf Auf die Probleme, nebenbei einzugehen, verbietet hat die mechanische Theorie sie lange zu haben schien. sich Kräfte, bestritt der Energie Grundbegriff die daraus erwachsen, schlechterdings; jedenfalls die Selbstverständlichkeit verloren, die Selten aber wird etwas neu zum Problem, ohne sich dabei umzubilden. So finden wir heute das ganze Gebiet voll Erregung und Streit; die Scylla und die Charybdis, zwischen welchen dahin zu segeln in der Tat ist; und es ist nicht zu leugnen, schwer und bis jetzt nur Wenigen gelungen daß die Verführung zu letzterer Auffassung mit der Zunahme unserer Kenntnis zunächst erheblich zugenommen hat" (S. 23). S. auch W. Roux: „Über die Selbstregulation der Lebewesen" 1902. ^ Coßmann, S. Elemente der empirischen Teleologie 1899, ferner König, »Die heutige Naturwissenschaft und die Teleologie". Beil. zur Es ist eine Allg. Z. 1900, No. 29 u. 30, sowie „Über Naturzwecke" 1902. überaus reiche, unablässig wachsende Literatur über diese Probleme entE. standen, ein deutliches Zeichen, wie sehr sie im Mittelpunkte des Interesses und der Arbeit stehen. ^ S. Ostwald, Vorlesungen über die Naturphilosophie S. 153: „Alles, was wir von der Außenwelt wissen, können wir in der Gestalt von Aussagen über vorhandene Energie darstellen; und daher erweist sich der Energiebegriff Er allseitig als der allgemeinste, den die Wissenschaft bisher gebildet hat. umfaßt nicht nur das Problem der Substanz, sondern auch noch das der Über die Bedeutung des Begriffs Energie aber heißt es S. 158: »Wir werden allgemein Energie als Arbeit, oder alles, was aus Arbeit entsteht und sich in Arbeit umwandeln läßt, definieren." Kausalität." Zum Erkenntnisproblem. 142 die Entscheidung aber liegt nicht bei allgemeinen Reflexionen, sondern bei Hauptrichtung, der welche Arbeit und Leben in Wirklichkeit So zeigte es die Vergangenheit, so wird auch einschlagen. in Zu- kunft der eigne Fortgang der Arbeit darüber befinden, wie sich das Verhältnis der Gegensätze gestalte, welche Weiterbildungen für beide Grundbegrilfe nötig werden, auch ob ihnen aufkommen mögen. klärungsarten Das gegenüber neue Er- aber darf die philosophische Betrachtung erwägen, wie der Überblick der Wirklichkeit heute den Stand der Begriffe zeigt, und welche Aufgaben er nahelegt. Erwägungen zum Kampf der Gegenwart. c) Das Problem im Gebiet der Philosophie. a. Die Philosophie muß vor allem darauf bestehen, daß der Mechanismus, selbst wenn er sich der ganzen Breite der Dinge bemächtigen nun und nimmer einen endgültigen Abschluß zu bilden könnte, vermag. Denn das, was für ihn das Letzte ist, das Nebeneinander der Elemente, wird der philosophischen Betrachtung notwendig zu einem schweren Problem. Stünden nämlich Elemente die ohne irgendwelche Verbindung gleichgültig nebeneinander, so wäre schlechterdings nicht zu ersehen, wie eine Wirkung des einen entstehen können. sollte Leibniz und einen Lotze die der Wechselwirkung Tatsache das nächste Weltbild gründlich umzugestalten. trieb, zum andern So vor allem in der Natur, wie denn einen dazu Und auch der Gedanke Leibnizens ist nicht wohl abzuweisen, daß kein Ding darin aufgehen kann, lediglich etwas für andere zu leisten, sondern daß es auch für sich selbst etwas sein muß. daß daher auch das als letztes Element Ergriffene irgend etwas Eignes zu sein dieses hat; die Verfolgung Gedankens drängt dahin, das Reich des Mechanismus zur bloßen Erscheinung einer andersgearteten Welt herabzusetzen. Auch beim Seelenleben würde, wer alles Geschehen auf den mechanischen Verlauf von Assoziationen zurückführen wollte, ratlos der Frage gegenüberstehen, wie dies ganze Getriebe als ein eignes Leben, als mein und Einheit ist dein erlebt werden könnte. und Zusammenhang Sorge zu Überall tragen, ist und irgendwie für dieser Aufgabe der Mechanismus nicht gewachsen. Läßt so der Mechanismus hinter sich ein ungelöstes Problem, so darf auch ganzen dem Tatbestande nach er nicht als Beherrscher der Wirklichkeit gelten, selbst wenn die Natur ihm ganz ge- Mechanisch — organisch. 143 Denn zur Natur gesellt sich das Seelenleben, dies aber bringt, hörte. namentlich auf der menschlichen Stufe, eine völlig andere Art des Geschehens mit Denn soweit das Innenleben über eine bloße sich. Begleitung der Naturprozesse hinauswächst und selbständige eine soweit in uns Geistesleben aufsteigt, genügt nicht mehr Art entfaltet, ein Zusammentreten einzelner Elemente, sondern hier befindet sich jede einzelne Erscheinung innerhalb eines Ganzen, und hier wird eine Verkettung nicht direkt zwischen den einzelnen Elementen, sondern durch ihr Verhältnis z. zum Ganzen hindurch gewonnen. Das Denken B. verläuft allerdings in einzelnen Vorstellungen, aber es ein bloßes Aneinanderreihen und Aufschichten ist nicht dieser, es verfolgt ein bestimmtes Ziel und wird dadurch innerlich zusammengehalten; es kann nichts dulden, was diesen Zusammenhang stört. diese neue Art bezeichnender als und logischen Widerspruchs. die Tatsache Widerspruch Ein ließe Nichts für ist die Macht des sich gar nicht empfinden, würde nicht im Denken die Vielheit von einer Gesamt- umspannt, und er könnte nicht so unerträglich sein wie er tätigkeit es wäre nicht das Verlangen nach Einheit von gewaltiger Stärke. ist, Zugleich erweist der bindung, als Widerspruch eine Mechanismus der völlig andere Art der Ver- aufbringen sie kann: er ist kein räumlicher Zusammenstoß, sondern eine Unverträglichkeit des Inhalts; so erscheint hier der Begriff schlechterdings unverständlich Prinzip der Anordnung mit Bedeutung, lichen des Inhalts, der dem Mechanismus ist. Der Inhalt bringt aber ein neues sich: das der Sachlichkeit, der sach- der gegenseitigen Determination. Das ist z, B. das Verhältnis der Merkmale eines logischen Begriffes; nur gröbstes Mißverständnis kann die innere Struktur eines solchen Begriffes mit dem Nebeneinander einer sinnlichen Vorstellung zusammenwerfen. Die Grundform der Verbindung ist hier die des Systems: jedes Ele- ment steht innerhalb eines Ganzen, unter dem Einfluß und der Triebkraft eines Ganzen, Elementen. in wechselseitiger Determination mit den anderen So gehört dem Mechanismus jedenfalls nicht das Ganze der Wirklichkeit. Daher verschwindet auch der Zweck lichen hat sie wenn Denn im mensch- nicht aus der Welt, die Natur für ihn keinen Platz mehr bieten sollte. Leben hat er zweifellos eine Wirklichkeit und eine Macht, nicht gemeinsamen er bloß in der Seele des Einzelnen, sondern auch im Leben Wissenschaft und bei Kunst, den großen Zusammenhängen, wie sie Recht und Moral, schließlich das Ganze Zum Erkenntnisproblem. 144 der Kultur bilden. ^ Damit, Innenleben wesentlich ist, ist daß es gehörig erwiesen, und es keit das Bild der Welt unweigerlich im Oesamtanblick die Sache schließlich Entweder als daß diese Tatsache verständlich wird. so zu gestalten, sich ist Handeln dem zum Ganzen der Wirklich- zwecktätiges ein auch Ja es notwendig — Oder. Wir pflegen heute die 'Welt als stellt auf ein ein Stufenreich aufsteigender Bildung zu betrachten, aber eine große Scheidung der Geister erfolgt bei der Frage, ein ob diesem Stufenreich das Höhere in Ergebnis des Niederen bloßes sei dem Höheren oder ob in ganze Erklärung finde, und daher aus ihm ein seine Neues und Ursprüngliches durchbreche, das sich nur aus einem tieferen Ganzen Der Gegensatz erlangt seine höchste Welt verstehen läßt. Spannung bei der Frage des Verhältnisses von Natur und Geistesleben. Ist dieses ein bloßes Erzeugnis jener, oder beginnt mit ihm der Die Beantwortung dieser Frage eine neue Stufe der Wirklichkeit? über Recht oder Unrecht des Zweckes. entscheidet auch Geistesleben mit seiner Innerlichkeit und Ganzheit von Ist da§ eigner Art und eignem Ursprung, so gehört es wesentlich zum Ganzen, so muß es von vornherein in der Bewegung wirksam sein und ihr eine Richtung zu sich geben; dann erhält das Weltgeschehen ein Ziel, und die spekulative Weltbetrachtung wird der Zweckidee nicht entkönnen ;2 raten Natur, so aber das Geistesleben ein bloßes Erzeugnis der alles entfällt und mit ihm auch der Zweck; dann Menschheit sinnlos ins Vage und Leere Ziel Welt und die die treibt ist dahin. ß. Das Problem in der Naturwissenschaft. Im Gebiet der Naturwissenschaft auf die allgemeinen physikalischen den Mittelpunkt des und chemischen Gesetze zurück- Daß aus dem Zweck „reale Kategorien" hervorgehen, hat Trendelen, ^ bürg bildet ob die dem Leben charakteristischen Erscheinungen Streites die Frage, einem hervorragenden in gezeigt; s. Kapitel der „Logischen Untersuchungen" Kap. XI. ^ So treibt es uns hier wieder zur Metaphysik gemäß jener Überzeugung Herbarts (Wke. 11, 461): „Im Denken über Natur und Menschheit drängt sich die Kraft des Geistes unvermeidlich zur Metaphysik hin, welche, ähnlich den Urgebirgen menschlichen schroffen, selten hinausragt." — zugleich die weite, , tiefe, unsichtbare Grundlage alles und Trachtens ausmacht, zugleich in einzelnen, erklommenen Spitzen über alle andern Höhen und Tiefen Dichtens Mechanisch — organisch. I45 führbar sind, oder ob in ihnen eine neue Art des Geschehens anerkannt werden muß. Tatsächlichkeit Diese Frage ist vor allem eine Frage der und gehört als solche in die Fachwissenschaft, aber es wirken in die Behandlung auch manche allgemeinere Erwägungen ein, an denen wir nicht vorbeigehen können. bar, daß die Eigentümlichkeit, mehr Lebens wieder heute wir nicht so den in das So viel ist unbestreit- Problem und Geheimnis Vordergrund gerückt des und daß ist, darüber hinauskommen wie unmittelbar Mehr und mehr scheint es ausgeschlossen, bloße Eigenschaft des Stoffes zu fassen, mehr leicht vorangehende Zeiten. das Leben als eine und mehr wird ihm eine Selbständigkeit zuerkannt. So, um einige hervorragende außerdeutsche Namen zu nennen, von Bergson, namentlich in seiner l'evolution creatrice^ (1907), so von Sir Oliver Lodge.^ Bei solcher Auffassung wird es zur Aufgabe, ein wesentliches und unterscheidendes Kennzeichen des Lebens herauszuheben; ein solches Boutroux findet in der Fähigkeit «ein System zu schaffen, in dem gewisse Teile gewissen anderen Teilen untergeordnet sind"; so würde „agens" und «Organe" geben, die zusammen eine «Hierarchie" es ein für die es in der anorganischen bilden, Welt keine Analogie gibt.^ Für Bergsons Fassung des Lebens sind folgende Stellen bezeichnend La vie est, avant tout, une tendance ä agir sur * L'evolut. CTeatrice pag. 105: la matiere brüte; fem er 197: La vie c'est-ä-dire la conscience lancee ä travers la matiere. Lodge * sagt »Leben und Materie" (deutsche Übers. 1908, S. 104) zu- sammenfassend: «Die Anschauung vom Leben, die ich im vorigen auszudrücken versucht habe, ist die, daß es weder Materie noch Energie sei, noch auch nur eine Funktion von beiden, sondern daß es in eine ganz andere sei, daß es in einer Weise, die wir zurnoch nicht durchschauen, imstande sei, mit der materiellen Welt in Wechselwirkung zu treten, aber daß es auch, abgesehen von dieser, seine wissenschaftliche Kategorie zu setzen zeit ursprüngliche Realität habe, wennschon diese sich der sinnlichen Erfassung entzieht. Es Natur und in ist abhängig von der Materie nach seiner Erscheinung in der uns hier und jetzt und nach seinen gesamten irdischen Wir- kungen; an und für sich aber ist es davon unabhängig, und sein Wesen ist und dauernd, während seine Wechselwirkung zur Materie disund zeitlich ist.« Ferner siehe S. 38: »Ich gebrauche das Wort Leben' in ganz allgemeinem Sinne. Denn wenn ich es nur in dem kontinuierlich kontinuierlich , engen Sinne, nach dem es nur die Stoffwechselvorgänge bedeuten würde, genomnien hätte, so wäre es natürlich absurd, ihm Existenz gesondert von der Materie zuzuschreiben. leicht Für diesen engeren Sinn könnte man sich viel- passend der Bezeichnung , Vitalität' statt ,Leben' bedienen." O. Beelitz, Die Lehre von Zufall bei E. Boutroux 1907, Eucken, Grundbegriffe. 4. Aufl. 10 ä S. S. 91. Zum Erkenntnisproblem. 146 Bergson entscheidenden Beweis für das Wirken des einen erblickt Lebens als einer psychischen Kraft zu pflegt bilden daß die Natur der Tatsache, demnach und gleiche Ziele auf abweichenden Fragen findet sich manche Ver- zu verfolgen scheint. ^ Wegen der Behandlung Bei schiedenheit zwischen merkenswert in in oder ähnliche Strukturen bei sehr verschiedenen Organismen gleiche ist dieser den einzelnen Kulturvölkern, namentlich be»Rolle, die das Prinzip der Diskontinuität hier die dem jüngsten französischen Denken als das 82 ff.) geschehen ist. philosophischen Literatur tritt (Moderne Philosophen der französischen S. Es läßt sich die- spielt". ^ selbe mit ihren Motiven nicht besser schildern, von Höffding Er sagt dort: „In die Diskontinuitäts- philosophie auf besonders interessante und energische Weise hervor. Diskontinuitätsphilosophie Es lassen sich drei verschiedene, für die maßgebende Motive unterscheiden. Die Erfahrung bietet Qualitäts- verschiedenheiten dar, deren Reduktion weder der Spekulation noch der gelungen Entwicklungslehre war. Comtes Positivismus ausdrücklich die Kluft anerkannt, welche von gebiete einander die Für Comte bezeichnete jede trennt. hatte verschiedenen Natur- neue Wissenschaft eine besondere, irreduktible Gruppe von Erscheinungen. — Hierzu kommt, daß ^ selbst in jeder einzelnen Gruppe von Er- «Le pur mecanisme serait donc oü nous l'entendons, demontrable par si Ton pouvait etablir que la vie fabrique certains appareils S. L'evolution creatrice 1907, pag. 59: refutable et la finalite, au sens special un certain cote, identiques, gentes. par des moyens dissemblables, sur des lignes d'evolution diver- La force de la preuve serait d'ailleurs proportionelle au degre d'ecarte- ment des lignes d'evolution choisies, et au degre de complexite des strudures similaires qu'on trouverait sur elles. Wie sich übrigens auch vom Standpunkt eines feineren, dabei tiefere Probleme bereitwillig anerkennenden Mechanismus dem Leben ein eigentümlicher Charakter zugestehen läßt, zeigt namentlich W. Roux. Er betrachtet als eine universelle elementare Eigenschaft der Lebewesen «die zur Dauerfähigkeit im Wechsel der Verhältnisse nötige Selbstregulation in der Ausübung aller Einzelfunktionen", er sieht darin „diejenige von allen Leistungen, welche die Lebewesen am meisten von allen andern Naturkörpern unterscheidet, indem sie die direkte Anpassung an die wech- Aus der unübersehbar langen, trotz Umstände zahllose Generationen gleicher Art her- selnden äußeren Verhältnisse bewirkt. des Wechsels der äußeren stellenden Dauer der daß auch die lationsfähigkeit einzelligen Lebewesen ist mit Sicherheit zu schließen, niedersten Lebewesen außer der Vererbung diese Selbstregu- haben" Archiv für Entwicklungsmechanik der Organismen (s. XXIV. Bd., 4. Heft [1907], S. 685). ? S. Harald Höffding, Moderne Philosophen (1905) S. 67. Mechanisch — organisch. 147 scheinungen der Kausalsatz nur unvollkommene Bestätigung zu finden Man zieht deshalb Hume wieder hervor und stellt seinen vermag. Empirismus gegen die Versuche mus auf, die Kant und der Evolutionis- — Endlich verweist man machten, denselben zu überwinden. auf das Bewußtsein der Initiative, des Vermögens, durch sein Denken und Handeln etwas Neues in Welt zu die und man betont setze i, mit Stärke die moralische Bedeutung dieses Vermögens. "^ Bei Mechanismus eine Neigung bestehen, Lebens vielmehr wird an scharfe Kritik geübt. er die Welt wie ein gegebenes daß verfehlt, keinerlei des Erscheinungen zurückzuführen, Kräfte kann Denkweise solcher charakteristischen auf solchem Versuche des Am Mechanismus scheint und geschlossenes System im Fluß Befindliches, daß behandelt, nicht als etwas die unterlebendige er daher alle Bewegung von innen her, sowie alle Möglichkeit eines wesentlichen daß er den Verbindungen der Elemente nichts Fortschrittes leugnet, 2 anderes zuschreiben seine Erklärungen sammensein will, was jedem einzelnen zukommt, ^ daß als den Elementen beizulegen pflegen, was ihr Zu- aufweist,* daß er nicht genügend beachtet, wie die genauere Erkenntnis des Lebensprozesses immer mehr die vermeintIsolierung der Elemente aufhebt.^ liche ^ hervorragendsten Vertreter dieser Diskontinuitätsphilosophie sind Djp. Renöuvier (f 1903) und E. Boutroux, dessen Werk de l'idee de la loi naturelle dans la science et dans la philosophie contemporaine (1895) 1907 in einer deutschen Übersetzung von Benrubi erschien. ' S. Bergson, L'evolution creatrice pag. 40: L'essence des explications mecaniques est en effet en fonction du present, ^ häufig de considerer l'avenir et le passe comme calculables et de pretendre ainsi que tout est donne. S. 57: „Man begegnet dem Satze, daß, was immer an Eigenschaften dem Ganzen zukomme, Sir Oliver Lodge, Leben und Materie (Übers.) auch in den Teilen zu finden sei. Atomen kann Eigenschaften Dieser Satz ist falsch. besitzen, die den Ein Aggregat von einzelnen nicht zukommen, auch nicht im geringsten Grade." „man konstatiert hier einfach das zu Erwägende dann den Atomen zu, in der Hoffnung, daß damit dem Fragen ein Ende gemacht sein wird." Bergson hat diesen Gedanken namentlich gegenüber der Entwicklungslehre Spencers verfochten, diese scheint ihm * und (s. S. Lodge a. a. O. S. 47: schiebt es L'evol. creatr. VI) evoluee, puis ä la darin zu bestehen: ä decouper la realite actuelle, dejä recomposer avec ces fragments, et ä se donner ainsi, par avance, tout ce qu'il s'agit d'expliquer. ' S. Bergson, L'evolution creatrice pag. 205: plus la physique avance, meme des particules en plus eile efface d'ailleurs l'individualite des corps et 10* Zum Erkenntnisproblem. 148 Mit solcher Wendung zum Leben und seiner vordringenden Bewegung tritt auch die Zweckbetrachtung in ein anderes Licht. Die völlige Verwerfung der Zwecke in der Natur wurzelte in der lange vorherrschenden Neigung, das Leben nicht als ein Urphänomen zu fassen, sondern es vom Leblosen abzuleiten, in schroffem Gegensatz zur älteren Denkweise, die (Jen ganzen Befund der Natur vom In einer gewissen aus erklärte. Rückkehr zu Lebendigen wenn auch in dieser, großer Verfeinerung der Betrachtung, treten nun die Tatsachen wieder mehr in den Vordergrund, die eine Richtung der Bewegung auf ein erst zu erreichendes Ziel, eine «Zielstrebigkeit" (K. E. sowie ein Zusammenstreben zu Die Schwierigkeit, scheinen. zeigen von Baer), einem Ganzen einzelner Elemente zu das irgend vorstellbar zu machen, ohne die menschliche Art des Erwägens und Überlegens in die Natur hineinzutragen, hat schon Aristoteles stark empfunden, muß sie noch größer erscheinen. uns Neueren dazu nicht keit dürfte führen, Aber alle Schwierig- Tatsachenkomplexe zurückzustellen und geringzuachten, weil sie sich nicht dem Rahmen des Mechanismus fügen. Haben sich doch die Theorien den Tatsachen, nicht diese jenen anzupassen. Das Hauptbedenken, das gegen den Vitalismus und die Teleologie auch in der neueren Form erhoben wird, ist, daß das von ihnen behauptete Gestaltungsprinzip «einfach alles, und zwar auf die- Weise" selbe ohne daß wir von den für die verschiedenartigen erklärt, zweckmäßigen Gestaltungen notwendig verschiedenen determinierenden Faktoren und deren Wirkungsweisen etwas erfahren können.^ Ausführung dessen In weiterer sagt Roux, der Probleme" keineswegs leugnet: «Es letzte ist verbleibende «stets überaus leicht, zweck- mäßig Erscheinendes von einem wirklich zwecktätigen Agens abzuleiten. Diese letzte Annahme bleibt uns immer noch, wenn die andere wirklich als dem nicht zureichend erwiesen scientifique commengalt par les decomposer; corps corpuscules tendent ä se fondre dans une interaction universelle. ^ xal was jetzt erst kurz nach Beginn exakter kausaler Forschung zwar vielfach so scheinen lesquelles l'imagination et ist, Ttt y.a,zu «pu'oiv äXX(i)v, xtv£; 199 a, 17: d ouv toc xaxi t:qv -ziyyriv ^vexa tou, SfXov S. Z, B. Phys. xaxa ttjv 6(ao(cü5 yflip <puaiv. Jtpos xa ouxe ^rjxi^aavxa xa ucrrepa a ouxe TOxepov vw xi^yri i] xivt ort ^jti Jtpo? aXXrjXa Iv Tot? xata -ziyyriv xai iv toi; Ttpdxepa. (AcIXioxa Se (pavepov ouxe ßouXsuga'jjLeva uotet. ini xwv !^towv xuiv od-ty SianopoZai öcXXw IpyaJ^ovxai o'f x'apayvat xa\ oi (Aup[XT]x£i; xai xa xotauxa. * S. W. Roux im Archiv für Entwicklungsmechanik der Organismen, XXVI. Bd., 4. Heft (1907), S. 687. Mechanisch — organisch. mag, aber doch nicht bewiesen werden kann. dagegen, solches »scheinbar Agentien abzuleiten. Zweckmäßige" von I49 Sehr schwer nicht es Erstere Lösung aber läßt alle in den verschiedenen Fällen verschiedenartigen Determinationen unbekannt, verlegt sie in Wir aber Wirkungsweisen nicht aufhellbares Prinzip. ein in seinen ist zwecktätigen möchten auch diese « Bestimmungsfaktoren « und ihre Wirkungsweisen erforschen. Gemeinsam ist uns beiden die Erforschung der physikalisch- chemischen Ausführungsfaktoren des Determinierten, denn daß das durch seelisches Wirken „Determinierte" durch physische Faktoren ausgeführt wird, geben die Sache mitten ja auch unsere Gegner zu" (S. 688). So ist im Fluß, aber aus Bewegung und Zusammenstoß Fortgang des Wissens mit Sicherheit hoffen. läßt sich ein y. Das Problem auf gesellschaftlichem Gebiet. Daß zum Verständnis des gesellschaftlichen Zusammenseins der Mechanismus nicht ausreicht, ist ohne Mühe zu zeigen, schwierig aber ist, ähnlich wie bei der Natur, ihm gegenüber zu einer positiven Be- Von den bloßen hauptung zu kommen. Einzelelementen her läßt sich irgendwelches Interesse für das Ganze, irgendwelche innere Ge- genwart des Ganzen, irgendwelche Hoheit und Selbständigkeit des Ganzen, wie B. des Staates, irgendwelcher geistige Charakter des z. Ganzen in keiner Weise begreiflich machen.^ Die mechanische Theorie müßte die Gemeinschaft in ein seelenloses Räderwerk verwandeln, in dem jeder nur seine eigenen Ziele verfolgt; eine gemeinsame Gedankenwelt wäre dabei unmöglich. Auch die Rechtsidee, worauf die Anhänger der mechanischen Theorie sich gern berufen, ist von hier aus nicht zu erklären, Gebilde erscheinen. Vernunft. ist könnte hier nur als ein mystisches Denn der Rechtsgedanke entwickelt sich nie vom natürlichen Einzelwesen, solches aber sie sondern nicht möglich nur vom Vernunftwesen her, ein ohne Begründung in einer Welt der Das Recht kann vom bloßen Individuum her zu entetehen nur scheinen, wenn dabei unvermerkt dem natürlichen Einzelwesen das vernünftige Geisteswesen untergeschoben wird. So geschah es namentlich in der englischen Aufklärung, wie denn die politischen und wirtschaftlichen Systeme eines Locke gängigen Widerspruch * Vortrefflich in und eines A. Smith einen durchsie arbeiten mit Größen der sich tragen: hat dies neuerdings Gierke ausgeführt: „Das Verbände", Rektoratsrede. Berlin 1902. Wesen der Zum Erkenntnisproblem. 150 Natur und behandeln sie Wer die Ver- wie Größen der Vernunft. mengung erkennt, der durchschaut zugleich die Unzulänglichkeit des hier gebotenen Ganzen. Aber mit dem Nein ist Der kennen. Begriff Gesellschaftslehre heißt nicht des Organischen überkommen, andersartigen Kultur über das Ja entschieden, nicht schon mechanische Lehre abweisen die ist organische aner- uns aus einer älteren, er trägt die Färbung der antiken und Weltanschauung. Die Vorkämpfer der organ- möchfen ihn davon befreien; ischen Lehre die können sie sich darauf mit Begriffen arbeiten, die der Lauf der Ge- berufen, daß wir schichte über die Enge der anfänglichen Fassung weit hinausgebildet Aber bei solchen Fragen hat. liegt alles an der besonderen Art des will uns nun scheinen, daß jenes Anfängliche dem Begriffe fest anhafte, um nicht leicht den Gedanken auf die Stufe älterer Es Falles. zu oft Denkart zurückzuziehen. Die Verwendung des Begriffs Organismus für das gesellschaftliche Gebiet ist zunächst eine bloße Analogie; mögen gewisse Übereinstimmungen zwischen einem organischen Lebewesen und einer gesellschaftlichen Ordnung bestehen ob sie das Wesentliche und das charakteristisch Geistige treffen, daran läßt sich recht wohl : Zunächst bildet der zur Aufklärung herangezogene Bau zweifeln. der Lebewesen selbst ein schweres Problem und eben wieder ein Gegenstand heute härtesten ist, wie wir sahen, Streites; von seinen philosophischen Definitionen, wie sie Aristoteles und, fügen wir hinzu, auch Kant und seine Nachfolger gaben, hat Lotze mit Recht gesagt, daß sie mehr das Rätselhafte des Eindruckes wiedergeben als eine Beim Begriff des Organischen bringt uns nicht, Erklärung enthalten. wie es wohl scheinen kann, die Natur die Sache sicher und fest entgegen, sondern es wird von uns eine eigentümliche Vorstellungs- weise in die Natur hineingetragen und wird, dort ins Anschauliche und Körperliche Umweg? gestaltet, dem Geist wieder zugeführt. Warum dieser Enthält er nicht die Gefahr eines Einströmens naturhafter Größen in das Geistesleben, oder doch des Einsetzens eines bloßen Bildes für eine Erklärung? Das Hauptbedenken aber ist das zähe Fortleben der griechischDenkweise mittelalterlichen in diesem Begriff; es droht, den not- wendigen Gedanken an eine innerlich überwundene Stufe zu binden. Die organische Theorie der älteren Zeit betrachtet den Einzelnen ganz und gar ihn als völlig ein in Glied des Ganzen, das Verhältnis sie läßt, zum Ganzen bei präziser Fassung, aufgehen, sie kennt Mechanisch — organisch. Recht Selbständigkeit, keinerlei keinerlei des 151 gegen Einzelnen das Das war von vornherein nur möglich durch eine Vermengung von Staat und menschlicher Gemeinschaft überhaupt; was Ganze. immer das Zusammenleben für den Menschen bedeutet, das ward für den Staat in Anspruch genommen; so konnten Ethik und Politik, individuelles und gesellschaftliches Lebensziel als völlig gleichartig gelten. In Wahrheit ist jene organische Lehre nicht einmal der treue Aus- druck des Staatsl6bens auf der Höhe der griechischen Kultur, sondern sie ist ein Gebilde der Auflösung Philosophen, ein Unternehmen, der beginnenden in lauter individuelle Lebenskreise zu widerstehen, sie ist der Versuch einer Restauration, erfolglos wie alle derartigen Versuche. Ja die Philosophen selbst haben am meisten dazu getan, ihre Forderung unmöglich zu machen, indem sie vor allem den Menschen über den eines neuen Lebensideals der wissenschaftlichen Forschung hinaus- bloßgesellschaftlichen Lebenskreis durch die Eröffnung hoben. Derselbe Aristoteles, der den Staat für früher (d. h. begrifflich früher) erklärt als den Menschen, sieht nur dem in theoretischen Leben mit seiner Richtung auf das große All ein wahrhaft glückseliges Leben. Und damit formuliert er nur die durchgehende Über- zeugung der gesamten griechischen Philosophie, zu deren Hauptverdiensten die Befreiung des Individuums von der gesellschaftlichen Umgebung gehört. Mittelalter. Die Hauptstätte der organischen Lehre ist das Hier ward in der Kirche das gesellschaftliche Ganze dem Individuum unbedingt überlegen, hier erhob es den Anspruch, dem Menschen alle Geistigkeit zuzuführen, hier bemaß sich alle Bedeutung der Einzelnen nach der Stellung im Ganzen, hier ward das Ganze zum Gewissen der Menschheit. Auch die wirtschaftliche Gestaltung des Mittelalters bildet ein Ordnungssystem, das aus sicherer Überlegenheit dem Einzelnen seine Stellung Gedankenwelt hat eine hierarchische zuweist Gestalt, indem Ja die gesamte von gewissen Zentralwahrheiten der Religion und Metaphysik den einzelnen Gebieten ihre Richtlinien vorgeschrieben werden, die sie nicht zu prüfen oder zu ändern haben. Für diesen Stand mag Vor- stellung und Ausdruck «organisch" Für die Neuzeit aber ist als nur weiterzuführen, angemessen erscheinen. nichts charakteristischer, als daß sich das Leben von solcher Bindung an einen sichtbaren Mittelpunkt befreit und über die ganze Fläche des Daseins gleichmäßig ausgedehnt hat: die Individuen sind geistig wie gesellschaftlich selbständig ge- worden, und die einzelnen Lebensgebiete wollen ihre Probleme selb- Zum Erkenntnisproblem. 152 sie wollen, jedes an seiner Stelle, auch um das Dem an die mittelalterliche Denkweise Gewöhnten behandeln, ständig Ganze kämpfen. muß das als ein kecker Abfall, als eine eigenwillige Auflösung aller Zusammenhänge erscheinen, wie es denn selbst freier nnd universaler denkenden Katholiken recht schwer zu werden Art und ein eigentümliches Recht des liche erkennen. In die die eigentüm- Wahrheit bedeutet jene Abwendung vom nicht die Preisgebung aller, hänge; pflegt, Protestantismus anzuMittelalter sondern nur die sichtbarer Zusammen- Größe der Neuzeit liegt in der Entfaltung und Ver- fechtung der Überzeugung, daß das Geistesleben als Ganzes an jeder gegenwärtig Stelle und sich zu sei voller Tätigkeit bringen lasse; so braucht der Mensch Zusammenhänge nicht erst von draußen her zu empfangen, sondern sie umfangen ihn von innen her; mit ihrer vollen Aneignung aber erhält er eben durch die innere Bindung eine sichere Überlegenheit gegen Bindung solche sich eigne Entscheidung nie und menschliche Ordnung. von außen erzwingen erst solche läßt, Da sondern eine und Zuwendung verlangt, so ist sie kein Gegen- sondern eine Zwillingsschwester der satz, keit, alle sichtbare Freiheit. Auch verleiht Wendung dem Leben den Charakter der reinen Innerlich- während es unvermeidlich ein Moment des Äußeren und Äußer- lichen behält, Ordnung solange der Einzelne an erster Stelle einer sichtbaren angehört. Persönlichkeiten wie Luther und Kant stellen uns deutlich genug vor Augen, wie sehr diese Umwälzung, dieser Fortgang von einem sichtbaren zu einem unsichtbaren Ganzen, diese Möglichkeit und diese Forderung, an jeder Stelle ursprüngliches und unendliches Leben zu erwecken, den Anblick der menschlichen Wirklichkeit umgestaltet Solche Umwälzung aber trägt einen Bruch mit der organischen muß nun als zu eng und beengend empfunden Der Mensch geht nicht auf in das Verhältnis zur gesellschaftlichen Umgebung und noch weniger in das zur politischen Lehre in sich; diese werden. Gemeinschaft; auch hat das Ganze, das uns umfängt, einen geistigen Charakter nicht aus als einen festen, aller Gefährdung entzogenen Besitz, dem die Individuen mühelos schöpfen, sondern was immer sich im geschichtlich-gesellschaftlichen Leben an gemeinsamen Vorstellungen, Einrichtungen u.s.w. gebildet hat, das verliert seinen geistigen Charakter sofort, wenn es nicht durch die Arbeit der Individuen, namentlich durch die großer Persönlichkeiten, immerfort mit neuem Leben erfüllt wird; wie überhaupt, so erhält sich auch im gesellschaftlichen Ganzen Mechanisch — organisch. die Geistigkeit nicht 153 durch ihr bloßes Dasein, sondern nur durch eine fortwährende Erneuerung, durch ein unablässiges Schaffen. scheint uns die Hauptgefahr der organischen Lehre, für allemal vorhanden betrachtet, daß Das sie als ein was immer neu aus freier Tat muß; sie will im Gegensatz zum Naturalismus dem Zusammenleben einen ethischen Charakter geben, aber sie gerät dabei in Gefahr, das Ethische selbst als ein Ruhendes und damit naturhervorgehen zu fassen. haft erlegen die ist: Es ist das dieselbe Gefahr, der die Romantik oft der Rückschlag gegen die bloße Reflexion führt unter Macht von Naturbegriffen. Warum sollen wir also die not- wendige Wahrheit einer so problematischen Form verketten, warum nicht für die Eigentümlichkeit geistiger Zusammenhänge Formen suchen, die der modernen Stufe des Lebens entsprechen? 3. Gesetz. Zur Geschichte. a) |er Begriff des Gesetzes steht heute im Mittelpunkt der wissen- I *—^ schaftHchen Arbeit; man streitet über seine Ausdehnung und man streitet über seinen Inhalt, besondere Fassungen kommen auf und und Feinde, finden Freunde die Auseinandersetzung zwischen Natur- und Geisteswissenschaften ist hier besonders lebhaft, ja erregt. weniger scheint hier gekämpft zu werden nichts als Um um die Eigen- tümlichkeit der einzelnen Wissenschaften wie um den Gesamtcharakter der wissenschaftlichen Arbeit. So breitet sich eine schier unermeßliche Fülle von Erörterungen aus; unsere Betrachtung kann an die Probleme, die dabei in Frage stehen, mehr nur erinnern als sie zu fördern hoffen. Der Begriff des Gesetzes ist vom Bereich des Menschen Natur gewandert, hat hier eine neue Gestalt gewonnen mit ihr zum Menschen in neues ein der Erscheinung, zurück, zu Licht daß um auch sein Leben und Handeln rücken. der zur und kehrt Er Mensch ist sein ein sinnfälliges eignes Bild in Beispiel das All hineinsieht und es, erweitert wie umgewandelt, aus ihm zurückempfängt. Dem einen dünkt das ein bloßer Zirkel der andere erhofft von und Anthropomorphismus, solchem Ausgehen und Zurückkehren eine innere Erweiterung des Menschen. Zu einem Hauptpunkt der Arbeit machte den Begriff des Gesetzes erst die Neuzeit, auf die bemerkenswerte Anfänge reichen aber in Der Ausdruck Naturgesetz geht zuerst nicht das Altertum zurück. Außenwelt, sondern auf die eigne Natur des Menschen, er bezeichnet das ungeschriebene Gesetz im Gegensatz zum geschriebenen. Über den geschichtlichen Ursprung des Terminus Naturgesetz handelt „Über Begriff und Begründung der sittlichen Gesetze" 1883 (Abh. der K. Pr. Akad. d. W.) und mit besonderer Umsicht und Gründlichkeit * F.. Zeller R. Hirzel «ypacpo? vojj.o? K. Sachs. Gesellschaft a'Ypacpo? vo'[jlos (Abhandlungen der der Wissenschaften, philologisch-histor. 20. Bd.). Klasse der Nach ihm bedeutet zunächst die altüberlieferte Sitte und Gewohnheit, diese Be- Gesetz. 155 Für die Natur als Außenwelt verwenden Plato und Ausdruck nur an vereinzelten Aristoteles den und geben ihm dabei keine Stellen technische Zuspitzung, ^ andere Bezeichnungen sind ihnen geläufiger den für Öfter haben den Ausdruck Naturgesetz zuerst die Begriff.^ wobei verwandt, «der Begriff der göttlichen Gesetze war dem zu konnte religiöse der Naturgesetze hinüberleitete" Vermittlung zur Vorstellungen Stoiker dienten; welcher zuerst es, Den (Zeller). Stoikern von der Gottheit begründete Gesetz zugleich das die als Ordnung der Dinge gelten, weil ihnen die Gottheit nicht so- eigne wohl eine jenseitige Macht als Der Ausdruck bedeutete. unter den Schule; die der Welt sich erhielt die Grenzen der Römern hat ihn öfter gleich der erste Philosoph Der Einbürgerung des Aus- Lucrez (foedera, foedus, leges naturae). deutung innewohnende Vernunft dann bald überschritt durch das ganze Altertum. Aber daneben kam (seit Thukydides) die andere der göttlichen, der ins Herz geschriebenen Gesetze auf.' 40 heißt es hier von der Revision und Reform der solonischen Ge- S. setzgebung, welche Kleisthenes ausführte: «Wie diese Reform nur durch die Macht des Demos gelang, so diente sie auch dessen- Zielen und Absichten, und es ist begreiflich, daß von an das Volk von Athen in seinen Gesetzen das Bollwerk des jungen Staates von Athen erblickte. Von hier an datiert der Kultus, der mit dem Gesetz und seinem Namen getrieben wurde. Die Weihe gaben ihm die Taten und der siegreiche Ausgang der Perserkriege" S. 50. «Wahrscheinlicher ist, daß der Name (i'Ypatpo? vo[i.O(;) erst im Gegensatz zum Y£Ypa(jL[x£voi; und aufkam, Xo'yo; daß er erst durch diesen Über den Gegensatz von vo'(jlo? noch genauere Untersuchung in sicher, Gegensatz seine schärfere Bedeutung erhielt." und «pu'ai; s, dort S. 82 ff. S. ferner die »Themis, Dike und Verwandtes" S. 386 vöaoiv opyava ye'Yovev, oxav atjxa aXX' ivavTitüv 15 caelo 268a, T015 —411. Die einzigen Stellen sind Plato Timaeus 83 E: ^ xptatv tov 10 ff: ü'pKTcat. [i.^ yäp xtijv tou? izapa o-fxov xa^^-a'^p Ix ipaut «puaew? TTfi xa\ o{ Xa[xßavT| 8t navta jj.kv vdfxou?. to HuÖ'aYopEioi, xeXeuxr yap xal [xeaov xa\ ap/71 xov xauxa 8k xov xtJ; xpia'8os. xal xaCta oiTtwv xa\ ttotcTv nXrj'S'uiJTi xata cpüatv, Ttäv ciptO-iiov Sio x:apa xtJ? ou'aew; £?X7)<pdxei; tSorcep Arist. de xol xa Travra iyti xoü rtavxd?, vo'jjlou? Ixe(vr)?, xal Wie vo'fxo; bei den Philosophen leicht einer- künstlichen Zubereitung angenähert und dem Wesen Ttpo? xa? iytaxeia; /ptüpieO-a xwv 9'Etöv entgegengestellt wird, zeigt u. a. aXXa $uXov, w? xo xXfvTjv xa\ X£')(^V7]V, x:^v 8' ou(Tiav [lIw xaxa ouaav xw aptö-jAW xouxw. Aristot., au[j.ߣßr]xd(; IxeJvtjv, .ri Phys. 193a, Ü7:a'p)(^ov, 14: xi^v oOx ay yeviid-on xaxa vdfxov Sta'O^atv xa\ 8ta[i£V£t Jtolvxa 7i:au)(^oucra <7\iVf)(iZ<;. Namentlich gehört hieher avccYxrj (meist im Plural), das sowohl in der ältp?i-:ri medizinischen Literatur als bei Demokrit, Xenophon (z. B. Memor. I, * 1, 11), Plato (z. B. leges 967 A), Aristoteles nicht selten vorkommt. Es dürfte der griechischen Forschung der Begriff des Naturgesetzes zuerst in der Astro- nomie und in der Medizin aufgegangen sein. Zum Erkenntnisproblem, 156 drucks war förderlich die im späteren Altertum übliche Personifikation der Gesamtnatur, indem Regelmäßigkeit ihres Geschehens als sie die den Ausdruck eines ordnenden Willens verstehen ließ. Einen tieferen Einfluß auf die wissenschaftliche Arbeit erlangte aber der Begriff des Naturgesetzes im Altertum nicht, namentlich wohl wegen des Über- gewichts einer und künstlerischen welche nicht zur Zerlegung in Naturbetrachtung, teleologischen elementare Vorgänge und zur Er- mittelung ihrer Regelmäßigkeiten trieb. Die Kirchenväter nahmen den Ausdruck auf und verstärkten seine religiöse Färbung, einem Augustin sind die Naturgesetze bloße Gewohnheiten göttlichen Handelns, Ge- wohnheiten, die zu Gunsten besonderer Zwecke jeden Augenblick verlassen ander So stören Wunder und Naturgesetze werden können. Im nicht. Mittelalter tritt ein- der Ausdruck sehr zurück; Natur- gesetz (lex naturae) bezeichnet hier das innere Moralgesetz, nicht die Ordnung der Außenwelt. ^ in Um so mehr hat die Neuzeit «Naturgesetz" den Vordergrund gerückt, an kaum irgendwelchem anderen Begriff hat sie so sehr ihr Selbstbewußtsein gefunden und ihre eigentümliche Art erwiesen. Gestaltung Ein Allgemeineres der Denkweise und die besondere der Arbeit verstärken einander dabei gegenseitig. Das Naturgesetz, als Ordnung des Geschehens, nicht des Sollens, als Aus- druck der Wirkformen einfachen der Elemente, Neigung der damaligen Menschheit für sich, hatte die volle weil es ein Verstehen der Wirklichkeit nicht aus jenseitiger Ordnung, sondern aus ihrer eignen Natur versprach, und weil es diese Natur ohne alle mensch- und Verfälschung bei sich selbst zu erschließen schien. Dazu kam das der modernen Wissenschaft eigentümliche Streben nach liche Zutat einer neuen, einer exakten Begreifung der Natur durch eine Zerlegung der Wirklichkeit in kleinste Elemente und eine Durchleuchtung von Der Ausdruck ^ leges naturae war so fremdartig geworden, daß der Beginn der Aufklärung ihn glaubte rechtfertigen und verteidigen zu müssen. So z. B. Clauberg op. omn. 103 : Est qui hie nodum in scirpo quaerat, quod quae imperant, non efficiunt, quae materiae, non possunt. Causa autem hujus appellationis leges sint tantura causae morales, utpote rationis experti,' (Naturae legum) est in ferri propatulo. ditis Deus leges imposuit morales, peccant, ita voluit res omnes Quemadmodum enim rebus ratione praequas abservando bene agunt, transgrediendo naturales certo semper ordine, moveri ac quiescere, quas quidem leges ipsae sariae, ligiösen zutage. non possunt non observare. Auch illae res, certis legibus utpote causae neces- hier liegt die Bedeutung der re- Denkweise für die Bildung und Verwendung des Begriffes deutlich Gesetz. 157 Die völlige Umwandlung des Weltbildes, die daraus hervor- daher. ging, hatte drei Hauptsttifen: Analyse, Gesetz, Entwicklung; das ist Ge- der einfachen Wirkformen der Elemente setz mit seiner Ermittelung das Rückgrat des Ganzen, erst mit ihm wird eine Präzision der Erkenntnis erreicht und eine vollständige Unterwerfung der Wirklich- den Gedanken angebahnt keit unter auf einfache Vorgänge so gewährte die sie zugleich die Elemente zu Gunsten wo Punkt, Natur Wie aber die Zurückführung zu machen schien, durchsichtig Möglichkeit neuer Kombinationen der Das Gesetz menschlicher Zwecke. ist der das Streben nach engster Verbindung von Theorie und Praxis, das der neueren Forschung von Anfang an innewohnt sich Erkennens Denn hier wird der Endpunkt des zum Ausgangpunkt des Handelns; aus vereinzelten und zufälligen Funden in wirksame Arbeit umsetzt ist die Technik eine selbständige und lebens- umspannende Macht nur mit Hilfe der Gesetze geworden. So laufen bei ihnen alle Fäden zusammen als dem Mittelpunkt der geistigen Arbeit; sie bilden den klarsten Ausdruck des modernen Verlangens nach immanenter und sachlicher Erklärung, nach analytisch-präziser Begreifung, nach einem aktiveren Verhältnis des Menschen zu seiner Naturumgebung. Aber zugleich war das Gesetz in dem neuen Sinne eine schwere Aufgabe und eine Sache mannigfachster Verwicklung. Im Streben nach Gesetzen verschlingt sich eng Erfahrung und Vernunft Regel- mäßigkeiten werden entdeckt, und es entsteht eine große Freude daraus, daß was im ersten Eindruck wirr durcheinander läuft, einer schärferen Betrachtung geordnete Reihen zu erkennen gibt Aber man verbleibt mehr oder weniger verwickelter Tatsachen, man möchte diese zerlegen und auf einfache, letzte, allnicht bei der bloßen Konstatierung gegenwärtige Elemente zurückführen, zugleich aber statt eines bloßen Nach- und Nebeneinander einen kausalen Zusammenhang erreichen; man strebt von empirischen zu rationalen, erklärenden Gesetzen, die notwendig und von beschreibenden zu allgemeingültig sind. Nur solche rationale Gesetze dürfen ausschließlich herrschen wollen, sie können keine Ausnahmen, also auch nicht das Wunder dulden. werden nach möglichster Einfachheit streben und keit als suchen. Ausdruck eines durchgehenden alle Sie Mannigfaltig- Geschehens zu verstehen Auch werden diese Gesetze auf einem präzisen Ausdruck, einer bestimmten Formel bestehen, da nur eine solche zur Beherrschung des Tatbestandes zu führen vermag. Diesen präzisen Ausdruck gibt Zum Erkenntnisproblem. 158 dem Naturgesetz vornehmlich So konnte Newton die Mathematik. Aufgabe echter Naturforschung darin die unter Verzicht auf setzen, Formen und die verborgenen Qualitäten die Naturerscheinungen auf mathematische Gesetze zurückzuführen, ^ und Kant die substantiellen behaupten, „daß in jeder besonderen Naturlehre nur so viel eigentliche Wissenschaft angetroffen werden könne, als darin Mathematik anzutreffen ist" (IV, 360 Hart). Aber solche Wendung schwere stellt Aufgaben und legt manche Irrungen nahe. Oft wird bloß empirischen Verallgemeinerungen beigelegt, was den nur Gesetzen strengster Fassung zukommt; kaum hat jemand mehr von der Ausnahmslosigkeit, der Unveränderlichkeit der Gesetze gesprochen dem sie nur eine Beschreibung derselbe Comte, als Erfahrung der bedeuten sollten. Zugleich gibt die bloße Regelmäßigkeit sich leicht wie einen völligen Abschluß, die Aufgabe scheint gelöst, wo sie eben nur bezeichnet Der Begriff des Gesetzes hat oft dogmatisierend gewirkt, nirgends ist. dem Gebiet der Biologie, wo Erscheinungskomplexe sich den Anschein strenger Gesetze mehr als auf und deren Forderungen sehr oft verwickelte gaben stellten. Zu solchen Verwicklungen aus der Durchführung der weiteren kommen Störungen aus einem mehr oder minder versteckten Fassung Fortwirken des älteren Gesetzesbegriffes ein überlegendes Wollen. 1 8. mit seiner Beziehung auf Das geschieht, wenn Denker des 1 7. und Jahrhunderts aus der Gesetzlichkeit der Natur eine gesetzgebende Gottheit glauben wenn in erschließen zu können. umgekehrter Richtung eine Aber es geschieht auch, pantheistische Denkweise die Gesetze wie lebendige Mächte behandelt und sie als einen Gegen- stand andächtiger Verehrung an die Stelle der Gottheit setzt, ^ Auch darin steckt ein Fortwirken der älteren Art, daß das Gesetz oft wie über den eine ^ S. den einzelnen Beginn der Vorgängen schwebende Macht behandelt philosophiae naturalis Missis formis substantialibus et qualitatibus occultis principia mathematica: phaenomena naturae ad leges mathematicas revocare. So zieht sich von Giordano Bruno durch die Neuzeit bis zur GegenBruno sucht das Höchste in inviolabili intemerabilique naturae lege, in bene ad eandem legem instituti ^ wart ein gewisser Kultus des Naturgesetzes. animi religione (de universo et immenso 653). Je skeptischer sich heute die Menschen zur Religion verhalten, desto blindere Verehrung pflegen sie dem Naturgesetz zu zollen. Gesetz. wird, EndUch gehört auch das ihnen die Bahn vorschreibt. ^ die daß, hieher, I59 kecker je Gesetzesformel auftritt, Behauptung eines Gesetzes und die sie desto leichter Eingang findet. an einem Gegegen den Geist der Sünde eine sache pflegen wir zu prüfen, ehe wir sie anerkennen zu setze zweifeln, das scheint So überträgt Wissenschaft. ein praktisches Gebot hat, zu einer Eine Tat- die sich Autorität, die ; das Gesetz als Unrecht auf das Gesetz des Geschehens: auch für dieses wird schleunige Zustimmung verlangt und keinerlei Widerspruch geduldet. Wie hätte ohne solchen kritiklosen Respekt das berüchtigte «eherne Lohngesetz" eine so große Rolle spielen können! Namentlich die Formel wirkt dabei Wunder. Mahhus die Geister bewegt, wenn rungszunahme nicht von der Bevölke- mathematische Formel gegeben bekannte die Wie viel weniger hätte er seiner Lehre «Man liebt die Sicherheit", so klagte schon Pascal; man hält hätte! aber leicht für sicher, was keck und selbstbewußt Aber so mißlich das sein alles mag, auftritt. menschliche solche Irrungen sind eine unvermeidliche Begleiterscheinung jeder großen Wendung, an den Gesetzen selbst Überblicken die wir also rasch dürfen sie intellektuelle nicht irre Bewegung, machen. der die Kampf um das Gesetz in der Neuzeit hervorgerufen hat und unablässig hervorruft. b) Der Kampf um das Gesetz in der Neuzeit. Die Naturgesetze haben ihre eigentümliche Gebiet der unorganischen Natur gefunden; Ausprägung im so war es eine Über- tragung der Größen und Methoden dieses Gebietes, welche das Vordringen des Gesetzesbegriffes mußte aber selbst — früher oder in andere Disziplinen begleitete. später — bemerklich werden, was an Problemen und auch Schranken liegt. Dabei ihm in Beim Gesetze ist alle Aufmerksamkeit den Formen des Geschehens zugewandt, die Kräfte und Ursachen bleiben im Hintergrund; wird solche Zurückstellung überall ^ möglich sein, und wird das Hervortreten dieses Mit Recht bemerkt darüber Sigwart (Logik II,', 512): Problems „Eine leere von Naturgesetzen so zu sprechen, als ob die bloße Formel eine magische Macht über die Erscheinungen übte und ihnen etwas zumutete, was nicht aus ihrer eignen Natur von selbst folgte. Gesetze können rhetorische Phrase ist es Gründe des wirklichen Geschehens sein, sondern nur die konstante Art und Weise ausdrücken, wie reale Dinge sich verhalten." nie Zum Erkenntnisproblem. 160 Ganzen verändern? Beim Gesetze wird die Wirklichkeit in lauter einzelne Vorgänge zerlegt und alles beherrschende nicht den Anblick des Ganze entfernt; jedes bildet das für sollte alle Beim Gesetze Gebiete taugen? Geschehen nur einen Sonderfall einzelne eines allge- meinen, alle Individualität wird hier für die Wissenschaft ausgeschaltet; wird das Individuelle sich überall mit einem so bescheidenen Platz begnügen, wird es nicht aller versuchten Gleichmachung eine namentlich will, wenn es nicht bloß das Geschehen als auch erklären beschreiben, sondern determiniert völlig un- Endlich erscheint beim Gesetze, vergleichliche Art entgegensetzen? und unweigerlich fest- gelegt; für freie Entscheidung, für eine Wahl zwischen verschiedenen Möglichkeiten gibt keinen hier es Platz. Werden dem alle einzelnen hin- sich Lebensgebiete fügen? Probleme also Hülle und Fülle, durch in alle durch aber das Gesamtproblem, wie weit die mechanischen Naturbegriffe die ganze Wirklichkeit unter sich zu bringen vermögen. Der Widerstand gegen den Gesetzesbegriff kann dabei eine schroffere und eine mildere Form annehmen: entweder wird der Gesetzesbegriff für ein besonderes Gebiet ganz und gar abgelehnt, oder er wird, unter Ablösung von der naturwissenschaftlichen Fassung, dessen Aus beidem zusammen erwächst eine höchst Besonderheit angepaßt. lebhafte Bewegung, die nicht wenig dazu beigetragen hat, die Eigen- tümlichkeit der einzelnen Gebiete in klares Licht zu stellen. Es beginnt aber die Ausbreitung des Gesetzesbegriffes über die Natur hinaus schon im 1 7. Jahrhundert, namentlich findet er schon Das 18. Jahrhundert setzt die Bewegung fort und führt sie tiefer in die einzelnen Gebiete ein.^ Ihren Höhepunkt aber erreicht jene erst im 1 9. Jahrhundert, nament- damals Eingang lich in die Psychologie. in seiner zweiten Hälfte. Es wirkte hier manches dahin zusammen, das Gesetz Mittelpunkt der wissenschaftlichen Arbeit zu rücken. ^ Mit besonderer Energie fochten. Er sagt gleich la signification la la in hat Montesquieu den les lois, ont leurs les intelligences lois, superieures ä l'homme a ses lois. den les lois, ver- dans rapports necessaires qui derivent de nature des choses; et dans ce sens tous a ses Gesetzesbegriff den Anfängen seines esprit des lois: plus etendue, sont in Vor allem das les etres ont leurslois: l'homme ont leurs lois, la divinite les betes Und etwas weiter: il y a donc une raison primitive, et les lois sont les rapports qui se trouvent entre eile et les diffe- rents etres, et les rapports de ces divers etres entre eux. Gesetz. Selbständigwerdeii fernerhin von wollten, desto der der Wissenschaften. einzelnen Philosophie 151 und Prinzipien weniger Je Regeln sie entlehnen mehr mußten sie darauf bedacht sein, in ihrem eignen feste Zusammenhänge zu finden. Gebiet durchgehende Begriffe und In den Gesetzen aber hofften sie solche zu finden; schien der unermeßliche Stoff sich ordnen schien auch mit ihrer Hilfe und gliedern zu lassen, eine Vergleichung verschiedener Reihen und Gruppen des Geschehens möglich zu werden. Diese Bewegung erhielt eine besondere Spannung durch das Verhältnis von Natur- und Geisteswissenschaften. Die glänzenden Erfolge der Naturwissenschaften haben auch ihre Expansionskraft, ihr Streben nach Beherrschung des ge- samten intellektuellen Reiches gesteigert, namentlich scheint die Ent- wicklungslehre Weltbegriffe zu liefern,, denen kein Gebiet sich ent- ziehen kann; so dringt die naturwissenschaftliche Denkweise mit ihren Begriffen tiefer und tiefer in die anderen Gebiete hinein. Aber zugleich werden diese zum Widerstände gereizt und zur Besinnung auf ihre Eigentümlichkeit getrieben, es entsteht ein Kampf, dessen lebhafter Fortgang die Unterschiede immer mehr zum Bewußtsein gebracht hat.^ * Ein anschauliches Bild von der Bewegung in der Sprachwissenschaft liefert B. Delbrück in der Abhandlung ,,Das Wesen der Lautgesetze" (Annalen der Naturphilosophie I, 277 ff.). Nachdem schon Fr. Schlegel und Bopp die Sprachwissenschaft in Vergleich mit der Naturwissenschaft gestellt hatten, ohne jedoch sie zu den Naturwissenschaften zu rechnen, gab Schleicher der Sache Sein Bekenntnis geht dahin (Die Darwinsche eine schärfere Zuspitzung. Theorie und die Sprachwissenschaft, S. 7): „Die Sprachen sind Naturorganisohne vom Wollen des Menschen bestimmbar zu sein, entstunden, nach bestimmten Gesetzen wuchsen und sich entwickelten, und wiederum altem und absterben; auch ihnen ist jene Reihe von Erscheinungen eigen, men, die die, man unter dem Namen „Leben" zu verstehen pflegt. Die Glottik, die demnach eine Naturwissenschaft, ihre Methode im Ganzen und Allgemeinen dieselbe, wie die der übrigen Naturwissenschaften." Demgegenüber haben, wie Delbrück näher ausführt, andere Forscher, unter ihnen namentlich Whitney, zur Geltung gebracht, daß sich bei der Entstehung und Veränderung von Sprachen nirgends dem Sprachstoff innewohnende Lebensgesetze, sondern immer nur menschliche Handlungen finden. Als ein solcher Ausfluß menschlichen Handelns und Wollens ist die Wissenschaft der Sprache, ist ist Sprache kein Naturorganismus, sondern eine Institution, stitutionen, welche die menschliche Kultur ausmachen. eine von den In- Damit muß auch das Sprachgesetz anders gefaßt werden als das Gesetz eines Naturorganismus. Delbrück kommt in seiner Untersuchung der Lautgesetze zu dem Ergebnis, daß, so sehr diese sui generis sind, kein Grund vorliegt, ihnen deswegen den Namen von Gesetzen abzusprechen. Eucken, Grundbegriffe. 4. Aufl. „Denn wir verstehen H auch bei Zum Erkenntnisproblem, 162 Von der Welt des Leblosen ausgegangen, hatte das Naturgesetz zunächst des Reichs des Lebendigen zu bemächtigen; auf wie sich viel in "Widerstand es dabei vollem Gange ist, stieß, und wie der Kampf eben jetzt wieder das hat uns im vorigen Abschnitt beschäftigt. Die Übertragung des Naturgesetzes auf die Seele war schon durch nahegelegt Descartes geführt; und Seelenleben alles wurde durch verwandelt Spinoza großartig hier sich in ein aus- Gewebe von Einzelvorgängen, die ganz nach Art der mechanischen Natur sich bewegen und wirken. Monade ihren eignen Leibniz läßt jede und unterscheidet von den «physiko-mechanischen" Gesetzen folgen Gesetzen der Körper die »ethiko-logischen" der Seelen (736 b, Erdm.). Psychologische Gesetze in strengerem Sinne haben namentlich die Engländer aufgebracht, in so die Assoziationsgesetze; Deutschland geht die Bewegung über Wolff zu Herbart fort, der die mathematische auch Formel in das Innere des einführen Seelenlebens möchte. Zugleich aber fehlt es nicht an Männern, welche die eigentümliche Art des Seelenlebens mit seiner inneren Einheit, seiner Beweglichkeit, und damit auch dem, seiner Individualität zur Geltung bringen was hier weisen sich an Gesetzen aufstellen läßt, deutliche eine Entscheidend für die Behandlung der inneren Welt Stellung der Gesetze in ihr eine Grenze 1 neue Stufe wird oder nicht. ist die Frage, ob in und die dem Geistesleben und selbständige Art der Wirklichkeit anerkannt Wo jenes geschieht, kann über einen wesentlichen Unterschied von allem naturgesetzlichen Geschehen keinerlei Zweifel sein. Die Naturgesetze sind die rein gefaßten Wirkformen des Ge- schehens, schlichte und einfache Tatsächlichkeit. Nun muß auch das Gesetz geistiger Art in irgendwelcher Tatsächlichkeit wurzeln; haltlos anderen Wissenschaften unter Gesetzen nichts weiter, als den Ausdruck für Gleichmäßigkeiten, welche zwar im Einzelfalle nicht rein hervortreten, von denen wir aber annehmen, daß sie stets rein hervortreten würden, wenn im Einzelfalle alle anderswoher kommenden störenden Einwirkungen entfernt werden könnten" (308). ^ So bemerkt Sigwart, dessen Untersuchungen über alle diese Probleme besonders klar und eindringend sind, über die Assoziationsgesetze (Log. IP, 553): „Die Assoziationsgesetze deuten nur bestimmte Richtungen an, in denen unsere Reproduktionen verlaufen können, oder in vielen Fällen verlaufen, bestimmte Tendenzen der wirklichen Aneinanderreihung von Bildern oder Wörtern u. s. w.; Gesetze, aus denen jeder wirkliche Vorstellungsverlauf als notwendig nachgewiesen werden könnte, vermögen sie nicht darzustellen." Gesetz. in der Luft schwebende Gesetze, die doch eine Wirkung tun sollen, Aber das Geistesleben, das den unentbehrlichen sind ein Unding. ^ Halt gewährt, es I53 wenn auch zu seinem für ihn, ist gleich keineswegs ein voller Besitz des Menschen, sondern ist hohes ein Damit werden Ideal. gehörig, zu- die Gesetze zu Normen, die nicht wirkungslos die aber Widerstand finden sind, Wesen innersten schwere Aufgabe, zugleich Natur und eine Ziel, und gegen ihn durchsetzen sich müssen. ^ Je nach der Eigentümlichkeit des intellektuellen, des ethischen, des ästhetischen Gebietes gestaltet sich der Widerstand und überhaupt der Lebensprozeß verschieden, wie wir Nur Sittengesetz Dies Problem hier sei mit einigen er die Moral über Worten alles das Sittliche und eines sicheren trieb ihn, einseitigen Aber diesen berechtigten Gedanken hat dadurch abgeschwächt. rechnet, der gibt und Wer die das gestellt das Moral zur Natur Schleier- der Charakteristische des Moral Menschen dem Begriff der Natur einen neuen Sinn und hat von allem bloßen Dasein zu scheiden; so Kant in besserem Recht als Schleiermacher.* * Anblick Haltes in der menschlichen Natur beraubt; überspannt stark sie scharf einen vom Natur- Einem Schleiermacher den engen Zusammenhang von Natur- und Sittengesetz zu verfechten. 3 macher unter Getriebe seelische hinaushob, mußte sich das Sittengesetz mit seinem Soll gesetz bis zu schroffem Gegensatz abheben. schien damit gestreift. den Vordergrund ge- namentlich durch Kant in ist Denn indem bracht. wissen. das vielbehandelte Problem des Verhältnisses von Natur- und gesetz alle ist schließlich Die unmittelbare Gleich- Mit Recht macht Husserl bemerklich, daß „jede normative und a fortiori jede praktische Disziplin eine oder mehrere theoretische Disziplinen als Funda- mente voraussetzt, ablösbaren in dem Sinne nämlich, daß sie einen von aller Normierung Gehalt besitzen muß, der als solcher in irgend- theoretischen welchen, sei es schon abgegrenzten oder noch zu konstituierenden theoretischen Wissenschaften seinen natürlichen Standort hat" (Logische Untersuchungen 1, 47). S. auch S. 164: „Der Gegensatz von Naturgesetz Regel eines tatsächlichen Seins und Geschehens als ist empirisch begründeter nicht das Normalgesetz sondern das Idealgesetz im Sinne einer rein als Vorschrift, (Ideen, reinen Gattungsbegriffen) in den Begriffen gründenden und daher nicht empirischen Gesetzlichkeit." ' Von neueren Untersuchungen darüber sei namentlich erwähnt Windelbands Abhandlung ,, Normen und Naturgesetze" in den „Präludien". » S. Werke zur Philos. II, 397-417. * Zeller „Über Begriff und Begründung der sittlichen Gesetze" (1883) kommt hinsichtlich der Ethik zu dem Schlußergebnis: „Ihre Sätze sind nicht 11* Zum Erkenntnisproblem. 164 Setzung von Natur- und Sittengesetzen entspricht antiken Ethik; sie hinfällig ist Geistesleben hat schwere Verwicklungen erkennen lassen. zu zeigen, daß, leicht faßten, der Verlauf immer wieder zur Anerkennung zwungen hat.i sie Die dem liche Auch wäre wo immer moderne Denker die Sittengesetze Naturgesetze prinzipiell als Stande der nachdem das Verhältnis des Menschen zum Lage, geschichtlichen dem geworden und widerspricht der welt- einer der Untersuchung abweichenden Art ge- Jahrhundert eigentümliche geschichtlich-gesellschaft- 1 9. Denkweise mußte das Streben erzeugen, sowohl das gesellschaftgeschichtliche Gebiet liche als das zu unterwerfen. festen Gesetzen Das Drängen auf Präzision vornehmlich unterscheidet Gesellschaftslehre, die Soziologie, großen Zahl der Hilfe wird die moderne von allen früheren Versuchen. Mit der Er- das Zufällige individuellen scheinungen ausgeschieden, werden Durchschnitte ermittelt und die Grenzen abgesteckt, innerhalb derer etwaige Abweichungen liegen, werden Regelmäßigkeiten innerhalb des sozialen Gebietes aufgedeckt ^ Je mehr aber welche das Forschung die Überraschung überwunden die erste Gewahren hat, von Regelmäßigkeiten innerhalb eines bis dem Zufall preisgegebenen Gebietes bewirkte, desto man gegenüber dem Begriff der Gesetze geworden, desto dahin scheinbar kritischer ist deutlicher hat sich der Unterschied zwischen bloßen gesellschaftlichen Tendenzen des Lebens und eigentlichen Naturgesetzen herausgestellt. Noch mehr Bewegung hat der Begriff auf dem wirtschaftlichen Gebiete engeren Sinnes hervorgerufen; nirgends hat der Streit darüber mehr Folgen für das Handeln und Leben als hier.^ Denn das der Ausdruck dessen, was irgendwo als Recht oder Sitte besteht, sondern der Forderungen, die als Normen der menschlichen Willenstätigkeit aus der Idee des Menschen hervorgehen." S. auch Siebeck: „Über das Verhältnis von Naturgesetz und Sittengesetz" (Philos. Monatshefte 1884, S. 321 ff.). ^ Comte liefert dafür ein hervorragendes Beispiel. Der große Empirist, der grundsätzlich will, die Gesetze nur als Beschreibungen verstanden wissen meint bei der Wendung zur Gesellschaft (cours de phil. pos. IV., 466): Cette generalite empirique, qui en toute autre science pourrait dejä avoir une valeur süffisante, ne saurait pleinement convenir ä la nature propre de la sociologie. ' In dem allen nimmt bekanntlich Quetelet eine hervorragende Stel- lung ein. ' Zur Geschichte des Begriffes bemerkt Neumann, dem wir besonders verdienstliche Untersuchungen über diesen Gegenstand verdanken (Jahrbücher für Nationalökonomie und Statistik, 3. Folge 1899, S. 152/3): „Geforscht hat Gesetz. Problem des Gesetzes steht 165 unmittelbarem Zusammenhange mit in wie sich der Staat zu den wirtschaftlichen Bewegungen der Frage, zu verhalten habe, ob bloß zuschauend oder selbsttätig eingreifend. der Bildete Prozeß wirtschaftliche bloßes ein Gewebe sich selbst regulierender Einzelbewegungen, so erschien alles Eingreifen als eine Störung, das laissez scher Weisheit selbst etwas In laissez faire, Wahrheit anderes als ist ein aller ein bloßer wurde zur Summe solches Denn neben Naturprozeß. jenem Gehenlassen stehen andere Möglichkeiten, Boden der Geschichte gegen politi- Gehenlassen an sich muß auf dem es andersartige Zustände erst durchgesetzt werden, es wirkt, wenn eingeführt, nicht wie selbstverständlich sondern es läßt sich zurücknehmen, es Willen getragen werden. fort, muß von einem fortlaufenden Dazu ist der Glaube an eine Selbstregu- lierung der wirtschaftlichen Verhältnisse durch die natürlichen Triebe und Kräfte der Individuen nicht möglich ohne einen Glauben an optimistischen die Vernunft der gesellschaftlichen Verhältnisse; jede Er- schütterung dieses Optimismus untergräbt auch den Glauben an das Allvermögen der Naturgesetze. Nun haben die wirtschaftlichen Ver- wicklungen des 19. Jahrhunderts jenen Optimismus aufs schwerste erschüttert, sie drängen immer stärker zum Eingreifen des Staates in den wirtschaftlichen Prozeß, sie entwinden damit das Gebiet den bloßen Naturgesetzen und verstärken die Bedeutung der ethischen wie der historischen historischen man nach Elemente.^ Die Anerkennung dieses ethischen und Moments verhindert keineswegs die Anerkennung wirt- wirtschaftlichen zeigen versucht ist und sozialen Gesetzen, wie an (So zuletzt in dem Aufsatz: anderem Ort zu Wirtschaftl. Gesetze nach und jetziger Auffassung. Jahrb. für Nationalökonomie und Statistik. N. F. 1898, Bd. 16), schon im Altertum und sodann, nach den allerdings auf anderen Gebieten erzielten Erfolgen Bacons und Newtons, namentiich seit der zweiten Hälfte des 17. Jahrhunderts, seit Locke und Hobbes, von denen der Erstere auch bereits den Ausdruck law hierfür gebrauchte, während gerade die Physiokraten da sie diesen Vorgängen folgten, nicht ganz frei von der Schuld zu sprechen sind, daß sie unter dem Einfluß damals allmächtiger naturrechtiicher Voretellungen die in Rede stehenden Gesetze des früherer , Geschehens von solchen des Sollens oder den ethischen Gesetzen nicht ausreichend zu trennen wußten." ^ Erwähnung verdient dabei, daß nicht bloß auf individualistischer, sondern auch auf sozialistischer Seite Neigung bestand, den Begriff des Gesetzes auf Kosten der freien Tat zu überspannen. seit alles Nur erhielt die Sache hier daß eine Gesamtbewegung des weltgeschichtlichen Lebens, jenWollens und Tuns der Individuen, mit unabwendbarer Dialektik die Gestalt, Zum Erkenntnisproblem. 166 Aber sie entsprechen dann schaftlicher Gesetze. den einfach nicht sondern sind nach Neumanns Definition Naturgesetzen, »der Aus- druck für eine infolge der Macht wirtschaftlicher Zusammenhänge aus gewissen Motiven sich ergebende regelmäßige Wiederkehr wirtschaftlicher Erscheinungen (Tendenzen oder Vorgänge)".^ Am meisten Bewegung hat in neuester Zeit das Problem der mehr und mehr geschichtlichen Gesetze hervorgerufen, um Mittelpunkt des Kampfes geworden. Je mehr zum es die Gesamtauffassung der Geschichte Beginn seit ist der Neuzeit überkommene die supranaturale Vorstellung von der Geschichte wich, desto mehr mußte dazu es drängen, innerhalb ihres Bereiches durchgehende eignen Bewegungen und feste Regelmäßigkeiten aufzuweisen. Die Aufklärung solchem Verlangen ihren eigentümlichen Stempel prägte Ihre auf. Geschichtsforschung „zertrümmerte das bisherige Geschichtsbild, wie es an den Monarchien, danielischen Augustin orientiert war, sie der Apokalypse an oder an deckte eine bisher ungekannte oder un- beachtete Welt auf, eröffnete unberechenbare Zeiträume der Geschichte, den Sündenfall von der Spitze verwies konstruierte einen weg und der Geschichte ganz anderen Urzustand Ausgangspunkt. als — Indem aber diese von Wunder und Vorsehung absehende Erklärung ein unendlich verworrenes Spiel menschlicher aufdeckte, Kräfte empfand man zugleich doppelt das Bedürfnis nach einem einfachen, normalen Gehalt der Geschichte, den man rechts, den Ideen des Natur- in der natürlichen Moral und Religion fand."^ Hatte die Philo- sophie zunächst die Neigung, die Geschichte der Vernunft entgegenso zusetzen, erwuchs bald ein Streben, in nunft und zugleich eine Gesetzlichkeit der ihr eine gewisse Ver- Bewegung aufzudecken. ^ Leibniz namentlich verficht die Idee einer durchgehenden Kontinuität große Wendungen und Umwälzungen hervortreibt. So hat es namentlich Marx in engem Anschluß an die Hegeische Geschichtsphilosophie ausgeführt. Aber auch hier entsteht der Widerspruch, daß eben das, was aus Karl gesetzlicher Notwendigkeit hervorgehen soll, zu seinem vollen Siege der Anerkennung durch den Menschen, der Aufnahme in die eigne Überzeugung Nicht zu bedarf. ruhiger Kontemplation, sondern zu energischer Aktion wird eben hier der Mensch aufgerufen. ^ S. «Naturgesetz und Wirtschaftsgesetz" Staatswissenschaft 1892, Heft * S. Tröltsch, (Zeitschrift für die gesamte 3). Real-Encyklopädie für Theologie und Kirche. 3. Aufl. unter „Aufklärung" S. 231. * in der S. über diese Fragen meine Behandlung der Philosophie der Geschichte „Kultur der Gegenwart". Gesetz. 167 der geschichtlichen Be^x'egung, Vico den Gedanken einer regelmäßigen Folge bestimmter Stufen in der Entwicklung der Völker und Epochen, immer stärker wird das Verlangen nach einer durchgängigen VerDas kettung der Ereignisse zum Zusammenhang eines Ganzen. 19. indem Jahrhundert führt die Sache erheblich weiter, Typen der Gesamtauffassung der Geschichte ausprägt, scharfe unermeßlichen Ausdehnung der Forschung der sowohl es mäßigkeiten aufdeckt. gesetzten Jenes geschieht empirische den in als in Regel- entgegen- zugleich und nahe verwandten Systemen Hegels und Comtes. Dort eine allumspannende Logik, hier eine langsame Anhäufung, der ein- zelnen Elemente, dort eine Bewegung durch schroffe Gegensätze, hier ein ruhiges Aufsteigen, das aber deutlich drei Hauptstufen (trois erkennen etats) hier wie dort ein läßt, Ausschluß aller Willkür, ein sicheres Fortschreiten, eine völlige Bestimmtheit aller Mannigfaltigkeit durch den jeweiligen Stand der Gesamtbewegung. Geschichte überströmenden Reichtum leicht ihren preßte und alle Irrationalität wegzudeuten der anderen Seite her Forschung Wurde so der von der Philosophie eine Gesetzlichkeit zugeführt, die beflissen Durchdringung des die zu engen einen in Rahmen war, so hat Stoffes mittels von der reichem Maße empirische Regelmäßigkeiten aufgedeckt. in Dabei wirkten tief in die Forschung hinein die großen Gegensätze Den gesetzlichen, ja speziell naturgesetzlichen des modernen Lebens. Charakter der Geschichte verstärkte die wachsende Einsicht in die Abhängigkeit des menschlichen Befindens und Tuns von äußeren Bedingungen, es verstärkte ihn weiter die Erkenntnis der Abhängigkeit vom Ganzen, vom sozialen „Milieu"; es widersprach der Individuen ihm die der Aufklärung entgegengehaltene Individualität und Positivität der Geschichte,^ es widersprach ihm nicht minder die Betonung der So vertritt * zeugung, ihr »daß seinen in z. B. Steffensen in der (Gesammelte Aufsätze, S. 278) die Über- Geschichte das durch und durch Indixaduelle, das in Formen, höchsten in Persönlichkeiten willenskräftigen und großen Taten und Leiden eines wahren Werdeprozesses offenbar wird, den unvergleichlichen Gesellschaften, zu oberst in der Menschheit selbst, in Reiz hervorbringt, den das geschichtliche Wissen für den menschlichen Geist hat"; sowie daß „nicht die pirischer Naturgesetze, Bestätigung von der Geltung allgemeiner em- sondern sammenstoßen der höchsten idealen Gesetzen, besser Gottes widerspiegeln", es vielmehr das so irdischen Natur, mit idealen Mächten, ist, „was uns in des ganz unverkennbare Zuinneren Menschen, mit welche dem die Unbedingtheit dramatischen, Gang des geschichtlichen Lebens die Seele erschüttert." tragischen Zum Erkenntnisproblem. 168 großen Persönlichkeiten, wie sie in Carlyle einen besonders prägnanten Ausdruck gefunden hat. Es ist nicht bloß die verschiedene Schätzung von Natur und Geist unserer Wirklichkeit, dem Inhalt die Frage nach Problem das in der des Geisteslebens, oder Rationalität es es Irrationalität ist minder nicht ist im besondern unseres Daseins, welche die Frage nach der Gesetzlichkeit der Geschichte verschieden beantworten lassen. Diese Gegensätze methodologischen und vielfach Klarheit neuerdings Windelband hat der Behandlung des die Forscher und hier das Einzelne den Unterschied geschichtlicher von Behandlung zum in natur- Ausdruck der geschichtlich bestimmten Gestalt gesucht, immer sich gleichbleibende Form, der einmalige, hier bestimnite Inhalt des wirklichen Geschehens wissenschaftliche bilden bewegt Dort wird das Allgemeine in der Form des Naturgesetzes, gebracht. ^ sich in heute auch welches Mit besonderer Energie und einleuchtender entzweit wissenschaftlicher dort die erscheinen Problems, — darf idiographisch Denken ist dem einen in — wenn man neue Kunstausdrücke Falle 26)"; ,;den festen (S. in «Das betrachtet. nomothetisch, in dem andern Rahmen unseres Weltbildes gibt jene aligemeine Gesetzmäßigkeit der Dinge ab, welche, über allen Wechsel erhaben, die ewig gleiche Wesenheit des Wirklichen zum Ausdruck und innerhalb bringt; dieses Rahmens sich entfaltet der lebendige Zusammenhang aller für das Menschentum wertvollen Einzelgestaltungen ihrer Gattungserinnerung" 2 (S. 38). Diese Überzeugung in geistvoller und selbständiger Weise weitergeführt;^ ist von Rickert sie hat überhaupt viel literarische Bewegung hervorgerufen. Gegen- über solcher Richtung auf das Individuelle verficht Lamprecht die Überzeugung, daß das Individuelle nur der künstlerischen Erfassung zugänglich so auch sei, in und daß das der Geschichte S. ^ Es hatte auch schon Paul dort Denken wie überall, nur auf das Typische gehen könne; „Geschichte und Naturwissenschaft", Rektoratsrede 1894. ^ „Geschichtswissenschaften" sagt wissenschaftliche (S. 1): in seinen Prinzipien der Sprachgeschichte und „Gesetzeswissenschaften" unterschieden; er „Wie jedem Zweige der Geschichtswissenschaft, so muß auch der Sprachgeschichte eine Wissenschaft zur Seite stehen, welche sich mit den allgemeinen Lebensbedingungen des geschichtlich sich entwickelnden Objektes beschäftigt, -welche die in allem Wechsel gleichbleibenden Faktoren nach ihrer Natur und Wirksamkeit untersucht." ^ S. „Grenzen der naturwissenschaftlichen Begriffsbildung" I und II. Gesetz. von hier aus entwickelt 169 von die Lehre er wicklungsstufen, die in bestimmter sozialpsychischen Eine nähere Erörterung an dieser Stelle Hauptproblem wird Hier nur zweierlei bemerkt. sei Würdigung der ist nicht wohl möglich; der Artikel Geschichte auf das Tatsächlichkeit Ent- Ordnung verlaufen.^ Zunächst, und daß zurückführen. auch Einmaligkeit der eine volle Geschichte nicht verhindert, gewisse Regelmäßigkeiten in ihr anzuerkennen. Art z. B., und wie sich Kunst u. Die wie sich der Ablauf der Entwicklung eines ganzen Volkes, s. die Bewegung von einzelnen Gebieten in verschiedenen Kulturepochen w, wie Religion, vollzieht, kann als Erzeugnis der bleibenden Art des Menschen sehr wohl etwas verwandtes, ja gleichmäßiges haben. Insofern könnten unbedenklich Ge- setze der Geschichte anerkannt werden. Aber sie würden dann nur die Form des Geschehens betreffen, der Inhalt würde der Individualiät der einzelnen entziehen. Epochen verbleiben und — Wie der "Geschichte viel sich damit aller Ableitung Selbständigkeit ferner die Einzelvorgänge in haben, Einzelvorgang zu fassen und wieweit ist, ganze Geschichte die das wird sich als ein verschieden gestalten je nach der prinzipiellen Fassung des Geisteslebens und seines Verhältnisses über, ob zur menschlichen Lage. Persönlichkeiten geschichtlichen Bewegung oder bilden. Diese Fassung entscheidet dar- Massenwirkungen Das alles den Kern der aber weist über das methodologische Problem hinaus und wird uns später zu beschäftigen haben. * Über den Streit um Gesetze der Geschichte orientiert vortrefflich Bemheim, Lehrbuch der historischen Methode und der Geschichtsphilosophie. 3. u. 4. Aufl. S. 91 ff. Zum Weltproblem. C. Monismus und Dualismus. 1. Auch bei der Wendung zu • den Weltproblemen Problem des Lebensprozesses, uns das stets Denn auch dort liegt die Entscheidung nicht gegenwärtig bleiben. abstrakt-begrifflichen bei wird des Geisteslebens, speziell Erwägungen, sondern beim Tatbestande der Wirklichkeit; dafür aber ist nichts wichtiger als die Frage, welchen Inhalt das Geistesleben zeigt hier liegt das Zentrum, bringen sind, und wo und welche Stellung es damit gewinnt; wohin seine besonderen Untersuchungen zu alle letzte Würdigung fahrung an der Breite der Dinge ermittelt Kämpfen hat; findet, was die Er- sucht doch in allen um die Weite der Welt schließlich der Mensch sich selbst, den Kern seines eignen Wesens. Auch die geschichtliche Betrachtung bestätigt das durch den Nachweis, daß es überall die eigentümliche Gestaltung des Geisteslebens war, welche die Theorien erzeugte und zur Macht erhob. a) Zur Geschichte und Kritik der Begriffe. Die Ausdrücke Monismus und Dualismus entstammen den letzten Jahrhunderten. 1700 (z. B. das Dualismus verwandte zuerst Thomas Hyde erschienenen Kap. IX, dem guten S. Schrift 164) Historia religionis (s. der in Persarum zur Bezeichnung eines religiösen Systems, Prinzip ein böses als gleichewig zur Seite diesem Sinne ward das Wort durch Bayle und Leibniz veterum (s. stellt; in den Artikel Zoroastre) Theodicee II, 144, 199) an weitere Kreise gebracht. Als Gegenstück zu „Monismus" gebrauchte es zuerst Wolff, aber zugleich übertrug er die Ausdrücke auf das Verhältnis von Körper und Geist: Monisten — das Wort nunmehr, die nur eine Art des ist Seins, von Wolff gebildet — heißen sei es Körper sei es Seelen, Monismus und Dualismus. 171 annehmen, also sowohl die Idealisten als die Materialisten, Dualisten welche Körper und Seelen als voneinander unabhängige dagegen, Substanzen Wolff betrachten.^ selbst Dualist wollte Beide sein. Ausdrücke blieben auf die Schule beschränkt, namentlich Monist das 19. Jahrhundert hinein äußerst selten. scheint bis in er- In weiteren Umlauf brachten das Wort zuerst Hegelianer als Bezeichnung ihrer eignen Denkweise, so erschien 1832 eine Schrift von Göschel »Der Dann folgte wieder eine Ebbe, bis die Monismus des Gedankens". und darwinistische Entwicklungslehre (Häckel druck ergriff und ihn sich anpaßte. System, das und Körper Weiter aber bezeichnet er jedes und Geist Natur Seele, den Aus- Schleicher) nicht einander, sondern beide einem überlegenen Dritten unterordnen und einfügen diesem Sinne werden In will. oft Monismus und Spinozismus als gleichbedeutend genommen. So führen uns das ' von und Natur Ausdrücke die jetzt Körper und Seele oder — Der Gegensatz, Geist. erhält eine besondere Schroffheit dadurch, eignen Wesens und daß betrifft, eröffnet sich uns in er wachsen daß dabei entsteht, den Kern unseres er scheint. Die Welt, so scheint von außen her durch Empfindung, von innen her durch ein Reich von sinnlichen Eindrücken und wird der von — auf im Fortgang der weltgeschicht- sinnliche Gedankengrößen; Verhältnis zwiefacher Weise: lichen Arbeit unablässig zu es, das auf Ausdehnung über das Weltall in die nehmen können, oder auch eine ein als ein als Reich unsinnlicher Reihe die andere tieferes Denken, selbsttätiges sich in auf- Eindringen den Gegensatz in einen bloßen Schein verwandeln? Zum Gegensatz der Betrachtungs- weisen kommt der wachsende Abstand des Inhalts Aus der Natur hat die Wissenschaft zu Gunsten beider Welten. einer präzisen Be- greifung und einer sicheren Beherrschung immer mehr alles seelische Element vertrieben, zugleich aber hat das Seelenleben im eignen immer weiter über die bloße Natur hinausgehoben und immer mehr zu einem selbständigen Reiche ausgebildet. So Kreise sich sich * Es ergab sich danach bei Wolff folgendes Scliema philosophischer Parteien Dogmatiker Skeptiker Monisten Idealisten Egoisten Dualisten Materialisten Pluralisten Zum Weltproblem. 172 der Verlauf der Geschichte das Körperliche immer seelenloser, läßt immer das Seelische Das müßte den Dua- erscheinen. spiritueller zum Monismus hin, da nicht nur die exakte Forschung den Zusammenhang von lismus empfehlen, aber zugleich wachsen die Antriebe immer deutlicher aufweist und immer weiter Seelenleben und Körper sondern auch ein zunehmender Drang nach ins Einzelne verfolgt, dem Menschen Einheit So verbietet. Nebeneinander ein verschiedener Welten unseren Begriffen immer weiter auseinander, was tritt Erfahrung immer enger verschlungen zeigt; so treibt es uns immer zwingender zur Umwandlung des widerspruchsvollen Bildes. die Die Hauptrichtungen solches Strebens was sie aber zeigt, lichem Bude; erloschen, und sondern es bleibt fordert immer ist zeigt die Geschichte in deut- nicht mit der besonderen Zeit eine Möglichkeit stets gegenwärtig als neuem zur Entscheidung von auf; bei allen Wandlungen der Begriffe behaupten sich charakteristische Denkund Lebenstypen durch den Lauf der Zeiten bis in die Gegenwart. Es geht aber die lebendige Geschichte, die die eigne Arbeit hineinreicht, in Frühere alles ist für uns zur bloßen befaßt, Jahrhunderts 17. nicht Auseinandersetzung Seelischen enthielt positive aber zu der alle Arbeit Geschichte, einer präzisen geführt. bis sich Denn viel mit zu Beginn des Fassung und deutlichen Die Vorstellung des mehr eine Verneinung des Körperlichen als eine Behauptung; 1 so war es nicht zu vermeiden, daß immer in sie bewegt von belebt.2 Mittelalter hatte Begriffe wieder das Bild einer nur feineren, lichkeit die geworden. Historie wohl haben schon das Altertum und das dem Probleme d. h. nicht hinter Descartes zurück; subtileren, luftartigen Der Körper aber schien eindrang. seelenartigen Kräften, die belebt, Körpergebildet, ganze Natur war innerlich Bei solchem Stande der Begriffe bediente sich die Erklärung der Natur fortwährend seelischer Größen und verschloß sich damit eine exakte Begreifung Psychologie unter den es erregte * der Vorgänge. Einfluß sinnlicher Andererseits geriet die und räumlicher Begriffe, keinen Anstoß, Wirkungen von außen in die Seele ein- So konnte Descartes mit gutem Grunde sich dessen rühmen, daß er d. h. bewußte Tätigkeit, positiv be- das Ganze der Seele als Denken, zuerst stimmt habe. * Bezeichnend dafür ist die aristotelische Definition was das «Prinzip der Ruhe und Bewegung in sich satze zur Kunst, die es außer sich hat. dessen, der Natur als trägt", im Gegen- Monismus und Dualismus. 173 fließen und Willensimpulse in räumliche Bewegungen übergehen zu lassen. Es war ein chaotischer Stand, der weder der Natur noch der Seele ihr Recht gewährte. überwunden, namentlich Erst die Aufklärung hat diesen Stand ist der eine durchgreifende Scheidung und Descartes, es Klärung Nun erst wird jedem Gebiet eine volle Eigentümlichkeit vollzieht Das Seelenleben wird zuerkannt. reines ein als Beisichselbstsein verstanden, dessen Einheit des Wesens (unitas essentiae) sich scharf von Einheit aller geht hier bloßen einer Zusammensetzung wie solche die Außenwelt scheidet, sitionis) einen seelischen Charakter; die seelische sich zurück und selbst schendes einfließen, kettet und verleiht ihr erst Bewegung kehrt immer zu Mannigfaltigkeit an alle sondern eignen Grunde Anregung kann alle auch in scheinbarer sich selbst. nur es aus seinem reizen, hervorzubringen; Leistungen gewisse beherr- ein kann nichts von draußen In ein solches Seelenleben Ich. compo- (unitas das Bewußtsein voran besonderen Tätigkeit aller bietet; so Abhängigkeit von draußen im Grunde bleibt es stets bei Solcher Selbständigkeit der Seele entspricht ein Selb- ständigwerden der Natur. nach Austreibung Die Massen und Bewegungen, alles seelischen Elementes verbleiben, ihr die bilden eine eigne Welt; die Bewegung, für die bis dahin eine Seele unentbehrdünkte, lich gelegt; wird nun den kleinsten Teilchen von Haus aus bei- das macht es möglich, aus der Zusammensetzung kleinster — bewegter, aber seelenloser — Teilchen faltigkeit alle der Natur hervorgehen zu lassen. Strebungen verschwinden damit aus ihr. sinnlichen Eigenschaften, mit der in den Dingen geborgt, als So ein selbst angehörig, Tönen u. s. w. die sie erscheint nun nicht als ihnen von der Seele Farben sondern sich scharf beide Abschluß dabei verbot Seiten, Aber so Verwicklungen diese Spaltung hervorrief, schritt, sie der fruchtbarste Anregungen brachte. beiden Gebiete ihre eigentümlichen voller Klarheit lisch, , vom Menschen in sie hineingelegt scheiden letzter und Ja, die ganze bunte Fülle der menschliche Fassung die Natur umkleidet, als unermeßliche Mannig- Alle inneren Kräfte herausarbeiten, nun so scharf, viele ist daß sich neue Fragen und ein gewaltiger Fort- Nun erst konnten die Prinzipien und Methoden mit erst ließ sich das Seelische see- das Körperliche körperlich verstehen, eröffnete sich eine exakte Physik und eine erklärende Psychologie. jetzt von den Dingen, nun Wie ein Schleier erst schien die Wirklichkeit sich fiel es uns völlig Zum Weltproblem. 174 aufzuhellen. Dazu brachte jene Scheidung nicht bloß der Begriffe, sie der Kultur, die seits Gedankengrößen, in lichkeit von jener Zeit an durch die Neuzeit geht. erhöhte Tätigkeit des Denkens, eine der Vernunft, ein kühn eine Klärung über ein Einer- Umsetzen der Wirk- Rationalisierung aller Verhältnisse, Bindung vordringende bisherige alle ein Messen des Daseins an Forderungen ein nach Streben andererseits kultur; eine entsprach der zwiefachen Richtung des Lebens und Selbständigwerden volles Intellektual- Außenwelt der gegenüber dem Menschen, eine engere Verflechtung seiner mit der Umgebung, unermeßliches Wachstum der Erfahrung, eine ein Bedeutung höhte der Faktoren, materiellen ein immer er- stärkeres Anschwellen einer Realkultur; wer könnte leugnen, daß diese zwei" Ströme durch das Leben der Neuzeit gehen, liche Spannung versetzen und durch in Gegensätze auseinandertreiben? ist die tiiefste die es in eine unaufhör- ganze Breite der Dinge Dieser Gegensatz des Lebens Wurzel des Dualismus der Begriffe und Lehren, aus ihm schöpft dieser immer neue Kraft, so sehr das Verlangen nach Einheit die Geister über ihn hinaustreiben mag. solches Verlangen Ein konnte schließen die war allerdings unabweisbar, denn ab- Denkarbeit bei hatte eine kräftige Analyse vollzogen jenem Dualismus und schärft, aber von der Analyse drängte es welcher Synthese, Einheit. Auch vom Gegensatz fehlte es nicht Zerspaltung der Wirklichkeit, zu die Begriffe nicht. immer wieder zu irgendwelcher Er dauernd verirgend- umfassenden an gewichtigen Gründen gegen jene Ihr widerspricht nicht nur der un- mittelbare Eindruck einer engen Zusammengehörigkeit von Seele und Körper, sowie die wachsende Erkenntnis der Abhängigkeit des Seelenlebens von körperlichen Bedingungen, ihr widerspricht auch die philo- sophische Forderung einer Einheit der Wirklichkeit, ihr widerspricht endlich die Tatsache der Kunst mit ihrer engen Verflechtung und fruchtbaren Wechselwirkung von Sichtbarem und Unsichtbarem, von Äußerem und Innerem. Alles zusammen ließ den Dualismus als einen bloßen Durchgang zur Einheit erscheinen; diese Einheit fand sich allerdings nicht fertig vor, sie gewinnen, und diese durfte Eindruck nicht scheuen. kühner geworden Es sind als je aber auseinanderging, drei So war durch geistige Arbeit erst zu dabei einen ist Gegensatz zum nächsten das Einheitsstreben in der Neuzeit zuvor. Hauptrichtungen, die Richtungen in welche des Materialismus, dies Streben Spiritualismus, Monismus und Dualismus. 175 Monismus: entweder wird das Körperliche, oder es wird das Seelische zum allumfassenden Sein, oder es wird beides zu Seiten, Erscheinungen, Ausdrucksweisen einer Wie es tiefer gegründeten Wirklichkeit. strengem Sinne Materialismus in einen durch Descartes erfolgten Klärung der Begriffe damals eine erst durchlief feste Bezeichnung tüchtigste, bei der Der Materialismus den Franzosen die den Deutschen die derbste Gestalt gefunden; seit so hat er auch gibt, erlangt. ^ Kulturvölker und nacheinander die großen Engländern die erst bei den geistreichste, bei hat und zu widerlegt oft Boden geworfen, ist er immer von neuem erstanden, und hat er immer von neuem weite Kreise gewonnen. Das bekundet doch mehr in ihm steckt als naive Gemüter wähnen, wohl, daß die ihn durch scharfsinnige Widerlegungen endgültig abgetan glauben und daß immer wieder Menschen dem längst durchschauten sich wundern, Irrtum verfallen. In zwingen, wenn bei Denn ständen. hängigkeit des Vorzug den Wahrheit wäre der Materialismus Seelenlebens von großen einer Bedingungen, körperlichen Einfachheit und und problematischeren Gemeinverständlich- zu Begriff als steht und es, Gerade die lassen. und Seelischen, Körperlichen schaft lange, als eine den der Materie, er ent- wo wir ihn zu fassen glauben; je präziser wir ihn aber nehmen, desto unmöglicher wird vom Ab- sowie Denn kaum gibt es einen schwie- Analyse der Begriffe unterbleibt. vorgehen Frage die Abhängigkeit läßt sich auch in anderer Weise verstehen, und die Einfachheit verbleibt nur so weicht uns, in so gewiß der Materialismus die unbestreitbare keit für sich anrufen kann, rigeren zu be- leicht ihm bloß theoretische Erwägungen fällt, unmöglich gemacht; hat mit aus ihm seelisches Leben her- moderne Klärung der Begriffe mit der die exakte Naturwissen- den Materialismus Recht sagt daher F. als Weltanschauung daß den A. Lange, Materialismus scharf denken ihn widerlegen, heiße. Aber es sind nicht wissenschaftliche Erwägimgen, es sind Kultur- und Lebenslagen, welche dem Materialismus eine Anziehungs- und Überzeugungskraft geben. reich in ^ solchen Zeiten, Der Ausdruck wo Materialist Wir erscheint Naturphilosophen Robert Boyle (so in stark und sieg- Kulturformen ihre volle finden überlieferte zuerst ihn bei dem Chemiker und der 1674 erschienenen Schrift: The and grounds of the mechanical philosophy), der eine Vorliebe für feste Termini hatte. Noch Giordano Bruno verwandte den älteren Ausdruck excellence »Epikureer". Zum Weltproblem. 176 Wahrheit eingebüßt haben und von vielen als ein tyrannischer Druck empfunden werden; der Materialismus dann erscheint das beste Mittel zur Befreiung von drückenden sowohl als Fesseln wie als ein, Rückgang auf einfache Grundlagen des Lebens, er scheint eine natürlichere und wahrere Gestaltung aller Verhältnisse zu versprechen. Auch bringt er die Bedeutung der materiellen Lebensbedingungen für das Ganze der Kultur mit besonderem Nachdruck zur Geltung. So er riß Geister fort die in den Bewegungen vor und in der französischen Revolution, so auch beim Sozialismus der Gegenwart. Was das Leben hervorgebracht so Leben widerlegen, es nur das auch negativ durch den inneren Widerspruch, positiver Weise, dem eine durch ihre eigne Entwicklung materialistische Gestaltung der Kultur durch die Gegenwirkung einer andersartigen Kultur. positiv verfällt, kann hat, widerlegt es aber sowohl in negativer als in Jener Widerspruch wurzelt darin, daß dort den materiellen Größen als eigne Leistung beigelegt wird, was ein überlegenes Geistesleben ihnen macht; aus wie dieses der in Welt uns sichtbaren unver- gleichlich mehr wahrnehmen läßt als die Sinne direkt zeigen, so macht es die materiellen Güter wertvoll als ein Werkzeug für die Betätigung und Entwicklung vernünftiger Lebewesen; wie vom Materialismus der dort Zuschauer, eignis behandelt. das Streben so wird unvermerkt hier eine zwecktätige hinzugedacht und ihr Erlebnis wie ein äußeres Er- Persönlichkeit und Indem aber die Arbeit die materialistische Lebensgestaltung von dem Träger des Lebens ablenkt, überliefert sie ihn einer wachsenden Verkümmerung und Leere; hat nun zugleich jenes Wachstum der Beziehungen nach außen einen gewaltigen Lebensdurst erzeugt, so muß ein schreiendes Mißverhältnis zwischen unserem Verlangen und unserem Besitz entstehen; das Mißbehagen, das daraus hervorgeht, wird schließlich mit Sicherheit über eine materialistische Lebensführung hinaustreiben. Die weltgeschichtliche Arbeit zeigt das vollzieht durch Materialismus. das Ganze ihres Verlaufs in großen Zügen und eine Überwindung des Der durch jahrtausendlange Arbeit, durch fruchtbare Erfahrungen und schmerzliche Enttäuschungen zu einem Innenleben geweckte Mensch kann unmöglich mit in dem Kinde und dem Wilden der materiellen Welt seine ganze Wirklichkeit sehen und in ihren Gütern seine Befriedigung finden. für ihn durch wonnen. jene Bewegung ein Die materielle Welt wesentlich selbst hat anderes Ansehen ge- Aus dem bunten Reich der sinnlichen Eindrücke ist jetzt Monismus und Dualismus. 177 Gewebe von Kräften, Gesetzen, Beziehungen geworden; nicht mehr die Handfestigkeit der Sinnesempfindung verbürgt uns die Wirkhchkeit des Ganzen, sondern die kausale Ordnung mit ihrer ein Verkettung schehens und Mannigfaltigkeit aller unter ihrer Auch Gesetze. einfache Einfügung die Ge- alles Außenwelt ist ins dem Denken entsprungene Größen, ideelle Größen, bilden ihren Kern. Wohl bleibt hier die geistige Tätigkeit Unsinnliche verwandelt, an einen ungeistigen Vorwurf gebunden, aber auch so verschieden von noch so fortgebildeten aller, sie weit ist Welcher Sinnlichkeit. Abstand ist zwischen der Welt des Naturforschers und der des Natur- menschen mit noch so geübten Sinnesorganen! Nicht minder verwandeln sich dem Kulturmenschen die äußeren Was sie heute ihm wertvoll macht, ist weniger der sinnliche Güter. Reiz und Genuß, als die Herrschaft über die Dinge, das Vermögen, diese nach eignem Wollen zu bewegen und damit das eigne Leben Unbegrenzte zu ins sowohl die Dinge So steigern. als sich genießt der selbst in Kulturmensch nicht den Dingen, sein Denken gibt dem Sinnlichen Wert und gestaltet es zu Gedankengrößen. Welcher Abstand liegt hier zwischen der Lust des Wilden am Glanz des Goldes und dem Selbstgefühl des großen Geschäftsmannes, dessen wirtschaftliche Macht den Erdball umspannt und von den sich dabei sinnlichen Wertzeichen ganz emanzipiert hat! So wirken in geistige Kräfte, die der Gestaltung eignen der materiellen Welt kann. Aber der Materialismus nicht verstehen zugleich leuchtet ein, daß, was dabei an Lebensentfaltung aufgeboten wird, als Abschluß unmöglich ist; leistet, was so viel an einem fremden Stoffe muß notwendig auch bei sich selbst etwas sein; werfung des Äußeren und alle vor peinlicher Leere, wenn Unter- alle Ausdehnung der Macht schützt nicht dem Geistesleben nicht irgendwelcher Diesen aber kann ihm alle Steigerung materieller Inhalt gegeben wird. und wirtschaftlicher Macht unmöglich gewähren. So muß der Versuch, das Glück von außen her zu begründen, schließlich große eine Enttäuschung und Erschütterung ergeben. Die materialistische Lebensgestaltung wird hat, aufs So muß härteste sich dem Glückverlangen, mit zusammenstoßen möglich ist sie selber angefacht und dabei zusammenbrechen. auch praktisch der Materialismus durch seine Entwicklung widerlegen. lösung das Aber alle solche Kritik und noch keine positive Überwindung. durch eine Eucken, Grundbegriffe. kräftige Entfaltung 4. Aufl. Eine solche selbsttätigen eigne kritische Aufist nur Geisteslebens; 12 Zum Weltproblem. 178 wo mit dies Aufgaben seinen die Gemüter da wird erfüllt, es kaum begreiflich dünken, wie der Mensch das ihm innerlich Nächste und die Quelle seiner eigentümlichen Größe mit dem Materialismus als etwas Abgeleitetes behandeln, seine eigne Existenz auf den Kopf sein stellen, Glück von außen her suchen kann. Vermag der Materialismus mit seiner Sinnfälligkeit und scheinbesonders baren Selbstverständlichkeit auf die breiten Massen zu wirken, so ist der Spiritualismus eher eine Sache einzelner vornehmer und auserlesener Geister Eindruck gegen sich, Denn er hat den unmittelbaren geistige Energie ist der von ihm ver- Kreise. ohne Weg nicht zu Ende zu gehen. suchte den Spiritualismus entweder ein als in Es zeigt aber Reich von lauter einzelnen Seelen, Leben und Sein eines Gesamtgeistes, jenes neueren deutschen Spekulation, wie da soll die am großartigsten Außenwelt sich gänzlich als eine Stufenfolge innerhalb Sinnliche wird hier niedere, statt oder als das bei Leibniz, dieses in der in bei Hier Hegel. Innenleben verwandeln, das Verhältnis von Geist und Natur wird nicht sondern die Neuzeit zwiefacher Gestalt: die Wirklichkeit erscheint als ein Gegensatz, des Geistes verstanden; das einer in sich selbst gegründeten Welt eine noch nicht zur vollen Bewußtheit gelangte Art des seelischen oder geistigen Lebens. Diese Denkweise braucht nur ausgedacht zu werden, minder wunderlich darzustellen, mag; um sich beim ersten Anblick scheinen uns doch das Innenleben die nächste und gewisseste Wirk- ist lichkeit, als sie und zeigt die einfachste Besinnung, daß wir diesen Kreis nie und uns in ein anderes Sein versetzen können, daß auch, was Außenwelt heißt, nur eine besondere, eine eigentümlich gänzlich verlassen gebundene Art des Innenlebens bedeutet. Aber so berechtigt und überzeugend der allgemeine Gedanke sein mag, leicht Die beim Versuch einer strikten Ausführung überspannt sich das menschliche Vermögen und wird unser Besitz überschätzt. Spiritualisten können nicht unternehmen, die Natur ganz und gar in Geist umzusetzen, ohne unser Geistesleben als Geistesleben schlechthin, als absolutes Geistesleben zu behandeln. Zu einer Stufe menschlichen Geisteslebens aber läßt die Natur sich nun und nimmer herabsetzen; jenem gegenüber erweist sie eine viel zu selbständige Art und verfolgt sie einen viel zu sie viel zu sehr ihren eigenen Weg, ihm hartnäckigen Widerstand. leistet Solcher Selbständigkeit der Natur und solcher Härte ihres Widerstandes hat sich der Spiri- Monismus und Dualismus. lyg tualismus nur gewachsen fühlen können, indem er das Geistesleben in bloßes Denken und Erkennen verwandelte und das Ungeistige als etwas verstand, das noch nicht voll aufgeklärt der Stufe der Unbewußtheit verharre. der Intellektualismus, dem aus Weltleben das noch auf ein kecker bloße Weltansicht eine machte, der damit die Wirklichkeit verflüchtigte Inhalts beraubte. sei, Aber das war und alles lebendigen Begreiflich ist solche intellektualistische Überschätzung des menschlichen Vermögens nur aus der Eigentümlichkeit beson- wo das Bewußtsein geistiger Kraft und das Voll- derer Kulturlagen, gefühl geistigen den Menschen Schaffens sich den Mittelpunkt als der Wirklichkeit fühlen ließ und ihn über alle Schwere der Dinge in kühnem Fluge hinaushob. Aber jene Schwere wird bald bemerklich der Kultur werden, jene Art aber aller Spiritualismus, fällt sich als zu erweisen. flach der sich als ein fertiges Damit System gibt. Das Mißlingen der Versuche, eine der beiden Lebensformen ausschließlich durchzusetzen, mußte dem Monismus zu Gute kommen. Aucfi er will eine Einheit, aber er will sie nicht durch Aufopferung der einen Seite an die andere, sondern durch Einfügung beider in ein umfassendes Drittes; lichkeit voll entfalten hier scheint jedes Gebiet seine Eigentüm- zu können, ohne aus der Gemeinschaft heraus- zutreten, hier entfällt die Schwierigkeit einer Wechselwirkung zwischen Körper und Seele, da dem Vorgehen auf der einen Seite unmittel- auf der anderen Zu Gunsten bar eins als Lebensgestaltung aber wirkt namentlich vermeintlich entspricht. des Monismus das hier erstrebte und gewonnene Gleichgewicht zwischen Natur und Geist, zwischen Äußerem und Innerem, zwischen Sinnlichkeit und Denken, zwischen realistischer und idealistischer Kultur; solches Gleich- gewicht scheint besonders geeignet, das Leben ins Weite und Große zu heben, den Menschen der Enge eines Sonderkreises zu entwinden und ihm an der ganzen Fülle der Wirklichkeit Anteil So hat der Monismus, namentlich seit ihm Spinoza zu geben. eine klassische Verkörperung gab oder doch zu geben schien, eine gewaltige Anziehungskraft geübt auf Dichter und Denker, auf Naturforscher und religiöse Naturen, er schien die Zauberformel, die überallhin Frieden bringe. Aber eine solche Zauberformel Seine zu denken in gestattet, ist er nur, weil er weil jeder den allgemeinen jedem das Gedanken sich eigentümlicher Weise zurechtlegt; so gewiß in jenem Gedanken eine unangreifbare Wahrheit liegt, in der Ausführung stellt sich alsbald 12* Zum Weltproblem. 180 der Gegensatz wieder ein, den es zu überwinden galt; es zeigt sich, daß auch Entweder diesem Problem bei — Oder menschliche Streben das vor ein gestellt ist, nicht friedlich die Gegensätze zusammen- zuschließen vermag. zeigt, Nach der Absicht des Monismus, wie ihn der Spinozismus müßten beide Gebiete in vollem Gleichgewicht stehen. So will es der neuerdings auch der «psychophysische Parallelismus", In Wahrheit ist eine jene Absicht zu genauerer Durchbildung bringt. nähere Ausführung des Grundgedankens gar nicht möglich ohne der einen Seite ein Übergewicht über die andere zu geb^. ist, Spinoza selbst genauer angesehen, an keiner Stelle Monist, sondern bald Materialist, bald Spiritualist, jenes in der Grundlegung, dieses im Abschluß seiner Denn Lehren; so zeigt es namentlich seine Ethik. zu Beginn die Natur Wirklichkeit, erscheint dort das Hauptgeschehen und als das Maß als während das Seelenleben zu einer Beim Ab- erscheinung, einem Reflex des Naturprozesses herabsinkt. ^ dem Materialismus ein Spirituawenn ein göttliches schluß des Systems aber wird aus Oder lismus. ist es kein aller bloßen Begleit- Spiritualismus, Leben die ganze WirkHchkeit durchdringt und zusammenhält, die der Mensch Natur zur Entfaltung dieses Lebens wird, Gott an intellektuelle Liebe zu teilgewinnen soll? Und der hinaus in den Kern des Unendlichkeit und der Zwiespalt über die Begriffe es nicht ein einziges, ist sondern ein zwiefaches Leben, das aus Spinoza wirkt: dann werden mag, die erstrebte Einheit Ewigkeit reicht Lebens hinein, Naturalismus, durch die einmal ein Wie immer Spinoza beurteilt ein Mystizismus. hat er nicht erreicht. Ebenso- wenig ist es späteren Versuchen gelungen, Natur und Geist Gleichgewicht zu bringen. hat es nicht erreicht; er Auch der psychophysische in ein Parallelismus macht entweder das Seelenleben zu einem bloßen Reflex der Naturvorgänge, oder diese zu einer Erscheinung des Geisteslebens; er verläßt damit die Neutralität und nähert sich entweder ^ dem Materialismus oder dem Spiritualismus. Mit vollem Recht bemerkt dagegen Herbart Metaphysik (Wke. aus Bestimmungen III, 198): „Da überdies alles in seiner Allgemeinen Psychologische bei Spinoza man wenig vom Ausgedehnten bestehen des Körperlichen gefolgert wird: so merkt davon, daß nach ihm das Denken unabhängig und wie könnte es anders sein in irgend einer Lehre, die ursprüngGedanken als Bilder des Ausgedehnten betrachtet? Eine solche unterwirft immer notgedrungen den Geist der Masse, vermöge des Verhältnisses der Abbildungen zu ihrem Vorbilde." sollte; lich die Monismus und Dualismus. Noch weniger ergibt jg! von jenem vermeintlichen Gleich- sich Denn gewicht aus eine charakteristische Gestaltung der Kulturarbeit. die Ausgleichung von Natur und Geist, welche namentlich künst- lerische Seelen anzog, erfolgte nicht zwischen als gleichberechtigten Außen- und Innenwelt Größen, sondern sie erfolgte gänzlich auf dem Boden des Innenlebens; wenn z. B. im Schaffen um zur Darstellung drängte, Innere eines damit sich so erhielt zugleich das Äußere ein inneres Leben; wurde hier durch gestaltet, ein befreit, menschliche Sein zwischen Geist und Natur deutlicher verläßt die nicht aber Denn Monismus der unterscheidet vom der ihn Materialismus nur dies, daß er das Seelenleben nicht däres, sondern als von -Haus aus primäres ein beilegt, Punkten entstehen läßt. feineren Materialisten; es wurde das zerteilt. Neutralität Entwickluilgslehre. und zum Weltall innigeres Verhältnis von der Enge kleinmenschlicher Art Noch zu finden, das Geistesleben ein kräftigeres Erfassen der Natur bereichert im besondern durch darwinistischen Goethe alles selbst als' ein sekun- Phänomen erachtet, es aller Materie nicht erst nachträglich an besonderen Aber kaum anders dachten von jeher die wie ihnen, so wird auch Wahrheit die sinnliche Natur zum All, den Monisten bemächtigen sich in Natur- begriffe der gesamten Wirklichkeit und wird alles selbständige Geistes- leben geleugnet. So muß, bei konsequentem Verfahren, auch die Lebens- und Kulturgestaltung ganz in die Bahnen des Materialismus geraten. Anders würde die Sache auslaufen, wenn mit der Be- seelung aller Elemente der Wirklichkeit voller Ernst gemacht würde; denn das müßte ein dem leibnizischen ähnliches Weltbild ergeben. Aber so weit pflegt der materialistische Monismus nicht vorzudringen, er glaubt den Elementen die Seele wie eine Eigenschaft, neben anderen beifügen zu können, ohne daß sie dadurch etwas wesentlich anderes werden. In Wahrheit läßt sich Seele nicht haben, sondern nur sein. Wird demnach der materialistische Monismus von allen Bedenken getroffen, die dem ausgesprochenen Materialismus entgegenwirken, so sind einem spiritualistischen Monismus bessere Aussichten zuzuerkennen. Ein solcher Monismus wird die Tatsache zum Ausgangspunkt nehmen, daß Innenleben nicht bloß an einzelnen Punkten, zerstreut und splittert, erscheint, zer- sondern daß es sich zu einem umfassenden Zu- sammenhange verbindet, daß sich auf der menschlichen Daseinsstufe ein den Individuen überlegenes Geistesleben und mit ihm eine Innenwelt voll eigentümlicher Größen und Aufgaben erschließt. Der Wende- Zum Weltproblem. 182 punkt der Wirklichkeit wird hier nicht zwischen Natur und Seele, sondern zwischen Ungeistigem und Geistigem gesucht; das Seelenleben hat an beiden Stufen teil, sofern es zunächst ein Stück der Natur bildet, dann Die Frage, wie sich Körper- aber ein Gefäß des Geisteslebens wird. liches und Seelisches zu einander verhalten, tritt hier zurück vor der, wie Eine Welt Geistiges und Ungeistiges miteinander umfassen kann. Diese Frage findet aber in diesen Zusammenhängen die Antwort, daß das Ungeistige nur etwas Untergeistiges bedeutet, daß dasselbe Sein, das die Natur und das natürliche Seelenleben im stände der Ver- einzelung und als ein Gewebe bloßer Beziehungen zeigt, im Geistesleben sich zu einem Ganzen zusammenzufassen entwickeln beginnt; erst und einen Inhalt zu damit scheint die Wirklichkeit ein Inneres Solche Erhebung von und zugleich ihre eigne Tiefe zu gewinnen. Untergeistigem zu Geistigem ist nicht eine bloße Forderung der Spekulation, sondern eine Aufgabe, die das ganze Menschenleben durch- dringt, denn ethische Bewegung, alle eigentümlich menschliche Leistung, vor allem die ein Aufstieg ist von der Natur zum Erhebung des Seins von der natürlichen zur wird hier das Problem vom bloßen Intellekt geistigen Geist, eine So Stufe. das Zentrum des in Lebens versetzt. Wenn aber dabei das Geistesleben zugleich als der Grundbestand und als das Ziel der Wirklichkeit erscheint, so besagt das keineswegs, daß die Gestalt, worin es dem Menschen vorliegt, im stände sei, sich der ganzen Welt zu bemächtigen und in der Natur sich einfach wiederzufinden, Denn so gewiß wie das der reine Spiritualismus wollte. das Geistesleben auch dem Menschen als etwas Übermenschliches und Allgemeingültiges irgend gegenwärtig sein muß, in seine nähere Gestaltung dringt unablässig Bloßmenschliches ein; wir haben nicht das Geistesleben an sich, sondern ein menschliches Geistesleben, d. h. ein Geistesleben, dessen übermenschlicher Kern uns immer nur durch eine menschliche Hülle zugänglich ist. Streben wir daher vom menschlichen Geistesleben her die ganze Wirklichkeit zurechtzulegen, so geraten wir unvermeidlich in eine zu enge, in eine Fassung; eine unentbehrliche Hülfe dagegen Unendlichkeit und ihrer Überlegenheit gegen Zwecke; sie bewahrt eine Sonderart, sie ist anthropomorphe die Natur mit ihrer alle kleinmenschlichen den Menschen vor einem Sicheinspinnen treibt in immer von neuem dazu, den Allgemein- gedanken des Geisteslebens von der bloßmenschlichen Daseinsform abzuheben. Aber alle diese Wirkung liegt innerhalb des Geistes- Monismus und Dualismus. und nur darin lebens, besteht die 183 Abweichung vom dogmatischen nun innerhalb des umfassenden Ganzen zwei verschiedene Ausgangs- und Angriffspunkte anerkannt werden. Eben daß Spiritualismus, dies aber ergibt einen anderen Typus des Kulturlebens, als ihn jener Das Geistesleben erscheint nun nicht bloß als die begründende vertrat. Tatsache, sondern auch als eine sich immer erneuernde Aufgabe; das menschliche Leben wird weit mehr zwischen Gegensätze erscheint weit mehr weit unfertiger, gestellt, es im Aufstreben begriffen; erst das ruft den Menschen mehr zu eigner Tat und Entscheidung auf und zur von ihm, fordert so wird Fortbewegung des Weltalls nicht nur es sich in seinen mitzuwirken, nicht das Intellektuelle, ein Ethisches weitester Fassung, immer die keinen Zweifel Kämpfen liegt, dieses, das Ethische, wo sich das näher gestalten freilich — Wie mögen, der Hauptstreitpunkt bei diesen und an welcher Stelle vornehmlich die Geister sich Das nämlich scheiden'. sondern zum Kern seines Strebens. Menschen und Zeiten leidet selbsttätig Gedanken zurechtzulegen; ist die Frage, ob selbständiges Geistes- ein leben und mit ihm eine neue Stufe der Wirklichkeit anerkannt wird oder nicht. Alle Verneinung oder auch nur Zurückhaltung gibt einem gröberen oder feineren Materialismus die Oberhand, mit dem Ja wird dagegen eine Überwindung und sichere Bahnen gewonnen. Wohin und nicht bloß an Scharfsinn ein Einlenken aber die Entscheidung Intelligenz, fällt, in neue das hängt sondern vornehmlich an der Kraft und der Klarheit, mit der das Geistesleben Manschen und Zeiten gegenwärtig ist, das greift damit in das persönliche Leben und Sein zurück. b) der Der Monismus der Gegenwart. Wer die geistigen Strömungen der Gegenwart kritisch erörtert, muß sich notwendig auch mit dem Monismus der Gegenwart befassen; nisses ist dieser doch über das besondere Problem des Verhält- von Natur und Geist hinaus eine mächtige, ja stürmische Be- wegung geworden, auf deren Beleuchtung und Würdigung sich nicht wohl verzichten läßt; je heftiger aber der Kampf an dieser Stelle entbrennt, desto mehr hat die philosophische Betrachtung nach ruhiger Abwägung zu streben. Der Monismus der Gegenwart ist nicht verständlich ohne eine Beachtung der breiteren Grundlage, von der aus er sich gestaltet hat. Diese die ist das Vordringen des Bildes der Natur in Begriffe Zum Weltproblem. 184 vom All und in die Gestaltung des Lebens. Es vollzog sich damit ein notwendiger Rückschlag gegen die ältere, einseitig religiöse welche die Natur wie etwas untergeordnetes transzendente Denkart, und wenn nebensächliches, und nicht zu gar bedenkliches behandeln Das energische Vordringen der Naturerkenntnis und auch pflegte. Umwandlung des Lebens gaben diesem RückWucht und siegreiche Kraft. Innerlich fiel dabei die dadurch bewirkte schlag gewaltige besonders ins Gewicht die Präzision der Naturbegriffe und ihr Zu- sammenstreben zum Ganzen einer Gedankenwelt; den Einflüssen, die davon ausgingen, konnten sich selbst die nicht entziehen, deren Streben der Das Hauptrichtung ist nach eine entgegengesetzte z. B. augenscheinlich bei Leibniz. Richtung verfolgte. Sein unablässiger gegen den „Naturalismus" hat nicht verhindert, an Hauptstellen in seine Gedankenwelt eindrangen und brachten. Oder zum ist kein es wenn solcher Einfluß, Kampf daß Naturbegriffe sie unter sich er die Lebens- wenn er alle Gegensätze in Unterschiede des Grades verwandelt, wenn ihm der Begriff der logischen Möglichkeit mit dem der gehemmten Kraft zusammenfließt? Im 19. Jahrhundert ging diese Bewegung weiter, mit lautem und stillem Wirken unterwirft die naturwissenschaftliche Denkweise sich mehr und mehr die Begriffe wie die Überzeugungen; steigerung allbeherrschenden Wertbegriff macht, wir empfinden gar nicht, wie eigentümliche Bejahungen neinungen das mit sich bringt. So idee die Gestalt eines Naturprozesses Verkettung, die daraus erwächst, weise alle Gegenwart, erhält und z. B. und Ver- die Entwicklungs- zerstört mit der strengen alle Selbsttätigkeit, ja ohne daß uns das irgend konsequenter- aufregt. In der Natur hat sich das Beharrungsgesetz, das sog. Gesetz der Trägheit, immer weiter bewährt; unbedenklich wird es auf und geschichtliche Gebiet übertragen, während hier der Forschung das geistige der Lebensstand gehen muß, immer neu aus ursprünglichem Schaffen hervor- um nicht sofort zu sinken. Glück die sinnliche Lust, ohne weiteres Glück als eine, wenn auch Die Naturstufe zeigt gilt als vielen auch das geistige feinere Art der Lust. Wenn so unauf- haltsam von außen und innen die Natur auf uns eindringt, so kann wundernehmen, wenn mehr und mehr die Natur als Welt und Wirklichkeit schlechthin behandelt wird, und wenn eine „naturwissenschaftliche Weltanschauung" sich unbedenklich und siegesgewiß es nicht nicht als einen besonderen Ausschnitt, sondern als das erschöpfende Bild des Ganzen der Wirklichkeit gibt. Monismus und Dualismus. 185 Alles Vordringen ließ indes diese Bewegung keinen vollen Sieg erringen, so lange noch der Mensch eine privilegierte, ja einzigartige Diese aber hat nun die schwerste Erschütterung Stellung einnahm. durch Entwicklungslehre die dem aufs engste mit ganz und gar Leben verkettete tierischen sie Menschen den und ihn dadurch von um so mehr mit Es gewann das die Gemüter ließ. überwältigender indem die Natur hineinzog, ihn als ein bloßes Stück in erscheinen ihr erfahren, weil Kraft, es in der Wendung zur Arbeit, emsiger und fruchtbarer Arbeit, eine unermeßliche zu von Tat- Fülle sächlichkeit eröffnet, sonst zerstreute Daten verknüpft und als Ganzes Wirkung zur langem gebracht zurückzukehren, eine Der Mensch schien nunmehr von hat. Wahn und eitler Selbstüberhebung zu seiner echten Heimat sein Leben aber damit schlichtere, frischere, festere Grundlagen, sowie Art gewinnen. Altes wahrhaftigere zu erschien damit als neu. Neues als alt, eine durchgreifende Wandlung Diese Strömungen und Stimmungen sind es, welche Monismus ergreift und zusammenfaßt, die Naturbegriffe scheinen ihm nur einer gewissen Ergänzung in der Richtung ward eingeleitet. der -moderne um den ganzen Umkreis der Wirklich- des Seelischen zu bedürfen, keit in sich aufnehmen und alles Leben beherrschen zu können. und Aussichten würden jedoch aus eignem Vermögen schwerlich der Bewegung zum Monismus eine solche Stärke verliehen und eine solche Macht über die Gemüter geAlle solche Vorteile geben haben, wie jene noch etwas anderes, in sie das Wahrheii und große Massen zu erregen vermag, entschiedenes ein dem Stande Zwischen der bestand leben überbrücken Nein. der Religion, Ein Religion vornherein versucht, solches gesellte Nein sich entzünden nicht entspringt zum Ja aber und dem modernen eine weite mehr und Kluft, mehr aber ist lange das dazu nicht zu wie ihn die kirchliche Gestaltung überlieferten von käme besitzt, Leidenschaften direkter ist aus bietet. Kultursie zu Unmögliche dessen ersichtlich geworden, mehr und mehr hat sich der Abstand in einen vollen und schroffen Gegensatz verwandelt. Und wenn Probleme lange Zeit nur die obere Schicht der Gesellschaft diese zu berühren schienen,^ so sind ^ So erschien es z. B. einem sie immer mehr auch in die Massen Pierre Bayle gänzlich ausgeschlossen, die Aufklärung je die Massen gewinne. daß Er hält für die Interessen und die Bedürfnisse der Gesellschaft, die nach seiner Überzeugung im wesentlichen zu allen Zeiten dieselben bleiben, einen gewissen Aberglauben unentbehrlich Zum Weltproblem. 186 gedrungen und erregen trotz aller Wandlungen offiziell aufrecht auferlegt wird, erhalten so immer und entsteht ein Wenn stärker. Druck und schwerer Der nun müßte Psycholog und ein kurzsichtiger Staatsmann viel aber zugleich namentlich der Schule gebieterisch Un Wahrhaftigkeit. lähmenden einer sie und Erschütterungen die alte Art der Religion sein, die Gefahr ein schlechter dem entginge, wie Unwille, wie viel verhaltener Zorn sich dadurch ansammeln und irgendwelchem Ausbruch drängen muß. Der Monismus aber zeigt einen Weg, einen scheinbar nahen und leichten Weg, auf dem ein solcher Ausbruch erfolgen kann; ist es ein Wunder, wenn er wie ein brausender Strom die Gemüter ergreift und un- schließlich auch nach widerstehlich fortreißt? Bewegung durchaus verständlich; sie würde nicht wenn nicht ihr Ja wie ihr Nein Wahrheitsmomente enthielte. Aber es ist etwas anderes, eine Bewegung aus der geschichtlichen Lage heraus zu verstehen und Berechtigtes in ihr zu würdigen, etwas anderes, ihr die Führung des Lebens zu geben. So die ist wirken, was sie wirkt, Was zunächst die Religion betrifft, so dem Stande der Art zu befreien und jetzt ist bei allen Kultur- Bewegung im Gange, sie von der veralteten völkern eine wachsende weltgeschichtlichen Evolution des Lebens gemäß zu gestalten; solche Bestrebungen sind minder einaber sie sind auch fach, fruchtbarer und aussichtsreicher, summarische Verwerfung der Religion, wie sie der als die Monismus zu Es kommt darauf an, ob die Religion über alle Form hinaus in inneren Notwendigkeiten unseres Wesens und unserer Stellung zum All gegründet ist oder nicht. Sollte sie vollziehen pflegt kirchliche es sein, so könnten alle Schäden des gegenwärtigen Standes in keiner Weise das Preisgebung einer Lebensmacht die des Verhältnis Menschen zum rechtfertigen, die sich Ganzen der Wirklichkeit zur Aufgabe macht, die seinem Leben Größe, seiner Seele einen Selbstwert und eine reine Innerlichkeit zu geben unternimmt. In dem das Priestertum und die Kirche zu schädigen, pflegen die Gegner der Religion zu übersehen, daß sie durch jene Verneinung Eifer, mit ihrer Preisgebung sich selber schädigen. „Les besoins dont et des tenebres, ils je parle ne sont point sujets aux vicissitudes de la lumiere ils sont les memes sous un siecle un siecle de science (s. den Artikel Franz von Assisi im sont de tous les tems; d'ignorance, et sans Dictionnaire). alles selbständigen Innenlebens am meisten Man gedenkt dabei unwillkürlich der Anekdote Monismus und Dualismus. 187 von dem Knaben, der keine Handschuhe erhalten hat, und dem nun in „Es geschieht arger Kälte die Hände erfrieren, der dazu aber meint: meinem Vater ganz recht, daß mir die Hände erfrieren; warum gibt er mir keine Handschuhe?" Sachlich des Hauptfrage, die bleibt Monismus entscheidet, die, über Recht oder Unrecht die ob die von ihm ausschließlich ver- wandten Naturbegriffe zur Umspannung der Wirklichkeit genügen. Namentlich von zwei Seiten erheben sich dagegen Bedenken, die einen von der Erkenntnislehre, die anderen vom Gehalt des Geistes- die weltgeschichtliche Arbeit lebens her, wie Erwägung kenntnistheoretische uns nicht von außen her zufällt, ihn zeigt. — Die er- einzuwenden, daß das Weltbild hat sondern daß wir es von seelischen Vorgängen aus und nach den Gesetzen unseres eignen Geistes zu vom Subjekt pflegt sich zunächst dem Forschen jene Solches Ausgehen bilden haben. auf Kant zu berufen, dessen überlegene Energie Richtung zwingend vorgeschrieben hat. Aber es nicht bloß Kant, ist es ist überhaupt kein einzelner Philosoph, sondern es ist die Gesanitart des modernen Denkens, Denn nichts ist ja Lebens, welche diesen Weg befiehlt. der modernen Kultur dem modernen Leben und eigentümlicher, als die Befreiung des Subjekts von der Gebunden- heit an die Umgebung, seine Befestigung im eignen Leben. nicht auf dabei mit Aufgebot einen Besitz Kraft aller der Welt verzichtet, leidenschaftlich dieser begehrt, Wird vielmehr nimmt das so vom Objekt zum Subjekt, von der Welt zum Menschen, geht es jetzt vom Subjekt zum Objekt, vom Menschen zur Welt. Eine solche Umwälzung muß auch den Leben eine völlig andere Richtung: ändern und sich damit in alle einzelnen Inhalt des Lebens wesentlich So auch Gebiete erstrecken. statt in wird Ergebnis aus dem Werden verstanden wird, sich Das das des Erkennens. Wirklichkeit verfeinern, beleben, Bild der wenn das wenn volle Anerkennung vergeistigen, daß wir jenes Bild von innen her zusammenfügen, daß nicht Außenwelt, sondern unsere geistige Organisation den Umriß findet, die wie die Grundlinien dazu liefert, daß eine oft sehr komplizierte Arbeit in Größen steckt, welche die naive Ansicht für einfach erachtet Zugleich wird klar, daß wir mit aller Arbeit nur einen menschlichen Durchblick der Wirklichkeit gewinnen, Forschung selbst wieder der einer tieferdringenden zum Probleme wird und seinen Wahrheits- gehalt erst zu erweisen hat. Neue Tragen und neue Sorgen tauchen damit unfertiger auf, unvergleichlich müssen wir uns fühlen als Zum Weltproblem. 188 zuvor, aber in aller Unfertigkeit gewinnen Dies wir eine Vertiefung der und auch unseres eignen Lebens. Wirklichkeit aber alles vorhanden; ihm ist ist für den Naturalisten und Monisten die Welt, wie sie sinnlich und handfest auf uns einzudringen scheint, die ganze und echte Wirklichkeit. heißt, erkenntnistheoretisch angesehen, und festhalten sich der kopemikanischen Denkweise der Neuzeit wie ihn die huldigen, mittelalterliche Scholastik besaß, für die gerade der Geringschätzung zu haben Das aber den ptolemäischen Standpunkt dem naiven Realismus verschließen, es heißt nicht Naturalismus nur So vertritt die Philosophie gegen pflegt. den Naturalismus das Recht jener modernen Wendung zum Subjekt, damit eine Wahrheit, die sich wohl verdunkeln, vertritt sie aber aufgeben Tiefer noch greift das Zweite, bei Inhalt ihm geht die Frage auf den Diesen Inhalt faßt der Naturalismus und der Wirklichkeit. Monismus viel zu knapp, er übersieht das Hauptstück erscheint: als nicht läßt. das uns anderen ein Stück, das Geistesleben. Alles Innere der Natur einfügen kann jener nur, indem er das Seelenleben lediglich ein als Vorgehen an den einzelnen Individuen behandelt nämlich mag er sich darauf wie fließend berufen, Dann Grenzen die zwischen Menschem und Tier sind, wie langsam sich auf dem Boden was früher als der Geschichte emporgearbeitet hat, des Menschen auch galt, wie Macht der Naturtriebe in seiner sehr Das alles bleibt. ein Stammbesitz der Kulturmensch sei unter der vollauf anerkannt und Bedeutung keineswegs abgeschwächt Aber es nicht ist Denn das menschliche Seelenleben verbleibt nicht wie das Ganze. das tierische bei jener Zerstreuung an einzelne Punkte, bei ihm erfolgt ein Zusammenschluß zu einem gemeinsamen Leben, und meinsame Leben entwickelt eine unermeßliche wesentlich dies ge- Tatsächlichkeit, neue Züge gegenüber der bloßen Natur erweist Zusammenschluß erst macht Geschichte und Gesellschaft im die Jener aus- zeichnend menschlichen Sinne möglich, auf diesem Boden entsteht Gedankensprache und Kultur, hier erwächst eine reiche Verzweigung eigentümlicher Lebensgebiete Wissenschaft Wie das Ganze, biete ihre eignen Gesetze, in Recht und Moral, in Kunst und so haben auch diese einzelnen Ge- Probleme, Menschen vor schwerste Aufgaben, sie Erfahrungen, ziehen ihn sie stellen den immer mehr an und machen zugleich unvergleichlich viel mehr aus ihm: aus einem bloßen Stück der Natur wird er mehr und mehr ein geistiges sich Monismus und Dualismus. Wesen, das die von Unendlichkeit innen 189 her miterlebt und als Persönlichkeit die Welt in eigne Tat zu verwandeln vermag. sittliche Eine so eingreifende Wendung kann sich nicht vollziehen ohne einen neuen Anblick der Wirklichkeit; mit jenem allen verkündet sich deutUch eine neue Stufe der Welt, deren Anerkennung das Gesamtbild des erweitern Alls wesentlich und muß. vertiefen Dies alles ist keine bloße Theorie, es hat im geschichtlich-gesellschaftlichen Leben der Menschheit es Die Wirkung. Hauptaufgabe, zur Wirklichkeit viel eingearbeitet, sich in alle Einrichtungen hin- entfaltet, umfängt uns mit bildender Kraft deutsche jenen inneren tausendfacher darin ihre Zusammenhang des Menschenlebens Anerkennung zu bringen, vollen in fand Philosophie spekulative Aufklärung weit überlegen, daß sie sie fühlte sich dadurch der die geistigen Inhalte und Werte aus jenem, nicht wie diese von den bloßen Individuen her erklärte. Der Naturalismus aber übersieht jene Wendung zum Geistes- Kulturstandes, Ausbildung eines eigentümlichen jenes leben; jene innere Wachstum des Menschen durch die Arbeit der Jahrtausende, der ganze Reichtum der dabei erschlossenen Wirklichkeit, für ihn nicht hängend von allem allem vorhanden gewürdigt; er charakteristisch Lebensgehalt. Was oder gibt ihre Größe anderes ein Verzicht suchte. ein ein Bild aber vom sind ist auf alles das, Alls All unter Absehen von allem Geistigen, von Verwerfen Das Bild des sie werden doch nicht zusammen- Menschlichen, Verengung und Verarmung, der Geschichte, sie dies alles die stärkste inneren Ertrages als worin die Menschheit wird ohne den Menschen und dann der Mensch unter Abschleifung aller Wir sprechen von überwundene innerlich Reaktion, wenn das Leben auf eine ältere, Phase zurückgeschraubt werden soll. Aber wie bescheiden sind alle fertig abgeschlossen, Eigentümlichkeit möglichst in es hineingepreßt. Versuche, es an einen Höhepunkt innerhalb der geschichtlichen Bewegung zu binden, gegen das Unternehmen, es ganz und gar an die vorgeschichtlichen Anfänge zu ketten und ihm alle Möglichkeit einer inneren Erhöhung, einer wirklichen Entwicklung zu nehmen? Von hier aus angesehen, wird die ganze eigentümlich menschliche Geschichte ein großer Irrtum, ein Abweg von der Wahrheit, indem sie dem Menschen immer stärker eine Innenwelt vorspiegelt, die doch nur eine leere Einbildung ist. Dabei erfahren wir das Verdrießliche, daß jene Verneinung eines selbständigen Geisteslebens oft wie etwas Selbstverständliches auftritt, Zum Weltproblem. 190 das nur Unverstand verkennen oder böser Wille verwerfen könne. Verneinende Richtungen waren Begreifen läßt sich das freilich wohl. besonderer Gefahr eines starren Fanatismus gegen Andersgesinnte. Nichts in stets kritisches Verhalten gegen sich ja eines notwendiger für ein ist eine gerechte Würdigung und selbst Dogmatismus, anderer als die Fähigkeit, sich in fremde Denkweisen hineinzuversetzen und von ihnen aus die eigne zu alles Jenseitige als als nicht betrachten. Dies Vermögen aber wo rasch der Kreis geschlossen und wird besonders dort gefährdet, vorhanden erklärt wird. Hume war gewiß Denker und Forscher groß und im Leben allem Fanatismus so fern wie möglich, aber gibt es einen krasseren Ausdruck eines Fanatismus, tellektuellen als jenes berühmte Autodafe aller in- anders- gerichteten philosophischen Literatur? Das Gleichgewicht des Geisteslebens war lange genug von der und der Theologie bedroht, Religion in einem Rückschlag dagegen wird es nun von dem ausschließlichen Herrscheranspruch der Naturwissenschaften gefährdet. Naturwissenschaften wie Diesen Anspruch aber erheben weniger die selbst, ihre als Gestaltung im Naturalismus und Monismus sie sehr daran zweifeln, ob der vorliegt. erachten, die Einheit der Gedankenwelt, läßt sich etwas großes als ob er nicht, indem er die Begriffe gewaltsam zusammenschweißt, das Leben als Ganzes inner- Seine Begriffe und Lehren folgen der Natur, wie die spaltet lich Philosophie, Dabei Monismus eben das erreicht, was ihm Hauptsache gilt, und was auch wir anderen als die zur mechanische Denkart sie faßt, das bloßen und blinden Tatsächlichkeit, All in wird damit ein Reich der dem es kein Handeln, sondern nur ein Geschehen, keinen inneren Forttrieb, sondern nur ein Nebeneinanderlagern, keine wahrhaftige Einheit, sondern nur eine Zusammen- setzung gibt. Bei und Werte halte ^ konsequenter Denkweise verschwinden, Jene Stelle (am Schluß der hätte auch müßten hier alle In- der Wahrheitsbegriff 12. Sektion d. Enquiry conc. h. u.) lautet: When we run over libraries, persuaded of these principles, what havock must we make? If we take in our band any volume of divinity or school meta- let us ask: Does it contain any abstract reasonings conceming quantity or number? No. Does it contain any experimental reasonings conceming matter of fact or existence. No. Commit it then physics for to the flames. instance; — For it can obtain nothing but sophistry and Illusion. — Würde ähnlich ein spekulativer Philosoph urteilen, so würden alle es Borniertheit oder Fanatismus nennen; geschieht es von der anderen vielen als Zeugnis eines starken Seite, so gilt es und unerschrockenen Geistes! Monismus und Dualismus. 191 und damit eine Wissenschaft keinen Platz. Was an geistiger Regung das hätte ruhig und urteilslos das Weltgeschehen über aufkäme, sich ergehen zu lassen. einem eifrigen Kampf Hoffnung auf ein dessen Statt finden wir den Monismus in um die Wahrheit begriffen und von freudiger Vordringen der Menschheit erfüllt, er hält in der Gestaltung des Menschenlebens die alten Ideale des Guten und Wahren fest, er schöpft den Hauptantrieb seines wissenschaftlichen Strebens aus der Überzeugung, dadurch mehr Wahrheit und mehr Vernunft in das menschliche Dasein zu bringen, hier ganz und gar die Wege des kurz er wandelt Gibt es nun wohl Idealismus. einen schrofferen Dualismus, als Materialist in der Weltanschauung und Idealist im Handeln zu sein? Beispiel der alten Erfahrung, So haben wir hier daß der Mensch das Gegenteil der eignen Absicht erreicht. in ein neues seinem Streben oft 2. Entwicklung. Zur Geschichte des Ausdrucks. a) |ie Ausdrücke für Entwicklung hat, wie den Begriff selbst, erst I -^ die Neuzeit allgemeinen Umlauf gebracht. in erscheint in unserer Sprache erst gegen das Ende des und findet erst in der zweiten Hälfte des Älter es ist M zuerst Grimm »Entwicklung" 1 7. Jahrhunderts 18. eine weitere Verbreitung. Auswicklung" (auch „sich auswickeln«), philosophisch hat wohl Jakob Böhme verwandt. Lexikograph der zuerst „Entwickeln" („der Stieler hat deutschen nach Sprache Stammbaum", 1691) „sich entwickeln" Haugwitz (im Soliman, 1684), sowie Hagedorn. Die Gelehrten des 1 8. Jahrhunderts sprachen oft von einem Entwickeln und einer Entwicklung eines Begriffes, Beweises und Satzes; „das Verfahren, wodurch ein Begriff ausführlich gemacht wird, heißt die Entwicklung Entwick- (Lambert). eines Begriffes" lung im Sinne eines Sichentwickeins, als Selbstentwicklung, gewann Boden erst mit dem Aufsteigen des deutschen Humanismus, dessen Verlangen nach einer inneren Beseelung der Wirklichkeit und nach Anerkennung bildender Kräfte in der Natur darin einen bezeichnen- den Ausdruck fand. Es genügen dafür die Namen Herders und Goethes. Im Titel 1777 eines Buches erscheint Entwicklung bei Tetens, in seinem veröffentlichten Hauptwerk „Philosophische Versuche über die menschliche Natur und ihre Entwickelüng". nun „Auswicklung«, das in den älteren wiegt, gänzlich zurück; auch „Auswicklung" zu begleiten „Entwicklung" drängt noch vor- Schriften Kants „Einwicklung", das pflegte, verschwindet als aus Gegenstück dem Sprach- gebrauch der Philosophie. Der deutsche Ausdruck war teils verdrängte, involutio und lateinischen teils eine Übersetzung fremder, die er neben sich duldete. Die Termini evolutio- explicatio-complicatio oder implicatio entstammen den Klassikern, aber sie werden dort nur methodologisch. Entwicklung. 1Q3 Werden verwandt.^ Ähnlich verbleibt es im Thomas von Aquino erscheinen nur explicitus und implicitus, und zwar in jener formalen Bedeutung. Nur die mystische das reale für nicht Mittelalter, bei Spekulation, die von den Schriften des Pseudo-Dionysius ausging, um ein inneres Verhältnis von verwendet die Wörter und Begriffe, zum Ausdruck zu bringen. So hat Scotus Eriugena involutus, convolutus, complicatio, replicatio. Wie Nikolaus von Kues, Gott und Welt der Philosoph an der Schwelle knüpft, so gebraucht er Neuzeit, an jene Denkweise an- d*er fortwährend die Ausdrücke explicatio und complicatio. Wenn er evolutio gebraucht, so glaubt er es erläutern zu Der Verlauf der Neuzeit macht die Ausdrücke immer sollen. 2 geläufiger. Evolutio und involutio enveloppement Lieblingswörter des 18. Jahrhunderts sind neben übernahm sie genannten »Einschachtelungstheorie". in developpement und auch Leibniz; bei Physiologie die dem Sinne der später so- Im Gegensatz dazu hieß dann die besonders glänzend von C. F. Wolff in der theoria generationis vertretene Lehre von einer Neubildung im Werden die der Epigenesis;^ die »Evolution" wurde hier verstanden und abgelehnt als eine bloß quantitative Steigerung Aber daneben behält Evolution auch den weiteren Sinn von Entwicklung überhaupt, so ist es namentlich bei außerdeutschen Völkern die gebräuchlichste Bezeichnung der neuesten Form der Entwicklungslehre geworden. b) Zur Geschichte des Begriffs und Problems. Es gibt kaum eine Überzeugung und Lehre, bei der die alte und die neue Denkart so weit auseinandergehen; die Beharrungs^ Bei Cicero heißt es z. B. (Top. 9) : tum definitio adhibetur quae quasi involutum evolvit id, de quo quaeritur. ' Niliolaus sagt (Pariser evolutio. Ausgabe von 1514, I, 89a): linea - Quomodo intelligis lineam puncti evolutionem? - est puncti Evolutionen! id est explicationem. ' C. F. Wolff hat sich über jene Begriffe namentlich in der deutschen Ausgabe und in der zweiten lateinischen Auflage von 1774 deutlich geäußert. Praemonenda § 50 heißt es in dieser: evolutio phaenomenon est, quod, si essentiam ejus et attributa spectes, omni quidem tempore, at inconspicuum, exstitit, denique vero, speciem prae se ferens ac si nunc demum oriatur, quomodocunque conspicuum redditur. S. auch Kant, Krit. d. Urteilskraft (V, 436 Hart.): „Das System der Zeugungen als bloßer Edukte heißt das der individuellen Präformation oder auch die Evolutionstheorie; das der Zeug- ungen als Produkte wird das System der Epigenesis genannt." Eucken, Grundbegriffe. 4. Aufl. 13 Zum Weltproblem. 194 den Idealen jener ebenso eng zusammen, wie die lehre hängt mit Entwicklungslehre mit denen der Neuzeit. So läßt wiederum der besondere Punkt einen Durchblick der gesamten weltgeschichtlichen — Wohl Bewegung erwarten. zeigt die älteste griechische Philosophie Höhe bedeutende Ansätze klassischen Kultur hat der Beharrungslehre das entschiedene Über- Entwicklungslehre, einer aber die der gewicht gegeben, da diese der künstlerischen Art jenes Volkes weit mehr in und entspricht Wirklichkeit gilt Maße feste Grundbestande nach und gefügtes von Die zu fassen geeignet war. in -Begriffe sie hier ihrem als ein unwandelbaren lebendiges, Ordnungen be- herrschtes Kunstwerk; aus der wirren Flucht der sinnlichen Eindrücke dies und klar zum Hauptziel das wird kräftig herauszusehen, der Diese Aufgabe ist nicht zu lösen ohne die Anerkennung Wissenschaft. eines zeitüberlegenen Seins; aus der Übereinstimmung mit ihm stammt Wahrheit der Begriffe, die alle Handeln und mitteilt Wissenschaft Werdens in die allem des Seins, Versetzung eine eines freilich So aus der ist die Welt des lebendigen Seins; durch- dem Werden voran.^ gängig geht hier das Sein dem aus sich auch beharrende Ziele richtet auf es vor hier vom Denken Eine nähere Aus- führung gibt dieser Denkart die Formenlehre, die Plato schafft und Zeitlos wirken die Formen als Urbilder und Aristoteles weiterführt. Grundkräfte der Dinge, durch den Weltprozeß, der keinen Anfang und kein Ende hat, gehen unverändert die Gestalten. Alle Veränderung kommt vom Stoffe, der, wenigstens im Bereich des Erdenlebens, sich der Form nicht ihr ergriffen Gestalt und verliert. Stoff ergreifen dauernd verbindet, sondern, eine Zeit lang von immer wieder entweicht und seine Immer von neuem muß daher die Form den gebändigt, ihr und bezwingen; das erklärt den unablässigen Wechsel, Werden und Vergehen. das rastlose einzelnen Lebewesen. geleugnet, gilt es zunächst von den Auch jenseit ihrer werden Verschiebungen nicht vielmehr Wandlungen Wandlungen So auch- im Auf- in den Stellungen der Gestirne, und Absteigen der Völker bereitwillig anerkannt. Aber auch dieses verwandelt sich einer näheren Betrachtung * Es sei dafür nur eine Stelle aus Aristoteles angeführt (de part. anim. 640a, 18): ^ b, der (S. 1: £:t£i y^^^<^'-? ^vexa tjJ? oüaia? eoriv, S'eoTt toioÜtov, -n^v ye'vEOtv üS\ Ausdruck für Wissenschaft wird PhyS. 244 b, 9): r o i^ «px.'i? ?>^'^'? xcl^ a.\X mit ttj; oOy^ f ouata l'veza -rf? ^evetew?. zoiauTTjV au[j.ßaiv£tv ivaYy.atov. Stehen ir.iTzr^rii ^pefifaat xa\ arrivat ttv Siavotav eTiioraaö-ai xai (ppovetv Selbst Beziehung gebracht Y£v£at; oüx eoriv tw y«P in Xe'yo(j.ev. Entwicklung. jg5 Denn aus aller Veränderung in eine Bestätigung der Beharrungslehre. ihrer Stellungen kehren die Gestirne nach Ablauf eines grofkn Welt- um die alte Bewegung jahres wieder an den Ausgangspunkt zurück, neu zu beginnen, der Wechsel Ähnlich im Grunde verändert also nichts. auch die Geschichte eine endlose Folge von Kreis- bildet bewegungen wesentlich gleichen Denn alles Aufsteigen der Inhalts. Völker geht nur bis zu einem gewissen Punkt, um Sinken umzuschlagen, von bis Elementarkatastrophen dann in Feuer ein oder Wasser wieder eine Verjüngung bewirken, so daß nun dieselbe Bewegung von neuem anheben kann. So eine ewige Wiederkehr der Dinge; was wir jetzt erleben, geschah schon unzähligemal und wird Demnach die Welt kein starres Sein, noch unzähligemal geschehen. sondern voller Bewegung, aber diese Bewegung, nach der Art von Tages- und Jahreszeiten, Rhythmus verlaufend und inmitten in festem alles Wandels feste Grenzen gebannt, kein echtes Streben geht es voll keinen gibt Ruhe. sicherer Fortschritt Überall Unendliche, ins das Leben hier ist keine in Unbestimmte, in? Hoffnung einer wesentlich besseren Zukunft. Dafür aber gilt die Überzeugung, daß die unmittelbare Gegenwart Ewiges erfassen und mit ihm das Leben erfüllen kann. nehmen, sie Die Tätigkeit tut das, selbst hat hier die indem sie sich selbst befriedigtes und des Aristoteles, Eine derartige Tätigkeit Stelle wird. Ruhe in sich aufzu- aus einem bloßem Streben gesättigtes ein in Wirken, „Energie" im Sinne gewährt an erster Anschauung des Wahren und Schönen, aber auch das die Handeln wird hier auf seiner Höhe zum Darstellen einer beharrenden Art und Natur. Denkweise Solche Gutes, in\ sieht Veränderlichen ein durchgängig im Unwandelbaren Schlechtes. ein Das Hauptmerkmal der Gottheit bildet hier die Ewigkeit, das Unberührtsein vom Strom der Zeit. Dem Handeln aber wird ein gehalten, an unwandelbarer Idealstand vor- dem es sich messen und auf den es sich richten So vornehmlich im Entwerfen von Idealverfassungen, schichtlichen Wandlung entzogen sind. soll. die aller ge- Die Überzeugung, daß unser Leben auf festen Grundlagen ruhe und sich innerhalb fester Grenzen bewege, selbst gestaltet die Arbeit aller Gebiete eigentümlich und verleiht der Logik und wissenschaftlichen Methode einen besonderen Charakter. Die Grundwahrheiten Urteilen gegeben zustellen, und abgeschlossen, zu einander in dünken es gilt hier in Begriffen und nur sie deutlich heraus- Beziehung zu setzen, in ihre Konsequenzen 13* Zum Weltproblem. 196 zu Die verfolgen. ward damit zum Hauptstück der Schlußlehre während der Neuzeit sich diese Arbeit, in die Urteile und Begriffe zurückverlegt hat. Dabei floß in die Lehren der Philosophen von Anfang an auch Stimmung des ganzen Menschen mit ein, das Verlangen, gegenüber dem bunten und ermüdenden Wechsel der kleinstaatlichen Verhältnisse einen beharrenden und bedeutenden Lebensgehalt zu gewinnen; die Wendung vom menschlichen Kreise zum All war zugleich ein Suchen nach innerer Erhöhung und Befestigung des 'Daseins. Solche Strömung verstärkte sich gegen den Ausgang des Die Sache Altertums und gewann neue Nahrung im Christentum. die ward nun vom Künstlerischen ins Religiöse gewandt; verlangte die Höhe des griechischen Lebens ein Ruhen innerhalb der Bewegung, so galt es jetzt, eine Ruhe in Gott im Gegensatz zu dem unsteten und sinnlosen Treiben der Welt zu finden, sich dahin als in einen Nicht sicheren Hafen aus den Stürmen des Lebens zu flüchten. streben wollte man, sondern fest dere Kraft und Kern Innigkeit Weisheit aller und sicher besitzen. Eine beson- gab solchem Verlangen die Mystik, den konnte sie darin finden, die ganze Zeit zu einem bloßen Scheine herabzusetzen und durch ein wachsendes Aufgehen ewige Sein jeden Tag »jünger" zu werden. in das ergriff in Dies Ideal den Zeiten des ausgehenden Altertums und des beginnenden Mittelalters um so mächtiger das Gemüt, als es der gesamten Lage der Kultur entsprach. Denn eine alte Art hatte sich hier ausgelebt, ohne daß fruchtbare Keime einer neuen erkennbar waren; selbst den Besten konnte es daher als Hauptaufgabe erscheinen, den vorhandenen Besitz treu zu wahren und gewissenhaft den künftigen Geschlechtern mitzuteilen. Unwandelbar schien vor allem als göttliche Offenbarung; aber auch auf den übrigen Gebieten, Philosophie und Medizin, in Recht und die religiöse Wahrheit, Staat, in schien alles erreicht, was dem Menschen irgend erreichbar ist; die Autorität eines Aristoteles und eines Galen kaum stand hinter der des kirchlichen Dogmas zurück. Diese Überzeugungen tragen Mittelalters, herstellt, das große Ordnungssystem des das überall unwandelbare Normen und feste Verkettungen innerlich wie äußerlich, namentlich auch in lichen Verhältnissen, das Leben in sichere Bahnen Trieb zur Veränderung aufkommen es fern, die Natur als ein läßt. den wirtschaft- bringt und keinen Solcher Denkweise liegt Reich allmählichen Werdens zu verstehen. Entwicklung. X97 vielmehr hat diese nur die Formen zu bewahren, die der Schöpfer ihr zu Anfang mitgeteilt hat.^ Die Neuzeit stand von vornherein zu einer solchen Beharrungs- Denn lehre feindlich. ohne einen Glauben an sie konnte keine selbständige Art entfalten die Bewegung und das Recht der Bewegung, im Eintreten dafür mußte sie ihrem eignem Streben Boden gewinnen. In Wahrheit war gegenüber dem Ausgang des Altertums die Lage der Menschheit erheblich verändert Neue Völker voll strotzender Jugendkraft waren entstanden, die langen Jahrhunderte des Mittelalters Vermögen gesammelt, das immer stärker nach Betätigung die Welt mit eignen Augen zu sehen und nach eignen Zielen zu gestalten; aus dem bloßen Empfangen und Überliefern treibt es zum Weiterbauen und Erneuern, ein verändertes Lebensgefühl eröffnet neue Ausblicke und Aufgaben, die Idee einer fortschreitenden Bewegung ergreift immer mehr das hatten viel drängte und sich zutrauen durfte, Leben wie die Arbeit Aber solchen Lebensdrang in eine sichere Bahn zu keineswegs leicht; die Geschichte der Entwicklungsidee das nur im Anschluß an ältere Bestrebungen gelungen es verschiedene Stufen durchlaufen hat. reicht in das Christentum selbst zurück. leiten, war zeigt, wie ist, und wie Die Anregung zum Neuen Mochte seine kirchliche Gestalt fest an der Beharrungslehre haften, seine Gedankenwelt enthielt auch fruchtbare Antriebe entgegengesetzter Art. wird die Geschichte weit mehr Zeit in als dem Dem Christentum Mitten Altertum. in die war nach seiner Überzeugung das Göttliche eingetreten, nicht mattem Abglanz, sondern mit ganzer Fülle seiner Herrlichkeit* beherrschender Mittelpunkt des Ganzen mußte es auf sich beziehen und alle artigkeit dieses Geschehens Zukunft aus sich litt alle als Vergangenheit entfalten. Die Einzig- keinen Zweifel, Christus konnte nicht immer von neuem kommen und sich kreuzigen lassen; so entfielen die unzähligen Perioden, die ewige Wiederkehr der Dinge; aus einem gleichmäßigen Ablauf von Rh>1hmen wurde die Geschichte ein um- spannendes Ganzes, ein einziges Drama; daß der Mensch hier zu ^ Um nur eine bezeichnende Stelle dafür anzuführen, so läßt Alanus de insulis (s. Baumgartner, die Philos. des A. d. J., S. 79) die Natur sprechen Me igitur tamquam sui vicariam rerum generibus sigillandis monetariam destinavit, ut ego in propriis incudibus rerum effigies commonetans ab incudis forma conformatum deviare non sinerem. Zum Weltproblem. 198 einer völligen Umwandlung aufgerufen wurde, das gab seinem Leben größere Spannung, als wo es nur eine vor- eine unvergleichlich handene Natur zu entfalten So galt. Wurzeln die liegen einer höheren Schätzung der Geschichte und des zeitlichen Lebens nirgend anders im Christentum. als Aber nur langsam geschah es, daß solche Wandlungen prinzipiell und deutlich ausgesprochen wurden; gefaßt es ist das vornehmlich von der Spekulation her geschehen, die damals mit einem Verlangen nach gemütvoller Aneignung Hand Hand in Vor allem ging. suchte sie die Welt zu Gott in ein engeres Verhältnis zu Was und bedeutet als der naive Glaube es all ihrem Geschehen, von Gott aus betrachtet? tat. ist die bringen, Welt mit Sie kann, so lautet die Antwort eines Augustin, nichts anderes als eine Selbstdarstellung des göttlichen Wesens einen ihr keit in sein. Damit aber gewinnt alle Mannigfaltig- Zusammenhang, auch inneren die verschiedenen Punkte der Geschichte können nun kein bloßes Nacheinander bleiben, sie werden Stücke einer durchgehenden Bewegung, weltumspannenden Tuns; auch das, was später Früheren irgend schon gegenwärtig sein; so ja eines einzigen eintritt, mußte beim sich der gesamte läßt Weltprozeß der Entwicklung eines Baumes aus seinem Samen vergleichen. ^ Die mystische Spekulation eines Dionysius, Scotus Eriu- gena w. u. s. steigert dies «Auswicklung" dessen zu als eine dahin, fassen, Ganze der Welt das was „eingewickelt" Entfaltung der Ewigkeit zu einem Leben unsichtbaren Einheit zu sichtbarer Vielheit. uns sollten freilich nahe zu rücken. * lehre in als eine bei Gott ist, der Zeit, der Ihre Ausdrücke und Bilder nicht verführen, diese Denkart der Das begründende Sein und die modernen zu bewegende Kraft Bei dem allen ist Augustin der führende Geist; für seine Entwicklungs- ist namentlich bezeichnend folgende Stelle (op. III, 148 D): Sicut in ipso omnia simul quae per tempora in arborem surgerent: ita ipse mundus cogitandus est, cum Deus simul omnia creavit, habuisse simul omnia quae in illo et cum illo facta sunt, quando factus est dies, non solum caelum cum sole et luna cum sideribus sed etiam illa quae aqua et terra produxit, potentialiter atque causaliter, priusquam per temporum moras ita exorirentur, quomodo nobis jam nota in eis operibus, quae Deus usque nunc operatur. Wie er sich aber die Entwicklung eines Baumes aus dem Samen vorstellt, zeigt V, 714 E: In illo grano seminis exiguo, vix visibili, si consideres animo, non oculis, in illa exiguitate, illis angustiis et grano invisibiliter erant — radix latet et robur insertum apparebit in arbore, jam est , est et folia futura alligata praemissus in semine. sunt et fructus, qui Entwicklung. 199 die Kette blieben gänzlich jenseitiger Art; Stufenfolge des Nacheinander war nicht des Geschehens und die dem eignen Boden der Zeit entsprungen, sondern in ihr hatte die göttliche Einheit sich zeitlos Wie auseinandergelegt. bedingt Höhere Fülle der Einheit. diese mit ihrer ewigen Bewegung, Zerstreuung aller und hat Unruhe überlegenen mystisch-spekulative die Gedankenwelt die moderne Entwicklungslehre gegeben. mit solcher hat Größe gegenwärtig sein, Forschung nicht hätte nicht die Gottesidee, die Leben und Glanz geliehen. ihr und Welt Allerdings mußte im Verhältnis von Gott Verschiebung Unend- Richtung auf Ewiges und die Die Welt würde der modernen Idee des absoluten Wesens, beträchtliche Sie eingeleitet. der Erscheinung göttlichen Wesens, höher denken als und ihrem Leben gelehrt liches das un- als ihr zurück zu jener aller Viel- Aber trotz solches Abstandes von der Welt, Ruhe so strebte auch das Leben letzthin nicht in die Welt hinein, sondern aus und heit galt, erfolgen, ehe das sicher erst eine fortschreiten konnte: die engere Verbindung von Welt und Gott durfte nicht dazu dienen, die Welt gänzlich in Gott verschwinden mußte dahin wirken, sie höheren Wert zu zu lassen, sondern ihr als dem Ausdruck göttlichen Seins einen Diese Verschiebung geben. aber ist dem bei bahnbrechenden Philosophen der Renaissance, bei Nikolaus von Kues (1401 — — 1464), erfolgt. Als die Auswicklung des unendlichen Lebens die neuere Spekulation pflegt durch lebendig sein, ja eben damit aber einen Trieb zu in sich tragen.^ Was Gott besitzt, un- das hat Nur ein Fortschritt ins Unendliche kann die Lebensfülle des absoluten * Seins zum Ausdruck bringen, 1514, der Ewigkeit muß sie an jeder Stelle nach Teilnahme am unendlichen Leben dürsten, begrenztem Fortschritt beim Gottesbegriff — muß ihm die Welt durch und die Unendlichkeit voranzustellen II, aeternae sapientiae, et ex hoc creatori s. sine fine. II, B. Nikolaus z. 188a): Posse semper plus et elice, von Kues (Paris. Ausg. von plus intelligere sine fine, est similitudo quod est viva imago, quae se conformat 187b: semper vellet id quod intelligit plus intelligere mundus totus non sufficit ei, quia non replet desiderium intelligendi ejus. — Der Begriff des Fortschritts ist dem Altertum et quod amat plus amare, et trotz der Vorherrschaft der Beharrungslehre keineswegs fremd, Plato und Aristoteles haben dafür die Ausdrücke smSoai; und iTrtötSo'vai, weit mehr aber trat r.poy.oTu hervor, das wir (z. B. bei Polybius) ganz ähnlich ver- das stoische wandt finden wie das heutige »Fortschritt". Der Gedanke eines Fortschritts ins Unendliche hat seine Wurzel bei den Piatonikern und Mystikern, er ist aber zur vollen Entwicklung erst in der neuen Philosophie gelangt. Seinen Zum Weltproblem. 200 die Kreatur Wachstum annähernd zu allmählichem in der ganzen Welt ein Streben nach aufwärts eingepflanzt. winnt, in erreichen. Wendung wird die Bewegung wesentlich gehoben und Mit solcher dem neben Künstlerische Indem zurückzudrängen. zum Ausgang Gegensatz direktem Religiösen Welt die Platz, sich ja Zugleich ge- des Altertums, es beginnt mehr und mehr das dieses ein als lebensvolles Kunstwerk darstellt, in dessen Harmonie sich alle Gegen- des ersten Anblicks auflösen, scheint sätze alle Bildung von innen hervorzubringen; das sie die Bewegung wie durch Entfaltung ihres eignen Wesens, her, Absolute bedeutet nun weniger ein eignes Reich, als es der Welt eine Tiefe oder doch einen Hintergrund gibt. Solche Verschiebung vom Theismus zum Pantheismus kommt zum Siege in Giordano Bruno; die immanente und künstlerische Gestalt der Entwicklungslehre hat von nun an die Oberhand. Form, welche bis zur Gegenwart in Das ist die den Ausdrücken und Bildern von der Sache überwiegt, das stille und stetige Wachsen der Pflanze wird hier zum Vorbilde für das Wirken und Aufsteigen der Natur von innen heraus. in seelenlose Die Aufklärung mit ihrer Zerlegung der Natur Elemente ist dieser Gedankenrichtung minder günstig,^ dagegen bringt der Rückschlag gegen die Aufklärung, wie ihn der deutsche Humanismus enthält, sie voll zu Ehren. Nicht die bloße Be- wegung, sondern das künstlerische Gestalten wird hier zum Haupt- Höhepunkt bildet Leibniz, s. z. B. 150a (Erdm.): In cumulum etiam pul- operum divinorum progressus quidam universi est agnoscendus, ita ut ad majorem chritudinis perfectionisque universalis perpetuus liberrimusque totius semper cultum procedat ff.; femer deutsche Schriften II, 36: «Der Kreaturen und also auch unsere Vollkommenheit bestehet in einem ungehinderten starken Forttrieb zu neuen und neuen Vollkommenheiten." Bei Wolff und seiner Schule galt als das höchste Gut perpetuus sive non impeditus ad majores, per fection es progressus. — Der Ausdruck Fortschritt dürfte erst in der zweiten Hälfte des 18. Jahrhunderts ein fester Terminus geworden sein. * Es fehlt hier aber keineswegs an wenig beachtete Richtung. S. z. (Nouv. ess. III, cp. VI, pag. 317a, B. die anregenden Gedanken nach dieser Stelle in Leibnizens Hauptwerk Erdm.): Peut-etre que dans quelque tems ou dans quelque Heu de l'univers ies especes des animaux sont ou etaient ou seront plus sujets ä changer, qu'elles ne sont presentement parmi nous, et plusieurs animaux qui ont quelque chose du chat, comme le Hon, le tigre et le lynx pourraient avoir ete d'une comme meme race et pourront etre maintenant des sousdivisions nouvelles de l'ancienne espece des chats. Ainsi que j'ai dit plus d'une fois que nos determinations des especes physiques sont provisionelles et proportionelles ä nos connaisances. je reviens toujours ä ce Entwicklung. 201 werke der Natur; damit wird ihre Veränderung zu einer Entwicklung von innen und her, Mannigfaltigkeit ihrer Gestalten scheint auf alle Über die Natur hinaus einen einzigen Grundtypus zurückzukommen. bemächtigt sich der Entwicklungsgedanke dann des Menschenlebens und des ganzen Alls; was „alles, in der Wirklichkeit vorkommt«, erscheint nun als „Entwicklung einer absoluten Vernunft" (s. Schelling I, Die nähere Durchführung dessen zeigt verschiedene Färbungen; 481). Romantik das ruhige Werden und Wachsen in den Vorder- stellt die grund, so gibt Hegel mit seiner kosmischen Logik der Entwicklungsidee mehr Immer aber Selbsttätigkeit. erfolgt Bewegung von die innen heraus, immer wirkt, auch an der einzelnen mit über- Stelle, legener Kraft das Ganze. Das eben es, ist was die der strengwissenschaftlichen tümlich stellt künstlerische Entwicklungslehre unterscheidet, die der Neuzeit von eigen- Denn diese gibt alle inneren Zusammenhänge auf und ist. das Problem ganz und gar auf den Boden des unmittelbaren Zusammenwirken der Elemente soll den lehren, und es soll aller Fortder Natur verstehen Gesamtstand Daseins: das empirische schritt in einem wird Entwicklungsidee die Diese Wissenschaft. Nacheinander erfolgen. zeitlichen zu einem Hauptstück das nächste Bild setzt diesem Sinne In modernen der der Dinge zu einer bloßen Erscheinung herab, von der es zu den echten Beständen durchzudringen Elemente Das gilt. aufsucht, geschieht, Gesetz das die indem Analyse einfache Wirkformen dieser ermittelt, dann aber die Entwicklung die Welt neu schichtlichen macht sie die und vom ge- begreiflich der neueren Wissenschaft zur Hauptvertreterin der Synthese, und erscheint sie als der gesamten Arbeit der stein aufbaut Werden aus den vorgefundenen Bestand So wird erst die Gunst des Denkens, ja die Vollendung und der Prüf- Forschung; kein Wunder, daß ihr des modernen Menschen gehört Es erscheint aber die neue Entwicklungslehre gleich beim entscheidenden Durchbruch der modernen Denkart; schon Descartes hat, wenn auch nur als eine Möglichkeit, den Gedanken, nach und nach daß der gegenwärtige (cum tempore, successive) Weltstand in der Zeit, entstanden Im Lauf der Jahrhunderte bemächtigt ^ sei.^ sich dieser Clauberg beschrieb im wesentlichen zutreffend (op. philos. 755) die Methode folgendermaßen: Hanc methodum Cartesiana physica cartesianische omnes res naturales non statim quales sunt in statu perfectionis suae absoluto (ut vulgo fieri solet ab aliis), sed prius agit de quibusdam tenens considerat Zum Weltproblem. 202 Gedanke aller einzelnen Gebiete und gräbt sich immer tiefer in den Stand des Wissens ein.^ Kosmologie weicht der antike Ge- In der danke einer Unveränderlichkeit des Himmelsgewölbes dem des allmählichen Werdens der Weltkörper und Weltsysteme (Kant, Laplace), der Befund der Seele wird nicht nach alter Art als fertig hingenommen, beschrieben und seit das Locke, sondern die moderne Psychologie strebt eingeteilt, Werden und Wachsen der Seele von Lebenserscheinungen ner genetisch Geschichte als erscheint zu verstehen; allmähliches ein schwindenden Anfängen her zu Überall befindlich behandelt. Bestände und ein bleibenden einem festen Ganzen, in als Fluß von ver- und ähnlich und Wandel gegen die ältere Art der Anblick war sie dort ein Herausheben der ist der Wissenschaft umgewandelt: menschliche die Aufklimmen unbegrenzter Höhe, werden auch die einzelnen Kulturwelten einfachsten unmittelbares Zusammenschliefkn künstlerisches ein Zusammenschauen zu der nun das nur scheinbar Feste in Fluß, dringt unermüdlich zu kleineren und kleineren Elementen vor und Mannigfaltigkeit, so bringt sie verwandelt die Wirklichkeit einen in noch mitten im Werden be- griffenen Prozeß. Dabei scheint sie zu einer weit engeren Berührung mit den Dingen zu trachtete; so heißt kommen, etwas in die sie früher die Entwicklung mehr von außen be- hineinziehen, voll es der Erkenntnis gewinnen. Aber so wirksam war, die moderne Entwicklungsidee schon lange zum Siege für das Ganze erst Darwin gebracht. des Lebens und der Arbeit hat sie Zunächst hat seine Leistung eine große Lücke ausgefüllt: die organischen Formen widerstanden bis dahin einer gene- tischen Erklärung hartnäckig, so blieb eine unüberbrückte Kluft zwischen den Weltbegriffen und den Erfahrungen des menschlichen Kreises, die beide der Entwicklung schon gewonnen waren. Wohl waren, namentlich durch Lamarck, bedeutende Anfänge zur Aufhellung gemacht, aber den Versuchen fehlte die Verbindung zu einem durchgebildeten Ganzen und zugleich eine zwingende Kraft. Darwin hat earundem principiis valde simplicibus et facilibus, deinde explicat, quomodo paulatim ex Ulis principiis, suprema causa certis legibus opus dirigente, oriantur et fiant, aut carte oriri aut fieri possint, donec tandem tales evadant, quales esse experimur dum consummatae et absolutae sunt. ' Einen wichtigen Abschnitt dieser Bewegung behandelt in vortrefflicher Weise H. Heußler: „Der Rationalismus des ziehungen zur Entwicklungslehre", 1885. 17. Jahrhunderts in seinen Be- Entwicklung. 203 von Deszendenz- und Selektionslehre jene mit seiner Vereinigung Lücke ausgefüllt und das Schlußstück des Ganzen Lehre namentlich dadurch stark, daß ist sie seine geliefert; aus genauester Durch- forschung ihres besonderen Gebietes Begriffe gewinnt, die einer unermeßlichen Ausdehnung nach allen Richtungen fähig scheinen; er wie Helmholtz sich ausdrückt hat, II, »alle 204), — (s. popul. wissensch. Vortr. 2. Aufl. vereinzelten Gebiete aus Wunderlichkeiten dem Zustande einer An- den Zusammenhang einer häufung großen Entwicklung erhoben und an die Stelle einer Art von künsträtselhafter lerischer in Solches Verdienst Anschauung bestimmte Begriffe gesetzt." erfährt keinen Abbruch dadurch, daß die Schranken der Selektions- und ihrem Kampfe ums Dasein zu immer klarerer Einsicht kommen; hat doch Darwin selbst diese Lehre nicht als die einzige Erklärung der organischen Formen lehre mit ihrer natürlichen Zuchtwahl Es bleibt dabei, daß durch ihn das Problem ausgegeben. neue Lage gebracht ist, eine in daß die Entwicklungsidee mit der Sicherung auf dem Gebiet des organischen Lebens zugleich die Fähigkeit er- zum Ganzen einer Weltanschauung zu erweitern. Für solche Wendung muß vornehmlich Spencers gedacht werden, langt hat, sich der zuerst von einer realistischen Denkweise aus die Entwicklungslehre ein eigentümliches Weltbild hat erzeugen lassen. ist ihm ein gilt Stoffes ihm Entwicklung, als Zusammenschluß und Zerstreuung der Bewegung; ihr von Bewegung in Aufnahme zunehmende Spezialisierung und Differenzierung, vom als Ganzen die (ab- vom Gleichartigen ins Ungleichartige, eine in des aber bei ihm in und Lockerung (disintegration) des Stoffes. Dort erfolgt eine Verschiebung bis einen (Integration) folgt endlosem Wechsel eine Periode der Auflösung sorption) in Als die allgemeinste Tatsache der zusammenhängenderen Zustand. Welt Entwicklung Übergehen aus einem zusammenhangsloseren einzelnen Weltkörper, die in Weltall menschliche Gemeinschaft, die Kultur, das Einzelwesen hinein; die Periode der Auflösung verfolgt die umgekehrte Richtung. Eine Verwandtschaft solches Rhythmus mit Gedanken der ältesten griechischen Philosophie, namentlich mit Empedokles, Lehre, die im Grundriß ist nicht zu verkennen. zeitlich Wenn Spencers voranging, der Darwins einen uni- versalen Hintergrund gegeben hat, so hat sie durch die Verbindung mit dieser unermeßlich an Fülle, Anschaulichkeit keit und Eindringlich- gewonaen. Trotz solches Vordringens der Bewegungslehre ist die Beharrungs- Zum Weltproblem. 204 lehre zu tief und zu eingelebt mit den Überzeugungen wich- fest tiger Lebensgebiete verschmolzen, als daß sie nicht vielfachen Im besonderen stand leisten müßte. nur einzelne Bestandteile die Religion, es ist Wider- die nicht herkömmlichen Vorstellungskreises, ihres sondern auch den ihr unentbehrlichen Gedanken einer ewigen Wahr- Aber auch heit bedroht die Überzeugung, daß sieht. hier befestigt als selbst, mit Überzeugungen material- oder doch naturalistischer Art istischer mehr und mehr weniger die Bewegungslehre es ihre, keineswegs notwendige Verquickung lichen Gegensatz sich ist, zur Religion enthält. ^ welche einen unversöhnAlles zusammen hebt die Entwicklungslehre weit über die Stellung einer besonderen Theorie neben anderen hinaus, sie hat die Führung des Ganzen übernommen und erzeugt einen neuen Lebenstypus, der ebenso unser Grundverhältnis lichste zur Wirklichkeit verändert. Nun als Art unseres Tuns aufs wesent- die mehr eine fertig vorhandene heißt es nicht Wirklichkeit anzueignen, sondern einer erst werdenden zur Vollendung zu verhelfen; die Tätigkeit verschlingt sich dabei enger mit der Um- gebung und gewinnt ihre nähere Gestalt erst aus der Berührung mit den Dingen. Sie darf sich der Welt zu stehen fällt sagen, und zu die alte Flucht aus nicht neben, sondern inmitten ihrer Bildung mitzuwirken. Nun ent- dem Strom der Zeit zu einer wandellosen Ewigkeit, sowie die Vorhaltung eines Idealstandes als eines unverrück- baren Zieles, vielmehr heißt Zeit zu folgen Es * es, ganz und gar der Bewegung der und das Handeln den Forderungen der jeweiligen dafür nur eine Stelle des hervorragenden französischen Theo- sei Er sagt in seiner vielbeachteten Rede Methode der Theologie (s. Bulletin de litterature ecclesiastique Nov. 1901, pag. 272): Vous savez avec quelle defiance justifiee fut regue logen Erzbischof Mignot angeführt. über die l'idee d'evolution, qui paraissait liee par il y a trente ans, de graves compromissions avec la philosophie pantheiste; depuis que l'analyse en a precise le contenu, on est ä peu pres unanime ä reconnaitre qu'une dans nos ecoles, d'entendre une conception germe dans saint avec certaine fagon religieuse et chretienne Augustin, et on deeouvre, avec Vincent de Lerins, qu'appliquee ä l'histoire religieuse, eile seraient restes forschung und peut de l'evolution est conciliable l'univers; on en apporter insolubles. de grandes Auch evolutionistische trouve clartes dans des problemes qui Reischle: „Wissenschaftliche Entwicklungs- Weltanschauung in Christentum" (Zeitschrift für Theologie und Kirche, scheidet scharf zwischen lungstatsachen. innert sein (s. le Evolutionismus Auch an Newman's als theory darüber Lady Blennerhassett , ihrem Verhältnis zum Jahrgang, Heft) 12. 1. Weltanschauung und Entwickof development mag hier er- Kardinal Newman S- 125 ff). Entwicklung. Das Lage möglichst genau anzupassen. aus träger Starrheit auf und bringt z. Von wart. in der lebendigen Gegen- Ergreifung des unmittelbaren Augenblicks und der Gestaltung nach seinen Bedürfnissen, eine Elastizität des Lebens, Wandlungen bereitwillig entgegenkommt. dem Ausdruck Hegels das Werden .,die so müssen auch die Ideale die Beweglichkeit temporis (veritas völligen älteren Denkweise hat Hauptziel Das filia). einem Relativismus, ein nicht gilt ja Bildet so werden auch »Kind der Zeit" ein unterwirft augenscheinlich aber mit dem so Wahrheit des Seins", teilen, die Wahrheit die Ziele veränderlich, so wird als Lebensgebiete alle frischesten Fluß, das gibt hier aus erwächst ein eigentümlicher Begriff des Modernen, weiteren nach rüttelt mit den Aufgaben erfüllt sie aller Verhältnisse die sie Gesetzgebung wie der Erziehung eine engere Beziehung B. der zur Zeit und als der 205 das Leben Hinfälligwerden solcher allen Schrecken verloren. mehr die Aneignung einer der Denn um uns vor- handenen und fertig abgeschlossenen Wahrheit, sondern die Erzeugung eines möglichst reichen Lebens mehr jene im eignen Kreise; dafür aber scheint Art mit ihrer unbegrenzten relative Anpassungsfähigkeit besonders geeignet. bei Bestrebungen inneren Beweglichkeit und alles und Wandlungen, auch modernen Lebens staltung des Das hat jene verbleibt die nicht äußere Ge- Verwandlung des Daseins in eine rastlos fortschreitende Bewegung aufs wirksamste unterstützt. Die Technik hat den Lebensprozeß in ungeahnter Weise beschleunigt, den Augenblick bedeutender gemacht, die Berührungen und mit ihnen die Wandlungen der Dinge unermeßlich gesteigert; bis in ihre Werkzeuge hinein griffen. ^ Mit ist jetzt die Arbeit in unablässiger Wandlung be- dem allen scheint der Sieg der Bewegungslehre end- gültig entschieden und mit ihm ein kräftigeres, freieres, frischeres Leben errungen. ^ * Die Konsequenzen dessen für die sozialen Probleme hat namentlich K. Marx in eindringlicher Weise dargelegt; er sagt (Das Kapital, I, 479): „Die moderne Industrie betrachtet und behandelt die vorhandene Form eines Produktionsprozesses nie während revolutionär, als definitiv. Ihre technologische Basis ist die aller früheren Produktionsweisen wesentlich daher kon- servativ war." * Merkwürdig genug ist, daß in derselben Zeit, wo die Entwicklungslehre so siegreich vordringt, der Forschung ernste Besorgnis über den bleibenden Fortbestand Wärme des nur von Lebens erwachen; das aber von daß und daß dem das Leben auf- der Tatsache heißeren zu kälteren Körpern übergehen kann, damit das Weltall einem Gleichgewicht zustrebt, bei aus, Zum Weltproblem. 206 Die Verwicklungen und Schranken der bloßen Entwicklungslehre. c) Das alles und hat seine Wahrheit Recht; töricht wäre sein es, sich einem solchen Strom von Tatsächlichkeit entgegenzuwerfen, kleinlich, an ihm einzelne Irrungen aufzusuchen und bei ihnen zu verweilen. Aber daß Welt und Leben darin aufgehen, daß der Kampf zwischen Bewegungs- und Beharrungslehre schon endgültig ausgekämpft ist, das dürfte mit allen jenen Wandlungen noch keineswegs entschieden sein. Es müßte wunderbar zugehen, wenn die Idee der Entwicklung selbst ohne wäre, wenn eine Strömung, welche die alle Verwicklung Zeit so überwältigend fortreißt, nicht viel Ungeklärtes enthielte, wenn das Sehen ausschließliche Menschen des nach einer Richtung ihn nicht nach anderen Seiten vieles übersehen besonderen ließe, seien es Ergänzungen, seien ts Widerstände. Nach dem Plane unserer Arbeit unsere Untersuchung namentlich dieses erwägen, was die Be- soll hauptungen und Wandlungen dem Lebensprozesse wie er leisten, unter ihrem Einfluß gestaltet, im besonderen, ob er dabei einen sich geistigen Charakter zu des Geisteslebens keit bewähren Daß wahren vermag. ist es, dem alles Unternehmen sich zu hat. der modernen Entwicklungslehre verschiedenartige Ten- in zusammenwirken, denzen an Das Problem der Möglich- das verraten schon die Bezeichnungen. Wer von „Entwicklung", von »Evolution" spricht, scheint anzunehmen, daß in sich die Dinge von innen her, nach einem Gesetz des Ganzen, sicherer Richtung auf ein Ziel entfalten. Das aber will der über- Zug der Gegenwart nicht, vielmehr erwartet er allen Fortschritt von dem Zusammentreffen von Elementen, die von Haus wiegende aus gegeneinander gleichgültig sind, Summierung kleiner Verschiebungen, hören müßte. besteht, und werden, wie Indessen hier sie mag fragt auf sich, die sowie von er verwirft einer alle langsamen inneren Ziele ob nicht eine Gegenwirkung dagegen Lehre vom Strahlungsdruck hingewiesen namentlich durch Arrhenius zu jenem Problem in frucht- So kommt Arrhenius zu dem Ergebnis (s. das Werden der Welten, deutsche Übers. S. 190): »Durch dieses kompensierende Zusammenwirken von Schwerkraft und Strahlungsdruck, sowie von Temperaturausgleich und Wärmekonzentration, wird es möglich, daß sich die Weltentwicklung in einem fortwährenden Kreislauf bewegt, bei dem wir weder Anfang noch Ende wahrnehmen können, und bei dem auch das Leben Aussicht hat, beständig und unvermindert weiter zu bestehen." bare Beziehung gesetzt ist. Entwicklung. 207 und Richtungen, er verwirft alles Wirken aus einem Ganzen. Aber wozu dann der andersartige Ausdruck, der unvermeidlich den Schein einer von innen her sicher und ruhig fortschreitenden Bewegung erzeugt? Gibt er nicht einem seelen- und sinnlosen Weltbilde ein freundliches Ansehen, zu viel und Zerstörungen, versteckt er nicht Verneinungen die die jenes Weltbild enthält? Solche Bedenken stören indes die Durchschnittsmeinung wenig. dem Gedanken Berauscht von Endlose, ins Dinge einer Entwicklung, fühlt sie kein Bedürfnis das Woher und Wohin geringer die Präzision, je Je ist die Begeisterung. die Tatsache nicht zu verkennen, daß in den leiten- Jedenfalls ist Systemen der Fassung überwiegt erscheint. Die modernen Entwicklungslehre und die als ist dadurch mit ihrer des Problems künstlerischen weit zurückgedrängt; mag er im Verborgenen stärker fortwirken als die die Herrschaft auf mechanische eine endgültige Lösung Entwicklungslehre ältere oder logischen Art ist machen. zu vager die Fassung, desto sicherer scheint die Sache, desto summarischer , Viele ohne über das Was und Wie, irgendwelche Sorge sich aller nach einer präziseren Fassung. begeistern sich heute für Entwicklung, den eines Fortschritts unbegrenzten Besser- und Besserwerdens eines von Hegel, meisten denken, Darwin übergegangen. Bei Darwin und im Darwinismus sind die beiden Hauptgedanken der Deszendenz und Die Deszendenzlehre der ist Selektion deutlich auseinanderzuhalten. von so verschiedenen Seiten her bestätigt und hat sich so unermeßlich fruchtbar erwiesen, daß über sie in kaum noch ein Streit besteht. Die Selektionslehre der Wissenschaft dagegen, die zeitweise die Forschung überwältigend fortriß, hat mehr und mehr Widerstand gefunden. Daß sich das ganze Reich der Formen lediglich aus einer Ansammlung zufälliger individueller Variationen, Beharren, durch ein blindes Zusammentreffen das hatte den überwiegenden gegen ^ E. und tatsächliches ohne irgendwelche innere Gesetzlichkeit, aufbauen solle, Zug der Philosophie von Anfang an sich,^ das hat sich mehr und mehr auch der Naturwissenschaft Es hier ist namentlich der unermüdlichen und scharfsinnigen Arbeit von Hartmanns zu gedenken, der ebenso von der Spekulation wie von den Tatsachen her die Unzulänglichkeit jener Lehre mit überlegenen Gründen dargetan hat. In seiner neuesten Behandlung der Frage: »Die Abstammungslehre seit Darwin" (in den Annalen der Naturphilosophie II, 3) faßt er S. 354 das Ergebnis der Forschungen der letzten Jahrzehnte dahin zusammen: «Die Zum Weltproblem. 208 als Gerade auf dem eignen Boden der Ent- unzulänglich erwiesen. wicklungslehre findet diese besondere Fassung mehr und mehr Widerspruch. Es sei hier, wo sich ein näheres Eingehen auf diese Probleme verbietet, nur an die Weismannschen Theorien, an die Entwicklungs- mechanik, an die Mutationslehre erinnert. die eigentümlichen Züge und Dieselbe Bewegung, welche die Probleme des Lebens wieder mehr muß auch dem Abschluß bei einer mechanischen Ent- hervortreibt, wicklungslehre und widerstehen eine dynamische empfehlen. So geschieht es vielfach in Wiederaufnahme und Weiterführung Lamarck- scher Gedanken, so geschieht es in gleichzeitiger scharfer Kritik einer bloßmechanischen Entwicklungslehre, die her und aus dem Ganzen leugnen muß. daß jene Lehre mit ihrer Verneinung gemacht, triebs alles wesentliche Fortschreiten Grunde im Entwicklungsgedanken dieses, alle Bildung von innen Es wird dagegen u. a. geltend alles inneren Fort- des Lebens und zugleich Nicht aufgebe.^ den minder auch daß jene Lehre nur dadurch zu einem leidlichen Abschluß daß gelange, sie schon den Elementen beilege, der Bildung sichtbar vorliegt. ^ was auf der Höhe Es stehen hier große Linien neben- Selektion kann überhaupt nichts Positives leisten, sondern nur negative, aus- schaltende Wirkungen entfalten. Die Entstehung neuer Arten durch minimale Abänderungen ist möglich, aber nicht erwiesen und, seit man den undulatorischen Charakter der minimalen Abänderungen kennt, weniger wahrscheinlich geworden die sprunghafte Abänderung ist jetzt in den Vordergrund getreten. Die Zufälligkeit weicht einer bestimmt gerichteten, planmäßigen Entwicklungstendenz aus inneren Ursachen, und diese bekundet sich ebensowohl in den Der Anspruch des Darkleinsten wie in den sprunghaften Abänderungen. ; winismus, zweckmäßige Resultate aus rein mechanischen Ursachen erklären zu können, ^ ist ganz unhaltbar." Bergson bemerkt (L'evol. creatrice, pag. 40) mecaniques est en effet : L'essence des explications de considerer l'avenir et le passe comme calculables en fonction du present et de pretendre ainsi que tout est donne. verficht die idee d'un elan originel de la vie, Er selbst passant d'une generation de germes ä la generation suivante de germes par l'intermediaire des organismes developpes qui forment entre les germes le trait d'union. Cet elan, se conservant sur les lignes d'evolution entre lesquelles il se partage, est la cause profonde des variations, du moins de Celles qui se transmittent regulierement, En general, quand des commence ä diverger ä partir d'une souche commune, elles ac- qui s'additionnent, qui creent des especes nouvelles. especes ont centuent leur divergence ä mesure qu'elles progressent dans leur evolution. Pourtant, sur des points definis, elles pourront et devront meme evoluer identiquement si l'on accepte l'hypothese d'un elan commun. ' S. Oliver Lodge, fach das zu Erklärende Leben und Materie S. 47: „Man konstatiert hier einund schiebt es dann den Atomen zu, in der Hoff- Entwicklung. 209 und der Kampf schwankt noch hin und einander, dünkt heute die Lage minder als begeisterten einfach, als sie her, jedenfalls weniger Darwin selbst Jüngern erschien. merkwürdig gfenug, gewinnt dieselbe Selektionslehre, Aber, die dem Gebiete ihres Ursprungs mehr und mehr kritisch behandelt auf und eingeschränkt wird, darüber hinaus, in der allgemeinen trachtung menschlicher Dinge, noch immer an Boden. auf überall weitverbreitet, ist zugreifen, die den möglichst einfache Be- Die Neigung Anfänge zurück- Menschen dem Tiere nahe verwandt zeigen, und Bewegung aufwärts nicht auf einen inneren Trieb, sondern auf die Notwendig- ein allmähliches Weitergestoßenwerden durch die äußeren keiten zurückzuführen, gebungen und sie als eine Lebensbedingungen bloße Anpassung an zu Um- die anderes Nichts verstehen. scheint dabei in Frage zu stehen als das natürliche Dasein, der Sieg im Kampf gegen die Mitbewerber. Das vermeintlich Höhere bringt dann nichts wesentlich Neues, sondern nur Kombinationen und Variationen der elementaren Lebenserscheinungen; damit daß dem Geistesleben Die Wandlung der Begriffe, die damit gebührt. ist auch gesagt, keinerlei Selbstständigkeit gegenüber der Natur erfolgt, reicht tief in wo alle Lebensentfaltung im Kampf ums Dasein zurückkommt, wo alle die Gestaltung der einzelnen Gebiete hinein; Erhaltung auf die geistige Betätigung ein bloßer Anhang des physischen Daseins wird, da wird zum Wert eines an sich Wahre nur der Werte das Nützliche, Guten zu einer leeren in dem Sinne einer Illusion, da sinkt der Begriff da der Lebenserhaltung Gruppierung der Vorstellungen bestehen bleiben. auch kann das förderlichen Ethik, Ästhetik, Erkenntnislehre müßten sich damit völlig verwandeln, sie alle müßten Lösung ihrer Probleme von den\ Ergreifen und Festhalten der die ersten Anfänge erwarten. Das Ganze wirkt mit der frischen Kraft einer eben errungenen Einsicht, es läßt in alten zerstreute seiner Daten in Erfahrungen Neues sehen, es bringt sonst einen aufhellenden Zurückwendung des Blickes Zusammenhang, es mancher Entdeckungen ist mit fähig. Die Naturbedingungen unseres Daseins, das Fortwirken elementarer daß damit dem Fragen ein Ende gemacht sein wird. Bergson findet den Fehler des Evolutionismus Spencers darin: »ä decouper la realite actuelle, dejä evoluee, en petits morceaux non moins evolues, puis ä la recomposer nung avec ces fragments, et ä se donner ainsi, par avance, tout ce qu'il s'agit d'expliquer." Eucken, Grundbegriffe. 4. Aufl. 14 Zum Wcltproblem. 210 Triebe inmitten und Langsamkeit die Kultur, aller Verwicklung und scheinbaren Vornehmheit der der Schwerfälligkeit geschichtlichen kommen nunmehr zur vollen Geltung; das Bild unseres Daseins scheint mit dem allen mehr Naturfarbe und LebensBewegung, sie wahrheit zu erreichen, zugleich aber gewinnt das Wirken zur Hebung der menschlichen Lage festere Angriffspunkte. Aber das alles ist in ein größeres Ganzes aufzunehmen und aus seinen Zusammenhängen zu würdigen, um Vernunft zu dienen ; rein der Wahrheit und und sucht es bei sich selbst den Abschluß will es aus eignem Vermögen die Gedankenwelt gestalten, so sind schwere nicht zu vermeiden. Sie kommen zurück, die besondere Art, in Irrungen Fehler der namentlich beim sich auf den iMenschen Geistesleben und Vernunft entwickelt, als den schaffenden und treiben- den Grund des Geisteslebens selbst zu behandeln; wird aber dieses so von vornherein zu einer bloßen Erscheinung gesetzt und aller Selbständigkeit beraubt, von der bloßen Natur keine Mühe kreise dieser so bereiten. am Menschen herab- kann seine Ableitung Wer nicht im Bann- Denkweise steht, wird alsbald den Zirkel in jener Er- klärung gewahren und die dort vollzogene Umwandlung des Geistes- Vernichtung lebens als eine erkennen. schließlich 'das Geistesleben selbst Die geistigen Liebe, Treue und Ein Gutes unter das Nützliche nicht sowohl verändert als zerstört Recht, Ehre, Werte werden durch jene Unterordnung — das wegen , seiner — Nützlichkeit, d. h. als ein bloßes Mittel für die physische und soziale Lebenserhaltung erstrebt wird, wird Charakter des Guten. damit innerlich verwandlelt und verliert den Ebenso müßte es dem Begriff der Wahrheit wenn er zu einer bloß zweckdienlichen Anordnung unserer ei gehen, Vorstellungen sänke; Wahrheit ist er mag dann alles mögliche andere er nicht mehr. werden, Einer solchen Erniedrigung des Lebens widersteht aber das innere Erlebnis, das Gewisseste von allem, was wir Denn mag über die nähere Fassung des Guten und Wahren viel Streit sein, und mag der Einzelne noch so wenig teil an jenen Größen haben, als bloße Lebensmöglichkeiten sind sie kennen. noch so Tatsachen, die sich schlechterdings nicht wegerklären lassen, aus dem Ganzen der Wirklichkeit etwas anderes machen. kommt hier in die Rede nisches Frage, sein kann. Getriebe und die Schließlich ob überhaupt noch von einem Geistesleben Wird unser ganzes Seelenleben elementarer Kräfte verwandelt, so in ein gibt mechaes kein Leben aus dem Ganzen, kein Denken, kein erlebendes Subjekt, so Entwicklung. müßte der Urteilende auch und alle geistige und nicht tut zum Verschwinden selbst Arbeit für ein Wahngebilde erklären. Verneinung selbst, bringen Solange er das Form der Aussage ihren nicht tun kann, widerlegt die bestätigt die Inhalt, sich 211 die als wissenschaftliche und allgemeingültige Wahrheit vorgetragene Verneinung, das Wirken eines dem Naturprozeß überlegenen Geisteslebens. Zu diesem Widerspruch, durch die geistige Arbeit ihre eignen Grundbedingungen aufzuheben, gebung aller bloße Natur genauer selbständigen betrachtet, unseres Lebens. wie der Menschheit, Verzweigung, sie und die Denn, auftritt. Aufbau der Kultur mit seiner die physische Existenz zu Dasei;!, Befreiung keine andere Aufgabe, hätten der daß jene Preis- Wendung allen Sinn und Wert Mühe und Arbeit des Menschen jene unsägliche aller Verwicklungen allem, und jene Bindung an Geistigkeit zerstört Alle sich vor ist Erhöhung eine als gesellen Wunderlich näheren Ausführung. erhalten, das als auf einem reichen sinnliche ungeheuren Umwege zu leisten, was das Tier so viel leichter und einfacher erreicht.^ Allts, was der physischen Existenz gegenüber einen Selbst- zweck und einen Selbstwert behauptet, müßte als unhaltbar verschwinden, irgendwelchen Nun Inhalt könnte ein aber sind wir einmal denkende solches Leben nicht und Wesen, urteilende bieten. nun haben wir den Mittelpunkt eines Selbst und müssen darauf alle Erfahrung beziehen und jene Inhaltslosigkeit sie als von da aus messen. So werden wir denn schmerzliche empfinden, eine Leere eine um so unerträglicher wird, als diese Zusammenhänge nicht Leere, die die mindeste Hoffnung einer Wandlung gewähren, uns vielmehr das Getriebe des Naturprozesses sinnlose es trostlosere Lebensgestaltung eine unablässiger Arbeit ohne allen Aufbietung aller inneren Es sei hier an erinnert (V, 65 Hart.): aus soll, der was festhält. Gibt mit ihrer Forderung Gewinn, ihrer fieberhaften bei Tieren vermeintliche Seite gerät diese Denkweise das Wort Kants aus der Kritik der prakt. Vernunft »Über die bloße Tierheit erhebt ihn (den iMenschen) das gar nicht, daß er Vernunft hat, dienen diese Kräfte zur Erringung des nackten Daseins? Auch nach der methodologischen ^ unbarmherzig als wenn sie ihm nur zum Behuf desjenigen Ja es müßte von hier der Instinkt verrichtet." Fortschritt in Wahrheit als ein Rückschritt gelten. Denn ist es nicht ein Rückschritt, wenn für die Erreichung desselben Zieles immer kompliziertere Mittel, immer mehr Mühe und Arbeit aufzubieten sind? Mit der Anerkennung neuer Gehalte und Werte aber wird die mechanische Evolutionslehre verlassen. 14* Zum Weltproblem. 212 in arge Verwicklungen, sobald Wir sehen dann zieht. Ästhetik u. s. zurückstreben Fehler, und Nun suchen. eine w. entstehen, evolutionistische die gewiß beging die begonnen Anfängen Anschauungsweise ältere den Daß das Geistesleben nicht vom sondern von geringen, halbtierischen Anfängen ist, daran hat, Rechtslehre, Ethik, tierischen Anfänge hineinzusehen und diese die in damit fälschlich zu idealisieren. Himmel gefallen den zu alle ihnen den Schlüssel aller weiteren Bildung in höhere Stufe die auch das Geistesleben an sich sie kaum mehr zu heute ist Aber zweifeln. "müssen jene Anfänge maßgebend für die Gesamtbewegung bleiben, könnte nicht der Lebensprozeß sich bei sich selbst erhöhen, könnten neue Kräfte nicht die Bindung an ihm hervorbrechen? in die Sind ferner die ersten Anfänge so einfach leugnet als sie dieselbe und klar, daß von ihnen Vermögen wir fiele? Licht hellstes sie notwendig heute einnehmen? So führt jener es ist auf sonst dunkle Gebiete uns unmittelbar vor Augen zu gestalten wir nicht hinein; Wahrheit bekräftigt In Anfänge die Entwicklung nicht sowohl, ersten wir Weg uns erst recht ins Dunkel Weg, sondern nicht ein gerader stellen, dem Stande, den nach ihr Bild ein Umweg, wenn wir von hypothetisch ausgedachten Anfängen aus höhere Stufen zu erklären suchen.^ Das alles wendet sich gegen und die mechanische Art der Entwicklungslehre, sofern sie nach ihren Leben gestaltet. naturalistische Maßen das ganze Aber auch der Gesamtgedanke der Entwicklung, wie er die Neuzeit durchdringt, enthält mehr Probleme, wärtig zu sein pflegt. * Zunächst wird viel zu leicht, als gegen- wo irgend Be- Neuerdings hat dies für das Gebiet der Ästhetik ebenso scharfsinnig in der Abhandlung «Die entwickelungs- wie überzeugend Volkelt dargelegt geschichtliche Betrachtungsweise in der Ästhetik" (Zeitschrift für Psychologie und Physiologie der Sinnesorgane, Bd. 29). Dort heißt es (Abdruck S. 7): »Es gilt zu bedenken, daß sich für die Beantwortung der Frage, was es heiße, sich zu den Dichtungen dichterisch, künstlerisch, ästhetisch verhalten, nur vom Standpunkte des gereiften gegenwärtigen Menschen aus eine sichere — Grundlage gewinnen läßt.« S. 8: »In Wahrheit ist um diesen kurzen Ausdruck zu gebrauchen die Ästhetik der Naturvölker kein methodisches — Mittel, sondern vielmehr eine der Sonderaufgaben der Gesamtästhetik." lungsgeschichtlicher Grundlage" Verhältnisses." ist allerdunkelsten S. 11: demnach und unzugänglichsten „Eine „Ästhetik auf entwickeine Umkehrung des richtigen Entwicklung. wegung vorliegt, ein 213 Fortschreiten, eine Entwicklung im Sinne eines Daß unsere Welt, namentlich der Bereich unseres Handelns, voller Bewegung sei, das stand unablässigen Aufsteigens angenommen. auch den Alten deutlich vor Augen; ihnen niedere Stufe, weil sie Vordringen sicheres enthielt als ein die Religion als eine Die moderne Überzeugung dagegen Hauptstück den Glauben an ein solches Aufsteigen; ihn hat weiter gestützt aber diese nur ein wirres Durcheinander, kein ihr in sahen. galt und aufgebracht, die Philosophie ihn spekulative und Spekulation sind Religion durcTigebildet. heute verblaßt und vielen gänzlich entschwunden, ihr Erzeugnis aber, der Fortschrittsglaube, ist verblieben; ist er, nach Wegfall jener Grundlagen, noch genügend fundiert, gibt ihn die bloße Erfahrung als eine unumstößliche Tatsache, kann sie mit ihrer Begrenztheit über- haupt einen unablässigen Fortschritt erweisen? im Es viel subjektive- Stimmung alle Veränderung als einen Fortschritt zu deuten. Lauf des Lebens an bringt, liegt und Sicherlich ist selbst Streben als alles den hier dem Menschen nahe, Er sieht, was der was zu- vergißt darüber, an Altem verloren geht; von da aus fühlt jede Zeit gleich sich Neuem Spiel. Höhepunkt des Ganzen, weil Übrige mißt; eine künstlerische Zeit sie am pflegt leicht eignen nach der Kunst, eine technische nach der technischen Leistung zu schätzen. Zu diesen dauernden Ursachen kommen zeitliche: nichts ist günstiger für den Fortschrittsglauben gefühl, wie es aufsteigende Zeiten durchdringt, wie es namentlich den Hauptzug der Neuzeit ergriffen, als ein starkes Kraft- Von erfüllt. und Gegenwarts- hier aus wird alles freudig was eine Mehrung des Lebens verspricht, dahingehende Erfahrungen einzelner Gebiete werden verallgemeinert. Vereinzeltes und Zerstreutes wird ergänzt und verbunden. Hemmendes dagegen übersehen oder zurückgestellt, selbst der Widerstand in durch den umgewandelt. inneren Lebenstrieb als ein Antrieb dem allen die bloße Erfahrung zu weiterer Tätigkeit verstanden, Einer solchen Be- trachtung und Behandlung des menschlichen Daseins droht schließlich notwendig ein Rückschlag, eine kühlere und art wird manches Stück jenes Fortschrittsglaubens zerstören, retardierende Momente vor Augen stellen, kritischere wird manches Denk- als wird eine vorübergehende Erscheinung erkennen lassen, was jener Glaube. zu einem bleibenden Gesetz erhob. So ging z. B. durch die letzten Jahrhunderte die Lehre von Bevölkerungszahl, einem ihr Stillstand bei unablässigen Anschwellen einzelnen Nationen der wurde wie Zum Weltproblem. 214 Wie jung aber Noch Montesquieu meinte, die Bevölkerung Europas habe gegen das Altertum abgenommen, und es empfehle sich, die eine ist merkwürdige Ausnahme behandelt und erörtert. diese Lehre! Vermehrung des Menschengeschlechts durch besondere Gesetze zu Dann siegte die entgegengesetzte Annahme, und die Gefahren einer übergroßen Vermehrung kamen bei Malthus zu starkem fördern. Die Zahlen Ausdruck. gaben einstweilen Annahme dieser recht, immer mehr Anzeichen, daß auf einer Höhe der Kultur die Zunahme langsamer wird und zum Stillstande, ja Rückgange kommt; das macht die Frage unabweisbar, aber neuerdings erscheinen gewissen ob sie nur für besondere Lagen vielleicht der Kultur, nicht aber Wie sehr aber muß die Verfolgung dieses Gedankens dauernd gelte. den Gesamtanblick der Geschichte verändern! Ferner greift das Problem über hinaus das Quantitative ins Wachstum der geistigen Minder des Vermögens? sie die Summe Menschheit, erhöht zuversichtlich, als die durchschnittliche Meinung darüber denkt, stimmt Qualitative. Bringt die Geschichte ein geistiges der von Lorenz nachdrücklich verfochtene Antagonismus zwischen geistiger Leistung und Tätigkeit eine geringere nach würde sich Fortpflanzungsfähigkeit — es und stärkere geistige schließt« sich in (Lehrbuch der Genealogie" S. 486/7), und meint, lichkeit nennt Lorenz Fortpflanzungsfähigkeit. «eine sehr beachtenswerte Tatsache, daß höhere »aller Wahrschein- eine Erfahrung, die man anderweitig beobachtet hat, auch genealogisch bestätigen lassen, daß der männliche Keim eine Wanderung von unten nach oben den oberen Ständen, oder wie man nach heutiger Organisation sagen könnte, in und Kulturvölker Überwältigungen, sondern vielmehr stellt nicht als sich als und in gesellschaftlicher den höheren Berufen Weiterführung dieses Gedankenganges höherer Kulturen vollzieht abstirbt." der eine ,; In Untergang Folge äußerer die natürliche Abnahme der Fortpflanzungspotenzen des höheren, kultivierten Individuums" dar; es erscheint ein «Unvermögen der Natur, das Geistige — um diesen gebrauchen — schlecht- Ausdruck nur im Sinne der hin fortzupflanzen" (S. 487). Kausalität zu So würde die Bewegung sich bei sich selbst erschöpfen, die Kulturen müßten sich ausleben und greisenhaft werden, eine Stagnation erfolgen, bis vor allem aber frische Menschen dann aus einem unablässigen Aufsteigen verschiedener Phasen verwandeln. wieder neue Anregungen, kommen; das Ganze würde sich in ein Auf- und Abwogen Was es dabei etwa an Fortschritt Entwicklung. 215 gäbe, das würde sich jedenfalls anders ausnehmen als im gewöhnlichen Fortschrittsglauben. eine Auch das gehört hierher, daß die verschiedenen Lebensgebiete verschiedene Art der Bewegung zeigen, und daß das Über- wiegen eines dieser Gebiete die ihm innewohnende Schätzung zur erheben Allgemeingültigkeit zu es Einen unaufhödichen Fort- pflegt. am meisten Technik und exakte Wissenschaft, obschon schritt zeigen auch hier an Verlusten und Rückgängen keineswegs fehlt; geistiges Schaffen im Sinne einer inneren findet eine volle Erhöhung des menschlichen Lebens Verkörperung nur an einzelnen ausgezeichneten Punkten, um dann rasch wieder abzunehmen; in scheint die Menschheit sowohl im Guten als im Bösen, wie Gegenwirkung in moralischer Hinsicht der Gegensatz fortzuschreiten, schroffer zu werden; die Religion endlich gibt als allem zeitlichen Wahrheit Wandel überlegen, sie ihre in Wirkung immer also Grundwahrheit betrachtet leicht diese an irgendwelchem früheren Zeitpunkt schon erreicht als und -kettet damit das Streben an die Vergangenheit. Jede dieser Arten hat aber die Neigung, von sich aus ein allumfassendes Ge- und Weltbild zu entwerfen. schichtsschritts voller Verwicklung, gemeingültig auftritt, Lage vorübergehender Endlich ist oft So ist das Problem des Fort- und was als selbstverständlich und allnur Erzeugnis das besonderen einer Art. muß die Entwicklung auch in der Richtung Bedenken erwecken, daß sie leicht dazu führt, die Bewegung ausschließlich als ein Werk ein zu kontemplatives und der Notwendigkeit zu verstehen Der bringen. Fortschritt passives scheint und den Menschen in Umgebung zu mehr an dem Menschen als Verhältnis hier zur durch ihn zu erfolgen, er scheint nicht eignen Eintretens und eigner Entscheidung zu bedürfen. So geschah es z. B. in der Entwicklungs- idee der Romantik, die ein stilles heraus alle und Gestaltung hervorbringen sicheres Wachsen ließ zu eigner Tätigkeit lähmte; so kann es auch geschehen, wegende Kraft in sinnliche Naturtriebe von innen und damit den Antrieb wo die be- und äußere Notwendigkeiten gesetzt wird. Hier wie da gefährdet die Entwicklung den ethischen Charakter des Lebens und zerstört sie die Grundbedingung einer echten Geschichte: ein immer neues Einsetzen ursprünglichen Lebens, ein Verwandeln Gegenwart. alles Empfangenen in eigne Tat und lebendige Während das menschliche Geistesleben seine Spannung durch den Zusammenstoß von und seinen Charakter vornehmlich Zum Weltproblem. 216 und Schicksal Freiheit opfert erhält, eine dem Schicksal auf. die Freiheit gänzlich das solche Probleme übersehen ein Durcheinander- Entwicklung wird läßt. Bewegung genannt, ohne daß dabei der Ur- oft alle fortschreitende sache des Fortschritts nachgefragt wird; ein ist und einer strengeren Fassung des Entwicklungs- laufen einer laxeren begriffes, Entwicklungslehre solche Es so könnte einen Naturprozeß, der aus zwingender Not- zeichnet Entwicklung wendigkeit vorwärts treibt — sei es durch ein Zusammenschießen — einzelner Elemente, sei es durch eine Bewegung des Ganzen dann und zugleich auch entfällt alle Freiheit zeichnend wohl hier ganz Im strengeren Sinne dagegen be- Platz für Freiheit verbleiben. menschlichen Alsdann Sinn. alle nur etwas geht , als- Geschichte im ausvor, es ist ge- wird aber nicht gehandelt; bei dieser Bedeutung des Wortes Unding. schichtliche Entwicklung ein Ja der Zweifel greift noch tiefer, er kehrt sich überhaupt gegen die Vorherrschaft der Bewegung, gegen die Verwandlung der ganzen Wirklichkeit in einen Prozeß. dem Flüssigwerden hältnisse, An dem Beweglichmachen aller Veraller starren Größen sah die Neuzeit zunächst nur den Gewinn: die Steigerung des Lebens, das Wachs- tum an Freiheit bleiben, daß auch und Schließlich aber kann nicht verborgen Kraft. vieles damit verloren geht, etwas verloren geht, ohne das geistiges Leben schlechterdings in Grundformen elementarsten seine nicht bestehen hinein verlangt kann. und Bis erweist nämlich das Geistesleben eine beharrende Art, ein Beharren nicht innerhalb der Zeit, sondern heute oder morgen ist ein gegenüber der Zeit. Ein Wahres für Unding; was irgend wahr ist, das für alle Zeit oder vielmehr ohne alle Beziehung zur Zeit; gilt mag die Behauptung unter besonderen Umständen nur auf eine Zeitspanne ist immer zeitloser Art, Wahre eine Befreiung von aller gehen, die Art, wie sie ausgesprochen wird, als geistiges Erlebnis enthält alles Auch was wir als gut erachten und schätzen, das hat seinen Wert nicht aus dem Gesichtspunkt einer besonderen Zeitlage, sondern Zeit unabhängig von So gewiß aller Zeit, aus einer zeitlosen die Begriffe der Zeiten vom Guten Ordnung der Dinge. sich ändern: Zeit als gut ergreift, das erklärt sie damit für schlechthin gültig. Aller Wandel menschlicher Verhältnisse was eine und dauernd vermag solche innere Überlegenheit des Geisteslebens über die Zeit nicht zu zerstören. Auch Begriffe wie Persönlichkeit, Charakter, geistige Individualität Entwicklung. bekunden eine Denn sie fordern die Gestaltung treues Festhalten des Überlegenheit gegenüber 217 beharrenden Art Bewegung; aller gegen Geisteslebens einer die Zeit. und ihr Mannigfaltigkeit alle des Handelns hat jene Art zum Ausdruck zu bringen und zu fördern. heißt, es von Grund aus zerstören. Bewegung selbst Ja die Beharrens. entbehrlichkeit des Ganzes zusammenfassen, Sie Sonst nämlich mögen die Seele wohl erfüllen und zerfällt sie Je sich als ein nämlich nicht über- Ganzes erleben, ohne in lauter einzelne Punkte; diese mit kaleidoskopisch wechselnden Eindrücken ergötzen, ein ihr nicht. läßt und ohne eine von daher bewirkte einen ihr überlegenen Standort Synthese. Un- bezeugt, innerlich angesehen, die blicken, in ein sie Bewegung verwandeln, das Geistesleben ganz und gar in So Ganzes und zugleich einen Inhalt geben Bewegung überlegene Kraft mehr drängt das Leben zur Oberfläche und mehr daher verschwindet, desto eine der Beisichselbstsein. verliert alles Diese zeitüberlegene Art des Geisteslebens erweist besonders deutlich der Aufbau einer Geschichte, d. h. einer eigentümlich menschlichen und Sinne ist Denn Geschichte im menschlichen geistigen Geschichte. bloßes Nacheinander von Ereignissen, ein keineswegs ein Dahintreiben des Menschen dem Strom mit der Zeit; das würde nie über die äußere Anhäufung von Wirkungen hinausführen, wie die Natur, der Bildung der Erdrinde, ist alle z. B. in sie zeigt. Vielmehr Geschichte menschlicher Art eine Gegenwirkung gegen die Flucht der Erscheinungen, ein Versuch, den Strom irgend zum Stehen Auch die primitivsten zu bringen, ein Kampf gegen die bloße Zeit. Versuche, Vorgänge und Taten überliefern, sie dem Gedächtnis der Nachwelt zu im Bewußtsein der Menschheil festzuhalten, zeigen einen solchen Widerstand gegen die Zeit; je mehr aber die Geschichte dem Menschen wird, je mehr seines Wissens, sondern eine desto mehr Selbsttätigkeit ist sie ihm nicht nur eine Erweiterung Erhöhung seines Lebens bringen soll, dabei aufzubieten; das aber verlangt notwendig einen zeitüberlegenen Standort. Um die Vergangenheit innerlich mitzuerleben, müssen wir uns von der Zufälligkeit der Gegenwart befreien, wenigstens nach solcher Befreiung streben; sonst würden wir in alles Frühere lediglich die heutige Art hineinsehen und in aller äußeren Erweiterung innerlich bei uns selbst verbleiben; ein Verständnis anderer Epochen aus ihren eignen Zusammenhängen wäre uns gänzlich versagt. Dazu möchten wir die Vergangenheit Zum Weltproblem. 218 zum eignen Leben in Beziehung setzen, ihren Reichtum in eignen Besitz verwandeln, an dem Großen sondern nicht bloß erkennen, in ihr sie Dafür aber gilt es nicht nur eine Gemein- uns selber heben. Epochen zu gewinnen, sondern auch an ihrem schaft mit früheren Bestände Wesentliches und Zufälliges, Wertvolles und Gleichgültiges zu scheiden; sollte das möglich sein ohne irgendwelche dem Wandel der Zeiten überlegenen Maßstäbe, ohne eine Versetzung der Arbeit auf einen zeitlosen Standort? für uns sofern Wert, zusetzen vermögen; das Letzthin wir als in sie die Geschichte hat eine zeitlose ihr Hauptertrag, ist nur in- Gegenwart um- uns aus der engen und armen Gegenwart des bloßen Augenblicks zu einer weiteren, überlegenen und zeitumspannenden Gegenwart zu führen. zeit- Es gibt keinen gefährlicheren Gegner einer echten Gegenwart als die Hin- gebung an den bloßen Augenblick. Bei solcher Lage der Dinge verbietet sich schlechterdings die Auslieferung des ganzen Lebens an die Bewegung; gewicht in extremsten So haben auch irgendwelchem Bleibenden gesucht. die Vorkämpfer der naturwissenschaftlichen Bewegungslehre irgendwelche Ergänzung der Bewegung anerkannt. der Lehre mag das Bewußt- von dieser erfüllt sein, die Arbeit hat stets ein Gegen- sein lediglich vom Beharren Das sowohl des Stoffes oder der Energie als in in der Unterordnung aller Erscheinungen unter unwandelbare Gesetze. Ohne solche Befestigung hätte ihre Arbeit den Charakter der Wissenschaft vedoren und wäre statt kausaler Begreifung eine bunte Erzählung geworden. Auch die Philosophen haben die Entwicklung nicht zur Zentralidee ihrer Gedankenwelt rpachen können, ohne ein der Veränderung überlegenes, ja umspannendes Beharren anzuerkennen. sie Hegel wäre sein System in lauter einzelne Punkte zerbrochen, und der Wechsel der einzelnen Phasen hätte ihm hätte ihm nicht eine zeitüberlegene Einem alle Wahrheit zerstört, Betrachtung eine Zusammen- fassung zu einem Ganzen geboten, alles Nacheinander in ein Selbst- Ganzen verwandelt und zugleich über den zeitlichen Ablauf hinaus in eine zeitlose Gegenwart gehoben. Ob das erstrebte leben Ziel dieses bei Hegel vollauf erreicht dem Streben aber ist ist, das ist nicht zu zweifeln, alle eine andere Frage, an Größe des Hegeischen Systems hängt eng mit ihm zusammen. Auch bei Comte, dem großen steht es ähnlich: zu realistischen Gegenstück Hegels, einem wissenschaftlichen System gelangt er nur Entwicklung. 219 Wohl durch Ausbildung und Voranstellung beharrender Elemente. bringt er alle bisherige Geschichte in Fluß und gewährt den früheren nur Stufen eine relative Aber Wahrheit. Positivismus scheint die absolute Wendung zum der in und endgültige Wahrheit erreicht; die Zukunft mag diese weiter entfalten, der Kern scheint unwandel- bar für alle Zeiten schichte rückwärts erfolgt gesichert. Auch die Durchleuchtung von diesem gänzlich als der Geerachteten fest So wird inmitten aller Bewegung eine bleibende Wahr- Höhepunkte. heit festgehalten. Für das gemeinsame Leben war freilich mit solcher versteckten Anerkennung eines Beharrens wenig gewonnen, der fortschreitenden Verwandlung des modernen Lebens in einen bloßen Prozeß wurde von da aus kein Gewicht ins fiel genügender Widerstand das tatsächliche Weit geleistet. stärker beharrender Größen Fortwirken und Mächte aus der älteren Lebensführung. In ihnen, die tief in den Bestand des Daseins eingebildet waren und den Menschen wie umfingen, seltjstverständlich hat die Bewegung stillschweigend bald Aber eine solche Lage einen Halt, bald eine Ergänzung gefunden. mit Unausgeglichenheit ihrer nicht auf die entgegengesetzter Strömungen Dauer verbleiben, im Vordringen befindet unverkennbar die Bewegung, so wird sie kann aber sich mehr und mehr das Feld einnehmen, ihre Konsequenzen hervortreiben, Feste alles auflösen, das ganze Leben in einen ruhelosen Prozeß verwandeln. Zugleich aber werden auch die Folgen eintreten, die das Ver- schwinden beharrenden Größen aller vornehmlich der Wegfall lebens aus aller dem Ganzen, selbständigen Geistigkeit, zugleich aber das Sinken einen völligen Sieg sowohl in mit sich Kräfte bringt, die Verkümmerung aller inneren Er- der Arbeit zur Der Triumph der bloßen Bewegung bedeutet höhung des Daseins. eine Preisgebung und inneren Zusammenfassung, alles Er- alles des Relativismus als Lebensinhalts, eine des Sensualismus, Auflösung des Daseins einzelne Augenblicke, einen Verzicht auf alle wahrhaftige Gegen- wart, Auch muß sich damit die Menschheit in lauter einzelne Lebens- kreise zersplittern und eine gemeinsame Gedankenwelt der und erhöhender Art mehr und mehr befestigen- verlieren. Läßt sich leugnen, daß der Anblick der Gegenwart uns die zerstörende stellt, Kraft dieser Wendung schon deutlich genug vor Augen und daß die daraus erwachsenden Fragen und Zweifel die Grundlagen des modernen Lebens zurückgreifen? bis in Ja wir haben Zum Weltproblem. 220 ein bunteres Autorität und ein bewegteres Leben und keine Tradition, wir gewonnen, uns beengt keine können mit voller Frische jedem Tempo des Lebens Eindruck folgen, den Augenblick ergreifen, das Aber in beschleunigen. und Geschäftigkeit droht aller Beweglichkeit das Leben sich uns an die bloße Oberfläche zu verlegen und seiner nach immer leerer zu werden; seelischen Art uns entweicht eine innere Einheit des Wesens und damit der einzig mögliche Halt gegen- dem Strom der Dinge; unfähig, unsere Selbständigkeit an ihnen über zu erweisen, werden wir wehrlos von ihnen hin- und hergeworfen. Zugleich zerrinnt uns alle wahrhaftige Gegenwart, da Lebens des bloße Zeit sich in unvermeidlich und eine sie ein Ruhen Erhebung über die Dafür bekommen wir bloße Augenblicke, deren enthält. ^ bunter Wechsel verlangt selbst das Leben in eine rastlose Flucht verwandelt und dem Streben die Richtung auf das unmittelbar Wirk- same, das Sinnfällige, das äußerlich Vorteilhafte gibt. Als notwendige Folge dessen jenes Haschen nach immer Neuem, Blendendem, Auf- regendem, jenes Spähen nach Sensation, Effekt dienerei gegen die u. s. w., jene Liebe- Launen und Stimmungen des Massenpublikums, dieses geringen Durchschnitts der Menschheit, jene unwürdige «Aktua- die den schönen Begriff des Aristoteles in sein volles Gegen- lität«, teil verkehrt hat!^ Der Zeit unserer Klassiker war das mit voller Deutlichkeit gegenwärtig. * Es sei nur an jenes Wort Goethes (aus den Gesprächen mit Eckermann) erinnert: „Jeder Zustand, ja jeder Augenblick er ist ist von unendlichem Wert, denn der Repräsentant der ganzen Ewigkeit"; auch des Wortes eines neueren feinsinnigen Geistes (W. Gidionsen) sei gedacht: »Nicht vom Tage sollst du leben, Auf und nieder schwankt die Welle Laß dein Inn'res fröhlich weben. — Stets verjüngten Daseins Quelle. Ist Ursprünglichkeit dir eigen, Darfst sie hegen, darfst sie zeigen. So nur spürst du in der Zeit Vorgefühl der Ewigkeit." ^ Der Ausdruck actualis ist eine Schöpfung des späteren Altertums (Augustin, Macrobius), im Mittelalter gewannen, von griechisch-lateinischen Übersetzungen des Aristoteles her, actus, actualis, actualitas - namentlich seit Duns Scotus — eine weite Verbreitung und gelangten von dort zur Neuzeit. Das Wort diente zur Wiedergabe des aristotelischen Begriffes der Energie oder Entelechie, der in sich selbst ruhenden und bei sich selbst befriedigten Tätigkeit im Gegensatz zu der noch unfertigen, erst anstrebenden Bewegung. Entwicklung. mehr uns aber damit Je die 221 Gegenwart entschwindet, desto lebhafter wird ein Sehnen unter den Händen eine unbestimmte in Zukunft, ein Erhaschen und Vorausnehmen dessen, was dort erwartet »Nie wird. als es die wie jetzt beeifert ist", so sagt Lotze Gegenwart in einer noch weit ruhigeren Zeit (Mikrokosmus 2. Aufl. II, 281), «so lebhaft der Widerspruch aufgetreten, das ganze Leben, das man ist und emsig mitlebt, doch im Grunde das wahre für nicht zu halten und von einem anderen schöneren zu träumen, das man leben möchte und leben wird, sobald uns jenes Zeit lassen und einen Zugang zu ihm öffnen wird." So zerfällt uns in einer Überspannung und Überstürzung der Bewegung das Leben von innen her, aus einem wahrhaftigen Leben wird es mehr und mehr ein bloßes Lebenwollen, eine Anweisung auf Leben, ja ein Schein des Lebens. Das kann unmöglich so weiter gehen, jener Verwandlung des Daseins in bloße Bewegung muß als einer Zerstörung völligen widerstanden so gewiß das Verlangen keit ihrer innersten Die Menschheit werden. muß jene gefährliche Krise überwinden und wird sie übei'winden, danach aus einer zwingenden Notwendig- Natur hervorgeht. Aber sie wird winden ohne eingreifende Wandlung des Daseins, sie nicht über- ohne nicht die Ausbildung eines neuen Lebenstypus, nicht ohne den Mut und die Kraft zu einem neuen geistigen Aufschwung. d) Forderungen für einen neuen Lebenstypus. So wenig das Problem, hier näher erörtern läßt,^ Richtung des in das unsere Untersuchung auslief, sich ohne irgendwelche Orientierung über die Weges würde unsere Betrachtung einzuschlagenden aller Kürze ins Leere zu verlaufen scheinen; so seien in einige Umrisse entworfen. — Vor allem gilt wenigstens jener drohenden es, Verflüchtigung des Lebens einen festen Halt^ entgegenzusetzen. solchen kann nicht Außenwelt die mittels unserer Seele erleben uns beweglich werden Bewegung gehörte. ^ und in da wir und daher auch das müßte, Denn hier Einen immer sie Festeste wenn das Seelenleben Eine Festigkeit aber gewährt unmittelbare Seelenleben. einander, bieten, gänzlich auch erst draußen nicht der das wogt Mannigfachstes durch- buntem Wirbel verdrängt die eine Erscheinung Es sei dafür auf meine «Grundlinien einer neuen Lebensanschauung" (1907) verwiesen. Zum Weltproblem. 222 die zu Es bleibt also nur die Hoffnung, andere. geistigen Tätigkeit vorzudringen, auch das übrige Leben die, sich fest in irgendwelcher gegründet, selbst Das haben große zu befestigen verspräche. Denker der Neuzeit in verschiedener Weise versucht: den archimedischen Punkt suchte Descartes im reinen Denken, Kant im sittlichen Handeln; beider Unternehmen aber wurzelt Bewegungen in weiteren des modernen Lebens, indem einerseits die wissenschaftliche Arbeit, andererseits ein ethisches Schaffen dem rneijschlichen Dasein einen festen Grund zu geben und seiner Verflüchtigung in bloße Erscheinungen entgegenzuwirken bemüht war. geleistet Beide Bewegungen haben Großes und fahren fort das zu tun; trotzdem wächst der Zweifel, ob sie den tiefsten Punkt erreichen und von hier aus das ganze Leben zu umfassen vermögen. Einmal nämlich treiben eine besondere Richtung und geben ihm intellektual istische, dort eine Färbung. Für unser moralistische, eine hier das Leben in sie eine besondere, Problem aber fällt noch mehr ins Gewicht, daß die Festlegung eines besonderen Punktes immer wieder von anderen Punkten zweifelt und bestritten werden kann; gegen dieses der das Handeln, gegen den Intellekt wenden, Intellekt her be- kann sich der Skeptizismus kann die Wissenschaft zu einem bloßen Vorstellungsgewebe herabzudrücken, Moral der Naturalismus die Naturtriebe zu verwandeln suchen. Die Gewißheit kann nicht besonderes Gebiet, ein Zusammenfassung zu einem Ganzen nicht eine der Erzeugnis ein in uns mögliche sondern nur eine höchste bieten; bloßer liegt im Geistesleben Verzweigung überlegene Einheit, und bricht nicht in dieser Einheit ein ursprüngliches Leben hervor, so kann unser Leben und Streben nie eine Festigkeit erlangen. Daß aber der Gedanke einer allumfassenden Einheit mehr als eine bloße Einbildung ist, das bezeugt die Bewegung zu einem Denn mag unser Persönlichsein, wie sie die Menschheit durchdringt. menschliches Persönlichsein noch so viel Bloßmenschliches an sich tragen und mannigfachsten Bedingungen und Einschränkungen unter- liegen, eine neue Art des Lebens, eine größere Tiefe der Wirklich- beginnt damit das Geistesleben erscheint hier nicht als eine besondere Betätigung, sondern als eine neue Art keit Wirklichkeit, sich als eine mit Handeln, sittliche Demnach entsteht neue Stufe des Seins, ihnen Betätigungen, das aufzuarbeiten; sowohl sich der die besonderen das wissenschaftliche Denken als unterzuordnen und einzufügen haben. eine Befestigung nur durch ein Vordringen des Entwicklung. 223 damit wird gesamten Lebens zu einer wesenbildenden Geistigkeit; auch der Kultur Theorie und Praxis überlegen dem Gegensatz von das vorgehalten, ideal ein und das jede von ihnen ist, eine in wesenhafte und wesenlose Stufe zerlegen muß. So rung des Daseins energische Aufrüttelung, eine gestattet lediglich Umkeh- ja Vordringen zu einem festen Punkte und ein ein Aufnehmen des Kampfes mit der Flucht der Zeit und der SinnlosigOhne ein Gegründetsein des Menschen keit der bloßen Bewegung. in einer dem nächsten Dasein überlegenen unmittelbar wäre die Sache völlig aus- gegenwärtigen Geisteswelt und sichtslos selbst das Streben und doch im Lebensprozeß danach nicht zu begreifen. Jene Zurückverlegung aber enthält die weitere Forderung, daß das Geistesleben nicht der Mensch sondern eine als als einem ihm überlegenen Geistesleben an teilhabend gelte, daß das Geistesleben dem Menschen ständig gegenüber geistiges als Leben und in seiner Substanz in anerkannt werde. als selb- Wenn damit Dasein weiter auseinandertreten menschliches der durchgehenden Menschen, Eigenschaft des bloßen Fassung, wird so zugleich Ver- eine und Bewegung und die .Ausbildung dem Gegensatz überlegenen Lebenstypus ermöglicht. Der Sub- ständigung zwischen Beharren eines stanz des Geistesleben die ist Veränderung und mit eine Ent- ihr — Der Begriff der Wahrheit dem Gegensatz des Theoretischen und des duldet kein Werden und keine Veränderung, wicklung schlechterdings fernzuhalten. auch dieser Begriff ist — Praktischen überlegen die Zugehörigkeit zu einer zeitlosen Ordnung der Zeit und ist für ihn unerläßlich. einen Lebensinhalt nur innerhalb durch allmähliche Erfahrung erringen; dazu aber be- Der Mensch hingegen kann Auch was c;r an Wahr- und der Beweglichkeit. darf er der Freiheit ihm nicht heit erreicht, sich des Besitzes ruhig ist sich ein für allemal erfreuen könnte, gewonnen, sondern es so daß er will immer neu gewonnen werden, es wird immer wieder zum Vorwurf des Kampfes. Auch in die Grundlagen unser geistigen Existenz Ungewißheit neuem immer von neuem zurück und So entstehen, immer von Überwindung. eine kräftige Lebens: die eine verlangt greift die deutlich ist geschieden, drei Arten und Typen des ausschließlich auf ein Beharren, ja einen ewigen Bestand gerichtet und sucht das menschliche Sein möglichst aller Bewegung zu entwinden; die andere ist gänzlich von der Bewegung erfüllt und will ihr nichts entzogen wissen; die dritte strebt über Zum Weltproblem. 224 den Gegensatz hinaus und möchte aus innerer Überlegenheit jeder zweite die Die gewähren. Recht ihr Seite erste moderne Gestaltung des beherrscht Lebens, die die antike, die dritte wirkt von Alters her innerhalb der geistigen Arbeit, aber prinzipiell anzuerkennen, sowie führen. Hier als ist sie erst Lebenstypus zu voller Kraft und Klarheit zu liegt die Aufgabe Die der Zukunft. alte Art war stark dem Geistesleben Festigkeit und Ruhe zu geben, es als eine unantastbare Ordnung über alles Mögen und Meinen der Einzelnen darin, wie der Massen hinauszuheben. sie veränderlich, sondern auch behandelte, die Ins Problematische aber Wahrheit nicht nur als dadurch, daß den Menschen als für geriet sie ihrer Substanz un- in fertig vorhanden daß sie Substanz und menschliche Existenzform in Eins So gilt der antiken Welt und mehr noch dem zusammenschob. Mittelalter die wissenschaftliche Wahrheit als endgültig abgeschlossen, so kennt auch das kirchliche Christentum keine Weiterbewegung der Damit wird aber der Besitzstand einer be- religiösen Gedankenwelt sonderen Zeit für immer festgelegt, alles Weiterstreben gehemmt, der Menschheit ein starres Joch auferlegt, das der Lauf der Zeiten immer drückender machen muß. Auch Wahrheit die selbst leidet Schaden, Wesen gesetzt Dagegen mußte ein Rückschlag kommen, die Bewegung erstritt indem Zufälliges der Zeiten und Menschen wird. ihr ins sich die Anerkennung ihres Rechts, der Mensch begann seine Schran- ken und die Bedingtheit seiner Leistungen zu empfinden, es begann modernen Lebens, dessen Größe, aber auch dessen jene Entwicklung Selbstverzehrung uns beschäftigt hat. Hatte die Beharrungslehre die menschliche Existenzform unmittelbar mit der Substanz des Geisteslebens zusammenrinnen umgekehrt Art; jenes das lassen, Geistesleben ergibt eine so den Erstarrung, unterwirft die Bedingungen dieses Bewegungslehre der menschlichen eine Verflüchtigung des Geisteslebens. An Versuchen zu Kompromissen hat es nicht gefehlt, das Ganze des Lebens half und hilft sich vornehmlich dadurch, daß das Neue, was der Lauf der Zeiten bringt, möglichst in das Alte hineingedeutet, in den geschichtlichen Bildungen Kern und Schale unterschieden, jener nach Kräften festgehalten, ist diese abgestreift wurde. Aber das nur eine Ausflucht, und zwar eine Ausflucht, der die historische ihrer Hervorkehrung der Eigentümlichund Unvergleichlichkeit der einzelnen Zeiten immer mehr den Denkweise der Neuzeit mit keit Boden entzieht. Wollen wir also nicht zwischen den Gegensätzen Entwicklung. 225 und uns von ihnen zerreiben lassen, so ist von innen und unter wesentlicher Umwandlung des Wirklichkeitsbildes Das aber wird erst möglich bei Anüber sie hinauszustreben. des Geisteslebens und einer schärferen Selbständigkeit erkennung einer stehen bleiben her Abhebung des menschlichen Daseins von ihm. Denn nur so lassen sich Der Mensch muß Beharren und Bewegung miteinander festhalten. im tiefsten Grunde seines Wesens in einer unwandelbaren Geisteswelt gegründet sein, und es müssen von da aus bewegende und Aber zugleich richtende Wirkungen ausgehen. Dasein höchst unsicher und unfertig, langsam wegung in weiter Fluß, zum und nur sein unmittelbares kommt eine Be- erst der Zeit läßt sich weiter und inmitten Aber die Bewegung vordringen. Ziele ist verliert sich, dank jener Grundlage, nicht ins Vage und Fremde, es vollzieht sich in ihr ein Erringen ist sie keine inmitten aller Wandlung Vom Menschen aus angesehen eignen Wesens, des bloße Veränderung. verlangt eine solche Überzeugung eine Zurückverlegung des Lebens Denn diese" hinter die Fläche der einzelnen seelischen Betätigungen. zeigen uns die Sache mitten im Fluß, namentlich Gedankenwelt als in unablässiger Wandlung erscheint hier die Aber begriffen. aller solchen Veränderung kann eine charakteristische Art des Grundlebens überlegen bleiben und hindurch behaupten, entfalten. So steht der Mensch zu- über der Zeit; sein Leben durch sich zeitüberlegene Wahrheit darin gleich Art, in der Zeit und indem es sich einmal einer zeitüberlegenen Tatsache zu versichern innerhalb sie der Zeit und eine in immer ihr zu Wahrheit als einer zugleich aber Herausarbeitung und deutlichere Entfaltung jener Wahrheit zu erstreben hier die zweiseitiger ist begründen, kräftigere ihre Daher hat. ist Wahrheit zugleich Besitz und Problem, jenes im innersten Grunde des Wesens, dieses bei der Verwandlung des Daseins in volle Selbsttätigkeit. Von hier aus wird ein Verhältnis zur Geschichte möglich, das den Gegensatz von Beharren und Bewegung in sich aufnimmt und Betrachten wir zugleich überwindet. z. B. unsere Stellung zu geschichtlichen Religion, etwa der christlichen. die menschlich-geschichtliche Zeiten behaupten. Form, die sie Unmöglich erhalten hat, einer läßt sich für alle Bei der gewaltigen Veränderung unseres äußeren und inneren Daseins würde unser Gefühls- und Überzeugungsleben wahrhaftigkeit geraten, Eucken, Qnindbegriffe. nicht nur unser Denken, sondern auch wenn 4. Aufl. sie in mit aller die Gefahr einer Un- Gewalt auf jene* ältere 15 Zum Weltproblem. 226 Art gestimmt werden leicht sollte; möchten wir unserer eignen Zeit Unrecht tun, wenn wir nur darauf bedacht wären, anderer Zeiten Recht wahren. zu — Aber die Entfernung von der unmittelbaren Lebensform braucht keine Preisgebung der Substanz zu bedeuten. Es kann unzulänglich gewordenen Existenzformen eine wahrhaf- in tige Art des Geisteslebens durchgebrochen sein, die gesamte Geschichte erfüllende Tatsachen leben fortfährt, eine Art, und nimmer losreißen welche zeitüberlegene, belebt und zu be- hat von der sich das menschliche Leben nun Dies Ewige aber würde zugleich seiner darf. menschlichen Gestaltung nach eine fortwährende Aufgabe es würde durch die alle Zeitüberlegenheit nicht durch seine ein starres bleiben, Beharren sondern vielmehr dadurch erweisen, daß es Zeiten, eingehen Eigentümlichkeit aller Zeiten kann, ohne sich in selbst daß es jede Zeit auf das ihr innewohnende Ewige zu verlieren, zu bringen und damit von der bloßen zu befreien vermag. Die Zeit aber würde gegen die antike Fassung dadurch gewaltig Zeit gehoben, daß innerhalb ihrer ein Fortschritt im Ewigen möglich wird. Wie sich auch und der Weltanblick die Stellung des Menschen zur Wirklichkeit verwandelt, wenn das Werden an die zweite Stelle weiter tritt, das läßt sich ohne hier in die antike Geringachtung zurückzusinken, nicht weiter Nur verfolgen. zum Schluß noch erwähnt sein. ein Punkt möge Jene Grundüberzeugung mit ihrem Ausgleich von Beharren und Bewegung kann nie den Tatsachen der Entwicklung widersprechen, wohl aber muß sie mit einer alleinselig- machenden Entwicklungsphilosophie, einer naturalistischen Evolutionslehre, hart Die zusammenstoßen. der Gesamtauffassung des eignen Wesens. letzte geistigen Entscheidung Lebens liegt hier bei und zugleich unseres Wie die Entwicklung im Ganzen der Wirklichkeit zu verstehen sei, das hängt am meisten davon ab, ob im Geistesleben eine neue Stufe des Lebens anerkannt, oder eine bloße Fortführung der Natur gesehen wird. Ist jenes der Fall, so gewinnt die Ent- wicklung das Ansehen, daß nicht der erfahrungsmäßig vorliegende Prozeß allen Fortgang aus sich selbst hervortreibt, nicht das ein bloßes Erzeugnis des Niederen bildet, sondern daß Höhere in die Be- wegung neue Kräfte aus weiteren Zusammenhängen eintreten. Damit erhält unsere Wirklichkeit einen Hintergrund und eine Tiefe, sie Bewegung aber ist dann nicht mehr ein Weiter- und Weiterhasten ohne Ziel und ohne Sinn, sondern sie wird getragen und umfaßt von einem Reiche hat einem größeren Ganzen sich einzufügen; die Entwicklung. ewiger Wahrheit. Ist 227 dagegen das Geistesieben ein bloßes Neben- ergebnis der Natur, so entfällt alle der Möglichkeit, Bewegung ein Gegengewicht zu geben und dem Leben einen Gehalt zu erringen, dann treibt die Menschheit Leere hinein. So ist wie die ganze Welt es auch hier, wie an allen Untersuchung, die Stellung zum Geistesleben, unaufhaltsam ins Hauptpunkten der namentlich die An- erkennung oder Verwerfung einer Selbständigkeit des Geisteslebens, welche über die Richtung der Gedankenarbeit entscheidet. 15- D. Zu den Problemen des Menschenlebens. 1. Kultur. I m eignen Gebiet des Menschen bildet den beherrschenden Mittel* punkt der Probleme die Kulturidee. Sie treibt aus sich eine reiche Verzweigung hervor, deren Gestaltung auf den Haupt- und Gesamtbegriff zu näherer Bestimmung zurückwirkt. Kultur führt Das Wie der den Problemen von Geschichte und Gesellschaft, zu Was zu denen der Moral, Kunst u. s. w. Als eine Einleitung zu dem allen sei zunächst der Kulturbegriff im bloßen Umriß das erörtert Zur Geschichte des Ausdrucks und Begriffs. a) vom Ausdem heute üblichen Sinne ist neueren Ursprungs. Unserer Gewohnheit gemäß beginnen wir auch hier Kultur in druck. Denn so nahe die Übertragung des Bildes von der Bestellung (colere) dem späteren Altertum wie der des Ackers auf den Stand der Seele Renaissance einen geschlossenen und abgegrenzten Begriff bildet lag, Die Kultur oder Georgik des Geistes wird ihm daraus erst Bacon. Aber dieser Versuch ein Hauptteil der Ethik. ^ Folge, er ward unmittelbar nicht Eine ausgedehntere Bewegung hat wohl des 17. Jahrhunderts ließ sie sich ^ S. selbst de augm. hervorgerufen. viel zu deutlich scient. VII, cp. 1: hatte zunächst keine aufgenommen und weitergeführt. erst die französische Ihr stolzes Kultur Selbstbewußtsein von niederen Stufen abheben, Partiemur igitur ethicam in doctrinas principales duas, alteram de exemplari sive imagine boni, alteram de regimine cultura animi, et quam etiam partem georgica anirai appellare consuevimus. haec regulas de animo ad illas conformando Der Ausdruck Georgik zeigt, wie stark das Bildliche des Ausdrucks empfunden wird. lila naturam boni praesCTibit, s. describit, auch cp. 3. Kultur. um nicht allgemeinere Reflexionen 229 über verschiedene Zustände der Menschheit anzuregen; das 18. Jahrhundert mit seinem Streben nach einer natürlichen Begreifung der Geschichte verfolgt solche Richtung mehr und mehr mit dem Gegensatz Aber so wenig es an Ausdrücken eines Natur- und Kulturstandes. für das Weiterkommen der Menschheit fehlt, verschiedene Bilder und Vorstellungen laufen hier neben- und durcheinander: Kultivieren, und weiter einen Polieren, Polizieren, Aufklären; ^ Zivilisieren, für das Ganze mit »Zivilisation" der Latein sich beschäftigt des dadurch geschaffen Standes dürfte haben. ^ In Ausdruck den Renaissance Ausdruck festen erreichten Deutschland civilisatio, ^ Turgot erst das besaß auch civilitas wird in ähnlicher Bedeutung verwandt,* aber die lebendige Sprache blieb davon unberührt und hatte bis in die Anfänge der klassischen Die Literaturepoche hinein nur schwankende Bezeichnungen.^ Aus der * (s. ent- Bayle schier endlosen Fülle sei hier nur einiges angeführt. oeuv. div. Haag 1727, 453a) hat cultiver leur esprit et leur raison; wenn I, ebenda 407a von toutes les societes, ou l'on cultivait I'esprit redet, so würden wir das kaum anders als mit „Kulturvölker" übersetzen. Aber zu- er gleich hat er civiliser lisees im Gegensatz zu (z. B. dictionn. 1465 se civiliser, 1472 b barbares). Leibniz (398a Erdm.) nations les eclaire; wo wir „Naturmensch" und „Kulturmensch" plus „Wilder" und peuples grossiers eclairees, „Europäer"; gegenüber, auch öfter nations civi- Bossuet hat in ähnlicher Bedeutung Montesquieu le stellt siecle les qui passe pour sagen würden, sagt er peuples eclaires aber hat er poli oder police (z. den B. les les polis, la Grece seul polie au milieu des barbares, un pays police, un royaume aussi police comme la France, les peuples polices, peuples bien Polices). Auch in England fehlt ein fester Ausdruck; so gebraucht A. Smith bunt durcheinander civilized und polished nations (s. z. B. the theory of moral peuples sentiments V, cp. * 2). S. Barth, die Philosophie der Geschichte als Soziologie, S. 253. Nach Paulsen (Gesch. des gelehrten Unterrichts in Deutschland, S. 78 u. 131) wurde zu Beginn des 16. Jahrhunderts von Wittenberg gesagt, daß es in termino civilisationis liege. ' Es bildet z. B. bei Kepler (II, 730) den Gegensatz von barbaries. In seiner zeigt z. B. der gediegene und gedankenreiche Iselin. „Geschichte der Menschheit" pflegt er dem „Stande der Natur" den „Stand * ^ Das der Sitten" entgegenzusetzen und spricht demgemäß von „gesitteten" Völkern. Aber nicht minder oft hat er „Polizierung" und „poliziert", er unterscheidet aber dabei, die spätere Sonderung von Kultur und Zivilisation vorausnehmend, zwei Arten der Polizierung: „die eine, durch welche der Gesellschaft die äußerliche Gestalt gegeben vcird", „die andere verbessert die Geister und die Gemüter" keit (7. Buch, 21. Hauptstück). Auch stellt er Barbarei und Menschlich- einander entgegen und verwendet „Milderung" (auch „Milderung der Zu den Problemen des Menschenlebens. 230 scheidende Wendung brachte für Deutschland die klassische LiteraturIhr Verlangen nach einer epoche. und einer künstlerischen Gestaltung des Daseins zu selbständiges Kulturideal, dem hätten ein enthielt viel daß nicht auch die Ausdrücke sich als So müssen. anpassen Belebung des ganzen Menschen denn auch geschehen. es ist Kultur wird nun zu einem festen und zum herrschenden Begriffe, Zivilisation grenzt sich davon als eine niedere Stufe ab, »Aufklärung" kaum durchgedrungen, die allgemeine Bedeutung und sinkt verliert, zur Bezeichnung der besonderen Art des 18. Jahrhunderts, zu einer historischen Kategorie; dafür hebt sich »Bildung", die bisherige Be- deutung ins Innere und gewinnt den Affekt der wendend, diese Verschiebung der Ausdrücke etwas näher Es sei sie den deutschen Sprachgebrauch ohne »Kultur" wohl erscheint aber er befestigt sich schon abzugeben.^ dargelegt, da Gegenwart beherrscht. begegnet uns zuerst bei Herder; allen Zusatz hier der neue bis zur Zeit. Gebrauch noch genug, Neben Kultur steht, als in um einen Fluß begriffen, bündigen Terminus so auch bei Goethe, lange noch Geisteskultur, aber allmählich gewinnt Kultur schlechtweg die Ober- hand. Die weitere Verwendung des Begriffes nimmt eine zwiefache gemäß den beiden Hauptströmungen im deutschen Idealismus: der künstlerischen und der ethischen. Bei den Dichtern und Humanisten überwiegt die ersk, Kunst und Wissenschaft in ihrer Richtung, Verbindung zum Schaffen literarischen sicheren Träger der Kultur, erscheinen hier als die das unterscheidende Merkmal eines als Kant und mehr noch Fichte dagegen machen zur Kulturstandes. 2 und „Erleuchtung" (auch „Erleuchtung der Geister") als unserem Goethe hat in seinen Jugendschriften „polierter" Mensch und „polierte" Nationen, und Kant spricht von „geschliffenen" Sitten") «Kultur« gleichbedeutend. — Volks klassen. ^ Namentlich wichtig ist für den Ausdruck die Stelle Ideen zur Philos. der Gesch. IX, 1 : „Wollen wir diese zweite Genesis des Menschen, die sein ganzes Leben durchgeht, von der Bearbeitung des Ackers Kultur oder vom Bilde des Lichts Aufklärung nennen : so stehet uns der Name frei ; die Kette der Kultur und Aufklärung reicht aber sodann bis ans Ende der Erde." Die Kultur hat als beherrschendes Ziel die „Humanität", die für Herder die volle und Harmonie aller Kräfte bedeutet, gemäß einer Überzeugung, enge Verbindung von Leben und Schönheit als Ideal verehrt. Das Unterscheidende des Menschen gegenüber der bloßen Natur aber ist Entfaltung welche die die Freiheit; so gehört diese wesentlich siehe bei ^ zum Kulturbegriffe. Näheres darüber Genthe „Der Kulturbegriff bei Herder". S. die gleich anzuführende Stelle aus F. A. Wolf. Kultur. und geben die Freiheit der Kultur Seele einen moralischen Charakter. «Die Hervorbringung der 231 Also kann nur die Kultur der Kultur. vornehmlich vernünftigen Wesens Tauglichkeit eines zu beliebigen Zwecken überhaupt (folglich der Natur in Ansehung damit ihr Kant definiert Kultur folgendermaßen: in seiner Freiheit) letzte der Menschengattung Ursache beizulegen wohl gar bloß hat (nicht seine eigne Glückseligkeit auf Erden, oder Ordnung und das vornehmste Werkzeug zu sein, Einhelligkeit der vernunftlosen Natur außer ihm zu stiften") (V, 464, Hart.). hat dies weiter ausgebaut Ihm wird die Freiheit, der Kultur. die ist Zweck sein, den man in Fichte und gemäß seiner Art kräftig durchgesetzt zum Inhalt «Übung aller die volle Selbsttätigkeit, zugleich So bedeutet ihm diese (Wke. VI, 86): Kräfte auf den Zweck der völligen Freiheit, der völligen Unabhängigkeit ihm von allem, was nicht wir selbst, unser reines Selbst diese Aufgabe alles Übrige in sich der Sinnenwelt, nichts von unserem Treiben, Erscheinung betrachtet, einen Wert, Religion, Wissenschaft ist". Wie hat «nichts in so schließt, Tun oder Leiden, als als insofern es auf Kultur wirkt" und Tugend werden ausdrücklich zu den höheren Zweigen der Vernunftkultur gerechnet (VII, 166); auch den dem Denker vor- Staatszweck bildet die Kultur, und der Staat, der schwebt, wird als Kulturstaat bezeichnet^ Die beiden Nuancen der Kulturbewegung stimmen aber darin zusammen, Kultur, als ein Bilden von innen her und eine Erhöhung des ganzen Menschen, von aller bloßen Ordnung der Gesellschaft deutlich abzuheben; zur Bezeichnung dieser so unterscheiden sich Zivilisation und dient Kultur nun wie Zivilisation; Niederes und Höheres, wie Beginn und Vollendung.^ ^ Der Begriff des Kulturstaates widerspricht zunächst der Fassung des Staates als eines bloß „juridischen Institutes". Auch zum nationalen Staat Welches ist denn das Vaterland des wahrhaft ausgebildeten christlichen Europäers? Im allgemeinen ist es Europa, insbesondere ist es in jedem Zeitalter derjenige bildete der Kulturstaat anfänglich einen Gegensatz; Staat in Europa, der auf der s. VII, 212: Höhe der Kultur steht." ,, Später hat gerade und Vaterland zu Ehren gebracht, aber nie war es das sinnliche Dasein, sondern immer der geistige Gehalt, der sie ihm beFichte die Begriffe Volk deutend machte. " Das erscheint schon deutlich genug bei Kant, s. namentlich IV, 152: „Wir sind in hohem Grade durch Kunst und Wissenschaft kultiviert. Wir sind zivilisiert bis und Anständigkeit zum Überlästigen, zu Aber uns für schon allerlei gesellschaftlicher Artigkeit moralisiert zu halten, daran fehlt Zu den Problemen des Menschenlebens. 232 In engem Zusammenhange mit jener Steigerung des Kultur- Aufkommen von «Bildung"; erst in der zweiten 1 8. Jahrhunderts wird es vom Äußeren aufs Innere, vom begriffes steht das Hälfte des Mit besonderer Lebhaftig- Körperlichen aufs Seelische übertragen.^ keit bemächtigten sich seiner die Romantiker, den Ausdruck «die Gebildeten" fester namentlich dürften Bei wie das Wort aus anfänglicher Fichte läßt sich deutlich verfolgen, Unsicherheit ein sie Umlauf gebracht haben. ^ in Terminus wird. „Bildung" wie „gebildet« haben sich dabei insofern eigentümlich gestaltet und von den anderen Ausdrücken abgezweigt, daß von ganzen Völkern nicht sowohl sie oder der Menschheit als von der höheren intellektuellen Schicht innerhalb eines Volkes gebraucht werden; bei „Bildung" wird mehr die eigne Tätigkeit, die selbständige Aneignung seitens des Individuums be- noch sehr viel. Denn die Idee der Moralität gehört noch zur Kultur; der Gebrauch dieser Idee aber, welcher nur auf das Sittenähnliche in der Ehrliebe und der äußeren Anständigkeit hinausläuft, macht bloß die Zivilisierung aus." Pestalozzi XII, 154 sagt in ähnlicher Tendenz: „Die kollektive Existenz unseres Geschlechts kann dasselbe nur zivilisieren, sie kann es nicht kultiDie vieren." spezifisch literarische Wolf verfochten, namentlich F. A. in Kultur hat mit besonderer Energie der berühmten Abhandlung, die das „Museum der Altertums-Wissenschaft" einleitet (1807). Der Unterschied von Kultur und Zivilisation wird ihm zum Mittel, die Griechen und auch die Römer über alle anderen Völker hinauszuheben. Als Hauptmerkmal echter Kultur erscheint Kultur ist dabei der Besitz einer allen der durch Ausbildung von Literatur Stand der Gesellschaft. S, S. 16: gemeinsamen Literatur; die und Kunst hervorgebrachte „Eine der wichtigsten Verschiedenheiten und diesen Nationen ist die, daß die ersten gar nicht oder nur wenige Stufen sich über die Art von Bildung erheben, welche man bürgerim Gegensatze höherer eigentlicher liche Polizierung oder Zivilisation, Geisteskultur, nennen sollte." S. 17, ,,jene höhere Kultur, die geistige oder literarische." S. 18, „Asiaten und Afrikaner werden, als literarisch nicht kultivierte, nur zivilisierte Völker, unbedenklich von unseren Grenzen ausgeschlossen." Jener ganzen Zeit sind „Europa" und „Kultur" eng assoziiert. Dieser Unterscheidung von Kultur und Zivilisation folgt auch W. v. Humboldt. * S. darüber Imelmann, Ausg. von Klopstocks Oden, S. 86; Paulsen Bildung in Reins Enzyklop. Handbuch der Pädagogik; Biese in d. N. Art. unter jenen Jahrb. für das klass. Altertum, Jahrgang 1902, S. 241. Der Ausdruck besagte aber weit mehr als heute nach der abschleifenden Wirkung des Jahrhunderts; das will auch bei Schleiermachers „Reden über ' die Religion an die Gebildeten unter ihren Verächtern" beachtet sein. über den Sinn des Ausdrucks bei den Romantikem mantische Schule", S. 420, 430. s. bei Näheres Haym, „Die ro- Kultur. So wird tont. * sie 233 wohl der Kultur als etwas Innerlicheres entgegen- — Die Abgrenzung von Kultur und gehalten. dings sehr ins Unsichere und zwar geraten, 2 Zivilisation ist neuer- insofern nicht ohne einen sachlichen Grund, als jene innere Kultur, die unseren großen Dichtern und Denkern vorschwebte, und die sich deutlich von aller bloßen Zivilisation abheben Boden mehr hat. keinen unserer Zeit in wollte, Auch gehen die Nationen festen auseinander; hier wo wir Deutschen von „Kultur" sprechen, sagen die Engländer und Fran- zosen „Zivilisation ".3 den über folgen; Doch das allgemeinen sich hier von „Kultur" Zweifel, die nähere Fassung aber weiter ver- besteht kein nicht läßt Sinn Kräftige völlig herrenlos, jeder ist mag ihr seinen Stempel verleihen. Mag aber der Begriff der Kultur heute sein,' sicherlich bezeichnet er ein altes noch Problem. so unbestimmt Auch die antike Welt konnte sich der Anerkennung eines großen Gegensatzes zwischen den Völkern, sowie der verschiedener eines Volkes nicht entziehen; die intellektueller Stufen Höhe innerhalb des attischen Lebens aber mußte sowohl das Selbstbewußtsein der griechischen Kultur steigern als innerhalb des griechischen Lebens eine schroffere Scheidung erEiner vollen zeugen. Würdigung des Kulturproblems wirkte hier freilich manches entgegen: die nationale Abschließung ließ den höheren Stand leicht als bloße Naturgabe eines besonderen Volkes erscheinen, zugleich setzte die geschichtliche Ansicht von einem endlosen Kreislauf der Dinge allem Fortschreiten enge Grenzen und hemmte leicht eine unbefangene viel Neigung, erkennen ; ' Erforschung der Anfänge. Aufsteigen aus Andererseits bestand einem rohen Naturstande anzu- der Scheidung der Menschheit in Griechen und Barbaren mußte aber fels, ein die Erweiterung des Horizontes und die engere Ver- Über die Probleme im Begriff der Bildung s. neuerdings O. Weißen- „Die Bildungswirren der Gegenwart." ^ Näheres darüber s. Barth, die Philosophie der Geschichte als Sozio- logie, S. 253. * Bei der Übersetzung eines Artikels von mir „Religion und Kultur" No, 3, pag. 114) wird zu Kultur angemerkt: in der Liberte Chretienne (1907, Nous n'avons guere l'habitude, en fran^is, d'employer ce mot Sans quelque d^terminatif: ,,la culture intellectuelle", „la culture des lettres". Zu den Problemen des Menschenlebens. 234 bindung der Völker entgegenwirken, In derselben Zeit wo aber, die mit Alexander begannen.* der Völker der Gegensatz verblaßte, Welt der Gegensatz von verschärfte sich innerhalb der griechischen Gebildet und Ungebildet, indem nun lediglich ein gelehrtes Studium den ererbten Kulturgütern vollauf teilnehmen an heit ist das spätere Im ließ. 2 Wahr- In Altertum voller Betrachtungen zum Kulturproblem. christlichen Altertum und im Mittelalter tritt diese Frage zurück, um in der Renaissance mit verstärkter Kraft wieder aufzuleben. Seit- dem steht die Kultur im Mittelpunkt der geistigen Arbeit; am Kampf um sie sind alle Gegensätze der Neuzeit beteiligt: der Idealismus von innen her aufbauen, der Realismus sie von außen her will sie zusammenfügen; künstlerische, intellektuelle, ethische Fassungen durchkreuzen sich und bestreiten einander die Oberhand; nicht an auch Im Verlauf des Mischungen mannigfacher Art. fehlt es 19. Jahr- hunderts hat ein Zusammenwirken von Geschichte und Naturforochung die spekulative ältere Behandlung dieser Fragen mehr und mehr weichen lassen; zugleich werden die exakt-wissenschaftlichen einer Bedingungen seelischen des Kulturlebens genauer erforscht,^ und während der Stoff massenhaft anschwillt, erzeugt das Bedürfnis eines Gesamtbildes neue Versuche einer Kulturphilosophie. Von den zahl- und Kontroversen reichen dadurch erzeugten Problemen seien hier nur diejenigen ausgewählt, welche das Lebens- und Geistesproblem unmittelbar berühren. ^ Das gibt nicht nur der Philosophie einen kosmopolitischen Zug, sondern verwandelt auch sonst die Denkweise. Bemerkenswert ist, was Strabo (Geographica, Sk Tou am Schluß des 1. Buches) von Eratosthenes berichtet: in\ -reXei oux ETratveaa? tou? Stya SiaipoGvxa; areav to twv avO'paxwv ü7:o{jLvr'|jLaT05 te "EXXrjva? xa\ ßapßa'pou;, — ßeXTiov etva( TcXfd-o? e'i? Taura. noXXou^ yap xa\ tcTv 'EXXTJvtüV etvat cprjaiv xaxou? xai «pexf y.ai xax(a: Staipetv tiIjv ßapßaptov aoTeiou?. Strabo verteidigt dagegen den Vorrang der Hellenen damit, daß dort gesetzliche Ordnung Tots (AEv ' und Bildung überwiege, bei den anderen aber das Gegenteil: iTzixpaztl TO vo[JLt{j.ov xa\ TO :iatS£(a; xa\ Xo'ytov o^xtiov, Tot; Se TavavT(a. Schon bei Plato und Aristoteles hat 7rai5e(a neben der Bedeutung der Erziehung auch die weitere der Bildung. aristotelische Zusammenstellung: («XoÜTos, euye'veta, aptTr^, TcatSeta), Pol. 1291b, xttl euyevEta, 1296b, 18: iXsudepfa, Bezeichnend dafür Reichtum, tiXoütoc, Adel, ist Tüchtigkeit, z. B. die Bildung 28 (s. ähnlich 1293 b, 37 natSeta TtatSeta, euyevsta, 1317b, 39: y^^°S ihm entsprechen TOnaiSeufievo; und a7:a(8euTos durchaus unserem „gebildet" und „ungebildet". * S. darüber das wertvolle Buch von Vierkandt, „Naturvölker und jcXouTo;, TtatSet'a). Kulturvölker. Bei Ein Beitrag zur Sozialpsychologie", 1896. Kultur. b) 235 Kritische Erwägung. Das Problem des Wesens und Wertes der Kultur. a. sich um so mehr verDenken mit ihnen beschäftigt. Der Begriff soll alles zusammenfassen, was den Menschen und die Mensch- Die Kultur gehört zu den Größen, die mehr wickeln, je heit unser sich über die bloße Natur hinaushebt, aber worin besteht dies Mehr Gelangt der Mensch nur zu einer größeren Selb- gegenüber jener? und Macht innerhalb eines gegebenen Daseins, und ver- ständigkeit mag er seine Umgebung nur weiter zu überschauen wie geschickter für sich zu verwerten, oder erscheint bei Art des Lebens, eröffnen sich ihm eine wesentlich neue neue Tiefen und gestatten ihm, ein neues Reich der Wirklichkeit aufzubauen? Dort würde nur eine Außenkultur, hier eine Innenkultur erreicht, dort eine bloße Zivili- sation, über jene kann kein Zweifel echte Geisteskultur, eine hier sein, 'die Möglichkeit dieser wird hart bestritten. Wie aber der Inhalt der Kultur, so ist auch ihr Umfang keineswegs sicher. Zweifellos liegt in eine Versetzung des mensch- ihr lichen Lebens in größere Tätigkeit, ja ein Gründen seiner auf eignes Tun, wie das auch schon der Ausdruck indem anzeigt, er an das Bestellen eines Ackers gegenüber der wildwachsenden Natur erinnert. Aber umfaßt diese Tätigkeit alles, was irgend dem Menschen eigentümlich ist, oder Möglichkeiten ist sie nur eine Seite des Lebens, neben der andere Daß bleiben? hier ein Problem vorliegt, bekundet schon die Unsicherheit über das Verhältnis der Religion zur Kultur: bald wird jene zu ihr gerechnet, Stande der Kultur zu und hängen; bald es scheint die Religion am scheinen sie Gegensätze, die einander durchkreuzen und hemmen, wie denn oft genug die einen von der Religion aus die Kultur, die anderen von der Kultur aus die Religion bekämpfen. Nicht Kultur. viel Dient anders steht es mit der Frage nach sie dem Werte der zur Bezeichnung alles dessen, was den Menschen über den Stand der rohen Natur hinaus zu dem der Gesittung und muß sie als der höchste aller Werte erscheinen, muß sich innerhalb ihrer alles begründen, was uns irgend Bildung führt, so und es schätzbar sein will. Aber zugleich ist die Geschichte voller Klagen über Schäden und Gefahren der Kultur, sie so sehr, daß Danaergeschenk die ganze Kultur wie ein steigern sich zuweilen erscheint. 236 Zu den Problemen des Menschenlebens. Namentlich in drei Richtungen ist die Kultur von alters her ein die Kultur Gegenstand harter Angriffe gewesen. Von der Religion menschlicher Kraft aus konnte als eine und Steigerung als Stärkung eine menschlichen Selbst- bewußtseins schwere Bedenken erregen. Ein frommer Sinn sah in dem kühnen Aufstreben ein Vermögens, ein der Menschheit Überschreiten Überspannen eignen naturgewiesener Mangel an frommer Gesinnung. Schranken, einen Die Mißstände und Rückschläge des Kulturlebens erschienen dann als eine Strafe für solchen Frevel. Eine derartige Überzeugung wirkt von Babylon her in der Erzählung vom Sündenfall und vom Turm, der bis zum Himmel reichen soll, sie erscheint in den Prometheussagen, Zuspitzung gegen einen sie ist, in der besonderen übermäßigen Wissensdurst, auch in den Faustlegenden unverkennbar. Auf dem eignen Boden der Menschheit aber griff oft der Zweifel um ob die Kultur dem Menschen das Glück sich; bringe, das sie ihm Verwicklung des Lebens, sie bildet in Wahrheit Sie erzeugt eine große zuversichtlich verheißt. künstliche Bedürfnisse aus, sie bindet den Menschen mehr und mehr an seine Umgebung, sie schafft ihm Arbeit und Mühe, sie erweckt unerreichbare Wünsche und wilde Leidenschaften, sie mag mit dem allen als ein Losreißen des Menschen von seiner natürlichen Grundlage erscheinen, äußeren Glanz innerlich unglücklich mache. das Auch ihn bei allem derartige Stim- mungen sind uralt, sie tauchen z. B. bei den alten Juden auf, wie Hosea und Jesaias zeigen. * Besonders voller Zweifel war das spätere griechische Altertum, ein Widerwille gegen das Raffinement der da- maligen Kultur, eine Sehnsucht nach einfachen Zuständen und schlichter Lebenshaltung griff weiter und weiter um sich. Stimmung namentlich diese in derberer, die Stoiker in Literatur gerät unter ihren Zum Ausdruck haben die Philosophen gebracht, die Kyniker etwas feinerer Art; aber auch die schöne Einfluß breitung im gemeinsamen Leben. ^ und bekundet damit ihre Ver- In der Neuzeit stellt namentlich Rousseau das ProJDlem aufs deutlichste vor Augen, mit seiner sensitiven, aufgeregten und aufregenden Art hat er es der modernen Menschheit zwingend auferlegt. ^ S. darüber Budde „das nomadische Ideal im alten Testament" (Preuß. Jahrbücher, Bd. 85.) "^ Anziehende Ausführungen darüber gibt E. Rohde „Der griechische Roman und seine Vorläufer". Kultur. 237 Das drohende Entweichen des Glücks hätte sich etwa ertragen lassen, wenn dabei Wachstum der ein Tüchtigkeit Menschen des Aber das war es nicht, vielmehr pflegen außer Zweifel gewesen wäre. den Klagen über das sinkende Glück solche über eine Minderung der Kraft und Tüchtigkeit durch den Fortgang der Kultur zur Seite zu Die Kultur, gehen. indem sie seines Handelns sie stellt hören so wir, schwächt den Menschen, ihn von anderen abhängig macht, sie erhebt die Wirkung im Zusammensein zur Hauptsache, gesellschaftlichen damit die Leistung vor die Gesinnung und droht bis in und UnMehr und mehr spielt der Einzelne nur eine ihm von der Gesellschaft zugewiesene Rolle, und es wird sein Leben mehr und mehr ihm selbst etwas fremdes, es hängt ihm nur äußerlich an; wie könnte er dabei eine Größe der Seele bewahren, ein wahrhaftiger, die innerlichsten Gefühle hinein das Leben ins Scheinhafte wahre zu führen. kräftiger, ganzer Mensch sein? Wohl fehlt es der Kultur nicht an Anwälten gegen solche Beschuldigungen. Jene Schäden, so heißt es, bloße seien Begleit- erscheinungen, Schatten, ohne die kein Licht besteht; nur der Mensch ziehe ins Kleine herab und mache damit groß und unangreifbar sei. menschlichen Lebenskreises; bunden, wird sie sich — Indes vom nicht sie ist zweifelhaft, was an sich die Kultur liegt innerhalb des an seinen Zustand kleirmienschlichen Getriebe ge- irgend ab- heben können unter deutlicher Scheidung von wesentlichem Gehalt und menschlicher Zutat, von Recht und Unrecht? So bleiben jene Bedenken einstweilen unwiderlegt, und zugleich bleibt die Frage offen, ob die Kultur ein Segen oder ß. Fluch für die Menschheit ein sei. Das Problem des Inhalts der Kultur. Darüber ist kein Zweifel, daß die Kultur das Dasein des Menschen in höherem Grade auf seine eigne Tätigkeit allgemeinen Begriff der Tätigkeit ist Umgebung stellt. Aber mit dem noch recht wenig gewonnen, an sich ziehen und sich umwandelnd in sie ergießen, ohne sich selber näher zu determinieren, die Tätigkeit kann ihre ohne dem Leben einen festen Mittelpunkt, eine beherrschende Haupt- eigentümliche richtung, eine aus Antwort sofort der die damit entsteht, ist nicht eine Durchbildung zu geben; Frage hervor. wächst so Die Aufgabe von der Arbeit der Weltgeschichte in aber, recht verschiedener Weise gelöst, mannigfache Arten der Kultur sind entstanden, von denen keine einzelne voll und dauernd zu genügen Zu den Problemen des Menschenlebens. 238 scheint, und die und Schätzungen mit ihren widerstreitenden Zielen unmöglich aneinanderlegen sich lassen. Es heben sich aber aus der Bewegung unseres gesamten Kulturmit ausgeprägter Gestalt namentlich kreises drei Arten der Kultur hervor: eine künstlerische, eine ethische, eine dynamische; im Griechen- tum, Christentum, modernen Leben sind Im Griechentum langt. bildet sie zur Verkörperung ge- den Kern der Kulturarbeit die Ver- bindung der Elemente, welche die Natur entgegenbringt, zu einem harmonisch geordneten, von innerem Leben erfüllten Ganzen. Verbindung, Ordnung, eigne Tätigkeit bereiten, Belebung kann dem Menschen sie entringt der Zerstreuung der sinnlichen Eindrücke ein beharrendes Weltbild, sie schlossenen die stellt Individuen in Gemeinschaft, Triebe der Seele, sie Diese nur seine und Flucht und zusammenhängendes das feste Gefüge einer ge- verbindet die einzelnen Kräfte und ohne irgend etwas davon aufzugeben oder abzu- schwächen, zu einem Gesamtwerk des Lebens, sie vollzieht an allen Wendung vom Chaos zum Kosmos. Durch solches Wirken sind Natur und Geist in eine enge und fruchtbare Beziehung Stellen eine gesetzt, ist ein kräftiges, tätiges, freudiges Leben geschaffen, ganze Umkreis des Daseins veredelt und durchgebildet. Fragen und Zweifel blieben nicht aus. ist der Aber auch Das Ganze ruht auf der Überzeugung, daß das Leben von Grund aus eine sichere Richtung besitzt, und diese Überzeugung geriet mehr und mehr Wanken; die Form, welche hier das Leben beherrscht, konnte zur Vernunft ins solche Stellung nur wahren, solange sie eine Seele in sich trug, die schien sie nicht und dauernd bewahren zu können; schließlich ge- wannen die Verwicklungen des Lebens so sehr die Oberhand, und schien der Mensch im innersten Kern seines Wesens so schwer bedroht, daß sein Grundverhältnis zur Welt und die Rettung seiner Seele zur dringendsten aller Aufgaben wurde. Dieser Aufgabe unterzog sich das Christentum, bei vollster An- erkennung des Nein unternahm es den Menschen zu einem über- legenen Ja zu führen, inmitten ungeheurer Erschütterung feste Pole zu wahren. Das dem Leben forderte eine unbedingte Konzentration auf eine Aufgabe ethischer Art, ein völlig neues Leben galt es gegen- über dem nächsten losigkeit jenes Dasein zu gewinnen, der Härte und Seelenwurde ein Reich barmherziger Liebe und kindlicher Hingebung entgegengehalten. In Entwicklung dessen ist eine gewaltige Vertiefung des Lebens erfolgt, unsichtbare Zusammenhänge taten sich Kultur. 239 eine große Weichheit der Empfindung und ein großer Ernst der auf, Gesinnung gingen Hand in Hand miteinander; ZeitUches und Ewiges, Endliches und Unendliches, Menschliches und Göttliches traten hier Aber auf dem Boden engsten Kontakt. in der Geschichte blieb Denkweise überwiegend transzendent und gewann kein sicheres diese Verhältnis zur Weltumgebung, neben einem Kreise reiner Innerlichr keit verblieb daher die übrige Welt unergriffen und ungeläutert, die Flucht in die Welt des Gemütes ließ leicht die Arbeit an den Widerständen des Daseins als nebensächlich erscheinen und gefährdete damit die männliche Kraft des Ganzen. zum Kern alles Strebens. Überwindung der Hemmung, der Aus- Diese Arbeit aber wurde der Neuzeit Der Gedanke einer treibung alles vollen Dunkels tritt hier in den Vordergrund, die eigne Be- wegung des Lebens, seine Steigerung ins Unbegrenzte wird hier zum Ziel aller Ziele, zum vollgenügenden Glück. Den Menschen scheint alles ' hier vornehmlich auszuzeichnen seine Überlegenheit gegen gegebene Maß, sein Vermögen, die eigne Kraft weiter und immer neue Wege zu bahnen, immer neue An- weiter zu steigern, fänge zu setzen. Die Bewegung, die daraus hervorgeht, gängig neue Bilder läßt durch- vom All, dem menschlichen Zusammensein, der Seele des Einzelnen entstehen, sie schafft eine neue Art der Arbeit, in welcher diese zuerst das Bewußtsein einer Weltüberlegenheit ge- winnt. Mehr als irgend sonst wird seines Daseins, überall erfolgt hier heit, ein Leben ein Unermeßliche Mut und in der Mensch zum Herrn Beleben alles Schlummernden, ein Befreien alles Gebundenen, überall wird das uns hier ein Aufrütteln aus träger Starr- rastloses Vorwärtsstreben, Kraft. Aber wenn die schwellen ins Ergebnisse dessen tausendfachen wohltätigen Wirkungen vor Augen stehen, vor Augen stehen uns auch die zahllosen Verwicklungen, die jene Belebung und Befreiung gebracht hat, an die freudig vordringende Vernunft hat sich soviel Unvernunft angeschlossen, mit den Erfolgen des Wachstums des Geisteslebens ist so viel kleinmenschliche Irrung und Leidenschaft aufgeschossen, daß die alleinseligmachende Kraft Immer der modernen Kultur uns sehr ins Unsichere geraten ist. weniger läßt sich auch der Zweifel unterdrücken, ob selbst beim Gelingen der Bewegung der Mensch ganz und gar in sie aufgehen kann. Denn als denkendes Wesen überschaut er die Bewegung, faßt er sie ein Ganzes und muß er von ihr eine bleibende Förderung seines Wesens verlangen; von hier aus wird ihm eine Kultur, die immer in Zu den Problemen des Menschenlebens. 240 nur ungestüm vorwärts drängt, nie einen zeitüberlegenen Besitz ergibt, und unerträglich werden. sinnlos Dies nacheinander entwickelt, aber es hat einander alles hat sich nicht einfach abgelöst; was äußerlich versank, behält eine innere Gegen- Nun wart und behauptet einen Einfluß auf das menschliche Leben. aber ist die Grundrichtung und der Gesamtcharakter jener Gestaltungen so verschieden, daß nur eine flache Denkart eine unmittelbare Ver- bindung für möglich erachten kann. Sie ist um so weniger mög- das geschichtliche Bewußtsein der Gegenwart uns die Unter- lich, als schiede mit vollster Schärfe gewahren läßt. So stehen die verschiedenen Lösungen wie fremdartig gegeneinander und führen, wenn auch meist miteinander versteckt, Kampf und Die künstlerische Kultur Krieg. eng und düster, die dynamische für form- ethische für erklärt die und ruhelos; der ethischen muß die künstlerische als flachoptimistisch und naturgebunden, gelten; die die dynamische dynamische wird wegung und Forttrieb finden. steht der als selbstbewußt und trotzig den anderen Formen zu wenig Be- in Und zwischen all diesen Gegensätzen Mensch der Gegenwart; wird und geistig herabgedrückt werden? er nicht von ihnen zerrieben Er kann jene verschiedenartigen Kulturtypen weder verbinden, noch auch zu Gunsten eines auf die anderen verzichten; er müßte, um jedem sein Recht zu geben und von seinem Unrecht zu befreien, eine sichere Überlegenheit gewinnen, aber er ermangelt nicht nur einer solchen, er sieht nicht einmal, in welcher Richtung y. Die Frage, sie zu suchen sei. Das unsichere Verhältnis des Menschen zur Kultur. Verwicklungen wie sich Mensch steigern weiter sich und Kultur bei Erwägung der Nur zueinander verhalten. zweierlei scheint hier möglich: entweder hat die Kultur dem Menschen Nun aber ist keines von Mühe ersehen läßt. Wäre die Kultur ein bloßes Mittel für das Wohl und den Zu- oder der Mensch der Kultur zu dienen. beiden möglich, wie sich ohne stand des Menschen, so müßte ihr Wachstum sein Leben immer angenehmer gestalten, so müßte das Mehr der Kultur zugleich Mehr des Glückes besagen. Das aber tut es nicht. menschlichen Behagen scheint die Kultur mehr schädlich zu sein. Sie erzeugt Mühe und Arbeit, sie ein Denn dem als nützlich unbegrenzte Wünsche und kostet unsägliche verwickelt in Sorgen und Aufregungen, sie umfängt uns mit festen Bindungen, sie verlangt Gehorsam und Opfer; Kultur. daß das 241 die Lust, die Annehmlichkeit des Lebens erhöhe, läßt alles behaupten. sich schwerlich Jene befindet sich weit besser, und es wird sich der Mensch weit eher zufrieden fühlen auf niederen Stufen der Kultur, auch werden weit eher Individuen geringer geistiger Regung dazu gelangen. hoher als Wäre zufriedenes und angenehmes Dasein das höchste Ziel, wie sehr müßten wir Kulturmenschen die brasilianischen Neger mit ihrem sorglosen Lebensgenuß beneiden! So wäre auch leicht zu zeigen, daß geistige Bewegungen, welche das Glück zum höchsten Ziele machten, wie der Epikureismus und der für den inneren Utilitarismus, geleistet sie haben. Innerhalb Aufbau der Kultur verzweifelt wenig eines gegebenen Kulturstandes mochten manche Härte mildern, mancher Not entgegenwirken: das Leben zu heben, Neuem die Bahn zu brechen, geht über ihr wesentlich Vermögen. So nur der andere Weg: die Kultur als Selbstzweck an- bleibt zuerkennen und den Menschen zu einem Fortgang zu machen. bloßen Mittel für ihren Eine solche Fassung hat für sich den Ein- druck innerer Größe: unvergleichlich wächst die Kultur, wenn sich bei jenem Selbständigwerden in ein sie Ganzes zusammenfaßt und mit der Kraft einer eignen, inneren Notwendigkeit wirkt; der Mensch aber scheint bei aller äußeren Unterordnung innerlich nur zu wachsen, wenn er alle Sorge um den eignen Zustand ablegt und sich ganz dem Strome des Weltlebens hingibt. Hegels System hat dieser Denkweise eine großartige Verkörperung gegeben. hinaus übt sie Aber weit darüber im modernen Leben eine nicht geringe Macht. In- menschlichen Verhältnisse und inWerdens und Vergehens der Geschlechter gibt heute Überzeugung vielen einen Halt und Trost, daß durch alles mitten alles Unerquicklichen der mitten die des Mühen hindurch die Kultur ihren sicheren Weg verfolge, und daß Gewinn auch dem Leben und Wirken des um sie bemühten ihr Menschen einen Sinn und Wert, sowie eine bleibende Dauer verleihe. «Viele werden vorbeiziehen und die Wissenschaft wird wachsen." Aber so anziehend dieser Gedanke, zum Siege vermag er nicht zu gelangen. Denn es gibt keine Kultur freischwebender Art; eine Kultur, die sich gänzlich bloßen Mittel vom Menschen ablösen und ihn zu einem herabsetzen wollte, würde selbst ins Leere fallen. Immer liegt die Kultur innerhalb des menschlichen Lebens, und muß sie diesem etwas sein, der Mensch behaupten haben, wenn Eucken, Grundbegriffe. sie 4. Aufl. seine muß in ihr ein geistiges Selbst zu volle Kraft gewinnen, ihn durch 16 Zu den Problemen des Menschenlebens. 242 Hemmungen hindurch hohe Ziele erreichen lassen soll. Eine unpersönliche, vom Menschen völlig abgelöste Kultur wäre ein Ge- alle und spenst ohne Fleisch Blut; soweit dies in unseren Vorstellungen eine Wirklichkeit erlangte, würde es uns ins Irre locken, unbekannten uns aufzuopfern heißen, Zielen das Leben einer Seele berauben. Und wie könnte die Hoffnung auf die Zukunft uns in den Mühen und Kämpfen der Gegenwart aufrecht erhalten und freudig stimmen, wenn diese Zukunft niemandes Sache, niemandes Freude, niemandes Förderung wäre? Unsere eigne Zeit immer uns stellt klarer daß vor Augen, jene Selbstaufopferung des Menschen an die Kultur sich schlechter- dings nicht vollziehen Denn immer stärker läßt. bricht aus allem und lärmenden Kulturbetriebe wieder das Verlangen nach Entfaltung und Förderung des lebendigen Menschen, nach Bildung hastigen der Seele, nach Rettung eines geistigen Selbst hervor; wir erkennen zugleich, nicht daß dies für die eigne Wahrheit und Tiefe der Kultur zu entbehren ist solchen Erfahrungen In tritt ihn dieses nicht nach seinen vor deutlich Augen, daß der Mensch kein bloßes Gefäß des Kulturlebens Bedürfnissen wie weiches daß ist, Wachs so oder anders formt, sondern daß er ihm eine selbständige Art entgegenzusetzen Kultur hat, alt nicht schreitet sicherm Zuge Die die nicht auf Befriedigung verzichten kann. fort, aus einem vielmehr ihr scheint in innewohnenden Zwange ihr alle in besondere Gestalt zu werden und sich auszuleben; immer wieder bedarf es neuer Anfänge, eines Hervorbrechens ursprünglichen Lebens, vor allem aber So erging es dem späteren Altertum; das Kulturleben kam erst wieder in Fluß, als neue Völker es aufnahmen und durch frische Kräfte verjüngten. Sollte auch der Gegenwart eine neuer Menschen. solche Verjüngung notwendig sein, sei es durch neue Völker, sei es durch neu aufsteigende, geistig noch minder verbrauchte Klassen? Wie dem sein mag, der lebendige Mensch behauptet seine Selbständigkeit gegen allen Versuch, ihn zu einem bloßen Werkzeug herabzusetzen. Aber auch die Kultur, so sahen wir, darf nicht zu einem bloßen Mittel sinken, wenn sie nicht einer Auflösung verfallen will. So befinden wir uns in einem schweren Dilemma, wir müssen darüber hinaus, aber zunächst sehen wir nicht, wie das möglich sein sollte. bald nach Im Durchschnitt des Lebens aber werden wir heute dieser, bald nach jener Seite getrieben, zwischen leerer Subjektivität und seelenloser Arbeit schwanken wir ratlos hin und her. Kultur. Alle diese Verwicklungen 243 treffen in der Gegenwart zusammen Vor allem peinlich wirkt die Unsicher- und steigern sich gegenseitig. heit, die über unser eignes Verhältnis zur Kultur besteht, das Fehlen umfassenden und leitenden eines zur eignen zur Sache, Zieles, Erhaltung zwingenden Notwendigkeit macht und dem menschliche Getriebe hinaushebt, das uns die unseres sie Kulturarbeit geistigen zugleich Selbst, über das zur klein- wir sonst wehrlos verfallen Schon das verhindert uns, nach einer neuen charakteristischen sind. Art der Kultur gegenüber den verschiedenen Gestaltungen zu streben, die von naher oder ferner Vergangenheit auf uns eindringen und uns einnehmen, ohne uns voll die daraus entstehen, uns schließlich der Wert und das Wesen aller fällt zu befriedigen. Kultur ins Ungewisse, notdürftig genug verdeckt gewandte Remit schönklingenden flexion Reden und ausgeklügelten «Gesichts- punkten" den Mangel eines Kernes des Ganzen. schließlich zwischen dem, was solches erstrebt wird, immer ausgeht, weiter wird der Abstand Ziel verkündet und was in Wahrheit als immer größer wird damit die Unwahrhaftig- als Dem muß widerstanden werden; die wachsende des Lebens. Unzufriedenheit zeigt schon im Gange ist. c) Unerträglich wird jene aufgeputzte Scheinkultur, wie sie namentlich von all unsern Millionenstädten keit den Wirren, In all genug, daß eine deutlich solche Bewegung Forderungen für ein wahrhaftiges Kulturleben. a. Die Notwendigkeit einer tieferen Begründung. Die Philosophie mag in solchen Bewegungen und Erschütter- ungen eine noch so bescheidene Rolle haben, der Aufgabe entziehen kann sie sich nicht. Ihre Sache wird es vor allem sein, die Richtungen herauszuarbeiten, die das Streben einzuschlagen hat, um uns das Leben wieder aus einem «Geschäft" zu einem «Dasein« zu machen (nach J. Burckhardt). Dazu aber bedarf Kultur ganz unser eigen sei es vornehmlich dessen, daß die und zur zwingenden Notwendigkeit unserer Selbsterhaltung werde, ohne unter die Kleinheit der bloßen Lust zu geraten. lebens Dafür aber bietet unsere Fassung des Geistes- und seines Verhältnisses zum Menschen einen gangbaren Weg. Denn mit dem Selbständigwerden des Geisteslebens, wie wir es vertreten, wird die Kultur, die seiner Entfaltung dient, von dem flachen Menschengetriebe befreit und auf eine tiefere Grundlage aufgetragen, 16' Zu den Problemen des Menschenlebens. 244 aber sie wird dabei nicht dem Menschen entfremdet, da er seiner eigentümlichen Art nach im Ganzen des Geisteslebens allererst sein echtes Wesen, die Möglichkeit eines wahrhaftigen Beisichselbstseins Bei Fassung solcher arbeitet er in der Kultur nicht für findet. fremde, sondern für eigne Zwecke, und vermag er auch in der weitesten Ausdehnung seines Strebens einen beherrschenden Mittelpunkt festSo zuhalten. ist Menschen und es das Geistesleben unmittelbar zu verschmelzen ohne sie die fertige Tatsache, uns bequemer Weise welches das ganze Leben Ideal, einander und damit das eine dem andern auf- Die Verbindung nämlich erscheint hier nicht zuopfern. hohes unserem Sinne, das den in die Kultur aufs engste verbindet, aufregt und in eine als sondern zufällt, ein als Bewegung Zusammenhängen erscheint die Kultur als unsere Mitarbeit an einer großen Bewegung des Alls, welche die Wirklich- versetzt. keit In solchen höheren Stufe, der Stufe des Beisichselbstseins, zuführt. einer Hinter unserer Arbeit steht und innerhalb unserer Arbeit wirkt damit die Kraft des Ganzen. Es keineswegs bloß eine ist leise Verschiebung oder gar eine bloße Veränderung des Namens, wenn so die Kultur von Forderungen, Erfüllung eine die allem als Entfaltung Denn das gestattet eines selbständigen Geisteslebens verstanden wird. echten Kulturstreben wesentlich sind, denen aber die durchschnittliche Fassung in keiner Weise genügt. So erst wird eine Selbständigkeit der Inhalte und der Werte Wäre die Kultur bloß ein möglich, welche die Kulturarbeit erfüllen. innermenschlicher Vorgang, so würde der Stand des Menschen zu ihrem ausschließlichen Maße, so gäbe es keine Zerlegung und Scheidung des uns umfangenden Chaos, so könnte nicht die Kultur dem menschlichen Dasein Ideale alle aufrüttelnde mit zwingender Stärke vorhalten, so fehlte ihr und vorwärtstreibende Kraft. Ganz anders sich die Sache, wenn in der Kultur eine dem bloßen stellt Menschen über- Bewegung anerkannt wird, die ihm den Kern seines eignen Wesens erst aufzuschließen vermag. Ferner kann erst die Begründung auf ein selbständiges Geistesleben der Kultur eine Größe geben. Denn wo das Leben ganz und legne gar auf den bloßen Menschen beschränkt bleibt, über seinen Zustand keit führt, geistern, da in ein Leben mit mag der Mensch noch raffinierte Unterschiede ihn nicht irgend dem Ganzen der Wirklichso ersinnen, sehr sich für in Größe be- Hochmut und Eitel- Kultur. keit sich 245 oder seinen Stand über andere hinausheben, bleibt alles klein, Erhabenheit, klein vornehmlich Größe, etwas, echte in. der Sache der Einbildung einer Größe. in Ehrfurcht gebieten das und im Unterordnen zugleich erheben könnte, entsteht innerhalb dieses bloßmenschlichen Kreises Dazu muß im Menschen etwas Mehr- nicht. dem er zugleich eine volle Überlegen- alsmenschliches durchbrechen, zuerkennen muß, und das er doch als irgendwie zu sich selbst heit gehörig betrachten darf; erst von da aus wird eine wahrhaftige Er- höhung seines Wesens möglich, auch die größte aller Befreiungen, die Befreiung des Lebens von der Enge des bloßen Menschen. Wie dies Übermenschliche im Menschen den Quell aller echten Größe bildet, so bewahrt es allein die Kultur davor, ein bloßer Menschen- dienst zu werden, Menschendienst gegen Einzelne, Menschendienst auch gegen Massen. Möchte uns stets jenes Kantische Wort gegen- wärtig auch «Alles, sein: Erhabenste, das den Händen des Menschen, wenn verkleinert sich unter Idee desselben zu ihrem sie die Gebrauch verwenden." Auch zur Ursprünglichkeit des Kulturlebens ist die Gegenwart einer ist neuen Stufe der Wirklichkeit Kultur nicht mehr als ein nicht zu entbehren. Denn menschlicher Zusatz zur Natur, so muß ihre Bewegung sich immer weiter von ihrer Basis entfernen, so muß ihr Bestand immer künstlicher, komplizierter und raffinierter Die Kultur wird dann das Leben immer starrer festlegen, ihm immer mehr Möglichkeiten verschließen^ es immer gebundener werden. machen. Mit dem allen würde und aller Ursprünglichkeit heit das in Ist sie die Zerstörerin aller Jugendfrische es ein Wunder, daß, wenn die Mensch- besonderen Lagen mit besonderer Stärke empfindet, sie dagegen sich aufbäumt und sich mit ganzer Seele zur Natur, zu den einfachsten Anfängen zurücksehnt, wie Kindheitsalter mit seiner Frische das Individuum in das oft und der Fülle seiner Möglichkeiten? Aber eine wirkliche Rückkehr zur Natur ist der Menschheit ebenso versagt wie dem Individuum die zur Kindheit, die Geschichte mit ihren Wirkungen läßt sich unmöglich streichen. So müßten wir uns also darin ergeben, daß die Kultur immer greisenhafter und starrer würde, daß die Menschheit im Großen erläge, demselben öden Spießbürgertum wie die meisten Individuen im Kleinen, wenn nicht etwas Neues ursprünglich durchbrechen, nicht frische Kräfte einsetzen, nicht neue Möglichkeiten aufgehen könnten. Sie können aber nur auf- gehen, wenn es eine geistige Tiefe des Lebens gibt, die inmitten Zu den Problemen des Menschenlebens. 246 und Abgelebten Vergriffenen alles neue Anfänge einer bloßmenschlichen Kultur einfache Größen erzeugt, im Einfachen eine neue setzt, Wenn es heißt, daß alles Große einfach ist, so erschließt. muß das wohl eine andere Einfachheit sein als die der natürlichen Welt Anfänge. wenn Endlich entbehrt die Kultur auch der nötigen Triebkraft, sie nur einer gegebenen Welt etwas beifügt, nicht eine neue, uns unentbehrliche Welt eröffnet Kräftig erregen und zwingend be- wegen kann uns nur die Erfahrung und Empfindung eines Widerspruches im eignen Leben, die Unmöglichkeit, bei ihm abzuschließen. Einen derartigen Widerspruch aber kann eine bloße Zusatz- und Luxuskultur nun und nimmer erzeugen. Das von ihr gewünschte Mehr könnte man ruhig ablehnen oder etwa wie Wind und Wetter geduldig über sich ergehen lassen, wie schnittsleben ja in Wahrheit das Durch- gegen die Kultur recht gleichgültig innerlich ist und Zwang denn als eine eigne Freude empfindet. Wenn es auf der Höhe des Schaffens anders stand, und wenn überhaupt solches Schaffen möglich war, so kam das daher, sie mehr als einen daß hier die Arbeit sozialen als die Erringung eines wahrhaftigen Lebens und damit eines Beisichselbstseins galt, und daß, einmal Verlangen aufgegangen war, die vorgefundene geistigen wo solches Lage als schlechterdings unerträglich, als Hemmung einer notwendigen Selbst- erhaltung erschien. Mit solchem Verlangen nach Selbsterhaltung fuhr eine leidenschaftliche Glut in das Streben, die keine Rücksicht auf Menschen kannte und zu jedem Opfer bereit war, vor keinem Hemmnis zurückwich. Durch alle Fragen zieht sich ein und dasselbe Problem, ein und derselbe Gegensatz: der einer echten und einer Scheinkultur. Echt ist die Kultur nur soweit, als sie den Zusammenhang mit dem begründenden Geistesleben wahrt und seiner Entfaltung dient, unecht wird sie, sobald sie unter die Zwecke des bloßen Menschen sinkt Der Kampf beider und auch das Geistesleben dahin herabzieht. durchdringt die ganze GeFormen hie Geist, hie Mensch schichte und läßt in ihr eU,'as anderes sehen als einen reinen Triumph — — des Geistes. Heute aber heit deutlicher erfaßt, tut es besonders not, daß die alte Wahr- die notwendige Bedingung echter Kultur klarer herausgestellt, die Scheidung der Geister für dieses oder jenes kräftiger vollzogen werde. Kultur. ß. 247 Die Notwendigkeit einer inneren Weiterbildung der Kultur. Daß wir einer Weiterbildung welcher Richtung wir der zu suchen sie Erörterung zur Genüge bisherige bedürfen, Kultur haben, das ließ auch schon in die Verschiedene Haupt- erkennen. gestaltungen wirken zu uns von der Geschichte her, von denen wir keine aufgeben und die wir auch nicht unmittelbar zusammenfassen können; was anderes bleibt da übrig, als ob nicht eine Lebensbewegung vorhanden uns danach umzusehen, ist und sich weiter ver- stärken läßt, die über den Gegensatz hinaushebt und ihm entgegen- genug ist, um sich über das zuwirken gestattet, die dabei universal Ganze des Lebens zu erstrecken und seinen Befund in ein Für oder Wider zu scheiden, und zugleich charakteristisch genug, um allem, was sie ergreift, eine eigentümliche Gestalt zu geben. Ein Urphänomen müßte das jedem Einzelnen gegenwärtig in ihr ergreifbar sein, und das zugleich das Ganze des Lebens ist, und bildender Wirkung über mit aufrüttelnder reicht. Ein solches beherrschendes Urphänomen ist nun nicht dieses oder am Geistesleben, jenes ist nicht diese oder jene Leistung, sondern es das Geistesleben selbst, wie wir es verstehen, die Bewegung der Wirklichkeit zu einem Beisichselbstsein des Lebens. Erst mit solchem Beisichselbstsein wird überhaupt ein wahrhaftiges Sein Übrige nur ein Schein davon; ein solches Sein kann nicht außer- ist nur innerhalb ihrer liegen, es halb der Tätigkeit, sondern indem jene Ganze ein in sich zu die Aufstieg einem beharrenden Ganzen einzelnen Betätigungen eines Lebens bloßen oder vielmehr es wird damit erst im Begriff des Lebens sonst spruch, daß einem Wendung wird Selbstleben Oder ist es nicht diese Lust. eine durchgehende * Für einer und muß ich erst deutlich einer wesenlosen, einer gebundenen Art, und Aufgabe daraus, alles weitere an Erst Von hier aus tritt alle Tätigkeit unter den Gegensatz einer wesenhaften und Widerstets der Begriff von Lebensinhalten verständ- von der niederen Stufe der selbständigen ein aber eine Selbständigkeit erlangt?^ auch der Begriff des Wertes scheidet sich hier lich, erreicht, der Widerspruch überwunden, der liegt. nie bleibt, entsteht, und dieses Damit erst wird vertieft hineinlegt. zu eine gewisse Innerlichkeit entsteht, Fremdes gebunden mit jener erreicht, alles die es entsteht zugleich übhche Vermengung, die auf meine systematischen Schriften, zu- nächst auf die „Grimdlinien einer neuen Lebensanschauung" verweisen. Zu den Problemen des Menschenlebens. 248 ineinander ungeschieden beides verfließen auszutreiben, läßt, Forderungen der Wesensbiidung scharf herauszuarbeiten und was die Bildung eines Wesens können, als Von der Kultur wird damit als echt nur unerläßlich durchzusetzen. gelten die sie was eine fördert, Weiterbildung der geistigen Wirklichkeit und zugleich unseres echten Selbst enthält; damit zu prunkend andere, so alles bloßen Menschenkultur, einer Soweit aber jene Wesensbildung gelingt, Befestigung und Vertiefung des Daseins auftreten es zu einer mag, sinkt Kulturkomödie. muß eine durchgreifende erfolgen; der Hauptaffekt des Lebens wird damit das Verlangen nach Wahrhaftigkeit, nach Befreiung von allem Schein. So entsteht ein eigentümlicher Typus des Lebens mit strengen Forderungen und mit aufrüttelnder Kraft; daß aber innerhalb dieses Kreises für mannigfache Bewegungen Platz verbleibt, das bringt die Tatsache mit sich, daß jene Wendung zu einem Beisichselbstsein des Hemmungen des mensch- Lebens sich unter den Bedingungen und muß; lichen Daseins vollziehen wird dadurch möglich, ja eine Mehrheit von Angriffspunkten Wir Menschen unentbehrlich. sind an das unmittelbare Dasein gebunden und bleiben auch für den Fort- Wir können uns gang des Lebens darauf angewiesen. nicht ein- uns der wesenhaften Einheit bemächtigen fach von jenem trennen, und von dort aus die ganze Wirklichkeit entwickeln, sondern auch wenn wir uns dorthin versetzt haben, bedarf es einer unablässigen Dabei stößt Befassung und Auseinandersetzung mit jenem Dasein, das geforderte Wirken aus dem Ganzen für das Ganze, das Getriebenwerden durch die innere Macht der Wahrheit, wie es echt- und Schaffen innewohnt, geistigem Leben Naturtriebe der Selbsterhaltung, zu Kräften einem zusammen mit dem hart den die Verflechtung mit geistigen grenzenlosen Egoismus eine steigert; völlige Wandlung der Gesinnung wird damit unerläßlich und erweist sich als Grundbedingung alles Geisteslebens echter Art; das hebt die ethische Aufgabe hauptet das über alles Wert. Aber zugleich hinaus. be- seiner Formgebung einen Was im Menschen an Geistigkeit aufstrebt, künstlerische eigentümlichen andere Wirken mit das hat zunächst ein rohes und seelenloses Dasein neben sich und verbleibt daher leicht in einem Stande der Halbwirklichkeit; erst weit über die eigentliche Kunst hin- das künstlerische Bilden, das ausreicht, bringt die verschiedenen wirkung, vermag in Seiten und Stufen der Berührung das Innere zu in Wechsel- gestalten, das Kultur. Äußere zu beseelen, das Leben bei 249 sich selbst zusammenzuführen. So gibt es keine volle Durchgeistigung des Lebens ohne die Kunst, ohne ihr bildendes und veredelndes Wirken vermag aller Eifer ethischen Aufschwunges es nicht vor Barbarei zu behüten. Endlich aber behauptet auch die Aufgabe der Lebenssteigerung ein Zum Geistesleben gehört Unbedingtheit, Unend- greifbares Recht. lichkeit, volle unan- Beherrschung der Wirklichkeit; der Mensch des un- mittelbaren Daseins aber steht unter zahlreichen Bedingungen Einschränkungen, er ist, Enge und Schwäche. und an jener Aufgabe gemessen, von kläglicher So bedarf notwendig einer Steigerung es seiner Kraft, einer Erweiterung seines Daseins, einer Schlummernden; ist es verwunderlich, daß Belebung alles dies besonderen Epochen das Ganze der Kultur zu bedeuten schien? Aus solchem Nebeneinander verschiedener Lebensrichtungen müssen schroffe Spannungen und harte Zusammenstöße erwachsen, und zwar keineswegs durch bloßes Irren und Mißverstehen der Menschen. Denn keine der Aufgaben läßt sich mit voller Hingebung ergreifen und mit voller Kraft verfolgen, ohne als Selbstzweck aufzutreten und im Augenblick des Handelns als die Hauptsache zu fühlen; so sich wird es begreiflich, daß im Ganzen des Menschenlebens nicht bloß ethische, künstlerische, dynamische Antriebe wirken, sondern daß sich eigentümliche Kulturtypen ausbilden und um die Herrschaft kämpfen. Abschwächungen und Kompromisse vermögen dagegen nichts, sie Aber wenn der Kampf drücken leicht das Niveau des Lebens herab. nicht zu vermeiden, ist, ja sein Nachlassen nicht einmal zu wünschen so wird um so wünschenswerter, daß etwas dem Kampf überlegen bleibe und einen Kampf gegen den bloßen Kampf unternehme. Das aber vermag nur die Belebung eines wesenhaften Seins, das durch alle Verschiedenheit hindurch sich selbst eriebt, das die verschiedenen Leistungen auf eine überlegene Einheit zurückbezieht, von da aus von dort aus zusammenzufassen mißt, winnen nun selbst alle Jene Bewegungen ge- strebt. eine Richtung auf die Entwicklung eines bei sich befindlichen wesenhaften Wirklichkeit; es wird hier ein und Geisteslebens Lebensraum geboten, einer in geistigen dem sie sich begegnen und auseinandersetzen können; etwaigen Konflikten stehen wir hier nicht wehrlos gegenüber, wir können zur Ausgleichung wirken, wir können der Festlegung bloßer die Ent- damit vor ein Ent- Teilkulturen wicklung einer Gesamtkultur entgegensetzen. Die Teilkulturen mit ihrer Arbeit treten Zu den Problemen des Menschenlebens. 250 weder— oder: dem Ganzen Ganzes ein finden — oder lösen sich sie , immer mehr fallen den Zusammenhang mit der Tiefe und sie — nur mit der Wendung zur Tiefe wird Leben das vom Lebensgrunde ab und ver- einer Vereinzelung? nach der Entscheidung Je Dort eine dorthin oder hierher ergibt sich ein schroffer Gegensatz. wesenhafte, hier eine wesenlose Art der Kultur; dort ein Insichauf- nehmen der Erfahrungen und Schicksale des ganzen Menschen und zugleich eine konkrete Gestaltung, hier eine freischwebende Betätig- ung und damit eine große Vagheit; dort eine Erhebung über alles zum Kleinmenschliche, mindesten ein tapferer Widerstand dagegen, hier eine Wehrlosigkeit der Geisteskultur gegen die bloße Menschendie Gegen- wesenhaften Geisteswelt bloßes Gesetzes- und Formel- So droht kultur. wart einer die Lebensbewegung ohne ethische wesen zu werden, zur Einengung und Bedrückung zu wirken, auch Pharisäismus auszulaufen; die künstlerische in einen selbstgerechten Gestaltung führt, auf sich allein das Leben gestellt, unvermeidlich ins Genießende, Weichliche, Spielende, die dynamische ins Selbstische, So hängt auch die Wahrheit der Teilkulturen daran, Wilde, Brutale. daß sie eine Wesens- und Gesamtkultur hinter sich haben, daß jene Zurückverlegung. der Kultur erfolge, die nur durch Anknüpfung an ein selbständiges Geistesleben möglich wird. Was die Idee einer zugleich wesenhaften und universalen Kultur, in ihrem Gegensatz zur ersten Kulturlage, an Folgen und Forder- ungen mit sich schäftigen. wird uns bei den nächsten Artikeln be- bringt, das Die Kultur wird sowohl in ihre Mittel in ihren Inhalt zu verfolgen sein: dort sind die schichte und Gesellschaft, hier die und Träger als Probleme von Ge- von Kunst und Moral in ihren mannigfachen Beziehungen zu erörtern; Punkt für Punkt wird sich zeigen, daß bei eine neue Sache jener Idee nicht und Aufgabe in bloß ein Frage steht neues Wort, Hier sei sondern nur noch das eine erwähnt, daß die gegenwärtige Lage die Forderung einer Zurückverlegung und dringlich macht. Diese Lage treffen zweier festeren Tatsachen ist kritisch Begründung der Kultur namentlich durch ein geworden. überaus Zusammen- Einmal sind die ge- schichtlich überkommenen Grundlagen und Inhalte der Kultur, soweit sie das Ganze und Innere des Menschen betreffen, sehr ins Unsichere sind das vornehmlich deshalb, geraten; sie Art als zu anthropomorph, zu kleinmenschlich empfinden und aus solcher Empfindung in Zweifel kommen, ob der Mensch überhaupt weil wir jetzt die ältere Kultur. 251 das sinnlich -natürliche Dasein irgend ob alles, überschreiten kann, was er an Mehralsmenschlichem zu erfassen glaubte, nicht ein bloßes Trugbild, ein Erzeugnis menschlichen tief in das der Welt Leben ein, dem das eine mit fällt Das greift sehr weit tiefer als diejenigen meinen, welche aus alle Geistigkeit entfernen und zugleich dem Menschen eine Denn in Wahrheit und wahren zu können wähnen. Idealität Wahnes sei. steht Punkt andern; es läßt sich unmöglich im und Subjekt erhalten, was für das Ganze und im Wesen aufgegeben wird. So sind wir aller unserer Ideale, ja unseres eigenen Wesens geworden, unsicher nicht mehr schöpfen wir aus einem gemein- samen Grundstock von Überzeugungen zusammenhaltende, erhöhende Kräfte; bei Verfall des aller subjektiven Regsamkeit ist richtende, ein innerer Lebens unvermeidlich, wenn jene Erschütterung weiter und weiter greift. Und in diese wankende und schwankende Zeit fällt hinein das Drängen der Massen nach vollem Teilhaben an Kultur und Glück, samt dem Anspruch, über das, was an der Kultur ge- stürmische und halt- wertvoll scheiden nach kraft sei, dem mit eignem Urteil zu entscheiden, zu ent- unmittelbaren Eindruck und nach der Fassungs- der Individuen, die von den weligeschichtlichen Bewegungen und Erfahrungen der Menschheit kaum irgend berührt worden sind. Nun macht jene innere Unsicherheit der bestehenden Kulturkomplexe, im besondern ihre schwere Belastung und Überunerschütterliche Wahr- mit Veraltetem lebtem, sie unfähig, solchem Verlangen eine und es damit in sichere Bahnen zu leiten; so Bewegung alles fortzureißen, wie sie schon jetzt vergröbernd und verflachend, verengend und verneinend wirkt. Über heit entgegenzuhalten droht jene eine solche Krise kann schlechterdings als ein nichts anderes hinausführen neuer Aufschwung des Lebens, eine Vertiefung des Geistes- lebens in sich selbst, die Entdeckung innerer Tatsachen Zusammenhänge. und innerer Von draußen kann uns das Heil nicht kommen; was an Stützen und Hülfen dort unwiderbringlich verloren ging, das können wir nur durch eine Verstärkung des Innern ersetzen, nur dadurch, daß wir bei uns selbst zu einer überlegenen Welt gelangen, uns darin befestigen, von daher unserem Leben einen Inhalt geben, von daher eine neue Kultur erbauen. tiefung und Befestigung, so kann die Gelingt solche Ver- bedrohliche Krise zu Erneuerung und Verjüngung des Lebens führen und durch einer alle menschliche Irrung hindurch dem Dasein einen größeren Wahrheits- 252 Zu den Problemen des Menschenlebens. gehalt verleihen. Besteht dagegen keine Möglichkeit einer solchen Vertiefung und eines Hervorbrechens ursprünglicher Kräfte, ist im menschlichen Dasein keine wesenhafte Geisteswelt neu zu beleben, so entfällt alle Hoffnung eines glücklichen Ausgangs, so müssen der menschlichen Selbstsucht und Leidenschaft Vernunft und Kultur unterliegen. 2. Zur Entwicklung des Problems. a) I Geschichte. |as Verhältnis des heutigen Menschen zur Geschichte ist voller -^ Verworrenheit: wir hängen an der Geschichte und zehren von aber zugleich fühlen wir unser Leben der Geschichte, aufs stärkste bedrückt durch sie und möchten uns dieser Last entledigen; in- dem wir aber das unternehmen, drohen wir der Leere des bloßen und Augenblicks zu verfallen, flüchten einen und Handeln solcher Gefahr doch dem anderen hin und her, eine Lage, die zielbewußtes und glückliches Schaffen unmöglich gedeihen läßt. Bewas trachten wir etwas näher, Das vor So schwanken wir zwischen dem wieder zur Geschichte zurück. 19. Lage führte. in eine solche Jahrhundert wird in seinem Verhältnis zur Geschichte vom Gegensatz zur Aufklärung mit ihrem Rationalismus beherrscht. Aus verworrenen Verhältnissen hatte die moderne Menschheit einen Ausweg durch ein Zurückgehen auf eine allen innewohnende Vernunft gesucht; nur ihre kräftige Belebung schien das menschliche Dasein von Veraltetem und Irrigem gründlich sowie befreien, das Leben von kindlicher Befangenheit und dumpfer Gebundenheit zu voller Mündigkeit und Klarheit erheben zu können. Die Vergangen- heit mit ihrer Autorität versank gegenüber der Forderung, das Leben und Wirken in eine zeitlose Gegenwart des Denkens zu unbeirrt durch die Überlieferung, meist in ihr, schuf die Vernunft eine »natürliche" Religion, eine Moral, «natürliches" ein Wirtschaftsleben, stellen; bewußtem Gegensatz zu eine ;, natürliche" «naturgemäße« Er- Gemüter überwältigend fortgerissen und in die Gestaltung des Lebens tief gewirkt; was es daraus an Frische, ziehung. Freiheit Das hat die und Selbständigkeit gewann, das konnte trotz fehdung und Verdunklung nicht wieder verloren gehen. Anfang an trug jenes Streben Problematisches in sich, aller Be- Aber von das im Zu den Problemen des Menschenlebens. 254 Laufe der Zeiten wuchs und endlich einen Das jugendliche Kraftgefühl, mit Rückschlag hervorrief. dem die Aufklärung einsetzte, gab ihr die freudige Zuversicht, einer absoluten den überkommenen Verhältnissen konnte Wahrheit nahe zu sie sein; siegesgewiß so nicht entgegentreten ohne ein festes Vertrauen auf das unmittelbare Walten und der Wirklichkeit im Menschenwesen. So einer Vernunft in schien denn in jedem Einzelnen die Vernunft angelegt und durch eine Selbstbesinnung leicht erreichbar; kräftige Erhebung zu Wahre zur voller Bewußtheit dünkte Herrschaft zu eine Klärung, Damit ward bringen. vornehmlich auf Denken und Erkennen gestellt, eine um das Gute und genügend, die Lebensarbeit die Kultur erhielt einen einseitig intellektuellen Charakter; beim Nachlassen des ersten Aufschwungs zog Räsonnement das Leben verstandesmäßiges ein mehr und mehr an sich, zwischen den Menschen und die stellte Dinge sein Erwägen und seine Zwecke und gefährdete damit immer einen stärker inneren Zusammenhang ward schließlich als drang schlug bildet die und eine Un- zu eng und seelenlos empfunden, der Lebens- um und verlangte mehr Inhalt sowie mehr Betätigung ganzen Menschen; des mit der Welt Die Wirklichkeit, die daraus hervorging, mittelbarkeit des Lebens. Hauptstück dieses neuen Lebens aber ein Wendung zur Geschichte. Denn was zu ein Durst nach mehr Grunde des Daseins, nach mehr Anschauung und mehr Lebensfülle, auch nach mehr Verbindung der Mannigfaltigkeit zu großen Zusammenhängen. Wie viel gesättigter ihr trieb, war vor allem Wirklichkeit, nach einem breiteren das Leben und dadurch ward, das zeigen Religion, die sonst ungenutzt verblieb, lichkeit, einzelnen Gebiete, alle Kunst und Wissenschaft; unendlich ist hier viel Recht mehr Wirk- dem eignen Tun ver- Das Ganze der Arbeit erzeugt eine historische Denkweise und verändert damit den Charakter des Lebens. Es reißt sich hier bunden. nicht der Mensch, wie in der Aufklärungszeit, los und stellt sich zu beherrschen, damit ihr Leben freie. ihr schroff gegenüber, sondern in er ersehnt ihn überströme von seiner Umgebung um sie wie etwas Fremdes eine innere Einheit mit ihr, und ihn von aller Kleinheit be- Damit gewinnt sein Dasein wie mehr Weite, so auch mehr Ruhe, aus den Dingen wächst dem Menschen eine Vernunft entgegen, deren Führung er getrost sich anvertrauen darf. auch die früheren Zeiten nahe und Verwandtschaft finden; läßt in Das rückt ihm ihnen die mannigfachste die eigne Zeit erscheint als die Spitze eines Geschichte. 255 Gesamtbaus, der alle Zeiten umfaßt; von solcher Spitze aus erscheint Frühere alles als ein wird Niederen allmähliches Ansteigen zur Höhe, auch in dem sowohl der Abstand und Gegensatz, die nicht als Annäherung und Vorbereitung gesehen; es kann mehr Verständnis und mehr Liebe finden, wenn die Schroffheit einer absoluten Schätzung, wie sie ung eigen war, dem Reformationszeitalter und auch der Aufklär- einer universaleren Keine Zeit hat solchen weicht. und versöhnlicheren Denkweise Umschwung der Behandlung stärker erfahren als das Mittelalter. Diese mehr relative Behandlung besagte aber zu Anfang keines- soluten Wahrheit. zum Relativismus und eine Preisgebung einer abDenn ein stolzes Selbstbewußtsein ließ die geistige Kraft sich allem Zustrom des wegs ein Sinken Stoffes gewachsen und seiner Assimi- Die Vernunft zog, wenigstens lierung fähig fühlen. in der Denk- weise der Philosophen, unter eigner innerer Erweiterung weit mehr die an Geschichte sich, als daß sie sich ihr unterworfen hätte. Diese Denkweise hat den großartigsten und durchgebildetsten Aus- druck in der Geschichtsphilosophie Hegels gefunden; zwischen Vernunft indem und Geschichte scheint Spannung überwunden, und gar zur Entwicklung der Vernunft die Geschichte ganz wird, diese aber in solcher Entwicklung ihr Welche Bedenken alle glücklich Konstruktion diese Wesen findet. der Geschichte erregen mag, die Überlegenheit der Vernunft und damit die geistige Aktivität wurde dabei kräftig gewahrt. Minder besorgt war dafür eine Be- handlung der Geschichte aus der Denkweise der Romantik. Denn hier schien ein unbewußtes Walten und Weben die Bewegung her- vorzubringen, aus der Vergangenheit schien dem Menschen ein Strom der Vernunft mühelos zuzufließen und ihn sicher mit fortzutragen; mit der Hingebung an diesen Strom schien sein Leben und Das schwächte die Aktivität in sichere Bahnen geleitet. und verkümmerte das Recht der lebendigen Gegenwart; indem man sich in vergangene Zeiten einlebte und diese dabei idealisierte, verSchon hier schloß man sich leicht den Aufgaben der eignen Zeit. Streben erscheint die Gefahr, daß der Erweiterung des Gesichtskreises durch die Geschichte nicht die Kraft des Zusammenhaltens entspricht, und daher der Mensch seines Lebens Schaden Dann kam von den die Problemen bei und Aneignens äußerem Gewinn im Kern erleidet. dem des 19. Jahrhundert inneren eigentümliche Wendung und geistigen Menschen des Zu den Problemen des Menschenlebens. 256 Schaffens zur Arbeit mit ihrer Richtung auf das Gegenständliche der Dinge; bei der Geschichte besagte das ein der exakten Forschung gegenüber Gesamtbildern. rissenen Wendung Diese Deutschland gewonnen, da wußtheit in siegreiches Vordringen Konstruktion der hat sie eine in nur um- besondere Be- hier gegen Über- das wiegen einer spekulativen Behandlung ihr Recht erst erstreiten mußte Namentlich erhob sich gegen die Hegeische Konstruktion der Geschichte erschien Jene nach, kreise nach mehr Weite, ein Verlangen vidualität. indem ihre Tatsächlichkeit und Indi- zu eng schon dem äußeren Gesichts- Begriffe im Grunde nur die europäische als und sich namentlich um den Gegensatz von Kulturwelt umspannten Altertum und Neuzeit bewegten; sie litt aber auch an einer inneren Enge, indem sie die Individualität und Positivität der einzelnen Er- scheinungen aufs stärkste abschwächen mußte, tischen Gefüge einordnen zu können. um sie ihrem dialek- Das neue Verlangen nach reiner und unbegrenzter Tatsächlichkeit sah darin eine Vergewaltigung und eine Verfälschung der Dinge; mit um so glühenderem Eifer wurde die historische Forschung als eine Befreiung davon ergriffen. Diese Forschung hat jenes Verlangen nach Weite und Tatsächlichkeit in vorzüglicher Weise in Arbeit und Leistung umzusetzen verstanden, sie hat für diese Arbeit neue hat Methoden ausgebildet, sie durch Inhalt und Form eine eigentümliche Denkweise erzeugt und wirkt damit stark auf das Leben der Neuzeit. Diese Forsch- ung will keineswegs Philosophie sein, geht doch ihr Hauptanliegen dahin, die Geschichte von aller Bevormundung durch die Philosophie zu befreien und allein auf die eigne Kraft zu stellen; aber die Arbeit hätte unmöglich so siegreich vordringen und die Hingebung des ganzen Menschen gewinnen können, ohne bestimmte Überzeugungen in sich zu tragen und anzuregen. Die Forschung kann nicht das Verlangen nach einem reinen Tatbestande entwickeln und verfechten, ohne zu gewahren, wie viel zwischen dem Menschen und jenem Tatbestande der Überlieferung ein energischer nommen, heit als liegt, ohne vielfacher Subjektivität sowohl der eignen Auffassung innezuwerden; so wird Kampf zur Austreibung Leben sein Gelingen läßt das gewinnen. Indem jenes Streben Subjektivität unter- mehr Ruhe und Klar- dieser nach Tatsächlichkeit eine grenzenlose Fülle individueller Bildungen aufdeckt, und indem sich ihm der Lauf der Zeiten mehr an Einen Faden reihen läßt, Gewebe durcheinanderlaufender, kaum nicht vielmehr ein unermeßliches Geschichte. Fäden entwirrbarer kann wird, ersichtlich 257 Unvermögen des das von innen her zu durchschauen und in einfache Menschen, das alles mehr zweifelhaft sein; so wird ihm eine geboten, nicht mehr darf er von sich aus die Tatsachen zurechtlegen und abrunden wollen. Aber indem Begriffe umzusetzen, nicht bescheidene Zurückhaltung herrschen zu dienen er statt zu meßliche Bereicherung, alter Enge. Gewinn und ohne VerAber Verwicklungen erschienen gar bald, und aus dem Das zunächst ein schien alles wicklung. Gewinn des Wissens drohte Die Objektivität, sagt Befreiung von gründliche eine Leben eine uner- erfährt sein hat, Beziehung zum Subjekt, werden. keineswegs einfach. der Dinge nur das, was ohne sie Bealle Wirken des Denkens sind, so ihnen zu verzichten, denn dieses ist einmal ohne wäre auf alles Innere bei nicht zu fassen ist Lebens zu des Verlust gefordert wird, die reine Tatbestand der ein reiner alles ohne ein Aufgebot eignen Denkens, ohne ein Nach- Auch eine Scheidung von Großem und Kleinem, von Wesentlichem und Nebensächlichem in der Geschichte die aus dem Ganzen einer ist nicht wohl möglich ohne Maßstäbe, erleben und Miterleben. Überzeugung hervorgehen müssen.^ Eine Geschichte aber ohne und ohne Abstufung müßte ein chaotisches Neben- und Durcheinander werden, das kaum mehr Wissenschaft heißen könnte. Innerlichkeit Wie wenig die Geschichte, bei aller Abweisung der Philosophie, gewisser Grundüberzeugungen entbehren kann, das bekundet mit voller immer stärker wogende Streit über den Aber woher Überzeugungen nehmen, nachdem alle Philosophie verneint Deutlichkeit der neuerdings Hauptinhalt und die bewegenden Kräfte der Geschichte. diese ist? Die Zeit hat sich Einmal beholfen. und abgeschwächt, ein zwiefacher Weise geholfen oder vielmehr in erhält sich unverkennbar, als Ganzes aufs schroffste abgelehnt wurde. aber führende innere Stellung unbedenklich fest. Notwendigkeit des der Es Intelligenz ist das bei ein scheinung im Leben der Gegenwart. * S. versteckt die Hegel wird perhorresziert, Innewohnen der Vernunft irgendwelches irgendwelche wenn auch Einfluß derselben spekulativen Denkart, in der Fortschritts, Geschichte, irgendwelche diesem Prozesse hält Stück einer allgemeineren man Er- Die pantheistische Denkweise, darüber die vortrefflichen, besonnen abwägenden und selbständig Werke von Arvid Grotenfelt „Die Wertschätzung in der Ge- urteilenden schichte" 1Q03, und „Geschichtliche Wertmaßstäbe in sophie bei Historikern und im Volksbewußtsein", 1905. Eucken, Grundbegriffe. 4. Aufl. der Geschichtsphilo- 17 den Problemen des MenschenleDens. 2[u 258 Bewegung der Neuzeit hervorging, und die früher eine die aus der feste Überzeugung, auch eine freudige Lebenstimmung hinter hatte, erhält sich in Grundlage unsicher wurde. wie Begriffe und Humanität u. s.w. verbleiben schritt, digen wurden Kräften und blaß alles Vernunft, Geist, schattenhaft geworden, aus leben- aus fruchtbaren Ideen Schattenbilder, um so unwahrer werden, Das Ganze muß Fort- dem Denken ge- liefern Nur ist nach Erschütterung wisse Richtungen wie Wertschätzungen. jener Grundlage sich mannigfachen Wirkungen, auch nachdem ihre leere mehr Grundwelche jener gewinnen, Seele solche Überzeugungen unsere überzeugung schnurstracks widersprechen. Der kräftigste Vertreter Redensarten. Widerspruchs solches Pessimismus, der ist immer 19. Jahrhunderts weiter im der je des Verlauf um sich gegriffen hat. Indem er das Dunkle und Unvernünftige unserer Welt und mit ihr der Ge- Empfindung bringt, schichte voll zur unbarmherzig den zerstört er verklärenden Glanz, den der Pantheismus dem Dasein' verlieh; viel zu energisch hat er uns allen neue Gruppen von Tatsachen vor die Augen gerückt, neue Durchblicke des Ganzen eröffnet, Glaube an alte als daß der die Vernunft unserer Wirklichkeit schlechthin Ein merkwürdiger Widerspruch bleiben könnte. ver- liegt hier vor: die Stimmung der Menschheit verdüstert sich, Menschen wie Schicksale machen einen trüberen Gesamteindruck, die Widersprüche des Daseins fallen der Zeit jene Dinge fest, um Halt, sie uns grell ins pantheistische Aber zugleich Auge. hält die Arbeit Denkweise mit ihrer Idealisierung der klammert sich daran wie an den einzig möglichen Der Pessimismus nicht völliger Nichtigkeit zu verfallen. und der Optimismus hier gewähren ein Beispiel für jene Entzweiung von Seele und Arbeit, für jene Zerklüftung des ganzen Menschen, woran das moderne Leben leidet Aber es gibt noch eine andere Weise, in der die Gegenwart dort des sich Problemes erwehrt: sie setzt den Zeiten gar keine be- stimmte Denkweise entgegen, sondern sucht ein Urteil über ein Maß selbst jene für sie lediglich zu entwickeln und zu erweisen; so ganz möchte versenken und daß einfühlen, sie lediglich Denkweise verstanden und gewürdigt werden. viel Bedeutendes geleistet; geschickt, fremden innerstes Wollen Zeiten und sie aus ihnen selbst zu gewinnen, an ihnen nie abzulauschen, volles Recht ihnen einen in eignen In dieser Richtung ist war eine Zeit so ihr aus sie sich ihrer zu bereit und auch so geben, ihnen Zusammenhang ihr nicht Geschichte. von draußen aufzudrängen, her 259 sondern ihn ihrem aus eignen Wirken und Wollen hervorzulocken, dabei mit gleicher Liebe in das Verschiedenste und Widersprechendste hineinzuversetzen. wir in Wahrheit bei ob trotz äußern, nicht tivität unsere Zeiten besser urteilen solchem Streben Abwehr aller Subjektivität die in Ob uns eignen Art ent- vermeintliche Objek- werden spätere darüber miteinfließt, wir als aller sich Eine Gefahr aber und eine selbst. Schädigung erfahren wir selber deutlich genug: es ist die Abschwäch- ung unseres eignen Wollens und Wesens durch jene Beflissenheit, fremder Art uns anzuschmiegen und anzupassen. Indem bei der unermeßlichen Erweiterung des Horizontes Mannigfachstes und Verauf uns schiedenartigstes verleiht, eindringt, uns bewältigt und seine Farbe wird unsere Seele zu einer Bühne, auf der alle möglichen Personen auftreten und ihre Rolle. hersagen; wir vergessen, daß die Ausdehnung unseres Vorstellungskreises keineswegs schon ein Weit- werden unseres Lebens bedeutet, und neigen dahin, gelehrtes Wissen für geistiges Leben einzusetzen; wir geraten in Gefahr, über jenem Mitleben, das schließlich immer ein Halbleben bleibt, ein eignes und Leben, ein klares Denken und festes Wollen mehr und mehr volles einzubüßen; die geistige Synthese, die wir an allen früheren Zeiten aufzusuchen bemüht sind, vermögen wir an der eignen nicht zu vollziehen. Am meisten wird unsere Schwäche bei ersichtlich fremdem Streben zu unserem eignen zu heit uns sicher zu Hause, wir fühlen gekommen dem Ver- Übergang von der Vergangenheit zur Gegenwart, von suche, einen In der Vergangen- finden. wir durchschauen, wir verstehen, wie das eine aus ist, wie alles dem anderen ent- sprang, entspringen mußte, wir verfolgen solche Betrachtung bis an die Schwelle die sich der Gegenwart; Verbindung uns mitteilen würdig genug verbleibt, ist hergestellt, und in nur ein einziger kleiner eignes Leben verwandeln. — dieser kleine Schritt, der Ertrag der langen Arbeit und kann Aber merk- Schritt will nicht gelingen, die Kluft Wissen und Leben kommen nicht zusammen. Ja der FortWissens hemmt die Verbindung der Geschichte schritt des historischen mit dem Leben. Denn je deutlicher die Wissenschaft die Eigen- tümlichkeit fremder Zeiten herausstellt, desto mehr zeigt sie die Ge- bundenheit ihres Gehalts an besondere Lagen, desto schärfer zieht sie ihre Grenzen gegen andere Zeiten und Denkweisen, desto zwingender verbietet sie ein einfaches Überströmen fremden Lebens 17» Zu den Problemen des Menschenlebens. 260 auch darin Diese Spannung zwischen Wissen und Leben kommt zum Ausdruck, daß die Forschung besondere Liebe weit- entlegenen Zeiten in das eigne. Denn feiert Leben eignen und dort spendet steht; mehr wir uns aber der Gegenwart nähern, je unabweisbarer wird diese Frage und desto peinlicher unsere desto Nirgends Unsicherheit. solche Kluft zwischen Wissen und Leben, ist Lebens und Vorbedingungen geistigen zwischen Religionsforschung wie fortgeschritten, Leben geistigem Wie weit ist heute selbst schroffer als auf dem Gebiete der Religion. die Triumphe glänzendsten die kommt minder zur Sprache, wie die Sache zum dort stehen präziser viel namentlich die großen Religionen und innerhalb ihrer die einzelnen Phasen Augen, unseren vor wie über dieser Tatsächlichkeit, unsere eignen Leben, wie bleiben, wie ist hier solange wir nicht Fülle eine hier wenig gewinnen wir aus Überzeugungen, religiösen groß uns umflutet Aber wie wehrlos sind wir gegen- anschaulicher Tatsächlichkeit! unser eignes Und unsere Hilflosigkeit! die zu Kraft eigner ihr für religiöses wird es sie Gestaltung des Lebens finden; daß wir sie aber finden, daran hindert uns vor allem indem Geschichte, die indem haften läßt, sie uns am bloßen Schein Besitzes eines uns durch unablässige Befassung mit fremden sie Dingen eignen Denkens und eigner Verantwortung entwöhnt, indem sie gelehrtes Kein Wissen für Leben Wunder ist schaftliche Bewegung wieder ein es daher, einsetzt. daß von Zeit zu Zeit eine leiden- gegen die Geschichte aufwallt, daß auch heute Zorn gegen den entnervenden Historismus mit Verstrickung in ein Halbleben um sich greift. Vergangenheit von euch und stellt Gegenwart; dann wird es wieder seiner „Werft die Bürde der das Leben ganz und gar in die und wahrhaftig, dann erst frisch wird es euer eignes Leben werden." Ja, wenn solches Abschütteln so einfach wäre und nicht mit der erstrebten Befreiung vieles verloren ginge, auf das sich nicht wohl hält die Geschichte uns weit fester uns fest auch gegen unseren Willen. ein Erzeugnis einer geschichtlichen eigentümliche Färbung, ihr Nein verzichten als jene trifft auch empfunden, sobald hat Wie rasch Lage und erhält B. die sie hält daraus eine Solche geschichtliche Bewegungen wird der Verlauf der Dinge über z. Wahrheit besondere Schäden, und auch geschichtsfeindlicher ist In Denn die Opposition ist selbst ihr Ja trägt das Gewand der besonderen Zeit. Bedingtheit läßt! Gegner meinen, Aufklärung, die deutlich sie hinausgeführt alle geschichtliche Geschichte. Bindung abstreifen gestalten wollte, und das Leben gänzlich aus zeitloser Vernunft selbst zu einer geschichtlichen Größe, einer histo- geworden, wie rischen Kategorie 261 viel berührt uns ihr in wie jetzt Wer das Ganze eine ferne Vergangenheit, ein vergilbtes Dokument! der Geschichte überschaut, dürfte eine Art Wellenbewegung zwischen Anschluß und Verwerfung gewahren und zugleich sich überzeugen, daß Verneinung genau die gut zur Geschichte gehört wie die so Bejahung, daß der leidenschaftliche Ansturm gegen die Geschichte mit seiner Neigung, das Gegenteil des Vorgefundenen zu behaupten, weniger eine echte Abhängigkeit verändert. Unterschied, ob das bewußte Streben des Menschen mit der Ge- erheblichen Beim Widerspruch wird das sie geht. aus lediglich als Empfinden jedes Einzelnen der unmittelbaren überzeugend Aber wird dartut. das Leben nicht eng und arm? solcher Absteckung zur Oberfläche gedrängt und legt, nur die Art der einen es Gegenwart zu wahr nur anzuerkennen, was sich dem Denken und gehießen, schöpfen und als - Immerhin macht oder ob es gegen schichte, Leben Unabhängigkeit gewährt, Erscheinungen zer- lauter einzelne in bei Wird es nicht wenn das dem bloßen Individuum Gegenwärtige als Maß alier Dinge dabei Leidet gilt? die liefert gewachsenen Geisteswelt diejenigen unter ihren Denkern, legenheit des Geisteslebens Ausbau ihrer ist sie nicht gelangt; die Natur verfochten, begriffen erlegen, gewiß mit aus dem Grunde, mit ihrem reichen vertiefenden Gegen die mächtig sind im Gedankenwelt immer wieder dem Einfluß von Natur- schichte ihren auch mit größtem Eifer die Über- die gegen Die Aufklärung Denn zu einer festgegrün- ein anschauliches Beispiel davon. deten, der Natur der geistige innere Selbständigkeit, Charakter des Lebens nicht schwersten Schaden? Erfahrungen unbegrenzte umfängt, Gehalt, scheint ihren glaubten uns Welt, die das innere festen weil sie die GeZusammenhängen, geringachten zu von her draußen dürfen. über- Leben nicht aufkommen zu können, wenn es nicht selbst einen Zusammenschluß übersubjektiver Art gewinnt; dazu aber bedarf es notwendig der Geschichte. ferner wart zu stellen, Muß das Leben ganz und gar in die Gegen- sich selbst zerstören, da die Gegenwart unablässig nicht der Versuch, und Ganze in ein Nichts zu verflattern und verwehen eine andere wird, das Heute alsbald in ein Gestern umschlägt, so schließlich das droht? Vor dem Äußersten behütet uns freilich der Umstand, daß, wie sich zeigte, die Geschichte den Menschen auch gegen seinen Zu den Problemen des Menschenlebens. 262 Willen festhält; aber steht es dann nicht so, daß, soweit wir uns der Geschichte entledigen, wir das Leben verflüchtigen, soweit es uns aber einen Gehalt bewahrt, wir dieselbe Geschichte widerwillig bejahen, Abschüttelung deren zur Kraft und Wahrhaftigkeit des Lebens unerläßlich schien? So befinden wir uns in einer höchst verworrenen Lage, ja einem unerträglichen Dilemma: wir können die Geschichte weder festhalten noch entbehren; wir geraten ins Leere, wo wir sie abschütteln, wir ver- einem Schattenleben, wo wir uns ihr unterwerfen. fallen schnittsart Die Durch- mag sich demgegenüber mit Kompromissen behelfen und an einem Mittelding von Freiheit und Knechtschaft Gefallen finden, eine energischere Denkweise wird die Unmöglichkeit eines Kompromisses durchschauen und eine Überwindung des Gegensatzes verlangen. Ist von der Geschichte möglich, die zugleich eine aber eine Befreiung Versöhnung mit der Geschichte bedeutet, kann das Leben eine Übergeschichtlichkeit erreichen und zugleich der Geschichte einen Wert belassen, ist ein Lebenstypus denkbar, der nicht haltlos zwischen dem Rationalismus des 18. und dem Historismus des 19. Jahrhunderts hin- undherschwankt, sondern Ausbildung einer selbständigen Art das in Recht eines jeden anzuerkennen und zugleich zu begrenzen vermag? Ohne eingreifende Umwandlungen des ersten Anblicks und energische Fortbildungen des Lebens wir, ist das sicherlich nicht zu erreichen; sehen ob der Zusammenhang unserer Untersuchung Anhaltspunkte dafür gewährt. 1 b) Forderungen und Ausblicke. Die nächste Frage Geschichte irgend zu ist die, ob das menschliche Leben entwinden und ihr zutreten vermag; die Beantwortung dieser Frage aber ^ sich der gegenüber- selbständig hängt daran. Alle Erörterung hier soll sich auf diesen einen Hauptpunkt beschränken, für das Weitere sei auf meine Darstellung der Philosophie der Geschichte in der „Kultur der G^enwart" (Band: „Systematische Philosophie") verwiesen. Das Auftauchen mannigfacher Streitfragen und die Leidenschaft, welche ihre Behandlung hervorruft, bekundet jedenfalls deutlich, daß uns der Inhalt der Geschichte wie unser Verhältnis zu ihr wieder unsicher geworden ist; wie hätte sonst in emporsteigen abgetan galt? neuester Zeit können, die Geschichtsphilosophie wieder die vor kurzem noch den meisten als so mächtig erledigt und Geschichte. 263 wie über das Ganze des menschlichen Lebens gedacht wird, notwendig ein Bekenntnis hält vom Kern der Mensch ganz und gar zur Natur tut, steht Überschritte er sie ent- Gehört zum guten Teil er es , zu einem nie eignen Leben gelangen. ferner die Natur nur durch einzelne Eigenschaften, Ganzen die nicht im — daß Ganzen. — so bleibt er unrettbar dem Strom der Zeit außer Frage und kann daraus verfallen dieses und Seins gegründet wären, so eines Lebens käme er vielleicht zu irgendwelchem Weiterstreben, aber nie zu einer wahrhaftigen Befreiung von der Zeit. gewährt das und Bestehen Die einzige Möglichkeit dessen Anerkennung die einer selbständigen den Hauptvorwurf unserer ganzen Untersuchung Geisteswelt, wie sie bildet. Denn wie schon beim Problem der Entwicklung ersichtlich wurde, ist die Erhebung über dem Ordnung loser Durchgängig wird die Zeit Geistesleben hier dem und ein Streben Wirken aus zeit- und wesentlich unentbehrlich. die Richtung auf ein zeitlos Gültiges gegeben, nie kann hier die Wirkung und Anerkennung auf dem Boden der Geschichte eine Wahrheit und ein Recht begründen, sondern die Wahrheit lichen will Leben her dargetan die Vergangenheit die hier unmittelbar, So kann sein. von einem ursprüngin diesem Gebiete nie Gegenwart ersetzen, und Gestern wie eine Frucht der Blüte entwachsen. Heute dem Denn was frühere nie das Zeiten an geistigem Leben erzeugten, das besteht keineswegs dadurch fort, daß es einmal da war; es gilt hier nicht das Beharrungsgesetz der Natur, wonach jedes Ding den vorhandenen Zustand einhält, bis es von außen her darin verändert wird. Ordnung, daß von neuem zugleich, in daß Vielmehr gilt hier die andere und immer weiter sinkt, was nicht immer eignes Leben und Tun verwandelt wird. Das besagt sofort sinkt alles Geistesleben aus unmittelbarer Gegenwart hervor- gehen muß, daß jede Verdunklung dessen eine Abschwächung seines Auch innerhalb der menschunterscheidenden Charakters bewirkt. lichen Erfahrung ist über die Gegenwart deutlich genug, daß weniger die Vergangenheit als diese über jene entscheidet, daß sich dem- nach mit der geistigen Art der Gegenwart das Bild der Vergangenheit unablässig verschiebt. Wie verschiedenes wurde am klassischen nachdem, was das eigne Leben Altertum gesehen und geschätzt, je an Aufgaben und Bedürfnissen enthielt. Die Scholastik ihm eine weltliche Kultur zur Ergänzung einer suchte in religiösen Lebens- ordnung, die Renaissance eine Unterstützung ihres Verlangens nach Leben und Schönheit, die Aufklärung schätzte an ihm, soweit sie es Zu den Problemen des Menschenlebens. 264 schätzte, die Klarheit flüchtete sich und Nützlichkeit/ der deutsche Humanismus zu ihm aus der Verwicklung modernen Lebens So erschloß das Alter- zu einer lauteren, einfachen, großen Natur. tum jedem verschiedene Seiten, aber es als gab und gibt auch viele, denen es inmitten aller Emsigkeit gelehrter Beschäftigung geistig gar nichts erschließen kann, nichts erschließt, weil sie Leben entgegenbringen. Daran also entscheidende Hauptsache immer der Besitz anders vollziehen geistige bildet sein, auch uns nicht zu, fällt die bleibt einer Gegenwart, einer sie will selbst. Eine von uns selbst ge- bloßer Augenblick, sondern eine Be- sie kein ist festigung gegenüber ihm kein eignes und so wir Lebenden und Handelnden als Gegenwart alles, Die Prägung aber kann niemand Gegenwart geistig ausgeprägten liegt dem Augenblick, ein Leben zeitloser Art. Ein solches Leben wäre aber nun und nimmer erreichbar, selbst ja das Streben danach würde eine Torheit, bestände nicht eine ewige Ordnung als neue Art der Wirklichkeit, und wäre eine sie nicht auch innerhalb unseres Lebensbereiches irgendwie gegenwärtig. Denn was hülfe uns jene Ordnung, wenn sie nicht auch in uns wirkte? Ohne das gibt es also keine Befreiung von der Geschichte, während die Wendung dahin einen sicheren Standort ihr gegenüber erreichen läßt. Da es uns zu solcher Anerkennung einer selbständigen Geisteswelt bei allen Problemen drängte, so kann ihre Forderung hier nicht Aber zugleich überraschen. erscheint eine muß, steht bei ihm in Befunde des Daseins. zerfällt in Das Geistesleben und stellt alle Mannigfaltigkeit Menschenleben ungeheure Verwicklung dem er letzthin irgendwie wurzeln schroffem Widerspruch mit dem nächsten beim Menschen: jenes Geistige, in in ist vor allem ein Ganzes umfassende Zusammenhänge, das individuelle Kreise, innerhalb derer die einzelnen Erscheinungen bunt durcheinander wirbeln; dort treibt die und Freude der Sache das Handeln, hier herrscht die Berührung mit geistiger Kraft innere Kraft natürliche Selbsterhaltung, die in der sich leicht zu einem unbegrenzten Egoismus steigert; der dort gefor- derten Ewigkeit widerspricht die strenge Gebundenheit des Menschen an die Zeit, der unaufhörliche Fluß aller Lebenserscheinungen samt dem raschen Versinken der Individuen; im Geistesleben gewinnt die Welt einen selbstseins, ^ und gestaltet sich zu einem Reiche des Beisichder Mensch hingegen scheint geistig leer mid wehrlos Inhalt Leibniz liebte die Alten Foucher de Careil, lettres et opuscules II introd. XXXIII) wegen la clarte dans l'expression et l'utilite dans les choses. (s. Geschichte. 265 Wie läßt sich eine so schroffe Kluft gegenüber der Unendlichkeit. überwinden? Das Erste ist Umkehrung innere jene sicherlich des Lebens, die Erhebung über die bloßmenschliche Art, die Versetzung auf den Standort; geistigen das tut in Wahrheit Arbeit, alle die auf das Ganze geht und zum ganzen Menschen wirkt; es braucht hier nur als Ganzes verstanden und in volle Tätigkeit aufgenommen zu werden, Aber was mit tausendfachen Wirkungen unser Leben durchdringt. Umkehrung und solche neue solcher Standort führt Es war eine das neue Leben zu genügender Entfaltung. weiteres ohne nicht Überspannung menschlichen Vermögens, wenn man von hier aus unmittelbar durch ein möglichst energisches Kraftaufgebot alle Geistigkeit hervorbringen wollte; diese Überspannung hat sich durch die viel zu blasse und schattenhafte Gestaltung der Welt gerächt, die Nachdem Schranken des Menschen daraus hervorging. deutlich genug vor Augen gerückt sind, werden wir nicht so die uns leicht wieder die Wirklichkeit aus freischwebender Tätigkeit zu konstruieren versuchen. So bedarf nach Entfaltung unser Streben einer zeit- überlegenen Geistigkeit einer wirksamen Unterstützung; eine solche liefert ihm aber die Geschichte. Nicht freilich die Geschichte, wie sie als ein ungeschiedenes Ganzes an uns kommt; denn dies Ganze als ein Reich von lauterer Vernunft, eine reine Entwicklung des Geistes- lebens zu verstehen, darauf haben wir einstweilen verzichtet. das schließt nicht aus, daß sich Aber innerhalb der Geschichte irgend- welche Eröffnung des Geisteslebens vollzieht, daß sich eine esoterische Geschichte von einer exoterischen bloßmenschlichen abhebt; in , jener eine Geistesgeschichte mag leben hervorbrechen, das durch allen ein von der selbständiges Geistes- Wandel der Zeiten hindurch auch zu uns spricht und unser eignes Streben zu fördern vermag. Am sinnfälligsten erscheint solches Geistesleben an einzelnen Höhepunkten, die klassisch heißen, weil an ihnen das Schaffen der bloßen und dem bloßen Menschen überlegen wird. Das wahrhaft Große waren dabei nicht einzelne Gedanken und Bestrebungen, sondern eine neue Art des Lebens gegenüber den Zwecken und Meinungen des Alltages; es vollzog sich dabei eine Umkehrung und Zeit mit ihr eine Erschließung geistiger Lebensquellen, geistiger Kräfte und Notwendigkeiten, eine Befreiung des Menschen von bloßmenschohne einen Zusammenhang mit dem übrigen Leben, nicht ohne mannigfache Vorbereitung und licher Art. Gewiß geschah das nicht Zu den Problemen des Menschenlebens. 266 eine enge Beziehung zur geschichtlichen Lage, aber nun und nimmer war jenes Klassische mit dem, was sein Wesen ausmacht, eine bloße Summierung und Weiterbildung vorhandener Elemente. Vielmehr ihm immer ein Bruch und eine Umkehrung, eine Versetzung auf einen neuen Standort, ein Gewinn eines neuen Lebens- erfolgte in raumes, ein Aufbauen einer geistigen Wirklichkeit. So pflegte denn auch sein Hervorbrechen schwere Erschütterungen mit sich zu bringen, und soweit es siegreich wurde, es das durch ist Kampf und Schmerz geworden, es hat seine Vorkämpfer auch da zu Märtyrern gemacht, wo das Märtyrertum nicht gerade durch Blut besiegelt wurde. Auch besagt die äußere Anerkennung, die das Große schließlich zu finden keineswegs pflegt, einen Sieg und reinen eine Umgestaltung der Denn jene Anerkennung ist zugleich ein Herabzum menschlichen Dasein und ein Anpassen an die klein- menschlichen Lage. ziehen menschliche Gesinnung; jedenfalls gelangt es hier nur mit einzelnen Wirkungen, nicht mit dem Ganzen seines Wesens zur Geltung. wird im Grunde So der Gegensatz nicht aufgehoben, sondern nur ver- und durch die ganze Geschichte bleibt echte Geistigkeit und steckt, bloßmenschliche Lebensführung in hartem Streit miteinander. Nun aber erscheint selbständiges Geistesleben nicht nur an vereinzelten Punkten, sondern diese Punkte suchen einen Zusammen- hang und möchten sich schließlich zum Aufbau eines allumfassenden Reiches verbinden. Dabei entstehen und harte Zusammenstöße. schwere Verwicklungen freilich Unter menschlichen Verhältnissen hat jede Eröffnung des Geisteslebens bemessene Schranken; wie sie das Problem nur an besonderer Stelle und nur angreift in besonderer löst, so wird sie das Ganze des Geisteslebens, das aus dem Grunde des Menschen wirkt, nicht voll befriedigen können; schließlich wird eine Gegenbewegung entstehen und neue Entfaltungen hervortreiben. Wiederum bringt das nicht bloß neue Ansichten und Richtung tiefsten Bestrebungen, sondern Erweiterungen und Vertiefungen des Lebensprozesses; es ist der Lebensprozeß und mit ihm die geistige Wirk- welche durch die Bewegung der Jahrtausende wächst; lichkeit selbst, es vollziehen sich in ihr Erzeugnis der bloßen Offenbarungen geistigen Lebens, die kein Reflexion der Tatsächlichkeit sprechen, daher erst sind, freilich sondern die mit der Macht einer Tatsächlichkeit durch Selbsttätigkeit anzueignender So wenig menschlichen diese Daseins Eröffnung geistigen einnimmt, Art. Lebens innerhalb geistiger, der die Breite geistigen des Arbeit Geschichte. 267 Höhe vor, ohne deren Erreichung voll zu befriedigen vermag. Was diesem weltgeschichtlichen Stande zurückbleibt, das mag die übt sie Macht und hält ihr eine sie und nicht wahrhaft zu fördern hinter Menschen und zeitweilig aufregen wird es auf fortreißen, schließlich überlegenen Widerstand stoßen und als unzulänglich befunden werden; indem er gewisse Lösungen jener weltgeschichtliche Stand wirkt negativ, als unzulänglich verbietet, gaben wirkt positiv, er und gewisse Anregungen stellt indem So kann dem Ganzen gibt. der Menschheit keine Lebensgestaltung genügen, welche nicht die und den moralischen Ernst seelische Vertiefung er gewisse Auf- in sich aufnimmt, die das Christentum brachte, aber auch keine, welche die Befreiung des Subjekts und den Gewinn einer inneren Unendlichkeit verschmäht, welche die Neuzeit errungen Demnach hat. liegen in der Geschichte, geistig angesehen, Anweis- ungen, Aufforderungen, Möglichkeiten; belebt sie können Zeit es ins sie wollen angeeignet und um zu vollen Wirklichkeiten für uns zu werden, aber sein, insofern jenes Geistige, so sehr besondere Nöte der das, Dasein hoben, seinem Kern nach zeitloser und daher bleibender Art es gilt ist; Eigentümlichkeit zu nur dieses Zeitlose dann ergreifen, kann seiner Kraft in es uns zur und lebendigen Gegenwart werden, dann ist die Geschichte kein bloßes Nacheinander und das Frühere keine bloße Vorbereitung eines Späteren, sondern dann hat jedes Große wie einen Selbstwert so eine unvergängliche Wahrheit, auch läßt sich dann über die Vielheit hinaus ein Ganzes Wenn so die Geschichte erstreben. aus einem bloßen Strom der Ereignisse zur allmählichen Eröffnung einer Geisteswelt, zum Gewinn einer zeitüberlegenen Gegenwart wird, so kann das Verlangen nach einem gehaltvollen Geistesleben durch (sie die kräftigste Unterstützung finden. und Nur es gilt vom eine Geistesgeschichte Zeitlichen von dem zum Ewigen durchzudringen sonstigen Chaos abzuheben. Dieses aber hat seine festen Bedingungen; es fordert zunächst, daß eine Tiefe des Lebens ein Ganzes können in jenseit den jenseit der nächsten Existenzform, und Denn nur so der einzelnen Funktionen wirke. Bewegungen Lebenstypen, mächtige der Weltgeschichte charakteristische Lebensströme aufkommen, die nicht an die Besonderheit des sichtbaren Ursprungs gebunden bleiben, sondern darüber hinaus ins Ganze wirken, wirken mit ausgeprägter Art, nicht mit vager Allgemeinheit. Nur so läßt im Wandel der Erscheinungen sich eine innere Einheit erkennen und in die Gegenwart überleiten. Zu den Problemen des Menschenlebens. 268 Zu solcher Überleitung und Aneignung gehört aber weiter, daß die eigne Zeit muß sich sie ein Geistesleben selbständiges aber ihre eigne welt- was in ihr an Selbständiggeistigem und Zeitüberlegenem erfassen, das ist, energisch herausarbeiten Wandel der Erscheinungen bloßen dazu Punkt des eignen Strebens geschichtliche Aufgabe, den springenden erreichbar entfalte; kräftig bei sich selbst konzentrieren, und sich zugleich über den sicher Unser erheben. selbst um bei anderen Charakteristisches zu geselbst Ewiges entdecken, um andere Zeiten und müssen wir inne werden, wahren, bei uns das Ganze der Geschichte auf ihr Ewiges zu bringen. sonders wird nur Hier be- dem gegeben, der da hat; hier ist besonders klar, daß die Vergangenheit die Gegenwart wohl zu erhöhen, nie aber zu ersetzen vermag. Soweit derart in Bewegung der Geschichte eine durchallen Wandel der Zeit hin- der gehende Aufgabe entdeckt und durch muß sich der Nun treiben wir nicht mehr durch zu einer zeitlosen Wahrheit vorgedrungen wird, Gesamtanblick des Lebens verändern. wehrlos mit dem Strom der Zeit dahin, sondern wir können ihm an ewiger Wahrheit eine Ruhe und Der Lebensprozeß wird sich nun durch die Erfahrungen der Geschichte hindurch immer gehaltvoller und immer gegenüber durch Teilhaben Festigkeit gewinnen. konkreter gestalten, das Geistesleben selbst in anschaulicherem Bilde vor Augen stehen, die besondere Art und Lage der Menschheit sich aufhellen, in dem ein allen Typus und eine be- charakteristischer harrende Art unseres geistigen Seins zur Entfaltung kommen. die damit das Festigkeit, gibt erlangt, selbst in ihm eine Die menschliche Leben im innersten Grunde Überlegenheit gegen die bloße Bewegung; dem Wandel wird es nun vor allem sich selbst erleben Mögen die Erimmer wieder in die letzte Grundlage zurückgreifen und den Menschen von neuem zum Probleme machen, was schon gesichert schien; mag, was an Ewigem in uns wirkt, in die Besonderheit der Zeiten eingehen und ihr gemäß und sich in seiner eigentümlichen Art bestärken. schütterungen des geschichtlichen Lebens sich gestalten müssen, trotzdem besagt es eine Wendung fundamentalster Art, wenn durch das Teilhaben an einer zeitüberlegenen Geisteswelt ein Ewiges im Kern unseres Lebens gesichert Aufgabe vor allem dahin gestellt wird, dieses in aufzunehmen und, was unser geistiges Wesen uns zuweist, Besitz zu verwandeln. und die unsere Tätigkeit in vollen Denn nunmehr läßt sich danach streben, in Geschichte. 269 der Geschichte Vergängh'ches und Unvergängliches zu scheiden und eine geistige Gegenwart zu entringen; sie erscheint nun nicht mehr als das Ganze, das seinen Zweck in sich selbst hat, sondern als eine bloße Seite des Lebens und Seins, die nur in Zurückbeziehung auf eine zeitlose Ordnung einen geistigen Gehalt und ihr irgendwelchen Sinn gewinnt. Nun und nimmer läßt sich demnach zu der älteren Art zurückwelche kehren, Ewige glaubte das uns und ergreifen als eine Verleugnung der lebendigen als eine Erstarrung, wart zugunsten Einem Zuge in können; die damit gewonnene Ruhe erscheint völlig durchbilden zu Gegen- Aber wir brauchen deshalb Vergangenheit. toter keineswegs der modernen Verflüchtigung alles festen Bestandes 2u verfallen, das Leben in einzelne Augenblicke aufzulösen, damit aber allen inneren Zusammenhang, alle überlegene Einheit preiszugeben. wenn unsere Verbindung mit einer umzukehren im Ewigen nehmen, Wirklichkeit vordringen, Beharrende werden alle inmitten sie Wahrheit einen sich unser Hauptstandort hindurch zu einer zeitlosen läßt Zeit aller Bewegung Die Vergangenheit festhalten. bloße Vergangenheit, wart so gestattet, durch zeitloser und durch eine Wesensbildung geistigen Charakter herauszuarbeiten das Leben Welt Denn ist ein überlegenes dann nicht mehr eine kann ein Stück einer zeitüberlegenen Gegen- und damit eine Sache eignen Lebens, unablässiger Arbeit bleiben. muß Die Wissenschaft Überzeugung eine eigen- aus solcher tümliche Behandlung geschichtlicher Erscheinungen entwickeln, die am und Zeitlichen das Bleibende, So geschah sucht. es z. B. am in Einzelnen Iherings das Ganze sieht großem Werk über den Geist des römischen Rechts, und zwar mit voller Klarheit über die Art des Verfahrens. Das Augenmerk ist hier «nicht das römische, sondern erforscht (3. das Recht, Aufl., Einl. IX), und und veranschaulicht am römischen" es wird demgemäß zur Aufgabe, „das Ver- und rein Römische von dem Unvergänglichen und Allgemeinen zu scheiden" (I, 15). Gewiß kann eine derartige philosophische Behandlung nur den Endpunkt einer langen wissenschaftgängliche lichen Arbeit bilden, aber wer sie in kleinmütiger Besorgnis vor ihren Gefahren abweisen wollte, den würde Hegels bekanntes Wort von der Metaphysik einen Tempel ohne ein Aller- treffen: er will heiligstes. Auch in das Leben des Individuums erstreckt sich die neue Zu den Problemen des Menschenlebens. 270 Nur so lange ist für den Art und gibt ihm eine neue Beleuchtung. Einzelnen das Dasein eine Flucht von rastlose Erscheinungen, als er eines selbständigen Innenlebens entbehrt, nicht irgendwie zu einem Ganzen persönlichen und Seins Individualität geistiger gelangt. Denn wo das geschieht und sich damit das Ereignis in ein Erlebnis zu verwandeln, der Mensch in Werk und Schicksal ein geistiges Selbst zu erleben vermag, da was uns irgend bewegt, nicht zieht, wie ein Schatten vorüber und versinkt sondern da vermag es Wurzel entfalten wie zu fördern, in den Abgrund des Nichts, uns- zu schlagen. in einer zeitüberlegenen Bleibendes zu Gegenwart sich ein- zuordnen. Dem Leben eine gehaltvolle Gegenwart zu sichern und es damit gegenüber dem Augenblick zu befestigen, das bleibt immer das Hauptziel; Liebe und solcher in Leid, in Gegenwart Daher haben Lebens wurde. fährt Glück und Unglück von jeher fort je ein zu wirken, was in Stück selbsteignen geisteskräftige Menschen über die Flüchtigkeit des Lebens zu klagen verschmäht, da es doch bei uns selber steht, uns jener Flüchtigkeit zu entwinden und unser Leben ins Unvergängliche zu und sich in «Ich bedaure die Menschen", stellen. «welche von der Vergänglichkeit sagt Goethe, viel Wesens machen Betrachtung irdischer Nichtigkeit verlieren; sind wir eben deshalb da, ja um das Vergängliche unvergänglich zu machen." So kann uns auch das bekannte Woil Dantes nicht als richtig gelten, daß das größte Elend darin ist bestehe, sich im Unglück vergangenen Denn war das Glück wahrhaftiges Glück, Glücks zu erinnern. es gar nicht zerstörbar, so so muß es durch alles Unglück hindurch mit lebendiger Gegenwart wirken. Auch die natürlichen Phasen scheinen in solchem ander. eine jede sie bleibt darin auf, spätere dem Leben seinen Gesamtstand. eine Jugend er- Zusammenhange nicht als ein bloßes Nachein- Diese Phasen leben sich weder gänzlich noch gehen Jugend, des Lebens, die Lebensalter, Daher ist in sich selber aus, Phasen vorzubereiten, sondern innerlich gegenwärtig und wirkt auf so wichtig eine frische wahrhaftiger Art; sie ist und freudige mehr als eine Sache sentimentaler Erinnerung, sie kann ein Stück einer weiteren Gegen- wart bleiben, eine unversiegliche Quelle frischen Lebens. Demnach ist der Mensch keineswegs ein bloßzeitliches Wesen; mehr Recht meinten tiefsinnige mittelalterliche Denker, daß er an der Grenze, dem Horizont von Zeit und Ewigkeit stehe und mit teil an ihnen beiden habe. Die Zeit ist für uns weniger ein starres Geschichte. 271 Schicksal als ein Problem; wie weit aber das Leben sie überwindet und Gegenwart erreicht, das hängt vor allem an eine überzeitliche der geistigen Kraft, die es aufzubieten vermag; bei uns selbst steht es ob der Schwerpunkt unseres Seins schließlich, oder ins Unvergängliche fällt. Allerdings hat und entbehrlichen Voraussetzung die Wirklichkeit ins Vergängliche dieses Tun zur un- die innere Gegen- wart einer geistigen Welt, auch die leidenschaftlichste Erregung des bloßen Subjekts kann nie einen geistigen Inhalt und mit ihm eine Zeitüberlegenheit erreichen, Schaffen zugleich und es Empfangen ein bleibt für den Menschen unsichtbaren aus alles Zusammen- hängen. Nach dem allen bedeutet unsere Abweisung des zerstreuenden und erschlaffenden Historismus keinen Rückfall in den Rationalismus. Das beiden gestellt, das von ist eine freilich ihm gestehen wir gern, daß, vor eine Wahl zwischen wir den Rationalismus vorziehen würden; denn mag entfaltete Leben noch so eng und einseitig sein, es und Wollen, während dem Historismus Nachbildung fremden Lebens genügt. Aber es bleibt genug, doch eignes Leben ein was uns vom Rationalismus scheidet. verleitete ihn, die Sein überspanntes Kraftgefühl Aufgabe zu unterschätzen; seine Verkennung des weiten Abstandes zwischen dem nächsten Dasein und der Tiefe des Menschenwesens von einer unmittelbaren Aufraffung ließ ihn warten, was in Wahrheit durchgreifende Vertiefungen ungen von einer Klärung konnte er nicht wohl verlangt; erhoffen, hätte er nicht die Vernunft schon in und vorhan/Jen er- und Umwälzalles Heil unserem Daseinskreise lediglich einer Freilegung bedürftig geglaubt. Das war überhaupt, weit über den Rationalismus hinaus, der Irrtum der Neuzeit, Wesen des Geisteslebens das Daseins zur Bewußtheit zu setzen; um in die bloße Erhebung des uns schien dasselbe wirksam, und dunkel, was in uns zur vollen Freiheit und Klarheit gelangt. Denn es wurden dabei die großen Widerstände und nur gebunden V^erwicklungen unseres Weltanblicks ebensowenig gewürdigt, als der Überwindung zur nötigen Tiefe vordrang. Ganz anders stellt sich die Sache, wenn das Geistesleben nicht als Lebensprozeß in ihrer eine bloße Aufhellung der Natur, sondern mit seinem Beisichselbstsein als eine wesentlich neue Art des Lebens verstanden wird. Wenn damit eine weit größere Spannung entsteht, so gewinnt auch die Geschichte an Bedeutung, nur wird dürfen, sie in ein Reich man nicht darauf ausgehen der reinen Vernunft zu verwandeln, son- Zu den Problemen des Menschenlebens. 272 dern zufrieden sein müssen, ihr irgendwelche in Eröffnung der Ver- nunft zu entdecken. Auch schichte war die Aufklärung einer Anerkennung der Ge- insofern wenig günstig, des Lebens der Wahrheit welche dort die Führung als die Intelligenz, einen hatte, zu engen und unduldsamen Begriff viel Eine bloß intellektuelle Wahrheit will unmittel- hat. bar als ausschließlich gelten, es verträgt sich hier nicht Verschiedenes nebeneinander; der Gegenwart Recht geben, heißt hier die ganze Wie sich die Lage völlig verwenn das Intellektuelle auf das Geistige aufgetragen wird, und wenn in der Geschichte nicht bloß Lehren und Meinungen, Vergangenheit ins Unrecht setzen. ändert, sondern Lebensentfaltungen und in ihr lichkeiten selbst Lebenskomplexe zusammentreffen, um Bilder der Wirklichkeit, bloß nicht sondern um Wirk- gekämpft wird, das dürften die früheren Ausführ- ungen zur Genüge dargetan haben. Entscheidend ist immer der Gewinn einer zeitüberlegenen hier Gegenwart mit ihrer Umkehrung des Lebens. Denn nur dadurch kann die mehr Geschichte nur dadurch Sache gelehrter Forschung werden, eine als läßt sich verhindern, daß die unbegrenzte Ausdehnung des Werdens und damit der geschichtlichen Betrachtung einen zer- Der Sieg einer geschichtlichen Be- störenden Relativismus bewirke. trachtung wohl der größte Nicht nur beim ist Forschung. Triumph Weltbau gesamten der und den bei neueren organischen Formen läßt diese Betrachtung allen vorgefundenen Bestand aus dem Werden verstehen, sie erstreckt sich bis in die elementarsten Vorgänge der weithin das leblosen Natur, Geschehen in umkehren indem einer selbst im Gebiet bestimmten Folge der Physik verläuft, sich Das menschliche Dasein aber erscheint in weit klarerem Bilde, seitdem die Gegenwart als das letzte nicht beliebig Glied einer langen Kette läßt. gewürdigt wird, Hauptrichtungen des Strebens manches was sonst als ein der Mensch bis fester Einsatz galt, in und nicht als veränderlich nur in den erkannt ist, sondern auch einleuchtet, wie seine seelische Art hinein von der Besonderheit seiner Zeit abhängt, wie verschiedene Zeiten verschiedene Menschen hatten. Ein unermeßlicher Reichtum des Lebens geht damit auf, das Verständnis wird weit präziser, indem es solchem Reichtum sich anschmiegt^ * Wir dürfen das alles als eine wesentliche Erweiterung Wir erinnern nur an Diltheys geistvolle Zeichnungen der Menschen verschiedener Jahrhunderte; auch Lamprechts Forschungen seien hier nicht Geschichte. 273 unseres Gesichtskreises, eine Befreiung von der Gebundenheit einer Aber der Gewinn an Wissen kann zu besonderen Zeit begrüßen. einem Verlust für das Leben werden, wenn Erweiterung eine Befestigung und Ewigen entgegenzusetzen. stärkung des das Zweite bleiben, sie nehmen, als er jener muß uns Die Geschichte darf nie das Erste werden. Überzeugung unser Daseinskreis dieser es nicht gelingt, dem Wachstum der Zeit eine Ver- sich weit Wohl wird bei unfertiger aus- dem Rationalismus und der konstruktiven Geschichts- philosophie erschien, aber woher sind wir denn dessen gewiß, daß bei uns der Kreis des Lebens sich schließt, und was schadet die bescheidnere Fassung, wenn dem Kleinerwerden des Menschen ein entspricht und das Leben an Tiefe Größerwerden der Wirklichkeit gewinnt, indem es sich minder einfach darstellt? Anhang: zum Begriff des Modernen. Der Begriff des Modernen bewegt und entzweit die Gemüter heute so sehr, daß einige Erörterung und Aufklärung nicht zu um- gehen des ist. Ausdrucks, Eine Aufklärung über die im Schwange zunächst fordert sehr unklare, wenn die nicht Geschichte irrige Meinungen sind. Das sachliche Problem reicht natürlich weit über die Prägung Den seelischen Stand der Gegenwart behandelt u. a. R. Baerwald, vergessen. „Psychologische Faktoren des modernen Zeitgeistes" (Publikation der Gesellschaft für psychologische Forschung). Es hat aber das Problem der Ab- hängigkeit des Menschen von seiner Zeit von alters her die Gedanken beschäftigt und sich schon Da sich für uns ein im 17. Jahrhundert zu einer Streitfrage zugespitzt. näheres Eingehen auf die Sache verbietet, so sei nur Walchs Philos. Lexikon angeführt (schon in der ersten Aufl. von 1726) Art. Sitten, S. 2377: „^t\\ nun solche Veränderung (nämlich der Sitten) fast unvermerkt geschieht, und wir es gemeiniglich nur gewahr werden, wenn es vorbei ist, daß diese und jene Sitten zu der und jener Zeit Mode gewesen, so pflegt man solches der Zeit zuzuschreiben. So haben einige einen genium seculi statuieren wollen, welcher nach den Zeiten die Gemüter der Menschen lenke und die Sitten der Menschen verändere. Dieser Meinung ist Barclajus, welcher in icone animor., pag. 505 (John Barclays icon animorum erschien 1614) sagt: omnia secula genium habent, qui mortalium animos in certa studia solet inflectere. Mit diesem stimmt überein der ungenannte Autor, der Germaniam milite destitutam geschrieben, und der sogenannte Pater Firmianus, von dem ein besonderes Buch unter dem Titel eine Stelle aus seculi genius, Paris 1663, Eucken, Grundbegriffe. 12 heraus(kam)". 4. Aufl. 18 Zu den Problemen des Menschenlebens. 274 des Ausdrucks zurück; wo immer daran lag, das Eigentümliche der Gegenwart abzugrenzen, da werden sich irgendwelche Bezeichnungen gefunden haben. ^ In ,/ modern" aber entstand beharrender Ausdruck, dessen ein Das Wort Geschicke etwas näher zu verfolgen sich lohnen dürfte. (abgeleitet modo von eben, jetzt) wird besonders geläufig sein, wenn innere Verschiebungen zum Bewußtsein kommen und die Menschen entzweien. Dann nennt sich der Freund des Neuen modern, um seine Überlegenheit gegen solche zu bekunden, die zähe am Alten haften; der Gegner aber stempelt den Ausdruck zu einem Schmähwort, das einen Menschen kennzeichnen und ohne Pietät Die Geschichte des Wortes Höhe sondere soll, der ohne Halt den flüchtigen Anregungen des Augenblickes erreichte, läßt ersehen, wann der Streit und welcher Punkt es folgt. eine be- vornehmlich war, der die Geister schied. Der Ausdruck erscheint im Übergang vom Altertum zum Mitteldem Grammatiker Priscianus, der im 6. Jahrhundert lehrte, alter bei dem Beamten Theodorichs (f um 575).^ In den folgenden Jahrhunderten findet es sich hie und da. ^ Zu einem eigentlichen Partei- und Streitwort wurde «modern« seit dem Ende des 11 Jahrhunderts, das aber auf dem Gebiete der Logik; es diente und bei Cassiodorius, . nämlich zur Bezeichnung der Nominalisten, d. h. solcher, welche den Begriffen des Denkens keine objektive Realität zuerkannten.* „Moderne" werden aber auch andere genannt, auch einfach die Gelehrten der * So hat z. B. Aristoteles wiederholt den Ausdruck o! vGv. bezefchnet vGv 1^ er deutlich (piXodo^fa, ^iQ-taaiv Toc die Platoniker seiner Zeit: y£y°^^^ ebenso auch lü69a, 26: y*P T^^T xaO^oXou, a (paaiv ol [ilv "^^ Met. Q92a, 33 (xaÖTri'jjiaTa tot; vüv Ta xaO-o'Xou oüaia? jiocXXov «px*? "O" ouafa^ etvai (xaXXov Sia to Xo^ixtü? ^T)T^v Ol 5c Jva'Xat ta xa^ ^xaorov, oTov nup xa\ ytjv, äXX'oü xo xotvov aöJjAa. * Cassiod. Variarum 4, 51 wird ein Architekt empfohlen als antiquorum imitator, * modemorum institutor. Ein Artikel der Historisch-politischen Blätter (139, 5 vom Jahre 1907) erwähnt einen Brief des Abtes Benediktus Avianensis (geschrieben zwischen modemos scholasticos, maxime apud Scotos [ijste Syllogismus delusionis, ut dicant trinitatem sicut personarum ita esse substantiarum. Mon. Germ. bist. Epist. Carol. Aevi, Tom. II, 563. * Prantl, Geschichte der Logik im Abendlande II, 82, führt die älteste Stelle an, wo die Nominalisten als modemi bezeichnet werden (non juxta quosdam modemos in voce, sed more Boethii antiquorumque doctorum in re discipulis legebat, nämlich Otto, seit 1106 Bischof von Cambray). 800 und 821), worin es heißt: Unde apud Geschichte. eignen 275 Einen bedeutenderen Gehalt und eine schärfere Zu- Zeit. ^ spitzung erhielt die Sache, als seit Johannes von Salisbury die Aristoteliker des 13. Jahrhunderts (also namentlich die großen Dominikaner, Thomas von Aquino, derselbe Mann, an den jetzt sich alles Antimoderne hängt) Moderne genannt werden, im Gegensatz zu der mehr durch Plato und Augustin beherrschten wie Albert der Große und Denkweise, die von der franziskanischen Theologie^ vertreten wurde. Diese «moderne" Denkweise erschien den Gegnern als eine Über- flutung der Theologie durch dialektische Sorgen und Spitzfindigkeiten.'^ Später überträgt sich der Begriff wie der Ausdruck Modernität auf Ockam und seine Schule, »Ockams Lehre blieb die ,moderne' Theologie bis in Luthers Zeit",^ auch Luther hat sich Aber es findet sich das Wort auch in vom gemeinsamen Leben eine devotio standen darunter eine solche, stark die den trägt vertraten »Innigkeit" Titel Aber Brüder modema und ver- welche neben der äußeren Haltung Werke von Johannes Busch über de origine devotionis modernae. ^ es dauert, lange, für das Bewußtsein lich ihr bekannt. anderer Bedeutung. betonte, eines der Das Mittelalter versinkt und Welt. zu klärt und bis die Renaissance eröffnet eine neue was feste in den Geistern vorgeht, Bezeichnungen annimmt. sich Natür- konnte der Renaissance „modern" nicht eine neue Art gegen- über dem Altertum, sondern nur eine neue Art in der Behandlung des Altertums bedeuten; da zugleich der mittelalterliche Sprach- gebrauch verblieb, so lief verschiedenartiges bunt durcheinander. Die epistolae ^ obscurorum virorum stellen das deutlich vor Augen. ^ Eine nähere Darlegung dessen hat hier kein Interesse, es auf Prantl verwiesen sein (s. z. B. II, Je mag dafür 116ff, 195, 241). ^ Von Roger Bacon werden Alexander von Haies und Albert genannt duo modemi gloriosi (s. den Artikel Scholastik von Seeberg in Herzogs Real- enzyklopädie). Der päpstliche Legat Simon de Brion, der tief in die Bewegungen welche damals die Pariser Universität aufregten und beinahe zerrütteten, erwähnt tadelnd die modema curiositas, quae plus solito innumeras multiplicat quaestiones (s. das ausgezeichnete Werk von Mandonnet: Siger de Brabant et l'Averroisme latin au XIII siede [1899} CCVIII, Anm. 1). * S. Seeberg, Herzogs Realenzyklopädie, 3. Aufl., XIV, 279. * S. den Artikel von Gustav Boerner: „Die Brüder des gemeinsamen Lebens in Deutschland" in den Deutschen Geschichtsblättem von 1905, Juni. ^ eingriff, S. namentlich S. 244/5. modernus bedeutet hier bisweilen nichts anderes als neu (modernus episcopus, modernus imperator), auch die ältere Bedeutung aus dem Streit * 18* Zu den Problemen des Menschenlebens. 276 mehr aber die Neuzeit mit dem Beginn des 17. Jahrhunderts eine Selbständigkeit und ein Selbstgefühl gewann, desto stärker mußte es von allem Früheren ab- die Gelehrten drängen, das Eigne deutlich zuheben und damit die Weltgeschichte anders zu ordnen und zuteilen als bisher geschehen Es war. wohl ist ein- namentlich der Aufschwung der Naturwissenschaften und die Blüte der französischen Literatur gewesen, welches der damaligen Zeit das Bewußtsein verliehen, Das ergab etwas neues und allen früheren Zeiten überlegenes zu sein. vor allem den Gegensatz der anciens und der modernes. kanntes Buch: des Parallele anciens des et modernes behandelt die Ausdrücke schon als eingebürgert, es so Hälfte lagen Reflexionen des Modernen War erfüllte.^ einmal (1688 der ff.) aber bezeich- ist 17. Jahrhundert nend für das Selbstgefühl, welches das zweiten Perraults be- Gegensatz seiner in geschaffen, über die eigentümliche Art des Antiken und nahe; wir wissen, wie Bewegung das viel hervor- gerufen und wie namentlich Schiller einer näheren Bestimmung dieser Begriffe hingebende und eindringende Arbeit gewidmet hat. Moderne Andererseits hatte das abzugrenzen, Das aber ist erst gebildet sein. bemerkt darüber trotz sich und dazu mußte der (a. a. gegen das Begriff spät Mittelalterliche des Mittelalters genug geschehen. selbst Bernheim O. S. 69): «Der Bann der Tradition dauerte doch noch lange einzelner Angriffe fort. Noch ein Sleidan, der bekannte Historiker des Zeitalters Karls V., nennt seine Chronik »De quattuor monarchiis" und hält trotz aller von ihm aufgeführten Zeichen der Auflösung des heiligen Römischen Reiches den Glauben an dessen Fortbestehen fest, weil eine fünfte irdische Weltmonarchie nach Daniels Prophezeiung unmöglich sei. Im 17. Jahrhundert kam et modemi), meist aber bezeichnet Anhänger der neuen humanistischen Denkart, z. B. poetae modemi; ex quo in Erphordia sumus modemi artista de via modemomm. Oft findet sich der Ausdruck nicht. ^ Es seien aus dem ersten Dialog jenes Werkes nur ein paar Stellen dafür angeführt: je pretens que nous avons aujourd'hui une plus parfaite der logischen Schulen erhält sich (antiqui es die ; connaissance de tous les arts et tous les sciences, qu'on ne l'a jamais eue. — Weiter spricht er von dem progres prodigieux des arts et des sciences, depuis cinquante ou soixante ans. Ferner: il ne faut que lire les joumaux de yeux sur les beaux ouvrages des academies de ces deux grands royaumes pour etre convaincu que depuis vingt ou trente ans il s'est fait plus de decouvertes dans la science des choses naturelles, que France et d'Angleterre et jetter les dans toute l'etendue de la savante antiquite. Geschichte. man zuerst lich 277 zu einer brauchbaren Einteilung des Stoffes. den Philologen und Literaten drängte Nament- das Bedürfnis auf, sich den augenfälligen Unterschied zwischen der klassischen und alterlichen Sprache und Literatur Bildung literarischen seit der Renaissance andererseits zum stehenden Ausdruck zu bringen, und oder oder von den Antoninen für die Literaturepoche von Augustus bis ins 15. Jahrhundert. Der Hallenser — 1707) war der in seinen Professor Christoph Cellarius (1634 Kompendien dieses Bezeichnung media es bildet sich so die medium aevum aetas mittel- der letzteren und der einerseits, auf Einteilungsprinzip gemeinen anwandte, indem er die unterschied es, Geschichte Historia im all- antiqua bis zu Konstantin dem Großen, und zwar so weit, nicht nur bis Augustus, weil, wie er ausdrücklich die innere erklärt, römischen Reiches noch weit über A und äußere Blüte des o^ustus' Zeit hinausreicht; Historia medii aevi bis zur. Eroberung Konstantinopels durch Historia nova. Und diese Einteilung nicht' ohne lebhaften Widerstand,^ durch." dem Mittelalter die Türken; drang allmählich, wenngleich So war „modern" auch gegenüber abgegrenzt; die weiteren Geschicke des Ausdrucks auf dem Boden der Neuzeit zu verfolgen, das würde ins Unbegrenzte führen und ist für unsere Aufgabe keineswegs nötig. So viel haben wir gesehen, daß der Ausdruck „modern" weit weniger ^ Wie langsam aie Sache sich durchgesetzt hat, und wie sie bis zum wenig bekannt. Ein Artikel der Revue des deux mondes vom 15. Januar 1907 über den hochverdienten belgischen Historiker Godefroid Kurth von Georges Goyau bemerkt über das Verhalten heutigen Tage Streit erweckte, der französischen Akademie ist zum Ausdruck Mittelalter folgendes: premieres editions du dictionnaire de l'Academie frangaise Les cinq contiennent au mot „moyen age" l'article suivant: „On appelle autheurs du moyen äge les autheurs qui ont ecrit depuis la decadence de l'empire romain jusque vers le X siecle ou environ." C'est seulement dans la 6e edition (1835) qu'on „Moyen äge, le temps qui s'est ecoule depuis la chute de l'empire romain, en 475, jusqu'ä la prise de Constantinople, par Mahomet, en 1453." Kurth selbst bekämpft den Begriff und Ausdruck Mittelalter sehr entschieden. In der Schrift Qu'est ce que le moyen äge tritt er dafür ein, einen einzigen Hauptschnitt mit dem Eintritt des Christentums zu machen, und so behandelt sein großes Werk Les origines de la civilisation moderne (3. Aufl. 1898) das Mittelalter als den Beginn der modernen Welt. In jener kleineren Schrift heißt es: Loin que le moyen äge soit intermediaire entre la civilisation antique et la civilisation moderne, le moyen äge est lui-meme le commencelit: ment de la civilisation moderne. Loin qu'il faille de depart de celle-ci aussi bas que l'epoque de stater au contraire qu'elle sort du christianisme. la faire descendre Renaissance, il le point faut con- Zu den Problemen des Menschenlebens. 278 modern ist, als elastischer Art Soviel man gewöhnlich und daß der Begriff sehr denkt, ist. zur Geschichte des Ausdrucks, nun einige Worte zur Daß der Begriff des Modernen so viel Bewegung und Streit kommt letzthin darauf zurück, daß es zum glücklichen Sache! hervorruft, Fortgang sowohl der Kulturarbeit eines Festhaltens des Alten als von Neuem bedarf. Wir würden schwerlich viel kommen, wenn wir immer von neuem beginnen müßten, wenn unsere Arbeit nicht geeignete Werkzeuge und leichteste Bahnen zu sicherem Besitz gewänne, wenn nicht vieles, was zunächst die volle Anspannung bewußter Tätigkeit fordert, ins Unbewußte und Gewohnheitsmäßige gebildet würde und damit freie Zeit für vordringendes Schaffen ließe. Wie nützlich, ja unentbehrlich ist z. B. der Philosophie der reiche Schatz von Begriffen und Kunstauseines Aufbringens weiter drücken, den die Arbeit vereinte Aber die Sache geht noch tiefer. von Jahrtausenden bereitet hat! Was an Wahrheit und überhaupt an geistigem Gehalt erreicht wurde, das konnte die Überzeugung und Hingebung der Menschen nur gewinnen, indem es sich über allen Wandel der Zeit hinaushob und alle Veränderung abwies; soweit wir Wahres echter Art besitzen, stehen wir über dem Fluß der Solcher Denkweise entsprang das Wort: Zeit. „Die Wahrheit war schon längst gefunden, Hat edle Geisterschaft verbunden; Das alte Wahre, faß' es an!" Das rechtfertigte die Hochschätzung des Alten und ung, die eigne Arbeit eng damit zu verketten, allen die Forder- und jeden schroffen Bruch zu vermeiden.^ ^ In der Philosophie ergab das den Gedanken einer philosophia perennis, der, schon in der Scholastik angelegt, mit besonderem Nachdruck von Agostino Steuco verfochten wurde (er schrieb de perenni philosophia libr. X, Bas. 1542); Leibniz nahm den Ausdruck auf, gab ihm aber aus der Idee einer stetig fort- schreitenden Entwicklung einen veränderten Sinn. Neuerdings hat namentlich Trendelenburg, wiederum in einer eigentümlichen Weise, den Gedanken der Stetigkeit der philosophischen Arbeit verteidigt: «Die Philosophie", so sagt er, »wird nicht eher die alte Macht wieder erreichen, als bis sie Bestand gewinnt, und sie wird nicht eher zum Bestände gelangen, als bis sie auf dieselbe Weise wächst wie die anderen Wissenschaften wachsen, bis sie sich stetig entwickelt, indem sie nicht in jedem Kopfe neu ansetzt und wieder absetzt, sondern geschichtlich die Probleme aufnimmt und weiterführt" (Vorwort zur 2. Aufl. der Log. Untersuchungen, S. Vlll). Geschichte. 279 Aber der Vertreter des Neuen hat dem manches entgegenzuwie äußere Dinge es tun, zur anderen, und anerkannt angeeignet und sein, in es will vermeiden der äußere wird Bestand und hältnis derselbe bleibt, immer von neuem der Aneignung wird eine gewisse Verschiebung sich schwerlich in von der einen Zeit Geistiges überträgt sich nicht so einfach halten. Auch wo lassen. sich das leicht dem Alten etwas Ver- man wird die Bewertung der einzelnen Teile verändern, anderes sehen und etwas anderes an ihm zur Dazu kom.men neue Lagen und Hauptsache machen. Probleme; der Mensch stellen neue kann diesen nicht gewachsen werden ohne auch innerlich weiterzustreben; Kulturen leben sich aus, neue Völker mit neuer seelischer Art; sollte das alles den Stand des erscheinen Geisteslebens gar nicht berühren? daß das überkommene Leben daß der eingeschlagene es ferner zweifellos ausgemacht, und steht, Weg uns gerade zum Ziele führt? Ja gibt wahrhaftiges Leben ein Ist unantastbarer Wahrheit in ohne eignes Entscheiden ohne Zweifel ein eignes Entscheiden, und ein und Kampf, ohne ein Umwandeln und Weiterbilden? Was an Verschiebungen erfolgt, mag zunächst innerhalb einer unantastbaren Welt zu liegen scheinen, ja die Veränderung wird lange Dann aber kommt ein wo die Spannung übergroß und eine Losreißung vom Alten hindurch überhaupt nicht empfunden. Zeit Punkt, für die Frische und die Wahrhaftigkeit des Lebens unerläßlich wird, wo die geistige Selbsterhaltung einen Bruch mit der Tradition und aus unmittelbarer Gegenwart zwingend verlangt. Ob und wann solche Umwälzungen nötig werden, darüber kann allein ein Schaffen die Erfahrung der Geschichte die radikalste von zeigt sie sie aber jedem Denn eine solche Umwälzung, viel- Unbefangenen deutlich genug. leicht belehren, allen, bildet das Eintreten des Christen- tums mit seiner von Grund aus neuen Schätzung der Dinge; das gute Recht und die ainer solchen Umwälzung dürfen auch die Reformation neue Wissenschaft für sich in Anspruch religiöse Leben der Neuzeit hätte seine Kraft nicht gefunden ohne nehmen. und seine ein selbständiges Neueinsetzen Das Innerlichkeit und ein Hervor- brechen ursprünglicher Kräfte, ebenso wenig hätte die neue Wissenschaft mit ihren völlig neuen Ausgangspunkten und Methoden sich allmählich aus der Scholastik heraus gestalten können. liche Leben darf es Das mensch- bedarf gewiß einer Kontinuität, aber nicht minder be- einer Diskontinuität, um in frischem Fluß zu bleiben und Zu den Problemen des Menschenlebens. 280 Nur das kann bei den Be- seine ganze Tiefe entfalten zu können. wegungen Sache des Streites sein, ob in ihr geistige Notwendigkeiten wirken und walten, oder ob dabei nur ein menschliches Bedürfnis nach Abwechslung in Frage steht. Das nämlich ist nicht zu leugnen, daß nicht alle Wandlung aus daß im menschlichen solchen geistigen Notwendigkeiten entspringt, namentlich im gesellschaftlichen Zusammenleben, auch ein Dasein, mehr Müdewerden des Menschen am subjektives Alten, ein Be- dürfnis nach Veränderung wirkt, wie es besonders greifbar die Tat- sache der Mode zeigt. einander, die Dabei entfernen die Zeiten sich weit von- einen fühlen sich indem wohl, ruhig die alten sie Wege verfolgen, andere zeigen eine eigentümliche Unruhe, ein Unbehagen an allem, was vorgefunden wird, eine Vorliebe für alles, was an Neuem auch sich dem mit Diese verschiedene Art hängt sicherlich regt. des Geisteslebens zusammen, jene Unruhe Stande bezeugt eine Kluft zwischen inneren Notwendigkeiten und äußerem aber solcher Lage bemächtigt sich leicht die Neuerungssucht Besitz, des bloßen Menschen und entwickelt eine Neigung, das Alte zu verwerfen, weil es alt, das Neue zu feiern, weil es neu ist. Demnach giU es zwischen Modernem echter und unechter Art zu scheiden, einem Modernen, in dem eine geistige Notwendigkeit wirkt, und einem Modernen, das bloßmenschlicher Lust und Laune Grundverschieden sind die Wirkungen und verdankt. sein Dasein Aussichten beider. Menschen nach Erzeugt die Bewegungen nur ein Verlangen der Veränderung, und Hin- ein Herschwanken der Stimmung, so mögen sie die Oberfläche noch so sehr erregen, sie dringen nicht durch bis zur Tiefe und gewinnen keine Kraft des Schaffens, derselbe Wind, der sie brachte, wird sie bald auch wieder verwehen, und der rasche Wechsel, der einem Extrem starke der ins andere umschlagen Ermüdung bewirken; Zeit, anders steht es, wenn die muß läßt, traurig das das an solchem Modernen Völlig leicht Menschen von schließlich eine Leben des Menschen wie haftet. ein Modernes echter Art eine Wendung des weltgeschichtlichen Lebens vertritt und ihrem Wahrheitsgehalt zur Anerkennung verhelfen möchte. in sich eine geistige Notwendigkeit, Dauer kein Widerstand solcher und Art wohnt eine zu deren hindern vermag. wunderbare Kraft. Denn dann trägt sie Durchdringen für die In einem Modernen Scheinbar vereinzelte zerstreute Vorgänge weisen dann nach derselben Richtung, die Geschichte. neue Denkweise, der Geist der Zeit dem an Wirkung, der eine Meinungen, Eingewurzelte ergreift die verschiedensten Ge- Weg in die entlegensten Winkel und übt auch einen findet biete, 281 sich ja als einen schroffen Gegner fühlt. Interessen selbstische verlieren ihre Macht gegenüber einer solchen Bewegung. — Schwierigkeit macht daß im nächsten Anblick der Dinge Echtes und Unechtes wirr nur, durcheinanderzugehen pflegt, des Flachmodernen auch verneinen zu Laune in daß auch das für der geistige während dürfen, schrittsgedankens die der Abweisung der glaubt Recht des Fort- flüchtigster Lage und andere das Erzeugnis Der Freund des Alten pflegt sich dann Anspruch nimmt. der Ordnung, der des Neuen als Vertreter mit eine Bewegung der Zeiten als Vertreter zu fühlen, jener dünkt sich moralisch, dieser der Freiheit intellektuell überlegen, jener glaubt besonders das Interesse der Gesellschaft, dieser das des Individuums zu wahren. Dabei eigentümliche Dialektik. Was alt ist, war einst neu; von Aquino galt einst kommen und Alter Sache in sich selbst eine trägt die auch Thomas «modern"; was heute neu als eines sich erwehren anderen ist, wird ins müssen. Das Moderne, das aufstrebt, zieht einen guten Teil seiner Kraft aus seiner oppositionellen Stellung; im Siege büßt und gerät in diese ein es Nachteil gegen neue Bildungen. Die Verwicklung, die aus solchem Kampfe hervorgehen kann, hat in der Gegenwart eine besondere Höhe erreicht. gegen alles Neue, ihn Seite findet sich der hartnäckigste Widerstand vertritt vornehmlich eine Auf der einen das römische System, große Weltmacht, dem Worte nach katholisch nennt, das aber in der Sache ist. Denn es setzt allen Eifer daran, die Bewegung der Menschheit in partikulare Bahnen zu leiten und das sich so unkatholisch wie möglich bei einer mittelalterlichen Gestalt endgültig festzulegen. aber erscheint die weiteste Verbreitung neueste Gestaltung des Kulturlebens schub. Das Tempo des Lebens eines leistet ihr Andererseits Flachmodernen, die den vielfachsten Vor- hat sich in unheimlicher Weise be- immer mehr Menschen drängen sich in die Groß- und Weltstädte zusammen; was dort zu Gehör kommen will, muß dreist, schleunigt, laut, ja schreiend auftreten, er zu bringen sich scheinen. auf der Neues, muß Neues, dem «Höhe der Bildung" sich fühlt. Prickelndes, Unerhörtes keiner entziehen darf, So der ein Sichüberspannen und Überbieten des Neuen, ein Schätzen des Neuen, bloß weil es neu ist, mag es an sich noch so leer oder töricht sein, dabei unendlich Zu den Problemen des Menschenlebens. 282 viel Schein, eine eitler des Lebens, Abneigung gegen keckem Verneinen, Lust an eine elende Talmikultur, ein allen Ernst und alle Tiefen in alles Versuch des Bildungspöbels, allem eine die geistige Bewegung der Menschheit unter sich zu bringen und sich zum Richter über gut oder böse, wahr oder unwahr zu machen. So wird das Moderne echter Art unmöglich vordringen können, ohne sich von Flachmodernen derartigem aufs zu allerschärfste und einen heftigen Kampf dagegen aufzunehmen. Sein eignes Recht aber können die Verirrungen des Flachmodernen nicht scheiden im Mindesten antasten. Unsere Zeit samtverlauf unserer Untersuchung, kommene Bahnen suchen hat; so zeigt es auch der Ge- eine solche und sie Selbstvertiefung des Lage stempelt schluß an das Alte zu einem bloßen und tum; für uns daß nicht über- ruhig weiterverfolgen kann, sondern daß energischer Selbstbesinnung zu ist, so geartet, gilt es auf allen sie in Lebens neue willfährigen An- unfruchtbaren Epigonen- uns selber zu stehen und den geistigen einen offnen Weg zu schaffen. Notwendigkeiten, die jetzt aufstreben, Dann wird sich mit frischer wart ein ewiger Wahrheit verbinden lassen, dann wird Festhalten und freudiger Ergreifung der Gegen- das eine das andere zu fördern vermögen. und Individuum. Gesellschaft 3. (Sozialismus.) a) Das Verhältnis von Gesellschaft und Individuum. a. Geschichtliches. t^eim Problem der Gesellschaft steht es heute ähnlich wie bei *-^ dem der Geschichte. Das 19. Jahrhundert hat einen Rückschlag gegen die Aufklärung gebracht, dieser aber voller hat, obschon noch in Wirkung, einen neuen Rückschlag hervorgerufen; so durch- kreuzren sich Wirkungen mit Gegenwirkungen und höchst verworrene Lage; sich ihr 7u entwinden erzeugen wird eine keineswegs leicht sein. Einige Worte seien zunächst den Ausdrücken gewidmet, soweit sie Aufklärung fordern. eine und Individuum Bildungen, aber die Neuzeit erst hat ältere sie Individualität geführt. Individuum hieß anfänglich etwas, das sich nicht trennen läßt; (XTOftov. die Mittelalter; spaltig." so ist bei Dieser Sinn älteste Cicero Individuum sind weiteren Kreisen zu- eine teilen oder Übersetzung von überwiegt wie im späteren Altertum^ so im deutsche Übersetzung (bei Notker) ist Aber schon dem Ausgang des Altertums bedeutet «un- indivi- duum auch das Einzelne als einzigartiges, von anderem unterschiedenes, in seiner Besonderheit ' So z. B. nur einmal vorhandenes;- das Mittelalter ver- Seneca de provid. 5: quaedam separari a quibusdam non possunt, cohaerent, individua sunt. '^ Bemerkenswert ist hier namentlich der höchst einflußreiche Boethius, aus dessen Kommentar tu Porphyrius (edit. Bas. 1570, pag. 65) folgende Stelle angeführt sein mag: Individuum autem pluribus dicitur modis. Dicitur In- dividuum quod omnino secari non potest, ut unitas vel mens; dicitur Individuum quod ob soliditatem dividi nequit, ut adamas; dicitur Individuum (üujus praedicatio in reliqua similiü non convenit, ut Socrates: nam cum illi sunt caeteri homines similes, non convenit proprietas et praedicatio Socratis in caeteris, ergo ab iis quae de uno tantum praedicantur genus differt, eo quod de pluribus praedicetur. Bei Porphyrius lautet die Hauptstelle (siehe Zu den Problemen des Menschenlebens. 284 wendet diese Bedeutung weiter und prägt auch 1 2. Jahrhundert) die Ausdrücke individualis (jedenfalls schon im Dem und individualitas. allgemeinen Leben führt diese erst Leibniz zu, auch hier ein Vermittler alter und neuer Zeit. Die Anfänge der Kultur zeigen das Individuum Stück als ein einer engeren oder weiteren Gemeinschaft, das in seinem Tun und Lassen durch solchen Zusammenhang wesentlich bestimmt wird. Die Weiterbewegung des Lebens pflegt das Individuum mehr und mehr zu verstärken, es gewinnt an Selbständigkeit, es beginnt nach einem Recht der überkommenen Ordnung zu fragen und ihre Vernunft zu dividuum endlich an einen Punkt, die Sache gelangt bezweifeln, Bindung abzuwerfen sucht und alle wo das In- eigne die Meinung zum Maßstab der Wahrheit, das eigne Wohl zum Ziel des Handelns Das erscheint solchen, die auf den Stand des Ganzen bedacht macht. sind, als eine verderbliche Zerstörung; so wird von ihnen ein kräftiger Widerstand geleistet und der Versuch unternommen, das Individuum bei Zuerkennung eines gewissen Rechtes wieder einem Zusammen- hange einzufügen und für seine Ziele zu gewinnen; geistige Arbeit soll was als natürlicher Besitz verlören ging. Zu solchem wiederherstellen, Zweck der Wiedereinfügung des Individuums ward zunächst der Begriff dem des Organismus verwandt, mit wir uns schon befaßten. Er scheint besonders geeignet, die Ansprüche des Einzelnen und der Gemeinschaft glücklich miteinander auszugleichen. digen Körper kräftiger es aber steht ist sich in Eigentümlichkeit ausbildet. seiner daß Glieder innerhalb des Ganzen zu verbleiben Glied aus allem Leben und aller vom Ganzen So kein Recht sich ablöst. gegen des Einzelnen auf das Weltall übertragen, wie von der Stoa geschah, mag Prantl, so Twv xaxä ?xa(JTOv, (le'po?. wv to a'9-potajjia alle hat, Das Betätigung der daß das einzelne Gestaltung herausfällt, sobald es duldet organische diese Wird Ganze. das es Fassung diese Lehre Bewußtheit zuerst mit voller ein besonderer Vorzug der Toiaüx«, oti I? JStoxrJTwv es Gesch. der Logik l. 629): axop-a ouv£(rn|xev Das Ganze um so höher, je differenzierter seine Teile sind. bildet eine unüberschreitbare Grenze, freilich Denn im leben- um so wertvoller für das Ganze, je jedes Glied Xe^e-cat oux av in -zä i'XXou Ttvo? izore ro auTo yivoixo Diese Definition geht durch die Kette der Jahrhunderte bis zu Leibniz, noch Jakob Thomasius, sein Lehrer, definierte: Individuum quod constat ex proprietatibus esse potest. quarum collectio est numquam in alio eadem Gesellschaft und Individuum. Einrichtung dünken, daß auch in 285 den kleinsten Dingen nicht zweies einander völlig gleicht, daß nicht zwei Haare, zwei Körner, zwei zusammenstimmen. ^ Blätter gänzlich Zu dieser organischen Lösung des Problems aber gesellt sich eine hierarchische, die, dem Ausgang der griechischen Philosophie ent- dem Boden des Christentums und im Mittelalter zur sprungen, ^ auf vollen Entfaltung gelangt ist und noch heute mächtige Wirkungen übt. Hier erscheint das All als eine fortlaufende Stufenfolge, das Leben von oben her in sicherem Fortgang nach jede Stelle hat von der nächsthöheren zu empfangen, fangene der niederen zuzuführen. eigentümlichen Wert und ein davon losreißt Werk, aber ins Leere, sobald fällt mitteilt; um das Emp- hier hat jeder Teil eigentümliches nur im Gefüge des Ganzen; er sie Auch bei der sich unten einen er hat er sich Dieser Gedankengang hat eine geschichtliche Ver- körperung nicht nur in der kirchlichen Hierarchie, sondern auch in der mittelalterlichen Lehnsordnung gefunden, indem hier jede Gewalt als von einem Höheren an den Niederen verliehen gilt. Die organische wie die hierarchische Fassung lassen dem Indivi- duum allen Wert aus dem Verhältnis zum Ganzen kommen, Wert für sich selbst wird ihm abgesprochen. ihm erst die Fassung, die sich als die ein Einen solchen gewährt mikrokosmische bezeichnen Hier wird das Individuum aus einem bloßen Stück der Welt läßt. zum Ganzen einer Welt, zu einer eigentümlichen Konzentration der Wirklichkeit, zu einer Stätte, wo die Unendlichkeit des Lebens unDas All wird damit eine Welt von Welten, Auch dieses aller umgrenzenden Zusammenfassung. mittelbar gegenwärtig es entzieht sich entstammt Bild dem der dabei voransteht. ist. spätesten Bei ihm Altertum, wiederum ist es Plotin, zuerst erscheint mit voller Klarheit der Gedanke, daß jeder Mensch eine eigne Welt bedeute und das All eigentümlich spiegle, «jeder einzelne sind wir eine geistige Welt.« Vom Neuplatonismus aus ist auch der Ausdruck Mikrokosmos, der bis auf Demokrit und Aristoteles zurückreicht, erst recht in Umgekommen. Im Mittelalter hat namentlich die mystische SpekuGedanken festgehalten, von hier kam er durch verschiedene Zwischenglieder (Nikolaus von Kues, Giordano Bruno) an die lauf lation jene ^ S. Cicero acad. quaest. II; dicis nihil idem quod sit aliud; Stoicum quidem nee admodum credibile, nullum esse pilum omnibus rebus talem, qualis sit pilus alius, nullum granum etc. est ' Am meisten hat dafür Plotin gewirkt. Zu den Problemen des Menschenlebens. 286 moderne Philosophie, um in Leibnizens Monadenlehre eine präzisere gewinnen. Gestalt zu In engem Zusammenhange damit steht die und Persönlichkeit in unserer hohe Schätzung von Individualität klassischen So führt auch Literatur. hier fortlaufende eine Kette vom Ausgang des Altertums bis zur Höhe der Neuzeit. Zwischen der organischen und der hierarchischen Fassung einerder mikrokosmischen andererseits besteht ein voller Gegensatz, seits, der einen Kampf unvermeidlich ein bloßes Glied, hier an ist es macht. Dort ein selbständiges ist das Individuum Ganzes, dort hat es den geistigen Gütern nur durch die Vermittlung einer Gemein- schaft teil, hier vermag es sie unmittelbar bei sich selbst zu erreichen. Damit muß fallen. Dort wird zur Hauptsache die Leistung schaft, hier auch der Lebens des Inhalt grundverschieden für die aus- Gemein- die Entfaltung des Individuums selbst in seiner Inner- und lichkeit. Auch Kraft erst von den Individuen empfangen und ihnen nie als Selbst- die Gemeinschaft wird hier zweck entgegentreten dürfen. ihre Es wird aber bei solcher Befreiung das Individuum seinem Leben einen lich ihre Gestalt bedeutenden Inhalt vornehm- durch ein unmittelbares Verhältnis zur Utiendlichkeit, zu den sei es, daß sich das mehr mehr künstlerisch oder mehr religiös gestaltet. So verschlingt sich mit dem Kampf zwischen Individuum und Ge- schaffenden Quellen des Lebens, geben, wissenschaftlich oder sellschaft das Problem, ob wir die Hauptaufgabe unseres Lebens mehr beim menschlichen Zusammensein oder im All zu suchen haben, ob an erster Stelle eine soziale oder eine kos- mische Lebensführung zu erstreben Wandel der Zeiten zu zum Verhältnis verfolgen, ist ist. Diese Fragen durch den hier nicht wohl möglich; wenden wir uns daher sofort zur Lage und Stimmung der Gegenwart. Es unterliegt aber unsere Zeit dem Einfluß dreier Strömungen von verschiedener Stärke und Breite: es sind das die Gesamtbewegung der Neuzeit zum Individuum, der Rückschlag des 19. Jahrhunderts zu gunsten der Gesellschaft, die Wiederbelebung des Individualismus gegen Ende des 19. Jahrhunderts. Die Emanzipation des Individuums ist wohl der hervorstechendste Zug des gesamten modernen Lebens. Der Einzelne erstrebte und gewann dabei sowohl ein unmittelbares Verhältnis zur Gottheit und zum All, Ganzen. als eine selbständige Stellung gegenüber dem sozialen Allmählich hat sich das von der Renaissance und von der Reformation aus über das ganze Dasein verbreitet, sich immer tiefer Gesellschaft und Individuum. eingegraben, darin es durchgängig frischer, 287 bewegter kraftvoller, Wie die neue Wissenschaft in ihre aufsteigende Bahn nur gekommen ist unter Zerlegung der überkommenen Größen, wie Zeit, Raum, Masse u. s. w., in diskrete Elemente, so ist auch dem modernen gemacht. Leben eine wachsende Selbständigkeit und Sonderung der Individuen Von der Behandlung der wesentlich. den Äußerlichkeiten von Sitte Fragen innerlichsten und Verkehr^ sich hat das bis zu immer mehr durchgesetzt. Es will das keineswegs alle gegenseitige Beziehung aufheben, aber die Verbindung werden, her aufgedrängt und freien den Individuen nicht von draußen soll sondern aus Vereinbarung hervorgehen. Individualisierung des Daseins sammenhänge. Vielmehr ist ihrer einen Verzicht auf der eignen Entscheidung Noch weniger bedeutet die auf alle inneren Zu- Höhe der geistigen Arbeit, bei Männern wie Luther und Kant, das Selbständigwerden des Menschen gegen die Menschen nur die eine Seite des Lebensprozesses, dessen andere die unbedingte, aber Gewalten bildet.. Unterwerfung unter unsichtbare freie Wer solche Männer als Verfechter bloßer Willkür lobt oder tadelt, der zeigt nur wie wenig er den Kern der Sache erfaßt hat. Im breiteren Strome der Zeit Verwicklungen und Bedenken. zum Individuum seit ist die Sache minder frei von In Deutschland gewann die Bewegung der Sturm- und Drangzeit vorwiegend einen künstlerisch-literarischen Charakter; wie das Individuum jener Zeit durch künstlerisches Schaffen über den Durchschnitt hinaushob sich und als »Genie" allem «Philistertum" weit überlegen fühlte, so hat sich öfter die selbstbewußte Erhebung des künstlerischen Individuums wiederholt. So zunächst in der Romantik, welche eben darin die Größe des Menschen fand, eine Individualität zu sein (Schleiermacher), und in Überspannung dieser Denkweise das unumschränkte Recht der «unendlich freien Subjektivität" verkündete, zugleich geneigt Kunst und Wissenschaft dem '^ politischen Leben weit voranzustellen,- ähn- Z. B. findet Ihering (Der Zweck im Recht II, S. 43Q) bei der ästhetischen Gestaltung des gem.einsamen Mahles einen höchst beachtenswerten Fortschritt der modernen Zeit gegenüber der Vergangenheit Kommunismus zum Individualismus". genossen gemeinsam waren, in der »Erhebung vom Während früher die Geräte der Tisch- erhält sie heute jeder für sich allein zu aus- schließlichem Gebrauch. ' tische Bezeichnend dafür sind die Worte Fr. Schlegels: „Nicht in die poliWelt verschleudere du Glauben und Liebe, aber in der göttlichen Weil Zu den Problemen des Menschenlebens, 288 Erscheinungen zeigt später das junge Deutschland, zeigt der liehe heutige Individualismus. Die Hochschätzung des Individuums teilt unsere klassische Zeit, und die leitenden Pädagogen, Pestalozzi wie Herbart, übertragen diese Denkweise auf die Erziehung. ^ das Individuum strebt nicht Gegensatz zur Welt und sozialen in Aber hier um in einem zur Selbständigkeit auf, Umgebung zu bleiben und sich das Bewußtsein stolzer Überlegenheit einzuspinnen, sondern es dehnt seinen Lebenskreis weiter und kehrt freudig dorthin zurück, weiter aus, wächst schließlich in Versöhnung mit zur weltumspannenden So Persönlichkeit stellt aller es Umgebung namentlich die und das Lebenswerk Goethes vor Augen, geistige Art Der erste Widerstand gegen den Vorrang des Individuums ent- dem sprang Idealismus selbst, indem der Gedanke eines weltum- spannenden, durch seine eigne Bewegung getriebenen Prozesses den Schwerpunkt des menschlichen Daseins von den Individuen Ganze der Menschheit verlegte. Wendung zur anschaulichen Welt. seiner in das Dann aber kam der Realismus mit Damit erschien eine un- absehbare Fülle von Aufgaben, deren Lösung eine Verbindung der zerstreuten Kräfte verlangte, die Menschen aus der bisherigen Ver- einzelung heraus zu engerem Zusammenschluß trieb und ein Arbeiten in Reih und Glied ihnen auferlegte. politischer Freiheit, Dahin wirkte das Verlangen das Streben nach einer von eigner Gesinnung der Bürger getragenen Ordnung, dahin Kraft und die Ausbildung nationaler Kreise, die alle Individuen mit überlegener Art umspannen und zu großen Aufgaben verbinden, dahin der ungeahnte Aufschwung der Technik, der die Verkettungen der Arbeit ausdehnt und die Arbeiter fester zusammenhält, dahin endlich das moderne Wirtschafts- leben mit seinen Riesenbetrieben, seiner Erzeugimg schroffer Gegenseiner sätze, Ansammlung gewaltiger Massen. Auch die moderne Beschleunigung des Lebens, das Einandernäherrücken der Menschen, der Wissenschaft und der Kunst opfere dein Innerstes in den heiligen Feuer- strom ewiger Bildung". * Namentlich Pestalozzi verficht mit großer Energie die Überlegenheit des Individuellen gegen das bloß Kollektive, er spottet über „Kollektivhand- lungen", meint : über ein „Kollektivgewissen", „Die sieren, sie über „R^mentsbekenntnisse" und kann dasselbe nur zivili- kollektive Existenz unseres Geschlechts kann es nicht kultivieren" (Wke. XII, 154). zuerst den Gegensatz herausgestellt hat. Hier muß immer der Ganze des Lebens von Individuum und Gesellschaft mit voller Klarheit große Einfluß Rousseau's gegenwärtig sein, der für das Gesellschaft und Individuum. 289 die tausendfache Verschlingung der Lebenskreise trägt viel dazu bei, die individuellen Züge abzuschleifen und der Summierung zu Massen- Im Zeitalter erscheinungen eine überwältigende Macht zu verleihen. der Telegraphen und der Presse, öffentliche eine Meinung und gewinnt umfängt das Individuum schon sie eine größere Stärke; sie seinem Werden und in als eignes Werk erscheinen, was zugeführt Eisenbahnen bildet sich rascher in ihm so läßt Umgebung ihm Wahrheit die hat. Endlich steigert, empfangend und zurückwirkend, auch die Theorie die Abhängigkeit des Individuums. Denn die neuere Gesellschafts- die „Soziologie" (Comte, Quetelet u. s. w.), lehre, bemüht, eifrigst ist Umgebung, die völlige Bedingtheit des Menschen durch seine soziale das „Milieu",^ zu zeigen; bis in seine Wünsche und Träume scheint er durch ihr selbst ein den Bedürfnissen in lich das beherrscht, des öanzen. schnitts, was die Gesellschaft an ihn heftiger Kampf des Individuums Zugleich des tritt bringt; gegen sie wurzelt schließ- Ganzen und damit liegt innerhalb der Begriff des gesellschaftlichen Durch- des mittleren Menschen, in den Vordergrund; es wird nach- gewiesen, daß die Abweichungen des Individuums, soweit meßbar, sich innerhalb weit engerer Grenzen bewegen, als der erste Eindruck mehr bei der Gleichheit als bei der Verschiedenheit der Individuen,^ und uns annehmen Analyse die großen läßt.^ des Dichter, individuellen weicht Statistik ein handliches * Milieu in So verweilt die Aufmerksamkeit Seelenlebens, diese der Massenbeobachtung, weit unserer Stärke die sich der in Werkzeug schafft. präziser Zuspitzung dürfte zuerst Philos. zoologique verwandt sein, von Lamarck in seiner Comte hat es von der Zoologie der Qesell- schaftslehre zugeführt, Taine aber es hier «mit besonderer Vorliebe verwandt. von ihm aus ist es in Deutschland zu einem Modewort geworden. Erst ^ Hierfür sei namentlich Quetelets „Anthropometrie" erwähnt. * Der Gedanke der Gleichheit samt dem des gleichen Wertes und Rechtes aller Menschen hat ältere Wurzeln, zur vollen Entwicklung aber ist er erst in den letzten Jahrhunderten gelangt. Dem klassischen Altertum ist und auch was sich zu seinen Gunsten im späteren Altertum regte, kam gegen die tatsächlichen Unterschiede der Menschen nicht auf. Die Wurzel er fremd, der Gleichheitsidee liegt in der Religion, für unseren Kulturkreis im Christen- tum. Es war das Verhältnis zu Gott, in dem alle Abstände der Menschen verschwanden, es war die Unendlichkeitsidee, der gegenüber alle endlichen Unterschiede gleichgültig wurden. Aber Konsequenzen für das irdische Dasein wurden daraus zunächst recht wenig gezogen, und in der weiteren Ge- schichte des Christentums trat der Gedanke des allgemeinen Priestertums weit Eucken, Grundbegriffe. 4. Aufl. 19 Zu den Problemen des Menschenlebens. 290 Das auch alles eine war nicht bloß eine äußere Verschiebung, innere es war Denn nunmehr wurde Wandlung des Lebens. zur Hauptsache an ihm, was wir für die Gemeinschaft leisten, nicht was wir im eignen Bereiche denken und alle Kraft zur Betätigung aufgerufen, Energischer wird tun. deutlicher die Bindung des Individuums an das Ganze hervorgekehrt. Auch eine eigentümliche davon aus. Die Verbesserung Gestaltung des Geisteslebens geht der gesellschaftlichen Lage wird das allüberragende Ziel. Die Moral zum Wirken für die Gesellschaft, zum Altruismus, die Kunst wird findet keine höhere Aufgabe als die eindringliche Vergegenwärtigung der gesellschaftlichen Hebung Zustände, die Erziehung des gemeinsamen Bildungsstandes dividueller Art. Einen Zusammenhalt gibt mehr die Entwicklung in- erstrebt als die dem Individuum hier namentlich die Arbeit, die Arbeit, welche weite Komplexe und feste Methoden entwickelt, damit aber stark genug wird, um einen Kampf mit aller Unvernunft des Daseins aufzunehmen und die Bedingungen des Daseins wesentlich zu verbessern. Daß das Ja auch ein Nein sich trug, der Gewinn von einem Verluste kam einstweilen kaum zur Empfindung. in begleitet war, das Wirkt solches Zusammenstreben der Kräfte mit seinen engeren Verschlingungen gegen das Individuum tut es offensichtlich die des 19. Jahrhunderts in versteckterer Weise, so mächtige Verstärkung, die der Staat im Lauf erlangt zurück hinter dem der Hierarchie. hat. Am zwingendsten trieben dazu Was von einzelnen Nebenströmungen das Mittelalter mühsam genug bewahrt hatte, das gelangte zu vollerem Durchbruch in der Reformation, und es war namentlich ihr calvinistischer Zweig, der daraus energische Folgerungen für die Gestaltung des Gemeindelebens zog. Von hier aus fand sich auch der Übergang in das politische Gebiet: unter Cromwell zuerst enthält ein Verfassungsentwurf das Verlangen des allgemeinen Stimmrechts (1647). Weiter wirkt dann für die Idee der Gleichheit die Aufklärung mit ihrer Berufung auf die allen Menschen gleiche Veniunft. So sagt z. B. Descartes (de methodo zu Anfang): Rationem quod attinet, quia per illam solam homines sumus, aequalem in omnibus esse facile credo. Rousseau hat endlich mit besonderer Energie den Gedanken der Gleichheit ins allgemeine Leben geworfen; der Gedanke der Menschenrechte dürfte aus Amerika stammen. Die Formel von der „Gleichheit alles dessen, was MenschenDas gesicht trägt", hat Fichte aufgebracht, s. z. B. IV, 423, VII, 673. 18. Jahrhundert bringt auch die Zusammenstellung von Freiheit und Gleichheit, und zwar wohl zuerst für das Gebiet des geselligen Verkehrs. So sagt z. B. schon Montesquieu in seinen lettres Persanes (erschienen zuerst 1721), — Bch. II: A Paris regne la liberte et l'egalite. Gesellschaft und Individuum. wirtschaftlichen die strengung Entwicklungen, da ihnen Individuums verloren des bloßen der Höhepunkt 291 gegenüber An- alle Aber dieser dünkte. durchgehenden Erscheinung. Punkt ist nur Es die wachsende Komplikation, die technischere Gestaltung der ist einer mehr Ineinandergreifen der einzelnen Kräfte und mehr Organisation des Ganzen verlangt, damit aber nach einer leitenden Spitze ruft. Das erzeugte z. B. mit Notwendigkeit eine stärkere Zentralisation im Unterrichtswesen. Und es fehlte dieser Bewegung welche Kultur, des Kulturlebens spricht zum Staates einer Kraft Gedankenwelt. Hauptträger der Kulturarbeit ent- der modernen Überzeugung von einem Innewohnen abso- Vernunft luter beseelende die nicht Die Erhebung des leitenden unserer Wirklichkeit; in Systematiker es Pantheismus, des kein Zufall, ist Spinoza daß die und Hegel, ent- schiedenste Vorkämpfer der Staatsidee waren, daß Spinoza nicht bei sondern beim Heil des Vaterlandes geschworen haben wollte, Gott, Hegel 'aber den Staat »wie ein Irdisch-Göttliches" verehrte. bünden Macht des So ver- gegen die Selbständigkeit des Individuums die sichtbare sich und Staates die unsichtbare der Gesellschaft; einen entflieht oder zu entfliehen glaubt, verfällt leicht wer der um so mehr der anderen. Aber wie der volle' einen Rückschlag ergibt, Menschen durch Sieg leicht eine Überspannung und damit hunderts eine Neuerhebung des Individuums sich davon die Selbstvergötterung subjektiver hervordrängt, geflissen unechter Stimmung zu ist vermeintlicher Denn ist Was hervorgerufen. unerquicklich genug, oft Genies mit der Verspottung jener Auswüchse ledigt. Umklammerung des so hat auch hier die und Gesellschaft gegen Ausgang des 19. Jahr- Staat so und das Sichaufbauschen Aber Weltüberlegenheit. nicht schon die Sache er- hinter allem Problematischen steckt eine Gegenwehr des Individuums und Subjekts gegen die drohende Einengung und Ver- kümmerung; was jene Bewegung zur Gesellschaft an Begrenzungen und Verneinungen enthält, lichem Bewußtsein. das bringt der Widerspruch jetzt zu deut- Eine Abschleifung der individuellen Züge, eine Gefährdung der Selbständigkeit, und Schaffens, verbunden. drückte, sie Ähnlich eine kleine Großes mehr sieht, der Individuen ein Stocken ursprünglichen Lebens scheinen mit jener Gesellschaftskultur untrennbar wie die Geschichte Gegenwart aber auch die in Gegenwart unter- der Geschichte nichts so scheint die Gesellschaft mit der Verkleinerung unvermeidlich auch bei sich selbst zu sinken. 19» Ge- Zu den Problemen des Menschenlebens. 292 wahren wir nicht deutlich genug, wie inmitten glänzenden aller Triumphe technischer Arbeit uns ausgeprägte Persönlichkeiten mehr und mehr entschwinden, zugleich aber das Niveau des gemeinsamen Lebens niedriger wird? gestaltung, daß muß das und sie den Zugleich sucht das Individuum sich lassen. Bindung möglichst abzulösen, und gänzlich „ausleben", entfalten viel von der gesellschaft- es will sich mit voller Freiheit es kehrt sein Unterscheidendes vom Durchschnitt irgend abzuheben. hervor und strebt sich Wie schwächt, ja unter- Gegenwehr reizen, sie der GesellWert ihrer Erfolge bestreiten die Seele zur widerstehen schaftskultur lichen nun kommt zur Empfindung, mit ihrer riesenhaften Entwicklung sie drückt; Die Arbeit, der Kern der neuen Lebens- die Seele kräftigen; sollte dem allen überspannt und verkehrt sein mag, es Zeit; mag es arm an positiver Leistung in hat eine Macht über unsere sein, in der Kritik und den Glauben an es stark, ist die Allgenug- samkeit einer bloßgesellschaftlichen Kultur hat es schwer erschüttert. Aber trotz solcher Erschütterung geht die Arbeit mit ihrer Richtung auf den Stand der Gesellschaft Individuum und mehr noch fort, so wächst ihr Druck auf das unsere Empfindung dafür. Demnach werden wir nach widerstreitenden Richtungen gezogen: die gesellKultur schaftliche behei"rscht verlangt unsere Seele. ergeben, oder Arbeit, ihr widerstehen sich läßt unsere eine Individualkultur Müssen wir uns solcher Spaltung wehrlos und nach irgendwelcher Einheit des Lebens streben? Die Probleme der Gegenwart. ß. Die Unzulänglichkeit einer bloßgesellschaftlichen Kultur. aa. Etwas anderes ist die es, Bedeutung einer gesellschaftlichen Kultur anzuerkennen, etwas anderes, in sie das ganze Dasein des Menschen zu setzen. Für das Mannigfachste zusammen. erstere wirkt in unserer Zeit das Deutlich steht uns vor Augen, wie von Anfang an der Mensch die eigentümlichen Züge seines Wesens nur in der Gemeinschaft entwickeln konnte, wie auch später finden wesentlich deutlich auch, wie die Wirkung des Zusammenseins weit das Leben des Einzelnen und den als man früher anzunehmen schaftliches Wesen funden. alles Be- von der Gestaltung des Zusammenseins abhing, ist, Grund Daß der Mensch pflegte. tiefer in seiner Seele hineinreicht, ein gesell- das hat erst jetzt seine volle Anerkennung ge- Aus den neuen Einsichten aber erwachsen sofort Aufgaben Gesellschaft und Individuum. Art fruchtbarster 293 Sind wir so sehr auf die Gesellschaft angewiesen und hängt unser Glück so sehr an ihrem Gedeihen, so wird es be- sonders wichtig, den Stand der Gesellschaft zu heben und alle ihr vorhandene Kraft zur vollen Wirkung zu bringen. Zusammenschluß die Menschheit hat im Kampf gegen die Unver- vordringen laäsen und mehr Glück nunft unaufhaltsam sein gebracht, die straffere Organisation das Handeln Hebel bei fälligkeit hat festere in Der engere in ihr Da- hat jedes Einzelne gehoben, Angriffspunkte gewonnen, den allgemeinen Verhältnissen ansetzt, indem es den nicht auf die Zu- der bloßen Individuen angewiesen bleibt. Die engere Ver- bindung im unmittelbaren Zusammensein hat reiche Quellen moralischer Gesinnung erschlossen, die Teilnahme für einander gesteigert, ein Bewußtsein durchgängiger Miteinanderarbeiten, Notwendigkeit, die mehr und zu einander zu fügen, Mannheit und Kraft in Solidarität erzeugt. sich Disziplin Auch hat das gegenseitig zu halten und zugleich mehr das Leben gebracht, das in der Vereinzelung leicht verweichlicht. War es bei solchen Erfolgen ein Wunder, daß die Hoffnungen und Gedanken die tatsächliche Leistung soviel geleistet hatte, alles leisten Indem und sie daß was Lebensführung das ganze Dasein des Menschen aus- sellschaftliche zufüllen weit überflogen, zu können vermeinte, daß die ge- alle Wünsche zu das versuchte, hat befriedigen sie allen eigentümliche Gestalt gegeben. sich zutrauen konnte. einzelnen Lebensgebieten eine Zum Inhalt der Ethik wird hier die Umgebung, der Altruismus, zum Ziel der Leistung für die soziale Erziehung die Bildung des Einzelnen für die Zwecke der Gesellschaft, die Kunst macht die gesellschaftlichen Zustände vorwurf ihrer Arbeit und die Wissenschaft sucht sondern will zum Haupt- mit ihr den weitesten Kreisen dienen, den Menschen nicht als isoliertes Individuum, vom Ganzen der Gesellschaft her «sozialpsychologisch" zu zum Maßstab der Wahrheit macht der Pragmatis- verstehen; selbst mus die Leistung für das Wohlbefinden der Menschheit. Indem in dem allen das Leben und Handeln direkter auf den lebendigen und empfindenden Menschen als Ganzes bezogen wird, scheint es eine größere seelische Nähe zu gewinnen, wird es mehr ins Unmittelbare, Frische, liegen je ja, hier so scheint alle unsicherer es. Wahrhaftige gestaltet; im Hintergrunde Verwicklungen der Religion wie der Metaphysik; die Neuzeit über diese Fragen wurde, kommener muß ihr solche Befreiung sein. desto will- Zu den Problemen des Menschenlebens. 294 Aber mag diese Bewegung noch so fruchtbare Ausblicke und Aufgaben eröffnen, nur so lange kann Zustimmung rechnen, zur Seite vertretenen Ja ihr Das Leben stimmter Art. gänzlich geht; läßt ist dem von sehr be- wie es dort geschieht, nicht, Umgebung, das Verhältnis zur in das bleibt, Nein aber dieses sich und freudige auf volle sie das Nein unbeachtet als die Entwicklung der in gegenseitigen Beziehungen verlegen, ohne daß zugleich die Selbständig- ist Nun aber wo geistiges Leben ursprüng- des einzelnen Punktes aufs stärkste herabgesetzt wird. keit das Individuum die einzige während das aufquillt, lich mehr Zusammensein nicht Die und gesellschaftliche und verwerten kann. verbinden als Stelle, Freihaltung Kräftigung dieses ursprünglichen Lebens wäre nun minder bedeut- sam und eine Einengung ließe sich eher ertragen, wenn das menschliche Leben auf fester Grundlage stünde und nur eine naturgewiesene Richtung ruhig zu verfolgen brauchte. Wahrheit steckt es nicht In nur voller Probleme im Einzelnen, sondern, zwischen bloßer Natur und Welt befindlich, hat es eine durchgehende Wendung, geistiger einen Aufstieg von halbgeistiger zu echtgeistiger Lebensführung erst zu vollziehen; so große Entscheidungen liegen ohne eine Aufrüttelung und standes können, erfolgen Tiefe zurückgreifen Die kräftige die notwendig auf die nicht ursprüngliche eine und damit auf das Individuum kommen. Lebensführung dagegen soziale ihm, in Belebung des gesamten Seelen- vorwiegend auf eine ist sie hebt und fördert, wenn sie das Leben angenehmer und Verbesserung der äußeren Verhältnisse gerichtet, sie und mildert freundlicher macht, sinkt alle geistige Be- wo sie nicht als völliger Selbstzweck behandelt wird; der behandelt. Feind aller Leben als ein Mittel Denn unvermeidlich Form er auch annehmen mag, ist ein unversöhn- echten Geisteskultur. für den Menschen erlangen, nie Als ein bloßes Mittel kann bei aller innere die Daher kann auf diesem äußeren Erweiterung keine innere Erhöhung des Menschen erfolgen, Verhältnis zum All, alle innere Selbständigkeit. hier fehlt alle Urerzeugung, kann nie etwas wesentlich Neues bringen, an denen sich Notwendigkeit mit solcher die Seele ergreifen und zu ursprünglichem Schaffen treiben. Wege den menschliche einer Selbsterhaltung sie sie das Utilitarismus, welche licher indem wirkt sie zerstörend, Lebens für Wohlsein geistiges am Kern des Gehalt geistigen tätigung, aber glättet, das Dasein nie alles unmittelbare Ein derartiges Leben hohe Ziele weisen, heben könnte, sondern es bannt den Gesellschaft und Individuum. 295 Menschen an seine eigne Zuständlichkeit und macht ihn zum Sklaven selbst, es läßt ihn sein Dasein zieren und schmücken, aber seiner Niederem, es vermag daher des Durchschnitts und Geist, Scheidung gründliche keine vollzieht es nicht den Menschen aus der Trägheit und Allgemeingültigem von Kleinmenschlichem entgegenzuwirken, die das Vermengung von Natur der nicht aufzurütteln, Höherem und zwischen gewöhnliche Dasein kräftig kennzeichnet; bei unermeßlicher Emsigkeit und Betriebsamkeit fehlt diesem Leben der rechte Tatcharakter, fehlt ihm ein unerbittliches Entweder - oder, Dies Leben der bloßen ein wahrhaftiger Gehalt und Sinn. ihm fehlt mag erträglich scheinen, solange der Blick und das Streben nur vom Einzelnen aufs Einzelne geht und von der bunten Fülle wechselnder Anregungen eingenommen wird. Aber man denke darüber hinaus und frage nach dem Ertrage des Ganzen, dann muß die Dürftigkeit, die Leere dieses Lebens augenscheinlich werden. Menschenkultur Wenn die Sozialkultur solcher innern Leere entrinnen zu können so pflegt sie das in der glauhjt, bindung der Elemente in der Vereinzelung vorliegt; Einzelnen das des Durcheinander der sellschaft öffentlichen Überzeugung zu daß die Ver- tun, etwas wesentlich Höheres entstehen läßt als so scheint individuellen Meinung sich zu weit B. z. Meinungen ein Träger das überragen, Wohl der Ge- dem so scheint in der bilden. In gegenüber der Wahrheit zu Wirklichkeit kommt der Schein einer inneren Erhöhung nur zu Stande, indem aus andersartigen Zusammenhängen Neues zugeführt wird; aus dem bloßen Durch- und Nebeneinander könnte nun und nimmer eine neue Stufe des Lebens hervorgehen; zu Unrecht wird hier, wie es tives unvermerkt Ziel als in freilich liegt, Gibt es umgesetzt. Qualitatives in das der der Neigung der Gegenwart natürlichen Selbsterhaltung, kein Quantita- anderes gibt es keine geistige Wesensbildung, so kann auch die Verflechtung der einzelnen Kreise im Zusammensein gesellschaftlichen stehen lassen, auch bei größter nichts wesentlich Neues ent- Ausdehnung nähert sich das Nütz- und Angenehme der natürlichen Lebensstufe in keiner Weise dem Guten. Ebensowenig ergibt die Bildung gewisser Durchschnitte der Meinungen, mögen sie sich noch so festlegen und noch so liche selbstbewußt auftreten, griff einer echten nicht die Wahrheit, die mindeste Annäherung an den Bealles menschliche Streben Gutes und Wahres werden immer schon glaubt, sie aus der Verbindung vorausgesetzt, mißt. wo man der Elemente ableiten zu können. Zu den Problemen des Menschenlebens. 296 Solche Überzeugung zwingt zu starker Skepsis gegenüber der bekannten Lehre von einer Summierung der Vernunft Sie sellschaft. ist philosophisch zuerst von der Ge- in vertreten. Aristoteles^ Die Gesamtheit erscheint ihm befähigter zu politischem wie zu künstder andere lerischem Urteil als die Individuen, weil der eine dies, anderes besser beurteile, im Zusammentreten aber eine gewisse Aus- dem Zorn gleichung erfolge; auch scheint ihm das Ganze minder und anderen Affekten unterworfen als das Individuum. den begrenzten Kreis eines Stadtstaates, den gemeinsame er aber an Ordnungen auch innerlich zusammenhalten, und unübersehbare Masse. So bleibt er Überlieferungen und nicht an jede beliebige auch in seinem Demokratismus weit geschieden. — Für dern durch das eines die Sache spricht zunächst die daß hervorragende Rousseau alte, große Publikum zur Anerkennung auch literarische nicht durch das Urteil der Techniker, Leistungen gewöhnlich gelangt sonsind, wegen eines moralischen Defektes jener, sondern weil sie sich nicht fest in Die vom Volksglauben gegenwärtige Erfahrung, Aristoteles zu Dabei denkt einen geschlossenen Gedankenkreis eingesponnen hatten. Unbefangenheit weiterer Bildung. — Weiter Kreise wiegt namentlich ungewöhn- gegenüber hier mehr als die höhere technische lichen Erscheinungen steht in einer gewissen Verwandtschaft mit jener Lehre von der Summierung der Vernunft die Überzeugung, daß es eine Berufung von der Zufälligkeit der Augenblicke und der im besonderen auch viduen, Ganze der Menschheit von der Enge der Parteien, das Vertrauen gibt, Indi- an das auf irgendwelchen Sieg des Guten auch innerhalb des menschlichen Kreises. Ohne solchen Glauben müßte Wirken nach ja, außen als zwecklos das lich wer in der Minderheit ist, einstellen; so durchdringt jener Glaube politische Auch Streben. liefert anfänglicher schroffer Verfolgung, es, minder als festgelegt Er- zum Siege kam trotz der Stein, den die Bauleute ver- oft als ein Eckstein das dazu verhalf sich nament- geschichtliche die fahrung Zeugnisse dafür genug, daß Großes warfen, hat sich alles erwiesen; was anders aber war das größere Ganze, die weiteren Kreise, die hatten und neuen Anregungen zugänglicher waren? Aber solches Durchdringen des Wahren vollzog sich kaum durch eine bloße Summierung menschlicher Meinungen, sondern dem Zwange unter * S. 62 ff. einer S. Pol. 1281b, 8, 34. geistigen Näheres s. Notwendigkeit, in welche jenes meinen gesammelten Aufsätzen Gesellschaft und Individuum. Höhere immer So machte. ist abhob und deutlicher 297 es schließlich unwiderstehlich Glaube an die Masse, sondern an eine es nicht der innerhalb der Menschheit waltende geistige Notwendigkeit, welcher Hoffnung auf einen Sieg der Vernunft auch im Bereich des Menschen rechtfertigt. Erst in Berührung mit einer solchen geisti- jene gen Notwendigkeit und Meinung ein kann leicht hinter sie als Recht gutes ihre Vertreterin und eine gewinnt die öffentliche sichere Überlegenheit; sonst dem Stande der einzelnen Individuen zurück- und weniger zur Vernunft als zur Unvernunft wirken. Es Zeiten, wo der Durchschnitt den Menschen erhöht, es gibt bleiben gibt aber auch solche, Masse wo er ihn herabdrückt. Jedenfalls tut die bloße es nicht. Dasein und gar in das unmittelbare die Sozialkultur sich ganz Indem hineinstellt, wird zum Haupt- unvermeidlich die Masse sie träger des Lebens machen, wird sie wohl oder übel der Art huldigen, mit der jene die großen Kulturfragen betreibt: hastig und aufgeregt, maßlos im Ja wie im Nein, haftend lichst starke geworfen, am sinnfälligen Eindruck, mög- Erregung suchend, zwischen Gegensätzen hin- und her- aller Zugleich Besonnenheit und Gerechtigkeit abgeneigt. wird das Individuum mehr und mehr zurückgestellt; selbst wo es unbestreitbar Großes wirkt, gilt es hier als ein bloßes Werkzeug der Gesellschaft,! als gleichgültig allem, in was an es Eigentüm- Nun hat gewiß auch die größte Leistung ihre geschicht- lichem hat. lichen und gesellschaftlichen Bedingungen und Zusammenhänge, alles Atmosphäre her- Schaffen aus, es wächst aus damit trägt einer besonderen unvermeidlich geistigen Färbung seiner die Zeit; ein Augustin wäre nicht möglich zur Zeit Kants noch ein Kam zur Zeit Augustins, ein heißt nicht das Ganze für und das Individuum zu einem gleichgültigen Werkzeuge machen. erklären Kraft nach völlig Aber solche Goethe nicht inmitten der Kreuzzüge. Bedingtheit anerkennen die erzeugende seiner Besonderheit Bei Zusammenhang stand das Große zum Durchschnitt allem inneren seiner Zeit ge- wöhnlich im Verhältnis des Gegensatzes, meist hat es seine Größe in der Art entu'ickelt, daß es eine Notwendigkeit seines eignen Wesens dem Ganzen der Zeit gegenüber siegreich durchzusetzen wußte, sieg- reich nicht in der Breite des Daseins, aber siegreich in der Sphäre ^ S. z. B. Comte, cours de phil. pos. IV, 269: Les ne se presentaient essen tiellement que conime les hommes de genie organes d'un niouvement predetermine, qui, ä leur defaute, se tut ouvert d'autres issues. Zu den Problemen des Menschenlebens. 298 Was dabei seine Leistung auszeichnet, ist vor der geistigen Arbeit. allem das Individuelle, Unvergleichliche ward Hilfe dessen und aufquoll, das aber regte und Unableitbare. möglich, das Geistige, das es in ihrer in Nur mit der Zeit sich mit Niederem Breite und Fremdartigem untrennbar zusammenfloß, davon abzulösen und zu wie kräftiger Gestalt zu bringen, klarer und erhöhenden Kraft zu erheben. welche Individualisierung, eine eigentümliche Bahnen trieb. zu einer aufrüttelnden es Dabei erfuhr das Geistige selbst Geschicke die Nirgends ist Menschheit der Denn das steht wohl außer Zweifel, daß ein Gebiete der Religion. Augustin und ein Luther nicht bloß zusammenfaßten, was die gebung ihnen darbot, sondern daß Lage gleich geistige Art haben. in sie die Um- Probleme der weltge- durchaus eigentümlicher Weise gelöst und zu- schichtlichen ihre in das deutlicher als auf dem ganzen Jahrhunderten zwingend auferlegt Jede Zeit von kräftigerer geistiger Regung enthält verschie- dene Möglichkeiten; welche von ihnen zur Wirklichkeit wird, das Schon das verbietet hängt vor allem an den führenden Individuen. eine Konstruktion der Geschichte aus einer Formel. War das Große einmal da, so konnte es alles irgend Entgegenkommende an sich ziehen, Aufstrebendes verstärken, Zerstreutes verGesamtbewegung erzeugen. Aber es war dabei nicht der Summierung, vielmehr hat es seinerseits die Summierung erst möglich gemacht. Denn die Summierung, das binden, eine Ergebnis ein Sichzusammenfinden, das den Vertretern der gesellschaftlichen Kultur so leicht erscheint, als blem. In liegen und ist in Wahrheit ein überaus schweres Pro- einer Zeit kann viel Verschiedenes, eine Summierung in sehr abweichender Höhenlage möglich in an einzelnen Stellen und ist aufstrebt, nicht sein; Richtung, zusammen Daß die Verbindung der gelingen will, das kann einem schweren Druck belasten; ein solcher Druck eignen Zeit. Das eben ist das auch was an Tüchtigem das findet sich oft nicht daher für das Ganze wie verloren. aufstrebenden Kräfte Widersprechendes ja mannigfacher eine liegt Zeit mit auf unserer Werk der Großen, durch glückliche Ausprägung eines geistigen Charakters und mutiges Vordringen eine Summierung in bestimmter und erhöhender Richtung anzubahnen und durchzusetzen; so waren sie die Herren, nicht die Diener der Zeit. des in Sprechen wir von einer Goethezeit, weil 18. Jahrhunderts eine humane und in der zweiten Hälfte künstlerische Denkweise der Art Goethes massenhaft verbreitet war, oder weil seine über- Gesellschaft und Individuum. 299 ragende Persönlichkeit Gestalten schuf und Ziele vorhielt, an denen minder Ausgeprägtes sich die in Höhe hob und zugleich mitein- ander zusammenfand? Wenn dem gegenüber die Sozialkultur die Abstufungen zurückstellt und nach möglichster Gleichheit strebt, so sicht der Besten, möglichst alle das Gesamtniveau die steigern, möglichst viele, auf die Höhe zu führen, ohne daß diese irgendwelche Minderung erfährt. als zu gewiß die Ab- ist Absicht Aber auch hier ist die Natur der Dinge stärker Unvermerkt wird der Stand des Menschen. (ier Maß der geistigen Bewegung, und es sinkt damit unvermeidlich die Höhe des Ganzen, es läßt sich die Arbeit Aufnehmenden zum vorwiegend auf die Wirkung bei anderen nicht sie Einbuße eine Denker in sich bei selbst ohne daß richten, Schopenhauer erfährt. teilte die solche ein, die für andere, und solche, die für sich selber denken, und erklärte nur diese für Denker echter Art; hat. er damit Recht, wie wir meinen, so kann über die Gefahr einer vorwiegend auf Mitteilung und Wirkung aus der Verbreiterung gerichteten muß Arbeit keinerlei eine Verflachung werden, Urerzeugung erfolgt, welche jener die Wage eine Dazu gesellt sich anzunehmen, was auf lichst sein; wenn nicht hält. die Neigung, sich nicht nur des Schwächeren und ist, sondern sich mög- seinen Standort zu versetzen und das Ganze des Lebens sicherlich edel nach seinen Interessen einzurichten. weise Zweifel recht Zeiten harter und weicher Denk- miteinander abzuwechseln, pflegen heute herrscht unzweifel- weiche Art und erzeugt die Neigung, den Schwachen haft die gut, den Starken als schlecht als zu denken, daher beim Zusammenstoß jenem ohne weiteres Recht zu geben. So nimmt eine weitverbreitete Zeitströmung Partei für die Kinder gegen die Eltern, für die Schüler gegen den selbstischer die als und finden. auf Auch sei alle Ordnung und brutaler Gesinnung. Gerechtigkeit Menschen durchgängig für den Untergebenen gegen den Lehrer, Gebietenden, untergeht, Erden leben" der drohende so hat würde alle Strenge Der keinen es hier nur ein Ausfluß kantische Satz: Wert schwerlich «Wenn mehr, daß Zustimmung Feminismus hängt damit zusammen, der nicht nur den Frauen zu ihrem gebührenden Recht verhelfen, sondern die Erziehung wie die ganze Kultur möglichst ihren Interessen gemäß »Coeducation" gestalten möchte. So wird die höchst problematische vornehmlich deshalb empfohlen, nur ja genau so viel damit die Frauen und genau dasselbe erhalten wie die Männer! Zu den Problemen des Menschenlebens. 300 Auf solchem Wege der Qleichmacherei muß die Kultur unvermeidlich ins' Welke und Matte geraten, sie wird vor aller kräftigen Art und aller ausgeprägten recht zurückscheuen, Individualität wie vor einem Übel und Un- was noch schwerer wiegt, das wird, sie was nach Ooethe's Wort »niemand mit auf die Welt lieren, und worauf doch Seiten zu ein ankommt, alles Mensch sei": die Bewegungen Derartige eine Zeit lang ertragen, der der Mensch damit nach in die Breite und Fläche lassen sich überkommene Lebensstand liefert einst- Aber zuweisen; erschöpft schließlich die Frage der reichste Kapital, allen Ehrfurcht.^ vom er- weilen eine Ergänzung, für einige Zeit läßt sich ganz wohl erbten Kapitale zehren. ver- bringt, sich auch das Urerzeugung ist nicht dauernd ab- sobald sie aber erscheint, werden die Schranken der ge- sellschaftlichen Kultur Die gesellschaftliche unverkennbar. konnte das Geistesleben auf den Menschen nicht stellen, Kultur ohne ihn innerlich zu erhöhen; sie konnte der Gesellschaft die höchsten Güter ohne mehr aus ihr zu machen. nicht anvertrauen, Aber mit eignen Erhöhung nicht bewirken, vielmehr zerstört sie mit der Schwächung und Stagnation des Geisteslebens die Bedingungen echter Größe und kann daher nicht verhindern, daß eine bloße Menschen- und Massenkultur eine wesenhafie Geisteskann Mitteln kultur überflutet Treten eine sie solche und unterdrückt schon nicht schmerzlich hervor? und im Ganzen heute sehen, deutlich und wie hervorragende Geschichtsschreiber uns frühere Zeiten sehen lassen, Außenkultur ein einer Erfahrungen solche Würden wir unsere eigne Zeit von innen her so möchte sich bewegendes inmitten vor Augen Bild alles Glanzes stellen. Die Menschheit wollte durch einen engeren Zusammenschluß und eine Entfaltung der Kräfte das Leben vollere genug, stark Wirken alles Geistesleben wollte sie einen selbst erhöhen, sie dünkte sich zu erzeugen, in rastlosem Turm bis zum Himmel führen. Nun aber erfährt sie bei allen äußeren Triumphen einen inneren Niedergang, Widerspruch gegen die Mattheit und Schlaffheit jener Macht über die Seelen gibt. „In die Höhe will es sich bauen mit Pfeilern und Stufen, das Leben selber: in weite Femen will es blicken und hinaus nach seligen Schönheiten, ^ Der kräftige Qleichmacherei ist es vornehmlich, welcher Nietzsche — darum braucht es Höhe! Und weil es Höhe braucht, braucht es Stufen und Widerspruch der Stufen und Steigenden! Steigen will das Leben und steigend sich überwinden". (Also sprach Zarathustra.) Gesellschaft und Individuum. vermag ja sie sich selbst nicht zu verstehen, es droht ihr ein innerer und eine Streit, lösung in wachsende Parteien, ein 301 mehr zusammenzufinden, sich selbst Überall Gegensatz Zerfall. des Leidenschaft Kampfes, Wo wir unter Zurückstellung aller Weltprobleme bei werden einig und wollten eine Auf- Entschwinden gemeinsamer Ideen und Ziele. von Einigung solcher uns selber die schönsten Früchte hofften, da ist eine Sprachverwirrung erfolgt, und wir werden immer mehr der Zerstreuung und Zerstückelung verfallen, wenn es Bewegung Einhalt zu gebieten, das menschliche Dasein wieder in größere Zusammenhänge zu stellen und ihm einen nicht gelingt, jener festeren Grund zu geben. Bis dahin beschäftigte uns das Problem der Sozialkultur übermit- einigen Worten der Stellung des Staates im modernen Geistesleben gedacht. Die stärkere Machtentwicklung haupt, es sei nun auch des Staates steht heute uns allen vor Augen, und es drängen nament- Verwicklungen zu noch weiterer Steigerung; damit lich die sozialen aber erwächst die Gefahr, daß mehr und mehr das ganze Geistes- und seine Stemplung er- leben unter den Einfluß des Staates gerät fährt, und diese Gefahr, nennen möchten, des Staates ist ist und die Gefahr ist Politismus, sittlich"^ indifferent, sie wir wir Entwicklung der Macht; nun ist, sie Die leitende Idee nicht gering anzuschlagen. bleibt die Macht keineswegs, wie wohl gesagt sie eines ist die an sich etwas böses, aber kennt kein höheres Ziel als sich selbst. Sie hat aus ihrer Natur das Streben, alle geistige Betätigung als ein bloßes Mittel für ihre Zwecke zu Selbständigkeit anderer behandeln, Wenn Lebensgebiete. sie anerkennt keine aber diese Gebiete vor allem daraufhin angesehen und danach gemessen werden, was sie für das staatliche Leben wert und leisten, so verlieren sie ihren Selbst- müssen zugleich an der Ursprünglichkeit ihres Schaffens schweren Schaden erleiden. Zugleich muß bei einer Beherrschung des ganzen Lebens durch die Staatsidee die eigne Art und die freie Bewegung des Individuums aufs stärkste eingeengt werden. Wenn der Mensch sein Sinnen und Denken vor allem darauf richtet, einen Platz im Staatsgefüge zu erringen und in diesem weiter zu kommen, wenn sein Wert daran hängt, was er in ihm bedeutet und leistet, so wird damit der Schwerpunkt seines Lebens nach außen verlegt, und Ursprünglichkeit des Lebens muß unvermeidlich Schaden erleiden. Ob das Gefüge des Staates mehr demo- die Selbständigkeit Zu den Problemen des Menschenlebens. 302 oder aristokratischer Art kratischer dabei wenig. Wie Hand in Hand gehen besagt ist, politische Größe mit geistiger Unproduktivität dafür kann, liefert die römische Geschichte das schlagendste Bei- Denn höchst merkwürdig ist und bleibt es, daß bei so viel politischer FCraft, Weisheit und Disziplin jenes Volk nicht einen spiel. einzigen großen philosophischen Gedanken, nicht eine einzige große künstlerische Tat aus eignem Auch wir Deutschen welche Jene tritt unsere Vermögen hervorgebracht hat. müssen der Gefahren zurückdrängende Macht der alles eingedenk jener Politismus, Staatsidee, uns namentlich im preußischen Staat entgegen. zeitweilige Unterordnung zwingende Notwendigkeit, schichtliche Aufgabe aller Aufgaben unter wenn lösen dieser sollte; Staat dabei hat danke der Macht aufs engste mit dem der damit innerlich sein, Entwicklung bei diesem Probleme mit sich bringt. veredelt. Gewiß war eine die Staatsidee eine seine große weltge- sich hier Pflicht der Ge- verbunden und Die Verbindung beider Ideen hat herr- liche Leistungen hervorgebracht, die das Deutschland der Gegenwart erst haben entstehen lassen. Aber alles dies darf die Gefahr einer Unproduktivität, auch geistigen eines Einschnürens der Individuen, und schablonenhaften Gestaltung des Lebens nicht lassen. Geistiges Schaffen und ursprüngliche Lebens- einer typischen übersehen führung wollen schlechterdings als Selbstzwecke behandelt sein, der Politismus aber, mag er noch so veredelte Formen annehmen, seinem Utilitarismus setzt sie mit unvermeidlich zu bloßen Mitteln und Werkzeugen herab. ßß. Die Unzulänglichkeit einer bloßen Individualkultur. Die Gegenbewegung gegen die gesellschaftliche Kultur, die das moderne Individuum Sorge vollzieht, entsprang zunächst weniger einer um den geistigen Gehalt des Lebens, als sie die Schädigungen abwehren wollte womit das Vordringen jener Kultur das Individuum bedrohte; doch standen tiefere Probleme im Hintergrunde und verschärften den Gegensatz. das Individuum es lediglich als — Die gesellschaftliche Kultur behandelt ein Stück ihres großen Räderwerkes, nach seinen Leistungen, sie muß sie schätzt es für ihre Zwecke beengen und einschränken. Dazu wirkt sie mit ihren Verzahnungen der Elemente, mit ihrer Anhäufung von Massen, ihrem lauten und fabrikmäßigen Getriebe übermächtig zur Unterdrückung vielfach und Abschleifung der individuellen Züge, sie gewährt keine stille Gesellschaft und Individuum. Ruhe zur Bildung eigentümlicher Art, Durch- erzeugt gewisse sie selbst zum Maßstab für Gut und Böse, für Wahr die sich schnitte, 303 und Unwahr machen. Gegen solche Bindung und Gleichmachung erhebt sich schließlich das Individuum kräftigerer Art und tritt dafür daß keineswegs der Mensch ein, lichen und heit das Verhältnis zur gesellschaft- die des Innerlichkeit Lebens, jenes Verhältnisses jenseit Dabei kann es das Zeugnis der Weltgeschichte dafür anrufen, liegt. daß in Umgebung aufgeht, daß vielmehr das Beste an ihm: die Ein- vorwiegend alle der Gestaltung gesellschaftliche Kultur eine Veräußerlichung und Mechanisierung bewirkte, und daß es keines- wegs bloß trotziges Selbstgefühl ein der Individuen, sondern das unabweisbare Verlangen nach mehr Seelenhaftigkeit des Lebens war, was einen Bruch damit erzwang. bringt Religion die dem Gebiet der Namentlich auf zur Kirche unver- gesellschaftliche Gestaltung meidlich die Neigung mit sich, die Leistung (Gottesdienst, Werke, vor die Gesinnung, pflichtungen) das des Beisichselbstsein Interesse der Religion Kirche nötig gemacht.^ Individuums des überhaupt Bekenntnisse, korrekte Ver- vor das persönliche Leben, vor zu Innern fromme religiösen die sog. so stellen; hat das eigenste immer von neuem einen Kampf gegen die Zu solcher Verfechtung der Selbständigkeit gesellt ein sich flammender Protest gegen die gleichförmige und schablonenhafte Gestaltung der Kultur, womit die Gesellschaft das Leben bedroht. Sind die Durchschnitte, die dabei entstehen, nicht recht geringer Art, Festlegung des Sind in nicht Lebens Wahrheit und bedürfen sie Wirkung das auf auf einer geistige nicht zu aber So gab wesentlichen Scheidung werden, der um das Menschheit, übrige nicht leicht zur und edle Mittelhöhe? Gesinnung selten, einer scharfen Ausprägung und kleinen Kreisen einer engeren Jüngerschar? in keinen Kraft sie beträchtlichen ihrer Ausbildung voller Freiheit, zu einer Ganze sicheren Befestigung es und führen nicht ein Fortschritt Höheres der Kultur ohne eine mußte vorausgeworfen nach sich ziehen zu können, eine Feuer- mußte dem großen Haufen voranleuchten und ihm den Weg säule Trotz aller Bedenken und Verwahrungen immer wieder ein Gegensatz esoterischer und exoterischer Lebens- durch die Wüste zeigen. ist ' Beim Begräbnis eines Zentrumsführers wurde seitens eines hohen rühmend hervorgehoben, der Verewigte habe die Sorge für seine Prälaten — Seele gänzlich den Händen der Kirche überlassen. daß eine innere Preisgebung des Lebens auch noch belobt wird? derartige Ist es nicht entsetzlich, Zu den Problemen des Menschenlebens. 304 führung entstanden, auch die radikalste politische Verfassung hat die Bildung schroffer sozialer Unterschiede nicht verhindert, Menschen sind, Einzelnen alle geistige Tätigkeit wenn sie sich damit selbst nicht mit individuell eignes Wesen kämpft? Und bleibt nicht für jeden matt und wie von außen angeweht, seiner gestaltet, Art individuellen wenn er nicht verflicht und um sein bei ihr Bilden heißt scheiden, differenzieren, indivi- war die Scheidung überall ein unentbehrliches Mittel Bewegung und Weiterkommen. Aus solchen Erwägungen gehen die Individuen von der Abwehr für zum vor Angriff und rücken Schranken deutlich vor Augen. ist bis in der Ehrgeiz, es Höherstehenden gleichzutun, eine unentbehrliche Triebkraft der Bewegung. dualisieren; so ja und der Sitte ist, wie einmal die die Äußerlichkeiten des Anstandes der gesellschaftlichen Kultur unmittelbaren Verhältnisses zur Wirklichkeit fähig, eines ihre Der Mensch, als denkendes Wesen, er ist kein bloßes Glied einer Verkettung, er kann sich der Unendlichkeit gegenüberstellen und mit ihr ringen, er wird der Enge der bloßen und kann von ihr zur eignen Wahrheit der Gewiß stößt solches Streben auf Hemmungen über Zuständlichkeit bewußt Dinge streben. Hemmungen, aber schon als Streben erweist es eine Überlegenheit des Menschen über den Kreis der bloßen Gesellschaft. Ist es nun nicht ein Widersinn, einem solchen Weltwesen das Geistesleben erst Maß dessen durch die Gesellschaft vermitteln und es dabei an das binden zu wollen, was der Zusammenschluß der Kräfte an Geistighat? keit erreicht Soll das Wesen, das aus seinem Grundverhältnis zur* Geisteswelt einen unendlichen Wert besitzt, sich seinen Wert erst von menschlicher Schätzung zusprechen der Menschen leben und damit verlieren? Existenz Soll erst alle und sicher fühlen, nachdem Güter, wenn nicht auf In und dem von Gnaden der Mensch sich einer Wahrheit, ja einer geistigen froh ihm mit Brief und Siegel verbürgt hat? erfolgen, lassen, soll es Unabhängigkeit der Gesinnung dem Markt die Gesellschaft sie Soll die Erzeugung geistiger des Lebens, so doch für ihn sollen damit jene Güter zu bloßen Marktwaren sinken? allen wegte Individuum, erscheint das Individuum, als der Vertreter d. h. das geistig be- der Geisteskultur gegenüber einer bloßen Menschenkultur, einer inneren Unendlichkeit gegen alle äußere Begrenzung, es erscheint widersteht, das aus der Erstarrung der Verflachung als eine Kraft, die aufrüttelt, notwendige Ziele vorhält, menschliche Streben immer neu auf seine wahren Grundlagen Gesellschaft und Individuum. 305 Und wenn solche Schätzung des geisterfüllten Individuums notwendig eine Abhebung vom gesellschaftlichen Durchschnitt zurückführt. mit sich so bringt, Unduldsamkeit gegen auch die diesem Durchschnitt eigne wird sie alles irgend Überragende mit sicherem Stolze Es gibt einen gemeinen Neid und Haß des Mittelmäßigen abweisen. gegen das Höhere als gegen eine Überschreitung und Herabsetzung der eignen Dürftigkeit; dies Höhere wird leidlich geduldet nur dann, wenn es Äußerung des alle bittet, Kraftgefühls behutsam unter- Daher steht die Tugend der Bescheidenheit beim drückt. um Ent- bescheiden duckt, für sein Dasein höflichst sich schuldigung Philister- Nahe verwandt ist dem das Unterordnen des Verschiedenartigsten und Verschiedenwertigsten unter die gleichen tum so hoch Ehren. in von Lob und Begriffe, das Operieren mit nichtssagenden Schablonen Tadel, jene laue und matte Art, die keiner kräftigen Liebe und keines kräftigen Hasses fähig ist, der Licht und Dunkel in einen grauen Nebel Demgegenüber zur Kräftigung des Empfindens, zusammenfließen. zur Scheidung der Geister zur 'Schärfung des Urteils, das zu wirken, ein gutes Recht, ja eine heilige Pflicht des Individuums. ist kann das Individuum Freilich rechtem Sinne überlegen nur in werden, wenn es eine Geisteswelt hinter sich hat und aus ihrer Kraft zu schöpfen vermag. Dies aber modernen Individualismus, wie Er stellt und das Individuum heißt es sich im Durchschnitt ausnimmt. gänzlich auf sein Zusammenhänge zu Bindung an Menschen, sondern auch streifen. So ihm verbleibt seelische Zustand, unmittelbares da das Leben gestalten; unsichtbaren alle flissen, von keineswegs die Meinung des ist er er Dasein besonders be- ist lockern, nicht nur die die an eine Geisteswelt abzu- anderes nichts das subjektive Befinden; als indem der unmittelbare dies zum Kern Lebens wird, verschmilzt der Individualismus mit dem Sub- alles damit eine gewisse Art von jektivismus. Augenscheinlich Wirklichkeit. Jene Zuständlichkeit läßt sich fixieren entsteht und steigern. Eigentümliches vermag sich schrankenlos auszubilden, Leben immer So neu aufzuquellen, eine große Leichtigkeit, Frische und Flüssigkeit, das Leben scheint hier ganz Stand sein auf sich selbst sich gestellt, unablässig und zu verändern. bei solcher Freiheit feiner, Auch der Begriff der Denn als Starrheit. wahr gilt nunmehr nur das, was die Seele des Einzelnen erlebt und was sie eben jetzt erlebt; so weicht der Begriff einer einzigen Wahr- zarter, intimer Wahrheit geworden verliert seine Eucken, Grundbegriffe. 4. als irgendwo sonst. sonstige Schwere und Aufl. 20 Zii 306 den Problemen des Menschenlebens. dem unzähliger Wahrheiten, jeder Mensch hat hier seine eigne Eine besondere Freude und Bewußtheit gibt dem der Wahiheit. heit Kontrast zur Gesellschaft, dem Lebensgefühle deren denn demgegenüber das Leben das halten, und Anordnungen Einrichtungen des Individuums oft widerstreiten; in Freiheit stets so heißt es und Fluß zu möglichst zu stärken und deutlich Eigentümliche er- her- vorkehren. Das alles konnte aber den Stand einer formlosen Erregung und unklaren Bewegung nicht überschreiten ohne sich irgend in geistige Arbeit Wendung umzusetzen, es fand solche Umsetzung durch die und zur Kunst Die Kunst Literatur. mannigfachen Ver- ihrer in zweigung wird hier zum Hauptmittel, das sonst unstet wogende und wallende Leben irgend zu fassen und festzulegen, durch die Fassung aber es zu verstärken, sich bei es selbst nach außen hin unabhängig zu machen. voll durchzubilden und Konzentration des Lebens in sich selbst und Steigerung seiner Kraft, das wird damit zur Haupt- aufgabe der Kunst. Sie die weit überlegen fühlt; über vornehmere einer die Kunst kann Zweckmäßigkeit bloßen aller wird die Seele einer individuell -aristokra- sich als die tischen Kultur, liegt wiegend auf sein individuelles Vermögen weil sie aus schnittslebens aller die Verworrenheit und weil sie selbst und den Menschen vorstellt, weiter aber deshalb, Vergriffenheit des Durch- Grundzüge menschlichen Seins heraus- einfachen sehen. Ewigjunges in praktisch-sozialen' das werden, ihm ergreifen und es damit aus aller Erstarr- ung im Konventionellen aufrütteln kann. Leicht überträgt das Bewußtsein Bahn. schüssige sich aber solche Abstufung des Lebens auf der Individuen und gerät dabei rasch auf eine abNicht nur, wer an der neuen Art mit eignem Wirken teilnimmt, sondern auch, wer sich bloß dazu bekennt, glaubt sich der übrigen Menschheit legen; entsteht es und der gesellschaftlichen Kultur über- eine Neigung, den Abstand hervorzukehren, das in der Absonderung ein GeGröße zu suchen. Und zugleich greift der Anspruch um sich, unbekümmert um alles, was da gilt, um Sitte und des Landläufigen zu tun, Gegenteil fallen, eine ja Gesetz, die eigne Art nach Lust sichtslos M auszuleben". und Laune zu entfalten, sich rück- Die Individualkultur mag das wollen, unter menschlichen Verhältnissen ist alles nicht solche Folge schwer zu vermeiden. Derartige subjektivistische Strebungen und Stimmungen spielen Gesellschaft und Individuum. 307 bekanntlich eine große Rolle in der neuesten Gestaltung des Lebens. Neu ist dabei freilich nur der Name, die Sache ist alt, uralt. Denn in periodischer Wiederkehr kommen immer wieder Lagen, wo wie das unmittelbare befriedigt Lebensgefühl von der dargebotenen Kultur nicht war und nun als Hülfe und Heil die völlige Emanzipation des Individuums verkündet wurde, wo seine unmittelbare Empfindung, selbstgefundenes sein Urteil, sein künstlerischer Geschmack eine Wendung zum Besseren bringen sollte. Wer Piatos Gorgias kennt, der kennt auch die nahe Verwandtschaft der Sophisten mit den heutigen Subjektivisten; in Deutschland brachte zuerst die Geniezeit, die Vorläuferin der klassischen Literaturepoche, eine derartige Emanzipation des Einzelnen; damals waren »Genie", »Kraftgenie«, «Originalgenie" Modewörter wie heute «Übermensch"; auch »schöne Seele" steht in Dann kam eine neue Welle der Romantik, deren nahe Verwandtschaft mit dem ästhetischen Verwandtschaft mit dieser Bewegung.^ in Subjektivismus der Gegenwart deutlich zutage liegt. Das Ganze gerecht zu beurteilen ist schwer, um eine Übergangserscheinung handelt, die weil es sich offenbar um so mehr Vernunft und Recht besitzt, je mehr sie sich weiteren Zusammenhängen einfügt und über sich selbst hinausweist, die um so mehr ins Unrecht gerät, Über das Aufkommen und die Schicksale des Ausdrucks Genie handelt und erschöpfender Weise Hildebrand in Grimms deutschem Wörterbuch. Hinzufügen möchten wir nur eine Stelle aus dem kürzlich veröffentlichten Briefwechsel zwischen Goethe und Lavater, die für die schärfere Abgrenzung von „Genie" gegenüber „Talent" von Bedeutung ist. Goethe ^ in mustergültiger schreibt (Schriften der Goethe-Gesellschaft, Bd. 16, S. 125) 24. Juli 1780:. „Bei Gelegenheit von Wielands Oberon brauchst du das Wort Talent, der Gegensatz von Genie wäre, subordiniertes. sein kann als als wenn es wo nicht gar, doch wenigstens etwas sehr Wir sollten aber bedenken, daß das eigentliche Talent nichts die Sprache des Genies." Lavater antwortet darauf (unterm August 1780) mit einer längeren Auseinandersetzung über den Unteischied von Talent und Genie (S. 130ff.), woraus nur folgendes angeführt sei: 5. „Nur Ein Wort von Talent und Genie. Zwei Worte, die ihrem Sinn und mögen wie schön und erhaben. Gehalte nach ungefähr so verschieden sein macht mit Leichügkeit, was tausend andere nur mit äußerster oder es macht mit Frohmut und Grazie, was andere nur gerecht und korrekt machen; Genie macht, was niemand machen kann. Alle Werke des Talentes erregen bewunderndes Wohlgefallen; Genie erweckt Ehrfurcht, erregt ein Gefühl, das der Anbetung nahekommt." Über „schöne Seele" bringen die beste Aufklärung die neuesten von Ippel besorgten Auflagen von Büchmanns Geflügelten Worten. Talent, mein' ich, Mühe und Langsamkeit machen können; — 20* Zu den Problemen des Menschenlebens. 308 je starrer sie sich festlegt hindert die hier und Verpönung Dazu ver- in sich selber einspinnt. aller bindenden Normen alle Höherem und Niederem, zwischen Scheidung zwischen scharfe geistiger Notwendigkeit und menschlicher Willkür, bunt wirbelt Mannigfachstes kaum durcheinander, vermeidlich Gefahr, die ist in Anerkennung des Höheren nachgiebig gegen das Niedere, in Abwehr des Niederen ungerecht gegen das Höhere zu werden. Trotzdem sich läßt auf Würdigung nicht wohl verzichten. irgendwelche. Warum kann eine auf das bloße Individuum und seine Zu- Aus zwei Hauptgründen das Individuum des unmittelbaren Daseins — und standlichkeit gestellte Kultur nicht genügen? nicht: 1. weil das allein steht hier in genugsam ist, 2. und hohler wird, je von ihm entwickelte Leben mehr Individuum empirische — weder unabhängig noch selbst- Frage weil das ist in um so leerer — Das Folgen hervortreibt. seine es Wahrheit eher als unabhängig. alles Denn Vererbung, Umgebung, Erziehung bedingen es nicht nur aufs mannigfachste, sie scheinen es gänzlich hervorzubringen; sie flechten ein daß ihm weder so dichtes Netz, kann. Seele, Sicherlich der das Jedenfalls ist reicht Individualismus es nicht deshalb List noch Gewalt entrinnen Bindung auch diese für schon druck keine Bindung empfindet. völlig frei, in jenes Innerste freischwebend der ausgibt. weil der unmittelbare Ein- Denn mag sich der Individualist der Welt noch so keck entgegenwerfen und sich völlig von ihr abzulösen scheinen, er bleibt doch im Schatten und Bannkreis dieser Welt gewöhnlich nur eine Seine vermeintliche Unabhängigkeit ist Der Indi- andere Art der Abhängigkeit, eine indirekte Abhängigkeit vidualist ist geneigt, das Gegenteil dessen zu sagen die Umgebung sagt ung vorschreibt; und zu tun, was und tut, so ist es diese, we'.he ihm die Kette ist nicht zerbrochen. die Richt- Der Individualist fühlt sich der Umgebung überlegen, aber den Abstand ermessen und genießen kann er nur, bleibt er auch hier an sofern er die anderen sie gebunden. der Unabhängigkeit, aber er Zuschauer im Auge behält; so Er wonnt im stolzen Gefühl muß dabei unablässig die anderen als und Bewunderer solcher Größe denken. Das Leben kommt also nicht zu einer festen Ruhe und einem freudigen Schaffen bei sich selbst, es steht nicht auf seinen eignen Notwendigkeiten. So muß es kann es die Beziehung zum Menschen nicht aufgeben, so vom Kontraste leben, vom Kontraste zehren, den Stand einer inneren Abhängigkeit so überwindet es nie Gesellschaft und Individuum. Auch gerät Wendung solcher bei von in Gefahr, einen Zusatz das Bewußtsein der aufzunehmen. Eitelkeit und Wesens sind schiede des Lebens 309 Größe Starke Unter- der Grad der Belebung da, der Geistigkeit zeigt weiteste Abstände, die ordinäre Gleichmacherei, deren Stumpfheit alles zusammenwirft, Auch die Individualität ja sei nicht dem schwächt! Denn Wahrheit und Durchbildung einzelnen Stelle nicht sie ist wird mit Recht verworfen. verdunkelt oder abge- irgend Schaffen geistigen unentbehrlich; zu seiner vollen gelangt es an der auf den Punkt seiner eigentümlichen Stärke, wo es vollauf seine eigne Natur entfaltet, so wird es nie der Widerstände Herr werden. Aber in dem allen muß eine überlegne Notwendigkeit des Menschen treiben und wahr. Lebensprozesses und walten, ein geistiger Zwang den nyr dann verbleibt die Sache gesund leiten; Sie verfällt sofort ins Künstliche und Ungesunde, wenn das Individuum darauf ausgeht, sich möglichst an jeder Stelle indi- und groß zu zeigen, wenn es den Abstand geflissentlich wenn es gar zur Sache reflektierenden Genusses macht, viduell hervorkehrt, dessen Ausführung reine Hingebung und selbstlose Liebe verlangt. Jedes Zurücktreten hinter die Sache, jedes Aufkommen eitler Selbst- bespiegelung schwächt die geistige Kraft und lockert den Zusammen- hang mit den inneren Notwendigkeiten, an dem alles Gelingen liegt. muß man haben, nicht danach streben" (J. Burckhardt). Gewiß kämpft unter der Fahne des modernen Individualismus «Originalität vieles, was solchen reflektierenden Subjektivismus mit seinem epikure- ischen Selbstgenusse und der bare Eifer der weit überragt; Größe ihrer Leistung nur verstehen aus dem Erscheinen neuer sachlicher Aufgaben, frischer Antriebe Wirklichkeit neue Seiten abgewinnen zu ihr eröffnen. setzt sie sie namentlich läßt sich der Ernst modernen bildenden Kunst, sowie die unverkenn- in innere des Schaffens, welche der und ein innerlicheres Verhältnis je bedeutender die Arbeit, desto mehr verZusammenhänge und Notwendigkeiten, unterwirft Aber das Schaffen einer überlegenen Wahrheit, befreit sie vom bloßen Subjektivismus und Individualismus. Unvermerkt wird hier aus einem Individuum gegenüber der Geisteswelt ein Individuum mit der Geisteswelt; einen einem solchen aber kann der heutige Sturm und Drang nur Übergang zu Ähnlich einer bewahrt auch reinen Individualismus höheren Stufe bei der Wahrheit der Frage des bedeuten. Lebensinhalts den und Subjektivismus nur eine unablässige Er- gänzung vor unerträglicher Leere. Streng genommen muß er die Zu den Problemen des Menschenlebens. 310 Seele in lauter einzelne Vorgänge, schließlich in bloße Stimmungen Da und vertreiben. auflösen, die in rascher Flucht einander jagen jeder Augenblick genau so viel Recht wie der andere hat, so hätte jeder seine eigne Wahrheit; mag, das was dabei zunächst ein Gewinn scheinen schließlich sich stellt als Das menschliche Leben erschöpft schwerer Verlust ein keineswegs ganz sich heraus. in lauter einzelne Augenblicke. Die Augenblicke mit ihren Erlebnissen ver- sinken nicht völlig, kehren zurück, so Seele; urteilen und messen, so Daher muß er heute als wahr keit sie sie stellen sich uns vor die muß der Mensch sie vergleichen und verbinden, er steht auch das Unwahrwerden gilt, dessen erleben, was ihm daher empfindet er die Flüchtigkeit und Nichtig- des ganzen Getriebes, daher überzeugt er heit für gestern be- sie über den bloßen Augenblicken. sich, daß eine Wahr- oder heute überhaupt keine Wahrheit ist, daß sein und jede Wahrheit verliert^ wenn es an die bloßen Augen- Leben alle blicke gebunden bleibt niederdrückt, als Stimmungen, das der Gibt es etwas, das mehr ermüdet und tiefer unablässige Umschlag der Meinungen und eifrige Verketzern dessen, was eben noch begeistert verehrt wurde, die Herabsetzung aller geistigen Sache bloßer Laune und Bewegung zu einer Mode? Der Individualismus möchte dem menschlichen Leben zur vollen Entwicklung seiner Kraft verhelfen und ihm möglichst den Charakter der Größe geben. und würdigen läßt. Das ist ein Streben, Steht der das sich vollauf verstehen Mensch an einem Wendepunkt des Alls, beginnt in ihm eine höhere Stufe der Wirklichkeit, so dies Höhere zu ergreifen und gegen durchzusetzen, es gilt, einem Berge zu leben. um gilt es allen Widerspruch des Alltages mit Marc Aurel So hat sich von zu sprechen, wie auf alters her, wo immer der Abstand zwischen den Forderungen des Geisteslebens und der Durchschnittslage der Menschheit zur deutlichen Empfindung gelangte, mit zwingender Notwendigkeit der Gedanke einer höheren Art des Lebens, Größe des Menschen entwickelt; er läßt sich von der Höhe der griechischen Kultur durch mannigfaltigste Wandlungen hin- einer inneren durch bis zur Gegenwart verfolgen. ^ ^ Aber wird der moderne Indi- Eine solche Verfolgung des Problems durch die verschiedenen Zeiten hindurch wäre eine anziehende Aufgabe. Den wissenschaftlichen Ausgangs- punkt würden dabei die eindringenden Untersuchungen des Aristoteles über den Großgesinnten (jjLEYaXdlu/o;) bilden. Hier sind die Begriffe noch mitten im Fluß, der Gedanke einer Größe innerhalb des menschlichen Kreises ver- Gesellschaft und Individuum. 311 Größe gelangen, wenn er alle Zusammenhänge und damit alle Möglichkeit einer ErweiterEs gibt kaum ung des Menschen zu einem Weltwesen aufgibt? vidualismus zu einer wahrhaftigen inneren einen härteren Widerspruch, als den Menschen zu einer überlegenen und zugleich eine selbständige Innen- Innerlichkeit führen zu wollen und welt schroff ja an erster Stelle jene selbständige Innenwelt ver- vielfach unerfreulich sein, tritt, Mag der heutige Stand zu bekämpfen. erbittert der Religion, die wir sollten doch als freie Menschen unsere Begriffe und Überzeugungen von höchsten Dingen nicht nach dem bilden, was die Umgebung uns zuführt, sondern nach dem, verlangt. Ohne eine Um- was die Notwendigkeit des eignen Lebens kehrung der ersten Lage, ohne Metaphysik gibt es keine selbständige Innenwelt, keine wahrhaftige Wo immer daher Größe des Lebens. dem Gemenge des modernen Lebens Gestalten merklich hervorragen, da ist eine Wendung zur Metaphysik mit im Spiel. So z. B. aus bei Nietzsche.. seinen In Begriffen drücklichst bekämpft, in seinen Welt dieser hinreißende es des nächsten Anblicks, die als Bildner Welt, er Metaphysik alle und eben als modernen nach- künstlerischer Metaphysiker der Stimmung, Gemüter gewonnen. Gewalt über die gesamten der als hat Stimmungen wirkt eine völlig andere hat er die Ähnlich Strömung zur Romantik. geht Die bloße Stimmung aber reicht nun und nimmer aus, eine Größe gegenüber den verflachenden und niederdrückenden Wirkungen der Umgebung auszubilden und durchzusetzen, die bloße sie gibt Aus ung, nicht der Wirklichkeit. nur eine Größe der Mein- nichts läßt sich nichts bauen, und Stimmung hat nichts hinter sich. Nicht anders steht es mit dem Verlangen nach Kraft. Ja gerade heute bedürfen wir gegenüber schweren Verwicklungen Aufgaben des Qesamtlebens viel mehr Kraft, Kraft, gesellschaftliche Kultur zu bereiten vermag, aber wandelt sich unvermerkt Menschlichen. fast in und großen als die bloß- durch ein bloß- den einer Größe im Gegensatz zu allem Die alte Weit denkt bei der Größe namentlich an eine dem Ruhe und Selbständigkeit, die Neuzeit und eine geistige Schöpfungskraft; so auch hier der Gegensatz der Ideale von Beharren und Bewegung. Das viele Gerede von Größe dürfte namentlich aus der Zeit Ludwigs XIV. menschlichen Getriebe mehr an ein überlegene überlegenes Leistungsvermögen stammen, wenigstens berauschen sich die Schriftsteller jener Zeit besonders Die bedeutendste neuere Untersuchung über historische an jenem Begriffe. Größe dürfte die Betrachtungen" sein. von Jakob Burckhardt in seinen „Weltgeschichtlichen Zu den Problemen des Menschenlebens. 312 subjektives Sichemporheben, ein Sicheinreden der Kraft, Sichr ein distanzieren von den anderen Menschen kommen wir nun und nimme: Die eigne Erfahrung der Gegenwart zu wahrhaftiger Kraft das deutlich genug. Mehr reden von läßt sich schwerlich; sind literarisches und unser Kraft, als zeigt wir es heute tun, wir dadurch kräftig geworden, zeigt unser politisches Leben eine Fülle starker, selbst- wüchsiger, ausgeprägter Persönlichkeiten, eine Fülle großer, erhöhender Schöpfungen ? Die Notwendigkeit einer inneren Überwindung des Gegensatzes. XX- Wenn weder die bloßgesellschaftliche noch die individualistische Kultur den Aufgaben gewachsen wenn ist, keine von beiden dem Leben einen wesenhaften Inhalt gibt, und wenn zugleich außer Zweifel daß nur eine klägliche Stumpfheit einen direkten Kompromiß steht, zwischen beiden versuchen, das Leben zwischen hier und dort verkann, teilen so müssen wir unbedingt dem Gegensatz überlegen Individuum und Gesellschaft sind notwendige Seiten und werden. Erscheinungsweisen des Geisteslebens, zu seiner Ursprünglichkeit bedarf es der Individuen, zu seiner Befestigung der Gesellschaft; Indi- viduum und Gesellschaft aber ziehen aus sich selbst, umfangen. ihre Kraft und Wahrheit nicht sondern aus den geistigen Zusammenhängen, die sie Das Verhältnis von Individuum und Gesellschaft wird dem Boden der Geschichte verschieden gestalten; die Gesellschaft hat für sich den Zug des Lebens, wo es nach Auflösungen sich auf und Erschütterungen vor allem einer Befestigung bedarf, wie beim Ausgang des Altertums. Individuen das stellt zwingend zur uns Augustin Was damals auch Anlehnung an mit voller die die Gemeinschaft Klarheit vor Augen. z. B. kräftigsten trieb, Die Be- wegung zum Individuum erhält dagegen die Oberhand, wo frisch aufstrebende Kräfte die überkommenen Ordnungen als zu eng und starr empfinden und nur in Befreiung von ihnen neue Bahnen zu Das war der Haupttrieb der Neuzeit bis ins Daß dann ein Rückschlag kam, und daß in 19. Jahrhundert hinein. der Gegenwart zugleich die Gesellschaft und das Individuum eine Verstärkung verlangen, daß eine praktisch-soziale und eine künstlerisch- suchen vermögen. individuale Art um den Menschen kämpfen, das zeigt mit besonderer Deutlichkeit die innere Zerklüftung unserer Zeit, das muß aber zu- gleich als ein zwingender Antrieb zur Erhebung über jenen Gegensatz, zur Wendung von einer bloßen Menschenkultur zu einer Gesellschaft und Individuum. Nur durch ein inneres Vordringen des Lebens läßt klüftung begegnen; das sich jener Zer- denn was überall von den echten Problemen, daß nicht Meinungen gegen Meinungen, besonders hier, gilt 313 umspannen vermag. Geistes- und Wesenskultur wirken, die jenen zu sondern Lebensentfaltungen gegen Lebensentfaltungen stehen. Die sozialdemokratische Bewegung. b) Von geistigen Strömungen der Gegenwart läßt sich nicht wohl handeln, ohne der Sozialdemokratie zu gedenken; so da aber über sie bis zum Überdruß verhandelt und geschrieben wurde, so viel empfiehlt Beschränkung auf die strengste sich was die das, sophische Betrachtung an Eigentümlichem vorzubringen philo- hat. muß aber für die sozialdemokratische Bewegung am meisten Ihr daß charakteristisch scheinen, drei verschiedene sie Strömungen zu- sammenfaßt und zur Wirkung verbindet: die demokratische, die ökonomische, die politistische;.es handelt sich einmal um die- Verlegung Lebens des Schwerpunktes des gemeinschaftlichen um sodann die Erhebung des herrschenden Seele jenes Lebens, endlich Staates den dem zu der Massen gunsten alle einzelnen namentlich schöpft Massen, um die Anerkennung des Durch ökonomische Umwälzung eine herbeizuführen und aufrecht zu erhalten, das bei die des einzigen Trägers von Vernunft und Macht. als Staat in Problems zur be- wirtschaftlichen ist der Zentralgedanke, Fäden zusammenlaufen. Das Ganze aber daraus Kraft, daß die einzelnen Bewegungen schon vor ihrer Vereinigung die Menschheit geweckt und begeistert hatten, und daß sonst bei ihre Verschmelzung nur zu vollenden Konsequenzen zurückschreckt. Strömungen Bei scheint, was unbestimmter Fassung verbleibt und vor seinen eignen sei in Kürze Demokratie Der weltgeschichtliche Verlauf jener betrachtet. denken wir keineswegs bloß an den Staat, Zusammenleben der Menschheit und an alles Verhältnis der Individuen zu den gemeinsamen Lebensgütern. Die Neu- sondern an zeit ist alles schon deswegen jener Richtung günstig, weil schwere Hemm- ungen früherer Epochen einer in Wegfall kamen. Im Altertum widerstand die Beschränkung Anerkennung der Gleichheit aller Menschen der Kultur auf einzelne Völker, Besten keinen Anstoß erregte; was eines unmittelbaren so daß die Sklaverei auch den dem Christentum die Eröffnung und eines gleichen Verhältnisses aller Individuen Zu den Problemen des Menschenlebens. 314 zu Gott an Demokratischem einpflanzte, die bis in die das wurde sowohl durch Anfänge zurückreichende hierarchische Gestaltung als durch die transzendente Lebensstimmung recht weit zurückgedrängt. Erst gehen. kam Zweigen der Reformation einzelnen in Entfaltung, es zu stärkerer um dann aber bald in die moderne Bewegung überzu- Die Neuzeit richtet den Menschen immer mehr und immer ausschließlicher auf das Diesseits, und zugleich macht ihr Hauptzug, die Aufklärung, zur Hauptsache an ihm etwas, das jenseit aller Unter- schiede der Individuen liegt: die abstrakte Vernunft, das reine Denken. Je mehr sich das zu voller Bewußtheit erhebt und auch in die Über- zeugung der Individuen eindringt, desto unwiderstehlicher wird es; immer mehr läßt damit das Menschsein alle gesellschaftlichen Unterimmer unabweisbarer wird die Anerkennung der schiede verblassen, was Menschengesicht Gleichheit alles dessen, trägt. Wohl enthält auch die Neuzeit Gegenwirkungen zu gunsten eines Aristokratismus. Große Unterschiede von von Besitz und von politischer Stellung, Bildung übermittelt die Geschichte; aristokratischer als alle Geschichte erweist sich bleibend die Natur mit ihren Unterschieden der körperlichen und schafft auch die moderne Kultur mit der technischen Gestaltung und seelischen Ausstattung; eine der wachsenden Verzweigung der Arbeit. eigentümliche Aristokratie Denn je mehr diese nimmt, desto mehr Gliederung und Abstufung erzeugt zu- desto sie, mehr Anordnung und Beherrschung bedarf sie, desto stärker wirkt zu sie stände gunsten eines neuen Aristokratismus. Dinge der hindern demokratischen Strömung Abstufung bald wird bekämpft, bald sie nicht wie als in jedoch nicht Alle das der menschlichen künstlich oder doch solche Wider- Vordringen der Überzeugung; die künstlich geworden als nebensächlich beiseite geschoben; jedenfalls wird ein starres Schicksal hingenommen, sondern durch menschliche Gegenwirkung tunlichst verringert. Mögen bei dieser Bewegung die kleinen Wogen vielfach zurücklaufen, die große Woge immer in der Richtung der Demokratie. Auch die Selbständigkeit und das Übergewicht der wirtschaft- geht noch lichen Fragen ist wiß war Sorge allen die erst auf um dem Boden der Neuzeit erwachsen. das Mein Ge- und Dein den Individuen zu Zeiten die alles überragende Hauptsache; nur ein arger Fehl- schluß konnte früher das antike Leben lediglich idealen Aufgaben zugewandt denken, weil die Philosophen das übermächtige Verlangen nach Geld und Gut aufs stärkste zu brandmarken suchten. Aber Gesellschaft und Individuum. eine Würdigung fand das wirtschaftliche Gebiet in der prinzipielle Lebensordnung antiken 315 volle Glückseligkeit Es fand es einmal nicht. nicht, weil die von der Entfaltung einer festen und begrenzten Natur erwartet wurde, diese Entfaltung aber nur eines beschränkten Aufwandes äußerer bedarf; Mittel es fand auch deshalb es viduum auf und auch die Gemeinschaft übertragen nicht, vom Indi- weil jenes ethisch-künstlerische Lebensideal unbedenklich bei dieser jene Grenze gezogen wurde. Das Christentum mit seiner Richtung der Gedanken auf eine übersinnliche Welt widerstand noch mehr einer Schätzung der wirtschaftlichen Güter. Die Theorie aber blieb bei ihm durchaus unter dem Einfluß des Altertums. Die Neuzeit da- gegen mit ihrem Verlangen nach Entwicklung aller Kraft und ihrem Streben zur unmittelbaren Welt stand von vornherein anders zur Sache. Die Güter gelten hier materiellen Hebel zur Bewegung der Kräfte, einzuleiten als weiterzuführen. Dazu wird das wirtschaftliche Streben Bedenken verblassen, indem ökonomie gestaltet. Schon die sich die in die die die veränderte den Über- zeigt So war es in den all- wenn schließlich die Theorie in Bewegung zum Kern und zum maß- vorbereitet, wirtschaftliche gebenden Typus des gesamten Haupttriebkraft aller die 17. Jahrhunderts Politik eines Großstaats findet. gemeinen Verhältnissen A. Smith veredelt, Ökonomie zur National- Renaissance Schätzung, die dann im Frankreich des gang unentbehrlicher ein als scheinen den Fortschritt sowohl Bildung nationaler Einheiten verstärkt und die durfch alten sie machte Kulturlebens Bewegung auch in Wissenschaft, und als die Kunst, Er- ziehung, Religion das Streben nach besserer Lebenshaltung erklärte. An entschiedenem Widerspruch gegen Ökonomischen lässige Wachstum deutung der solche Führerstellung des hat es nicht gefehlt, aber andererseits hat das unab- einer technischen materiellen und verfeinerten Kultur die Be- Güter immer weiter gesteigert; auch das Anschwellen des Realismus, der die Abhängigkeit des Geisteslebens von Naturbedingungen deutlich vor Augen stellt und von außen her ableiten möchte, unterstützt das weiter. in alles Innere Erzeugt nun vollem Gegensatz zu A. Smiths Optimismus die neueste Gestalt- ung der Arbeit schwere Verwicklungen auf wirtschaftlichem Gebiet, so kann es nicht Wunder nehmen, wenn von der Lösung dieser Verwicklungen, von der Herstellung einer neuen ökonomischen Ord- nung Heil Die für das ganze dritte Strömung Leben erwartet wird. ist der Politismus, die Schätzung und Zu den Problemen des Menschenlebens. 316 Wie viel im Überschätzung des Staates. 1 9. Jahrhundert dazu trieb, das hat uns schon früher beschäftigt; die Neigung, in allen Dingen den Staat voranzustellen und ihm die Leitung aller Kulturarbeit zuzuweisen, noch immer im Wachsen. sichtlich ist und abgeschwächt, verblaßt jedenfalls nicht, daß Auch hier bringt nur zu vollem und starkem Ausdruck, Sozialdemokratie die in die meisten beherrscht was, Zufällig ist es Deutschland mit seiner Neigung zur Staats- omnipotenz die Sozialdemokratie besonders rasch vordrang, während bei sie den angelsächsischen Völkern langsamer Boden gewinnt. Die Verbindung des Demokratismus, Ökonomismus, Politismus an sich keineswegs nötig, ist Frage ja die liegt nahe, ob sie nicht ob im besondem nicht die vom Demo- harte Widersprüche enthalte, kratismus verfochtene Freiheit der Individuen mit der Zwangsgewalt Wie immer es aber mit des Staates unversöhnlich zusammenstoße. der Berechtigung jener Verbindung stehen mag, zunächst geschichtliche Tatsache, ist sie eine und mit der Macht einer Tatsache ergreift sie die Zeitgenossen. Auch haben jene Hauptrichtungen trotz aller Differenz eine innere Verwandtschaft, die namentlich in merklich Abgelehnt wird wird. durchgängig der Verneinung beJenseitige alles und Metaphysische, also auch eine selbständige Qeisteswelt; das Ganze will durchaus immanent, will bloße Diesseitskultur sein und wird damit bloße Menschenkultur. Diese Grundüberzeugung spricht aus dem Glauben sie spricht an die Masse, aus der Voranstellung der wirtschaftlichen Güter, sie erscheint in der Erhebung des Staates zum Daher Träger der Vernunft. Gedankenwelt eine in religiöse ist die Meinung Überzeugung verbinden, das religiöse Fahrwasser überleiten zu können. bloßmenschliche Charakter ihr verfehlt, ist Bewegung jener mit jener sie wohl gar Denn der säkulare, wesentlich, keineswegs von den Individuen nur nebenbei er angehängt. ist Es um partielle Theorien, die so oder so zu wenden sind, sondern um eine Gesamtgestaltung des Lebens und handelt sich hier nicht eine allumfassende Gedankenwelt, die zum ganzen und seine ganze Seele verlangen. Menschen sprechen Das vornehmlich gibt der Be- wegimg heute ihre Kraft, daß sie den ganzen Menschen fordert und alle Mannigfaltigkeit seines Strebens einer allbeherrschenden Idee unterwirft Lebensentfaltungen sind nur Lebensentfaltungen gewachsen, bloße Kritik, sich ihnen alle mag sie noch so geschickt und geistreich sein, verhält gegenüber wie der flüchtige Schatten zu einem festen Gesellschaft und Individuum. So Körper. sei auch hier die Kritik auf das Notdürftigste dessen was besonders beschränkt, 317 und die Lebensanschau- die Philosophie ung betrifft. — Augenscheinlich ist zunächst der schroffe und unversöhnliche Gegensatz des Ganzen unserer Überzeugung mit dem in Bewegung wirksamen jener Wir widersprechen Lebensideal. bloßen Menschenkultur; entschiedenste aller aufs wir tun das, weil wir im Menschen zwei Welten zusammentreffen sehen, und weil nur die Ergreifung der höheren unserem Leben einen Sinn und Wert zu geben und es Bewegung zu rechte in greifung aber verlangt eine energische Weltanblicks, ersten Menschen, so ist setzen vermag. Er- Jene Umwandlung nicht nur des mehr noch des eignen Wesens des sondern kräftige Aufrüttelungen, Erhöhungen, Erneuerungen; nur zu einer Geistes- und Wesenskultur zu gelangen und dadurch dem Menschen eine innere Größe zu geben. Aus solcher Überzeugung widerstehen wir dem Demokratismus, weil er eine fälschliche Idealisierung des sinnlichen Menschen vollzieht und die Geisteswelt dem bloßen Menschentum unterzuordnen geneigt ist, widerstehen wir ferner dem Ökonomismus, weil sein Bauen von außen nach innen eine Leugnung selbständiger Probleme des Innenlebens ent- und hält, weil er mit der Herstellung eines sorgenfreien, behäbigen Zustandes das volle Glück des Menschen gesichert glaubt, verwerfen wir endlich den Politismus, weil er die Selbständigkeit der Persönunterdrückt lichkeit und damit Ursprünglichkeit des geistigen die Schaffens gefährdet, auch den Selbstwert der geistigen Güter bloßen Zweckmäßigkeiten aufzuopfern bereit Überall ist. hier in allem äußeren Vordringen ein inneres Sinken, eine Behandlung der Hauptsachen als So Aber Nebendinge, ein geistiges Kleinwerden des Menschen. ein die voller und Gegensatz eine entschiedene Macht über die Menschheit gewinnen konnte, wie Leidenschaften viel sondern auch erregen, edle Gemüter gewinnen konnte. Zuspitzung nähere Verneinung. bloße Verneinung läßt unerklärt, wie das Ganze so Leben das viel viel es nicht bloß die Aufopferung erzeugen, Es müssen hinter dem, dessen gefährdet, allgemeinere Probleme wirken, die auch wir anderen nicht ablehnen können, die uns nicht ruhen und rasten lassen, bis sie irgendwelche, wenn nicht Lösung, so doch Beschwichtigung gefunden haben. Ein solches allgemeineres Problem steckt mus; es ist die Frage in dem Demokratis- einer größeren Verbreitung der Kultur und einer gleichmäßigeren Verteilung ihrer Güter, einer kräftigeren Teil- Zu den Problemen des Menschenlebens. 318 nähme der einzelnen Individuen am Geistesleben. Nach aller Arbeit der Jahrtausende steht es damit noch immer kümmerlich genug. Wie gering ist trotz aller Fortschritte noch immer das, was der weit überwiegenden Mehrheit von den Schätzen der Bildung dünn die Schicht, ist innerlicheren Kultur teilnimmt! lichem wenig wie Christentum, das zufällt, wie Bewegung zu einer höheren und Über tausend Jahre wirkt bei uns die an der es ist uns dieser in nach mensch- Maße langen Zeit zu einer umgestaltenden Macht, zu festem inneren Besitz, Viel zu sehr ist zu wesendurchdringender Überzeugung geworden! es bei allem Gerede von Fortschritt und Geistesleben ein bloßer Überwurf über ein Dasein geblieben, zu viel von bloßen Naturtrieben beherrschtes wenig sind die großen Gegensätze und Spannungen, aber auch die großen Möglichkeiten, die unser Leben für das Bewußtsein der Einzelnen herausgearbeitet. Nunmehr beginnen wir - schon das ist eine Wendung zum Besseren — enthält, es als eine Unwahrheit zu empfinden, in der Lebens Mehrzahl Menschheit wohl daß eine höhere Art des der Individuen innerlich fem solches Gefühl einmal wirksam irgend und fremd ist, aber bleibt; ist der ein wach geworden, so wird es irgendwie zu be- wenn im Kampfe nach solchen Zielen die Schranken menschlichen Vermögens sich noch so bemerklich machen, friedigen es sein; selbst besagt einen gewaltigen Unterschied, ein Schicksal Teilnahme aller ob die bisherige Lage wie Kampf für eine stärkere aufgenommen und damit die Menschheit der Schuld hingenommen, oder ob ein möglichst entlastet wird. Diese Erwägungen werden durch eine Wahrnehmung verstärkt, der sich kein Unbefangener verschließen kann. manche Zeichen mus des Lebens Unsere Zeit enthält eines Oreisenhaftwerdens: ein raffinierter Epikureis- weiter greift Führung berufene Kreise zeigen und weiter um sich, manche zur sich geistesträge ohne ihrem Leben einen wertvollen und abgestumpft; Inhalt zu geben, halten sie hohe ist es verwunderlich, daß die Überzeugung immer mehr Boden gewinnt, daß es heute fast mehr noch als neuer Ansprüche aufrecht; Ideen neuer Menschen, frischer und einfacher Individuen, aufstreben- der und geistesdurstiger anerkennt, braucht noch bekennen und die von halten, das Bevölkerungsschichten keineswegs sich zur bedarf? Wer das Sozialdemokratie zu ihr erstrebte Erneuerung für die richtige zu Verlangen nach Erneuerung aber wird er verstehen. Der Ökonomismus droht mit seinem Anspruch auf die Führung Gesellschaft und Individuum. des Lebens es in 319 abschüssige Bahn zu wo ein selbständiges kann nur da die Gemüter gewinnen, er leiten, problematische und eine wo seelische Probleme fehlen. Aber die wirtschaftHebung ließe sich nicht als eine Befreiung von aller Not be- Innenleben und liche grüßen, lastete Schwere licher die Sorge nicht um die Lebenserhaltung mit pein- Menschen. auf vielen Es wäre gewiß kein Glück, wenn der Tisch des Lebens den Menschen fertig bereitet würde und sie nur zu genießen brauchten, wenn alle Sorge und aller Kampf Aber entfiele. es bleibt um daß diese eine Sorge tieftraurig, die Lebenserhaltung so sehr überwiegt und das menschliche Sinnen und Denken so zwingend kommt festhält, wie gewöhnlich es Es geschieht. damit ein schwerer Druck auf das Leben, der zu innerer Kleinheit lichkeit und Erniedrigung wirkt und mit seiner stumpfen Alltäg- und frische alles freie Großes geboren. Not viel gibt eine Armut, Aufstreben Aber mit hemmt. Recht sagt Gewiß hat die Pestalozzi: zur Emporbildung der menschlichen die »Es Kräfte und 'zur Grundlage seines Glückes und seiner inneren Größe dient. Aber es VIII, 98). gibt auch eine Armut, die zur Verzweiflung führt" (Wke. Vieles in ist der Neuzeit zur Bekämpfung solches Druckes und solcher inneren Erniedrigung geschehen; dürfen wir behaupten, viel mehr geschehen könnte, daß nicht bloß in den daß nicht noch Gesinnungen der Individuen, sondern auch hältnissen nicht manches sich anders den allgemeinen Ver- in und besser gestalten ließe? Das Bedenkliche des Politismus kam wiederholt zu Erwähnung; nicht nur der Freiheit der Einzelnen, auch der Seele des Gesamt- lebens drohen von daher Gefahren. gehen sollte, erträglich Jahren. so „Wenn alles nach Vorschriften müßte das Leben, das so schon schwer, völlig un- werden", so meinte schon Plato vor mehr als zweitausend Aber warum dringt denn vor, gerade auch die Staatsidee heute so mächtig Kreisen, welche besonders in um die Freiheit be- Doch wohl, weil das Individuum bei der Zerreibung der überkommenen Zusammenhänge und dem völligen Unsichersorgt sind? werden der eignen Stellung irgend welchen es seine irgendwie auch Existenz schützt sehen will. hinaus auch in Das das greift weit Innere festen Halt ersehnt, weil vom Ganzen über alle geschätzt und ge- wirtschaftliche und Ganze des Lebens. Ein Fragen solches Verlangen nach mehr Halt und Schätzung hat beim Zusammenbruch des Altertums nicht wenig dazu beigetragen, der christlichen Kirche die Herzen zu gewinnen; heute aber scheint es sich wieder mit Zu den Problemen des Menschenlebens. 320 Hüten wir uns, solche Bewegungen gering und verborgen im Untergrunde des Lebens neuer Kraft zu erheben. zu achten, weil sie still Denn erfolgen. bereiten, die sie sind in es, denen sich die seelischen Lagen dann plötzlich mit unwiderstehlicher Kraft hervorbrechen und das Ganze des sichtbaren Lebens in völlig neue Bahnen treiben. Verschiebungen, Innere Ausdruck gestattet Umwandlungen, wenn molekulare heute sind ist, im Gange; welche menschlicher Verhältnisse daraus hervorgehen wird, das dieser Gestaltung liegt einst- weilen in tiefem Dunkel. Auch die Einheit der Gedankenwelt, die in der sozialdemokraBewegung wirkt, sei nicht zu gering angeschlagen. Wohl muß bei unserer Abweisung aller bloßen Menschenkultur die be- tischen sondere Art dieser Einheit mit ihrer Vergötterung des Menschen sie allein macht es möglich, daß die Verzweigungen der Arbeit sich gegenseitig unterstützen, Mensch als Aber Einheit bleibt Einheit, eine verhängnisvolle Irrung erscheinen. in Tätigkeit tritt. und daß an jeder einzelnen Stelle der ganze Das einzige System, das außerdem heute eine allumfassende Einheit bietet, ist der kirchliche Katholizismus, der aber mit seiner strengen Bindung an die mittelalterliche Denkweise unvermeidlich in einen immer schrofferen Gegensatz zu den Beweg- ungen der Zeit und den Bedürfnissen des modernen Menschen, zu den inneren Notwendigkeiten des Geisteslebens gerät. ja Auf dem eignen Boden der Neuzeit besaß die Aufklärung eine Art von Lebens- und zugleich einheit ein allumfassendes Ideal; seit ihrer Erschütter- ung befinden wir uns in starker innerer Zerklüftung, die immer Im besonderen pflegen die, welche von der unerträglicher wird. Freiheit aus das Leben gestalten möchten, spruch zu verfallen, daß fähigkeit gegen des Menschen eine allein und ihrer sich leerer sich ihre Freiheit * gegenbringen, um welche eine so solche sie da- Schätzung des mit Feuereifer bekämpfen sicherer dünken, Gedankenwelt gestaltet. in Würde, Leistungs- genug rühmen können, daß zu begründen vermag, ung und Verflachung trägt. nicht Weltanschauung, Menschen dem wunderlichen Wider- praktisch die Größe, sie Indem je negativer, je sie so aller Vernein- der Weltanschauung ihre Sympathie ent- untergraben sie selbst den Boden, der ihr Streben Solche Unklarheit oder vielmehr Gedankenlosigkeit ist keiner durchgreifenden Wirkung fähig. So ist entscheiden eine schwere Krise nicht zu verkennen; müssen, ob die heutige Kultur und es wird sich Gesellschaft die Gesellschaft und Individuum. Kraft enthält, Zusammenfassung und geistige Erhöhung zu vollziehen und damit der Auflösung Widerstand zu des Lebens leisten, eine innere 321 oder ob sie eine solche nicht aufzubringen vermag. Ersteren könnte der Angriff nur dazu dienen, die Kultur auf ihre eigne Falls Tiefe zu bringen und sie der Kleinheit des menschlichen Getriebes zu entwinden; im anderen Falle müßte die heutige Kultur und Gesellschaft untergehen, und sie würde solchen Untergang dann ver- dienen. Die heit Welt geistige und steht sicher fest selbst, .sowie stirne über den Wandlungen könnte sein, daß Geistigkeit und erst eine bloßen der krasse Wirken zur MenschErdoberfläche. Verneinung eine Auflösung aller erforderlich wäre, ihr über solchen Wandlungen, wie die Ge- aller unsichtbaren es Zusammenhänge um der Menschheit auf dem Wege eines indirekten zum Bewußtdem Leben wieder zu dem Wahr- Beweises ihre Unentbehrlichkeit wieder nachdrücklich sein Ja selbständigen zu bringen und dadurch heitsgehalt zu verhelfen, der heute schmerzlich vermißt wird. Eucken, Grundbegriffe. 4. Aufl. 21 4. a) I Probleme der Moral. Die unsichere Stellung der Moral in der Gegenwart. |ie Fassung wie '-^ Sicherheit. die Schätzung der Moral ist Un- heute voller Einmal erscheint gegenüber der Erschütterung der religiösen und philosophischen Überzeugungen die Moral als ein fester dem sich alles zusammenfinden kann, was sonst auseinanderfällt. Denn wenn alles ins Wanken gerät, es bleibt der Mensch und das Verhältnis zum Menschen, unser Zusammensein Punkt, an stellt uns Aufgaben, die niemand bestreiten kann. Bewegung zur ethischen findet sich viel So entstand eine Kultur, über den besonderen Kreis hinaus Bewegung, das Wohlsein der Mitmenschen zu fördern und zugleich dem eignen Leben einen wertvollen Inhalt zu geben. Als Moral erscheint hier der Altruismus, das Wirken für andere, das Zurückstellen der eignen Interessen vor denen der anderen. Diese Bewegung bildet ein Hauptstück der gegenwärtigen Kultur, breite Wirkungen zur Hebung von Not und Leid, zur Milderung von Strenge und Härte, zur Humanisierung des menschlichen Daseins sind von ihr ausgegangen und gehen unablässig von ihr aus. Aber inmitten solcher Leistungen Bedenken und Widerspruch. Moral Altruismus nur deshalb als tieferen verbleibt Vielleicht so im Prinzipiellen viel uns bei der zusammen, weil die finden leicht wir Probleme der Sache zurückgeschoben, wenn werden. Ist es nicht geleugnet denn ausgemacht, daß Moral mit Altruismus, mit einem Handeln für andere zusammenfällt? Der Ausdruck Altruismus stammt aus der Gedankenwelt Comtes, das heißt aus einem Systeme, das alles Beisichselbstsein der Seele und gar in das Verhältnis die hier der Moral anderen gelten? setzen die In zur preisgibt Umgebung läßt; sollte gegebene Fassung ohne weiteres auch für uns Wahrheit heißt Moral und Altruismus gleich- Moral im Umfang einengen und Erschöpft denn und das Leben ganz aufgehen das soziale und im Inhalt verflachen. humane Wirken den ganzen Um- Probleme der Moral. kreis der Moral, tragen wir nicht auch große Aufgaben in uns selbst, Bildung der eignen Seele, auch in der 323 in dem Verhältnis zur Welt und zu den Dingen ? Durch das Ganze unseres Lebens geht das Ent- — oder, ob das Handeln unserer eignen weder Es kann sachlichen Ziele dient. oder ob es einem Lust, B. das Schaffen eines Künstlers z. durch Beweggründe verschiedenster Art geleitel sein. Der Schaffende kann Vorteil, Ruhm, Anerkennung suchen, er kann die Wünsche und Launen des ihn umgebenden Publikums zu befriedigen trachten, er kann endlich gegen und allein der inneren Notwendigkeit des lediglich wenn es sein muß, diese mit heroischer Kraft Schaffens folgen und, Widerspruch der Umgebung allen durchsetzen; ist bis Treue gegen sich solche nicht auch ein Handeln moralischer Art? zu eigner Gefährdung selbst und Sache die Ähnlich kann den Forscher, ähnlich den religiös Überzeugten die geistige Selbsterhaltung in den härtesten Widerspruch zur Umgebung führen und ihn ihre Ruhe, ihr Behagen gründlich zu erschüttern zwingen; ja es mag die ganze Be- wegung zur Geistigkeit mit ihren Ansprüchen, Sorgen und Zweifeln als und eine Störung des Gleichgewichts Wohlseins mittelbaren erscheinen; moralische Aufgabe anzuerkennen? ist als eine Feindin trotzdem Trifft nicht in des uneine ihr aber dieses zu, so ist die Moral sicherlich etwas tieferes und besseres als der bloße Altruismus. Auch das spricht wider diesen, daß er die Moral nicht aus der eignen Tiefe der Seele zu begründen, nicht sie zu einer Sache machen versteht geistiger Selbsterhaltung zu Zu Gute kommt ihm aber bei den Menschen die Unsicherheit, die heute über einer solchen Begründung Von waltet. der Vergangenheit her wirken auf uns zwei Gedankenwelten mit einer innerlicheren Art der Moral: die der und Religion Verhältnis die immanenten Idealismus. eines Dort war es das zu einem weltüberlegenen Sein, hier war es die eigne Vernunft des Menschen, woraus lebenumspannende Aufgaben und eine ethische Bewertung Beide Arten aber sind in ihrer Grundlage all unseres Handelns hervorgehen dem Geistesleben erschüttert, sondern sollte. der Gegenwart nicht nur auch ihrem Inhalt nach vielfach bedenklich geworden. gänzlich verschwunden, aber auch die einer immanenten Vernunft ist Die Welt der Religion ist für viele mehr und mehr verblaßt; zugleich aber erscheint dem Hauptzuge der Zeit die religiöse Moral als zu weich und zu passiv, die Ver- nunftmoral aber auch als zu rauh. als So zu abstrakt und mit ihrer strengen Pflichtidee bleibt als der einzige unbestrittene Punkt die 21* Zu den Problemen des Menschenlebens. 324 soziale Moral mit ihrem Altruismus, dieser aber hat sich uns als viel Demnach bleibt zunächst nur die Tatsache zu eng und flach erwiesen. festzustellen, daß unsere Zeit überhaupt keine Moral dem vorhandenen Stande der weltgeschichtlichen Evolution in ihr besitzt, welche des Geisteslebens entspricht, daß sie einer charakteristischen, ihre Ihrem innersten innersten Bedürfnisse befriedigenden Moral entbehrt. Wesen nach angesehen, ist die Moral uns heute mindestens ebenso unsicher als die Religion. ^ Wie sehr solcher Mangel an einer eignen Moral die Kraft der Moral in unserer Zeit herabsetzt, wie sehr er es den Gegnern der Moral werfen und Karikaturen von ihr zu ent- erleichtert, und mit der Verspottung dieser sie selbst für widerlegt abgetan zu halten, das stellen uns zahlreiche Eindrücke der Gegen- wart mit voller Klarheit vor Augen. Wir werden solchen Verwick- lungen nicht gewachsen werden, wenn es nicht gelingt, durch Selbst- besinnung und Selbstvertiefung des Lebens wieder zu einer eignen, selbsterlebten Moral zu Hier gelangen. liegt das vielleicht alier- dringendste Bedürfnis der Zeit. b) Moral und Metaphysik. Weithin reicht heute die Neigung, die Moral von den Problemen der Weltanschauung zu selbst erfassen. gänzlich abzulösen Viele sehen darin zugleich eine Vereinfachung des Lebens, bilder ist und unmittelbar eine bei sich große Befreiung und manche geschichtliche Vor- werden zur Unterstützung dessen herangerufen, im besondern es der große Name Kants, der dies Unternehmen sanktionieren soll. Gewiß bestand von altersher die Neigung, Verwicklungen alle der Weltprobleme von sich zu werfen und sich auf ein rechtschaffenes Leben zurückzuziehen; der Einzelne mag ein gutes Recht haben, das zu tun, aber hat es auch das Ganze der Menschheit ? die Setzt nicht Wendung des Einzelnen voraus, daß unabhängig von ihm eine Moral gesichert und anerkannt ^ Trotz solcher Unsicherheit ist? Im besondern kann nur eine im Fundament bücher und Kompendien der Moral in Hülle und bietet Fülle. unsere Zeit Lehr- Und warum auch wenn er zu dem Worte Hamlets, es gäbe Menge Dinge im Himmel und auf der Erde, wovon nichts in unseren Kompendien stünde, bemerkt: «Gut, aber dafür stehen auch wieder eine Menge von Dingen in unseren Kompendien, wovon weder im Himmel noch nicht? Lichtenberg hat wohl recht, eine auf der Erde etwas vorkommt« (s. Vermischte Schriften [1801] II, 356). Probleme der Moral. gänzliche Verkennung Kants ihm probleme von die Absicht zuschieben, alle Welt- sich zu werfen und tischen Arbeit zu in Denn flüchten. 325 den sicheren Hafen der prak- Denken bewegt sein sich nicht um den Gegensatz von Theorie und Praxis, sondern um den von wo und praktischer Vernunft; theoretischer Frage kommt, da handelt es sich immer aber die Vernunft in um Weltzusammenhänge; vom Ganzen der so verzichtet Kant nicht auf letzte Überzeugungen anderen einer den Punkt, der über sie entscheidet, an er sucht nur Wirklichkeit, Stelle als die alte Spekulation, nicht zum Mittelpunkt seiner Gedankenwelt, Ordnung der Dinge, eines intelligiblen Reiches der Ver- einer neuen nunft zu machen. Kant Metaphysiker durch ein macht die Moral er ohne sie zur Erscheinung ist ein Metaphysiker eigner Art, aber er und durch, die Alltagsweisheit ist von dem Vorrang der praktischen Arbeit über das Denken hat an ihm keinen Bundesgenossen. In Wahrheit brauchen wir das Phänomen der Moral nur etwas um zu gewahren, daß es mit dem nächsten genauer zu betrachten, Weltanblick aufs schroffste Lebens und Strebens immer versteckt wieder verschieden eine Sobald wir an einer Handlung entdecken, die Ablösung des daß als moralisch ge- Gesinnung, die wenn auch und auf Umwegen, zu den Zwecken der Selbsterhaltung zurücklenkt, Nun nichtet. enthält sie vom bloßen Ich, ein Hinauswachsen über die natürliche Selbsterhaitung. priesenen Wie zusammenstößt Moral gefaßt werden mag, zeigt gilt ihr freilich Charakter moralischer uns als ver- schon die Natur gewisse Ansätze einer Befreiung des Lebens von der bloßen Selbsterhaltung, aber diese bleiben zerstreut Wendung, und mit fremdem verquickt; so bedeutet es eine Umwälzung, wenn die neue Art des Handelns eine reine Ausbildung erlangt und die Herrschaft über das Leben fordert Neue Größen, neue Werte treten damit ein; bedarf es nicht einer neuen Welt, um ihnen Kraft und Zusammenhang zu geben? ja eine Einen moralischen Charakter kann ferner das Handeln nur behaupten, liches wo es aus freiem Entschlüsse hervorgeht und ein ursprüng- Leben betätigt. Wo irgend sich daher herausstellt, daß ein vermeintlich moralisches Tun aus bloßer Gewöhnung, mechanischem Zwange, autoritativem Drucke hervorgeht, scheidung und Zuwendung auszeichnende Art Nun und in sich trägt, fällt aus der nicht eine eigne Ent- da verliert es sofort seine moralischen Sphäre heraus. aber bietet jener Selbsttätigkeit und jener freien Entscheidung Zu den Problemen des Menschenlebens. 326 die natürliche Welt mit den mindesten Platz, ihrer ihr durchgängigen Kausalverkettung nicht Gefüge widerstrebt jeder Lockerung. Be- steht also nur ein Reich der Natur, und bleiben seine Ordnungen maßgebend auch für das Geistesleben, so ist kein Platz für irgend- welche Moral, die mehr als eine Polizei des gesellschaftlichen Lebens sein will. Wo die moralische Forderung volle Selbstbewußtheit gewinnt, da erhebt den Anspruch, sie allen übrigen überlegen zu sein, da "verwirft mäßigkeit und stellt Leben Sie steht hinein. Zwecken unvergleichlich Erwägung der bloßen Zweck- sie alle unmittelbar in das menschliche ein Absolutes und fällt mit dem Wort: «Was dem Menschen, wenn er die ganze Welt gewönne und an seiner Seele?" Aber wie ist eine neue Art der Wirklichkeit sich alle hülfe es Schaden das möglich, und bedeutet es nicht eine ungebührliche Überspannung, müssen litte wenn hinter jener Schätzung nicht Denn im nächsten Dasein steht? Zwecke zueinander fügen und nacheinander be- messen; hier gibt es nichts Absolutes, das sich unvergleichlich über andere hinaushöbe. alles So enthält trachten, die die Moral, von welchen Seiten her wir Forderung einer neuen Welt, sie be- es liegt in ihr eine Um- kehrung des nächsten Anblicks der Dinge und damit eine Metaphysik. Wir werden also die Metaphysik nicht los, wenn wir uns zur Moral begeben; machen wir Ernst damit, von physische fernzuhalten, so drücken licher Flachheit herab. ihr alles Meta- wir sie unvermeidlich zu kläg- Andererseits hat es gewiß einen guten Grund, die Moral nicht an die verwickelten Gedankengänge der älteren Spekulation zu binden, jenseit ihrer Einen bietet sie nicht als ein sekundäres Phänomen von einer begründeten Weltanschauung abhängig zu machen. Weg zwischen solcher Scylla und Charybdis hindurch uns wiederum unser Begriff des Geisteslebens als der Wendung dem Zusichselbstkommen des Weltprozesses, der Erringung eines Wesens und Sinnes der Wirklichkeit zu einem Eigen- und Innenleben, gegenüber allem erhaltungen der sinnlosen Gewebe der Beziehungen und Selbst- Mit Anerkennung neuen bloßen Punkte. dieser Welt sinkt die Natur notwendig zur zweiten, zu einer niedrigeren Form des Seins. Wie aber die höhere von unablässiger Selbstttätig- keit getragen werden muß, so erst zu erwecken und von ihr selbsttätig anzueignen. aber als solche selbsttätige ist sie auch an jeder einzelnen Stelle Nichts anderes Aneignung der Geisteswelt ist die Moral, Probleme der Moral. 327 sie ist damit ein Durchdringen des Lebens zur Wesen- und Wahrhaf- tigkeit, das Gewinnen eines neuen, unendlichen Selbst, ein Unendlich- verden von innen Denn das erkannten wir als der Geistesstufe her. daß hier jedes Einzelne unmittelbar am Gesamtleben wesentlich, teil hat, nicht erst durch die Vermittlung des Punktes es zugefiihrt erhält. Bei solcher Fassung ist die Moral an erster Stelle eine Beweg- ung innerhalb des eignen Lebenskreises, jetzt Weltcharakter einen selbst unmittelbar eine erweist, Streben zu unserem ein Aber indem eine Erringung des eignen Wesens. Selbst, erscheint dies Wesen der Arbeit an uns in Bewegung der Welten. Das ist es, was uns die engste Verbindung von Moral und Metaphysik verlangen was uns eine läßt. Moral ohne Metaphysik ein als heißt, Unding erscheinen Die Moral verlangt nicht etwa bloß zu ihrer Erklärung Welt- begriffe, sondern durch ihr eignes Dasein entwickelt sie unmittelbar eine neue Welt und umfängt uns damit in einleuchtender Gegen- Die Verbindung der Moral mit der Metaphysik verwerfen wart. kann nur, wer entweder unter Metaphysik jene alte Schulmetaphysik versteht, die aus vermeintlicher Denknotwendigkeit zur vorhandenen Welt eine neue hinzu ersann, oder wer die Moral zu einer bloß- Ordnung, zu einer Polizei des Lebens herabdrückt. gesellschaftlichen Denn dafür bedarf es allerdings keiner neuen Welt, aber es ist dann auch jene Lebenspolizei nur dem Worte nach Moral. Nach unserer Überzeugung sinkt alle Moral zu einem bloßen Schein, wenn nicht das Geistesleben, Aneignung dessen sie vollzieht, den Kern der Wirklichkeit bildet. Die dargebotene Fassung der Moral ist den Problemen und Schwierigkeiten gewachsen, mit denen die Moral zu tun hat, und an die sich Irrung und Mißverständnis knüpft. hier viel Die Moral ist an erster Stelle Lebenserhöhung, Gewinn eines wahren Selbst gegenüber einem nur scheinbaren, Aneignung der ganzen Unendlichkeit. Aber diese Erhöhung entwickelt sich nicht vom unmittelbaren Dasein aus durch eine bloße Steigerung der Natur, sondern im Gegensatz zu jenem sie erscheint ergriffen sein, Aufgabe, eine Forderung, ein Gebot. grenzungen und Verneinungen damit sie will als eine Aber was dies Gebot an Be- enthält, dient letzthin der Lebens- bejahung; der Pflichtgedanke, der hier entsteht, entspringt dem eignen Wesen, nicht von außen her. So eine Bejahung des Lebens, die keine Vergötterung der bloßen Natur und Selbstheit bedeutet, sondern sich durch ein entschiedenes Nein davon abgrenzt. Zu den Problemen des Menschenlebens. 328 Bei dieser Fassung besagt die Moral nicht eine bloße Leistung gegebenen Welt, sondern das Erringen einer neuen Welt, in einer nicht einen Kampf sondern einen Kampf um um eine neue Art des Wirkens, innerhalb der Welt, ganze Welten; es handelt sich nicht um eine neue Art des Seins, die sich freilich unablässig sondern Wirken umsetzen muß. Im Menschen nunmehr verschiedene Stufen der Wirklichkeit, ja ganze Welten, und seine Entscheidung gehört dazu, welche davon für ihn zur Hauptwelt werde. Ja, indem er nunmehr an seiner beein in entsprechendes begegnen sich sonderen Stelle die höhere Stufe der Wirklichkeit zu behaupten hat, indem die neue Welt hier nur durch seine Tat zur vollen Verwirklichung kommt, Handeln wächst sein über den einzelnen Punkt hinaus und gewinnt Bedeutung auch für den Weltstand. sicherste Befreiung von der bloßen Ichheit, ein So die Weitwerden der Seele, die Erhebung über alle bloße Zweckmäßigkeit, eine unvergleich- Größe und Würde des Menschen. liche Mit der Größe zusammen freilich auch schwere Verwicklungen. Denn in jener Weise läßt sich die Aufgabe nicht steigern, ohne daß in der menschlichen Lage weiteste Abstände und härteste Wider- Vor allem hält die natürliche Welt den Menschen fest beim bloßen Ich, die Bewegung zur Geistigkeit dringt stände ersichtlich werden. demgegenüber wenig durch, sie zu bloßem Scheine zu sinken. droht ein bloßer Vorsatz zu bleiben, Deutlich erhellt, wie, an der Kraft des bloßen Menschen gemessen, etwas Unmögliches gefordert wird; muß der Mensch mehr werden als bloßer Mensch; wie könnte Wendung im Weltleben erfolgen ohne die Kraft einer Welt? muß eine Weltkraft von vornherein im Menschen wirken, es So muß dem Tun ein Empfangen, dem Aufsteigen ein Gehobenwerden entsprechen, in der Freiheit selbst muß eine Gnade ersichtlich werden. In dem allen vollziehen sich große Wandlungen des ersten Anblicks; so eine das anfängliche Ja wird unerträglich, aber aus dem Nein erhebt sich Große Forderungen und schwere Erschütterungen, ergreifend und verwandelnd, viel Unfertigkeit und viel Unsicherheit, viel starrer Widerstand und lähmende Hemmung. Aber inmitten der Zweifel und Widerstände ein neues Ja. gewaltige Lebensfluten den Menschen ein Aufrechterhalten des Lebens, die Eröffnung größerer Tiefen, das Gewißwerden einer inneren Unendlichkeit. zeigt die Moral, Wenn irgend etwas, so daß unser Leben nicht gleichgültig deutendes in ihm vorgeht. ist, daß Be- Probleme der Moral, c. (Ethische Moral und Kunst. und ästhetische Lebensanschauung.) Daß Kunst und Moral von und Zusammenstoß vielfachem menschlicher Folge 329 Irrung, Spannung altersher in feindlicher stehen, einen hat es keineswegs bloß Grund der ist in eine Sache. Beide Gebiete scheinen das Leben unter entgegengesetzte Aufgaben Die Moral verlangt eine Unterordnung und Schätzungen zu stellen. unter allgemeingültige Gesetze, dagegen die freieste Ent- die Kunst faltung der eignen Individualität; die Moral spricht mit dem strengen Gebote der Pflicht, die Kunst beruft zum freien Spiel der Kräfte; die Moral hat ihre Stätte in der reinen Innerlichkeit und ist geneigt von der sichtbaren Leistung gering zu denken, die Kunst schätzt Weg zur Verkörperung findet Zur richtigen Wür- und es vorteilhaft sein, nur, was den digung dieses Gegensatzes Streites ein^n Blick auf die geschichtliche weil halb, zur Ablösung das augenblicklicher wird Bewegung zu werfen, schon des- des Problems von der Zufälligkeit Stimmungen dienen mag. a. Die Geschichte des Problems. Merkwürdigerweise hat das alle anderen Völker in künstlerischer Leistung überragende griechische Volk in seiner Gedankenarbeit der Kunst keinen bedeutenden Platz die Anklagen gegen die Kunst Denkern: gegeben. als der größte Künstler unter den Die verschiedensten Plato. Kein Geringerer führt Richtungen verbinden sich ihm zur Bemänglung der Kunst. einem zum wesenhaften seines Strebens Sein Verlangen nach und unsinnlichen Sein drückt ihm die Kunst Schein des bloßen Scheines herab, anstößig wird ihm femer der bunte Wechsel ihrer Gestalten, wie ihn vornehmlich das Drama zeigt, anstößig kreises, die Unlauterkeit des mjrthologischen Vorstellungs- der sie beherrschte, anstößig endlich die fieberhafte Erregung des Gefühlslebens, die er immer weiter vordringen sah. durch solche Anklagen ging die Kunst ihren Führung des antiken Lebens. Virtuosität verlief, liche Spielerei, bald in Unbeirrt Weg und behielt die Aber je mehr sie sich in subjektive barocke Überspannung, bald in weich- und je mehr die Glätte der Form einen bedeutenden Inhalt verdrängte, desto stärker wurde der Rückschlag einer herben und harten Moral, desto mehr wurden Kynismus und Stoizismus Zu den Problemen des Menschenlebens. 330 Zuflucht eine dem Genüsse des Schönen zu selbständigen Wertes die Verbindung mit der aufsteigenden religiösen die Seelen, stolzer huldigen verschmähten. Die volle Kunst erst ung, hat in die Kunst vertieft. Wirklichkeit Nach Plotin dabei Plotin des Niedern wältigtwerden die Seele, eines namentlich bei sie welche Anerkennung vollzieht den sich auch die Aufgabe der beim Schönen durch das Höhere, Be- ein des Körpers durch den Gedanken; das Schaffen versenkt des Stoffes durch sich nicht in hat Beweg- Verinnerlichung, die erreicht; erfuhr, hat sondern es bleibt bei sich selbst und geht Stein, von Seele zur Seele; nur als Übermittlung des seelischen Standes Werk einen Wert. Die Kunst ist nicht eine bloße Nachahmung der Natur wie bei Plato, sondern sie sucht die in hat das sichtbare jener wirksame höchste Vernunft abzubilden und kann dabei ganz wohl mehr stimmung erreichen läßt solche die als religiöse Grund- Schätzung des Schönen weit mehr der Be- trachtung des Alls zugute kommen, Eine künstlerische Schaffen triebe. Aber die Natur. Ganze des Lebens, aber sie als daß sie zu künstlerischem Stimmung durchwaltet hier das scheut vor greifbarer Gestaltung eher zurück, als daß sie dieselbe suchte. Das Christentum konnte den Schwerpunkt des Lebens nicht vom Künstlerischen ins Moralische verlegen, ohne daß die Schätzung und Stellung der Kunst zunächst den schwersten Abbruch erfuhr. Auch die besondere Art der Kunst des ausgehenden Altertums konnte die Abwendung von ihr nur unterstützen. Aber was in der Breite des Daseins sich oft recht unerquicklich ausnimmt und nicht selten in eine Geringachtung aller Form verfallen läßt, das hat auf den Höhe- punkten die entschiedenste Gegenwirkung gefunden; die Verinnerlichung des Seelenlebens durch die Religion hat auch hier die Kunst in neue Bahnen geführt. So vornehmlich bei Jesus selbst. überhaupt von den Begründern der geschichtlichen Religionen Was gilt, daß nur das Vermögen einer hervorragenden schöpferischen Phantasie sie einer unsichtbaren Gegenwart geben, das gilt Welt eine anschauliche und ja sie überwältigende zur Hauptwelt des Menschen machen ließ, ganz besonders von Jesus; bei ihm gewinnt jenes Bilden eine besondere Wärme, Zartheit und Innigkeit. Daß er der Menschheit ein Reich Gottes als ein Reich echter Liebe und kindlichen Vertrauens mit deutlichen Zügen vorhielt, damit schlummernde Gefühle erweckte und den Gemütern eine tiefe Sehnsucht einpflanzte, das hat dem mensch- Probleme der Moral. Dasein liehen solche künstlerische eine Verklärung gebracht; eine dem Entdecken der mit besonderer Klarheit hervor in tritt Reinheit, auch 331 Unschuld und Hingebung des kindlichen Lebens und dem wunderbaren Gestalten einfachster nissen menschlicher Seelenstände. So ist hier bei Zurückstellung aller Kunst der seelischen Kunst eine sichere Bahn sinnlichen in Naturvorgänge zu Gleich- bereitet. vom GriechenSo bei Gregor von Nyssa, so auch bei Augustin. Wohl Später wirkte der Schönheitsgedanke immer stärker tum her. haben über diesen kunstfeindliche Stimmungen eine große Macht gewonnen, seine Wendung zum Christentum ward zum guten Teil durch einen tiefen Überdruß an einer formalliterarischen Bildung, durch ein Verlangen nach einem Inhalt des Lebens hervorgetrieben. Aber der in eignen Gedankenwelt hält er am Schönen einen Aufstieg zur allumfassenden Einheit bildet als Werk und Zeugnis ein staltet sich ethischer ihm Art, schließlich dieser Einheit und verstehen fest, das alle Vielheit lehrt. So ge- das ganze Weltall zu einem Kunstwerk zu einer Ordnung, die Gerechtigkeit und Liebe zu voller Ausgleichung bringt; zugleich ist Augustin selbst ein hervor- ragender Meister des Worts, seine Sprache hat die ganze Kraft und Weichheit eines zwischen den Gegensätzen des Daseins hin- und her- bewegten Geistes in sich aufgenommen, durch ihn hat die lateinische Sprache einen wunderbaren musikalischen Klang erhalten, und ist sie zu einem angemessenen Gefäß einer lichkeit in sich selbst vertieften Inner- geworden. Wie das mittelalterliche Kirchensystem durchgängig eine gewisse Ausgleichung der großen Gegensätze brachte, so hat es auch dem Schönen einen Platz innerhalb seiner Ordnung gegeben. So im Gesamtbau des Lebens, indem die Sorge für die Ordnung und Harmonie des Ganzen Gestaltung, indem hier sehr hervortritt, so auch in der näheren die Kunst der Verherrlichung der Religion und der Kirche in mannigfachster Weise zu dienen hat. Die Neuzeit mit ihrer größeren Lebensenergie und ihrer Schärf- ung aller Gegensätze zersprengt die mittelalterliche Ausgleichung, während ihres ganzen Verlaufes hat der Kampf und Gegensatz nicht aufgehört. Ihr Beginn in Renaissance und Reformation gibt dem Gegensatz sofort den stärksten Ausdruck. In der Renaissance ge- winnt eine ästhetische Welt- und Lebensanschauung zuerst eine volle Bewußtheit. Das Schöne wird hier zum Hauptwerkzeug der Ent- wicklung des Lebens, das wichtigste Mittel zur Herausarbeitung aller Zu den Problemen des Menschenlebens. 332 zur Selbstaneignung und Selbstgenießung des Menschen; die Kraft, Höhe Kunst lehrt das Leben sich selber finden, seine eigene Zugleich wirft das Leben reichen. überwiegend der nächsten Wirklichkeit zugewandt, erstrebt es sich; in er- unsichtbaren Bindungen von alle ihrer äußeren kenloses Glück. man empfindet und inneren Unterwerfung ein volles und schranBei starkem Lebensdurst und stolzem Selbstgefühl leicht die Moral als von draußen auferlegte eine Fessel, als eine starre Satzung" und widerwärtige tiger die Individualität, desto berechtiger scheint Hemmung; je kräfsie, Hemm- solche ung abzuschütteln und allein der eignen Neigung zu leben. So der Immoralismus der Renaissance, ein Hauptgrund ihres Zusammenbruchs als nicht es und denen die Kunst, sich zugleich dafür eines ihrer Höhe aber fehlt mit Einsetzung des ganzen Wesens erfaßt, zu einem ethischen Lebenswerke Michelangelo pflanzt die künstlerische fort, Auf weltbeherrschender Macht. an Gestalten, welche den Gegensatz überwunden haben, zu gedenken. Bewegung sich — in gestaltet; Von der es genügt Renaissance das Barock und Rokoko auch zieht die Renaissance die Geister immer von neuem zu sich zurück. Die Reformation hat ihre Stärke in der entschiedenen Hervor- kehrung der Moral und der Steigerung der persönlichen Verantwortlichkeit, weit über den Kreis ihres eignen Bekenntnisses hinaus War hat sie damit einen gewaltigen Ernst in das Leben gebracht. schon solche Verinnerlichung unmittelbar der Kunst nicht günstig, so kam dem dem ein hinzu, daß jene mit der Fülle ihrer sinnlichen Gestalten Menschen den Zugang zu Gott zu erschweren schien, zu unmittelbares Verhältnis zu gewinnen die allüberragende Hauptsache So konnte ein heftiger Zorn gegen alles Bild und allen Schmuck als gegen eine Verdunklung der lebendigen Gegenwart Gottes in der Seele und als eine Veräußerlichung und Verweichlichung des Lebens entstehen. Aber wenn damit kunstfeindliche Stimmungen weithin Boden gewannen, so hat auch hier auf der Höhe des wurde. Schaffens die Kunst in anderer Richtung mannigfachste Förderung sie ist nur mehr von der sinnlichen Anschauung abgeund tiefer in die Seele zurückverlegt. Die Namen eines Luther empfangen, löst und eines Bach genügen, um das zu zeigen. Die verstandesmäßige Art der Aufklärung mit ihrem Dringen auf logische Klarheit und kluge Zweckmäßigkeit, auch mit ihrer un- geschichtlichen Denkweise war der Kunst wenig günstig und stellte Probleme der Moral. sie weit hinter die Moral zurück. Diese selbst aber gewann dabei Um so stärker wurde das Verlangen nach keine besondere Tiefe. Schönheit, als durch das Aufsteigen des Epoche begann. 333 Neuhumanismus eine neue Es strebt aber auf seiner Höhe, bei den führenden deutschen Dichtern und Denkern, Gutes und Schönes zusammen und fördert sich stein gegenseitig. Daß Kant Selbständigkeit zum Eckdem Schönen eine die moralische Idee des Lebens machte, hat ihn nicht verhindert, und einen Selbstwert zuzuerkennen; er zuerst hat es gegen das Gute wie das Angenehme mit voller Deutlichkeit abgegrenzt, er hat es allen ein im eignen Innern der Seele begründet und es über bloßen Nutzen und Genuß sicher hinausgehoben. Goethe «die großen Hauptgedanken der So konnte Kritik der Urteilskraft seinem bisherigen Schaffen, Tun, Denken ganz analog" finden. Goethe war bei aller Größe seines künstlerischen Schaffens von selbst aber einer Geringschätzung der Moral und zugleich von dem Bekenntnis zu einer ästhetischen Weltanschauung weit entfernt viel Dazu war ihm zu sehr sein künstlerisches Schaffen ein ernstes und mühsames Suchen seines eignen innersten Wesens, ein gewissenhaftes Arbeiten an sich selbst; eine laxe Auffassung dieser Probleme hat kein Recht, sich auf Goethe zu berufen, wenn man ihn im Ganzen seines Wesens nimmt, nicht auf einzelne Äußerungen festlegt So wenig er die Kunst und eine künstlerische Kultur durch »konventionelle Sittlichkeiten", wollte, er durch „Pedanterie gab dem Menschen und Dünkel" beschränkt wissen in der Forderung, die Ordnung der Welt mit Freiheit zu ergreifen und sich setzen, eine selbst Grenze zu seine moralische Aufgabe, die sein ganzes Leben und ihm überall eine hohe Aufgabe stellt umspannt Schiller endlich, zwischen dem Dichter und dem Denker stehend, war unablässig um eine Versöhnung von Gutem und Schönem, der „Freiheit in der Erscheinung" bemüht; „die hohe Reinheit des sittlichen Standpunktes bei der vollsten Anerkennung des künstlerischen Lebens in seiner Selbständigkeit ist das Eigenartige, Denkweise" (Kühnemann). Die ja Einzigartige Ströme haben der sich Schillerschen dann wieder geschieden, die Romantik gab dem Vorrang der Kunst und der ästhe- Lebensanschauung einen besonders zugespitzten und selbstbewußten Ausdruck, Fichte und die anderen Führer der nationalen tischen Bewegung wirkten kräftig zur Verstärkung der Moral. Die soziale und technische Kultur, die im Verlauf des 19. Jahrhunderts immer mehr zur Herrschaft kam, ist mit ihrer Richtung auf die gesellschaft- Zu den Problemen des Menschenlebens. 334 Wohlfahrt und auf die Zweckmäßigkeit des Lebens die Kunst liehe erhebt Anhang zu behandeln nebensächlichen als einen nun sich die moderne Kunst, dem das Ganze des Lebens, sie mehr Dagegen geneigt. einen erstrebt Einfluß auf mehr Freude, Leichtigkeit, mehr Individualität verheißt, und hält oft der Moral eine ästhetische Lebensanschauung als allein So hat zur Zeit entgegen. berechtigt einmal wieder die Entzweiung der beiden Gebiete die Oberhand. Die geschichtliche Betrachtung zeigte, daß der Gegensatz durch die Jahrtausende geht, nicht eine Stimmung des bloßen Tages be- Immer wieder rückte die Moral der Kunst eine Verweich- deutet. lichung, Ermattung, Auflösung des Lebens vor, aber zugleich mußte sie dafür den Vorwurf der Härte, Schabion enhaftigkeit, Seelenlosig- keit entgegennehmen. Aber auch davon überzeugten wir uns, daß, Höhe sich suchte; bei den schaffenden Geistern war der Gegensatz, wenn auch was der Breite des Lebens einander in aufgehoben, so völlig nicht deutlich, daß das geistige floh, auf seiner doch sehr gemildert; daß die Schuld des Zwistes weniger an der Sache In liegt. ihnen ward in Leben keiner Seite entbehren kann, und und Kunst Wahrheit können Moral als die am Menschen eigne Aufgabe großem Sinne nehmen, ohne sich gegenseitig als bedeutend, ja unentbehrlich anzuerkennen, sie können jenes nicht, ohne sich in nicht in umfassendes Ganzes des Geisteslebens hineinzustellen und dort ein eine Verständigung anzustreben. Wo die Moral unmittelbar das ganze Leben einnehmen wollte, da pflegte sie zu einem System von Regeln und Vorschriften zu zum Menschen sprachen, ihm zugleich aber für werden, die streng Erfüllung ihre auch viel einen straffe Lohn Konzentration überwiegende Fassung voller innerer entzünden. Leicht zu hohen als ist verhießen. damit eines Gebotes Viel geleistet ließ Aufrüttelung, worden, aber die hier die Moral nicht Aneignung kommen, sowie Liebe und Freude ward hier der Mensch zwischen einem Bewußt- Schwäche sein hülfloser hin- und hergeworfen. und einem In der Tat selbstbewußten Pharisäismus war es immer nur eine gewisse Mittelhöhe des bürgerlichen oder kirchlichen Lebens, der die bloße Moral genügte; weder der Durchschnitt der ersten christlichen Jahrhunderte noch der der Aufklärung hatte bei allem moralischen Eifer einen bedeutenden geistigen Gehalt. Gefahr eines sie in weitere Die Moral selbst konnte die und Äußerlichwerdens erst überwinden, wenn Zusammenhänge hineingestellt wurde. Geschah dies Starr- Probleme der Moral. aber und wurde keit, eine Weg zur Aneignung einer neuen Wirklich- sie ein von Geboten, son- ihr nicht eine korrekte Erfüllung galt es in dern 335 innere ursprünglichem Erneuerung des Leben, Menschen, konnte so Vordringen ein zu keiner Weise der Kunst in sie Denn jenes Neue ließ sich nur mit Hülfe einer künstlerals Ganzes fassen und in lebendige Gegenwart entbehren. ischen Tätigkeit stellen, auch konnte es die Breite des Lebens nicht gewinnen ohne das bildende, Inneres und Äußeres zusammenflechtende Wirken der Kunst. wenn Ja daran alles neue Welt und ein neues eine liegt, Leben gegenüber den kleinen Zwecken des bloßen Menschen und dem Alltagsleben zu erreichen, so muß die Kunst mit ihrem stillen und Wirken aus der inneren Notwendigkeit der Dinge, sicheren mit ihrer inneren Befreiung der Seele, mit ihrem Vermögen, uns die ganze Unendlichkeit zu nahe innerlich und zu eignem bringen Leben zu machen, unmittelbar als moralisch gelten. Ebenso wenig kann eine Kunst, die groß von ihrfer Schwerlich war Aufgabe denkt, die Moral verachten. und sich selbst ein je schaffender Künstler ersten Ranges einer ästhetischen Lebensanschau- ung und zwar deshalb nicht, zugetan, ein Sondergebiet vom übrigen Schaffen seine ganze Seele, und weil legen mußte, Schaffens In um Dilettanten; Genuß vom Ganzen den reflektierenden und als bei haben diese die, sie genug oft den auch theoretischen Erörterungen wenig hold, kaum empfinden, daß jene Ablösung ihnen gegenüber wehrlos, der Kunst daraus einen bloßen Wahrheit hat die ästhetische Lebensanschauung ihre Künstlern aufgedrängt, ja das nicht nur eine gewisse Technik hinein- Heimat weniger bei den Künstlern genießenden in er die Mühen, ja die Unzulänglichkeit dieses zu stark empfand, viel zu saugen. weil er nicht die Kunst als Leben ablösen konnte, weil er des Lebens sie nicht sowohl erhöht als erniedrigt. Das Angewiesensein von namentlich Kunst und Moral aufeinander wird da zu voller Anerkennung gelangen, wo unsere Welt nicht als eine fertig abgeschlossene, sondern als eine erst im Werden befindliche, ja als eine solche gilt, in der nicht nur Vorhandenes auszubauen, sondern eine neue Stufe der Wirklichkeit zu erreichen ist. Dazu bedarf rüttelung des es eines selbständigen Entscheidens, einer Auf- Wesens, eines energischen Tuns, welches das ganze Dasein umfaßt; deutlich ist hier, daß wir nicht an erster Stelle zu behaglichem Genießen, zur Kontemplation, sondern zu Wirken und Zu den Problemen des Menschenlebens. 336 Schaffen, zur Aktivität berufen sind. und glücklichen kräftigen Aber zugleich bedarf es eines künstlerischen wenn Bildens, die neue Welt uns nicht in vagem Umriß verbleiben, und wenn sie das Ganze unserer Seele gewinnen soll; auch die Kunst ist eine unentbehrliche zum Aufbau eines neuen Lebens. Helferin Die Probleme der Gegenwart, ß. Der moderne Ästhetizismus. aa. Wie wir prinzipiell über den denken, Ästhetizismus bedarf Den Ästheti- nach diesen Darlegungen keiner weiteren Erörterung. zismus der Gegenwart aber klagen wir einer inneren Unwahrhaftig- und das Leben viel zu viel Dunkel und viel zu viel Unvernunft, und es regen uns die großen Uns keit an. zeigen heute die Welt als daß wir aus tiefster und mit voller Hingebung unser Dasein überwiegend in Genuß verwandeln und eine Harmonie des Ganzen mit reiner Freude Widersprüche des Daseins viel zu sehr auf, Seele Der Ästhetizismus erleben könnten. druck des heutigen Lebensgefühls als weniger ein wahrer Aus- ist ein Versuch, seiner Schwere und seinem Ernst zu entfliehen. Das aber kann er nur, indem er sich mit dem modernen Subjektivismus verbindet und in solcher Verbindung Stimmungen erzeugt, die gewiß beachtenswert sind, alle als Zeichen der Zeit denen aber alles schöpferisches Vermögen und seelenerhöhende Kraft gebricht. und Ästhetizismus Ein Zusammenwirken von Individualismus ließ das Schlagwort einer «neuen Ethik" entstehen, in weiblichen Kreisen viel Einfluß gewonnen hat. das namentlich Auch eine der- Bewegung sei nicht von vornherein verschrieen, sondern in Gründen unbefangen gewürdigt. Was die Gesellschaft Moral nennt, ist nicht mehr als eine gewisse Ordnung des Zusammenlebens, der Sitte und Gewöhnung einen Heiligenschein verliehen haben, die artige ihren daher bei auftritt, der aller Unzulänglichkeit leicht mit starker Selbstbewußtheit wie Diener leicht arroganter als Lauf geschichtliche der Herren sind. Nun ändert Dinge die Art des Zusammenseins, Änderungen können notwendig werden, die starre Festhaltung der überkommenen Art kann einen peinlichen Druck erzeugen, kann Recht in Unrecht, Unrecht in Recht verwandeln. den gegenseitigen Verhältnissen und verschoben, daß eine Revision jener in Die Neuzeit aber hat in der Art der Arbeit so viel gesellschaftlichen Ordnungen Probleme der Moral. 337 und damit der konventionellen Moral in verschiedener Richtung notwendig ist. Aber dieses anerkennen heißt nicht der hastigen und summarischen Art beipflichten, mit der von den Vertretern und vielleicht noch mehr den Vertreterinnen eines ästhetischen Subjektivismus die schwierigen und verantwortungsvollen Fragen erledigt werden. nächst Moral ist Zu- etwas anderes als ihre sichtbare Vertreterin, selbst die gesellschaftliche Ordnung, und moralisches Verhalten etwas anderes Wie gering wurde auf der Höhe des moralischen als soziale Korrektheit Schaffens von jener Korrektheit gedacht, wie entschieden wurde hier abgelehnt, das bloße Mittel zum beherrschenden Zweck zu machen! Aber auch bei das ist nicht alle Ordnung wie gesellschaftliche Druck verrufen werden. ist keineswegs Unzulänglichkeit aller Weil gewisse Einrichtungen problematisch geworden wertlos. sollte Mittel sind, ungebühriicher ein Denn wie die menschlichen Dinge stehen, unentbehrliches Mittel, das Leben auf ein gewisses Niveau sie ein zu heben und den zerstörenden Kräften, am Werke die unablässig genügenden Widerstand zu leisten. Nur ein grenzenloser, man möchte sagen kindlichnaiver Optimismus, den man liebenswürdig sind, nennen möchte, wenn mit seiner die Halbgebildeten be- er nicht man dem stechenden Flachheit gefähriich wäre, kann wähnen, daß Menschen nur schrankenlose Freiheit zu gewähren brauche, um das ganze Leben zu seliger Harmonie zu führen. Daß der Mensch zur Zügelung seiner Begierden ist wer über müßte auch sie klagt, angenehm schmeckt; Ordnungen sozialer klagenswert sein, es mag bedarf, be- aber doch nicht die Schuld der Ordnungen; Medizin verwerfen, die nicht alle würden wir nicht leicht das Gegenteil der wenn wir einem verschönernden Bilde zuliebe alle Schranken einreißen wollten? L'homme n'est ni ange ni bete; Absicht et erreichen, malheur veut que qui veut le Wenn von Tange faire fait bete (Pascal). la »neuer Ethik" geredet wird, so müssen wir auch einem solchen Mißbrauch des Wortes Ethik widersprechen. gleichgültig sind die Wörter nicht, ihr Mißbrauch Ganz kann zu einer Verdunklung echter Probleme wirken. Unter Moral hatten wir uns gewöhnt an willkürentzogenen Ordnung, und Gewissen zu denken. Was aber die die Anerkennung Hochhaltung von Pflicht einer der ästhetische Subjektivismus mit seiner „neuen Ethik" nichts anderes als ein feinerer Epikureismus, des Individuums, das sich von aller Eucken, Orundbegriffe. 4. Aufl. als ein bietet, ist Selbstgenuß Hemmung befreit; wer darin 22 Zu den Problemen des Menschenlebens. 338 sein Genüge findet, der sollte die Ethik genau so gut wie die Reli- gion Bausch und Bogen für eine Verirrung erklären und aus in seiner Gedankenwelt streichen, nicht aber sollte er durch ihren Namen Denkweise einen schimmernden Aufputz geben. seiner andersgearteten Ein schroffer Gegensatz ist hier unverkennbar. Ist der Mensch nicht mehr als die Summe seiner natürlichen Neigungen und kommt alle Lebensweisheit darauf hinaus, unser Dasein Herr zu werden? Ist rezeptiver oder es aktiver Art? Höchste ist in ein leidliches uns eine geistige Kraft, unser Verhältnis zur Wirklichkeit vorwiegend oder Güter, aller in Tat zu verwandeln und zugleich unser selber freie in Neigungen diese oder steckt Gleichgewicht zu bringen, über solchen Abschluß hinaus? unser subjektives Glück das Ist uns treibt innere eine Dieser Gegensatz Notwendigkeit steht den seit Stoikern und den Epikureern mit voller Klarheit vor Augen, und er schließt jede Die Vermittlung aus. modernen, präziser als die alten Epikureer aber dachten haben sich nicht als Vertreter einer sie neuen Ethik gegeben.^ Der Punkt, an dem der moderne Subjektivismus mit anderen Überzeugungen am zusammenstößt, härtesten ist die Behandlung des Sinnlichen, besonders in der geschlechtlichen Sphäre. Verwicklungen namentlich Christentum, vielfach keit vorliegen, niemand wird in seinen noch eine Geringachtung, fort, die in katholischen ja eine Im Fassungen, wirkt Verachtung der Sinnlich- den Stimmungen des ausgehenden Altertums und seinem Kampf gegen eine entartete Sinnlichkeit wurzelt; hier ein Stück Manichäismus in das * ist es, es ja ist Christentum eingedrungen und wirkt bei aller äußeren Strenge zu innerer Verflachung. Verflachung Daß hier können. bestreiten Denn eine wenn die Hauptsorge des Lebens darin gesetzt Mit besonderer Kraft und Tiefe hat sich über diese Probleme der aus- gezeichnete schwedische Philosoph Vitalis Norström in seinem Buch tausendjährige Reich" (deutsch 1907) ausgesprochen; er sagt z. B. S. 31 : „Das „Selt- same Schwärmerei, die auf der Ersättigung der Sinne ein stabiles seelisches Gleichgewicht zu begründen meint! Armselige Weisheit, die kein höheres Ziel kennt, als sich dauernd, wenn man so sagen kann, einen guten Tag zu — wofern sie überhaupt machen! Diese Welt des allgemeinen Genießens würde das Beste nicht aufkommen lassen, dessen der möglich wäre Mensch fähig ist: die Erhebung über den Genuß, die Selbstüberwindung. Sie würde das ausschalten, was unsrem gewiß verbesserungsbedürftigen Dasein doch eine gewisse Weihe gibt; das, was ein Landsmaim Zolas, der — edle Alfred de Vigny, gefeiert hat als die „majeste dessouffrances humaines.« Probleme der Moral. Kampf einen wird, dem mit Sinnlichen 339 zu führen, das Sinnliche wenn wer es möglichst abzuschwächen, zu erniedrigen, zu kasteien, in solchem Ertöten der Sinnlichkeit weit gebracht ein noch so als Vorbild erwählt wird. handlung harter Mensch, als Held ein Denn was wird denn und sei gefeiert er und mit solcher Miß- und Dazu muß jene Unter- der Sinnlichkeit für eine innere Läuterung der Seele Aufbau für den und leerer hat, geistigen Lebens gewonnen? drückung der Sinnlichkeit, wie alles Naturwidrige, schlimmere Schäden herbeiführen, als sie zu heilen unternimmt; die Natur pflegt sich für Aber mit der Abweisung einer eine Mißhandlung schwer zu rächen. derartigen Askese die Sache nicht schon erledigt, ist und sinnliche geschlechtliche Aber zugleich fest. Seite zeigt den Menschen der Natur mehr als irgendwie sonst hält hier die Natur aufs engste verwachsen, ihn sie liegt nicht dem ästhetischen Subjektivismus scheint. Die so einfach, wie es oft hat er sich mit der Entwicklung geistigen Lebens weit über sie erhoben und damit die schlichte Naivität auch auf jenem Gebiete verloren; so wird ihm das Sinnliche zum Problem, vom das es sich in ein voller Freiheit, verschieden beantworten läßt; den Zwecken des Geisteslebens unterzuordnen und Maß zu Wer finden? soll unbekümmert um höhere Zwecke des und Laune des Einzelnen ausleben, oder nach Lust Geistes, es sich Geistesleben aus sich unter hat in ihnen Berufung auf das unantastbare Recht der Natur sich für jenes entscheidet, der pflegt zu übersehen, daß wir bei unserer gesteigerten und vielfach überspannten KuUur nicht mit der reinen Natur zu tun haben, daß die heutige Sinnlichkeit vielfach eine raffinierte, ja entartete ist So können wir schon zur Scheidung dessen, was an der Natur echt oder unecht ist, geistiger Arbeit nicht entbehren, eine einfache Kapitulation vor dem, was sich heute sinnlich nennt, ist schlechterdings ausgeschlossen. bb. Die Stellung der Kunst im modernen Leben. Siegreich dringt im modernen Leben die Kunst wieder vor und mächtig bewegt sie die Geister; so ist es kein Wunder, daß sie alle Abhängigkeit verschmäht und auf voller Selbständigkeit besteht. solches Verlangen l'art pour l'art.* ^ S. 326) findet In dem Der Ausdruck hatte, (s. wie ihn einen Ausdruck in der Ein bekannten Formel was sie an Verneinung enthält, wird ihr kein darüber Büchmanns Geflügelte Worte, 21. Aufl., zuerst Victor Cousin 1818 Vorlesungen an in Sorbonne gebrauchte, einen ganz unverfänglichen Sinn : II faut der de la religion 22» Zu den Problemen des Menschenlebens. 340 Freund der Kunst widersprechen. Die Kunst soll nicht fremden Zwecken dienen, sie darf nicht der Moral, reichung und damit zu leisten der Politik, der Religion Hand- einer Tendenzkunst sinken, die nur für einen Augenblick blenden mag, nie aber wahre Förderung bringt Schwieriger ist es, jenem Wort einen positiven Sinn zu geben. geschieht das nicht selten gleichgültig gegen allen Stoff und Inhalt sein der Vollendung ihrer Form befassen; so allein und könne selbst völlig Heute mit der Behauptung, die Kunst müsse und sei ungehindert ihre eignen sich sie allein mit ganz bei sich Wege verfolgen. Aber liegt eine solche Ablösung vom übrigen Leben in ihrem eignen Interesse, ist? kann bei sie Die Gefahr liegt ihr das Höchste leisten, dessen sie fähig nahe, daß mit dieser Wendung die Kunst zur bloßen Formbeherrschung, zu virtuosenhafter, glänzender, blendender Technik wird, daß sie so weder den ganzen Menschen hinter sich hat, noch zum ganzen Menschen und zur Menschheit zu wirken vermag. Eine solche Kunst mag weit mehr in der sinnlichen Erfahrung entdecken, sie mag unser Empfinden in ungeahnter Weise bereichern und verfeinern, sie mag in Überwindung von Widerständen schwelgen, aber seelisch kann sie uns wenig bieten, und das Geistesleben wird kaum erhöhen. sie War nicht dies den großen Kunst- werken, die dauernd zur Menschheit sprechen, aller Gegensatz von Inhalt und Form haben sie nicht in in der Vollendung der eigentümlich, Form zugleich zu vollem Ausdruck gebracht, was das Innere des Lebens erfüllte? die daß ihnen überwunden wurde, Muß nicht Kunst die Probleme der Menschheit aufnehmen und in ihrer Weise zu lösen suchen ?i Auf jene Innerlichkeit kann am wenigsten pour la religion, de la morale pour la morale, de l'art pour l'art. Erheblich wurden die letzten Worte zum Bekenntnis einer Schule und zu einem Zankapfel der Parteien. — Hinzufügen möchten wir nur, daß auch Comte sich einmal mit jenem Schlag\i'ort befaßt, aber es sehr äußerlich wendet. Cultiver l'art pour l'art lui-meme bedeutet ihm nichts anderes als ne se proposer habituellement d'autre bout reel que de divertir le public (cours de phil. pos. VI, 167). ^ Auch über dies Problem hat sich Norström in vorzüglicher Weise später erst So bemerkt er z. B. über das oft als Beispiel eines bloßen SchönGriechentum (Das tausendjährige Reich, S. 73): »Man bildet sich oft ein, die Grundkraft des griechischen Lebens sei ein unwiderstehlicher, zwangloser Trieb zur schönen Form gewesen, also ein Bedürfnis, das an sich schon schöne Dasein noch mehr zu verschönern. Statt dessen geäußert. heitskultus angeführte aber dienten sittliche bei Kräfte zu ihnen die Gebilde der Kunst wesentlich dazu, befreien, dunkle Bewußtseinsinhalte gefesselte aufzuhellen und Probleme der Moral. Er der nordische Mensch verzichten. 341 besitzt nicht die natürliche Leichtigkeit der sinnlichen Darstellung wie der Bewohner des Südens, er das der Innerste sich selbst unausgesprochen Seele Daher wird ihm die Kunst Tiefe verschlossen. Mittel, Weg von innen nach außen; so bleibt ihm schwer den findet leicht zu finden, Eigentum sein und ein in seine eigne unentbehrliches vollen Besitz zu nehmen, die Kluft im eignen Wesen irgend zu schließen. So kann ihm auch die vollendetste Form als bloße Form nun und nimmer genügen. Wer einen Inhalt der Kunst als etwas ihr fremdes und gefähran eine Gedankenleistung, eine liches verschmäht, der pflegt dabei Idee abstrakte Aber zu denken. fällt denn Geistesleben mit dem Denken zusammen, gibt es keinen anderen geistigen Inhalt als eine Gedankengröße? Der alte Intellektualismus mochte das meinen, heute aber wollen wir. nicht mehr Intellektualisten sein; also jetzt die veralteten wie könnten uns Maße binden und uns hindern, in tieferem und' weiterem Sinne nach einem Inhalt für den ganzen Menschen zu streben? Ja wir sehen sogar in der Zurückstellung des Inhalts eine Ge- fahr für eben die Selbständigkeit der Kunst, zu deren Gunsten sie verlangt wird. Mit dem Unabhängigwerden vom Stoffe wird nicht eine Unabhängigkeit schlechthin wird ist, leicht wenn nicht anderen, zu zeigen; erreicht. Eine überwiegende Formkunst zu einem Virtuosentum, das vor allem darauf bedacht das aber so doch sich selbst das eigne Vermögen eine Vorliebe für das Abweichende, erzeugt Paradoxe, Überspannte, und mit dem Suchen dessen gerät man leicht, indem man seine Freiheit beteuert, nur in eine andere Art der Ab- hängigkeit, eine Abhängigkeit von anderen und auch von der eignen Stimmung. Schaffen Nur da besteht wahrhaftige Unabhängigkeit, wo das ganz und gar aus einer innem Notwendigkeit des eignen Wesens hervorgeht; dazu aber muß der Mensch etwas auszusprechen, zersplitterte Arbeit weiter: „Tatsächlich um gemeinsame praktische Ziele zu sammeln." - Und gilt von aller großen, echten Kunst, daß sie die Form dem Inhalte, den sie wiederzugeben sucht, mehr oder weniger unterordnet und gegen die Lust oder Unlust, die sie — bei dem Künstler und anderen — erweckt, sich gleichgültig verhält, wenn nur die Bilder den Inhalt mitteilen. eher, Die echte Kunst öffnet uns die Tiefe der schöpferischen Phantasie um uns von dem ,heiteren' Dasein zu befreien, als um uns tiefer in dasselbe hineinzuführen.« Zu den Problemen des Menschenlebens. 342 ja Der bloßen Virtuosenkunst aber fehlt eine zu offenbaren haben. solche Notwendigkeit. Mit Worte einem zur nicht von ihr kann ihr nur eine oft des Verhältnisses der modernen auch sei Daß geschlechtlichen Sphäre gedacht. Kunst die Kunst sich zurückzieht, sondern sich eingehend mit ihr befaßt, aber jenes unkünstlerische Denkweise verargen. Daß sie mit sichtlicher Vorliebe in den Vordergrund rückt und möglichst lange bei ihm und so raffimehr ein Zeichen sitt- verweilt, es so reflektiert niert bis zur Eckelhaftigkeit ausmalt, das ist Könnens, das kann durch keine ästhe- licher Fäulnis als technischen tische Theorie verteidigt werden. Wie viel bei der bildenden Kunst im Fluß und im Streit sein mag, an hervorragenden Persönlichkeiten und an glänzenden ungen auf fehlt es hier wahrlich dem Gebiete nicht. der Literatur. Minder günstig steht die Sache Anregungen und Aufgaben und und neue über seine Stellung im All steigen auf, Alte ist der Mensch höchst unsicher geworden, der eigne Kreis der Menschheit Bewegungen und Wandlungen, dazu bietet Qedankenmassen versinken hier die Zeit in Hülle Fülle. Leist- gestattet ist voller die wachsende Hast des Lebens keine genügende Selbstbesinnung; so ist uns das Dasein Hätte hier verworren und wir selbst sind uns vielfach ein Rätsel. nicht das literarische Schaffen die Aufgabe einer Klärung, hätte es nicht, was auf uns eindringt und Aussprache zu bringen, aus sich in uns regt, zur deutlichen dem Chaos der Erscheinungen, das uns umwogt einfache Grundlinien herauszuheben, uns das Leben mögGanzes zu fassen und zugleich es weiterzuführen? Dazu lichst in ein aber bedürfte es einer inneren Überlegenheit über die Gegensätze der Zeit, einer energischen Synthese, die nicht aufnähme ohne auch auszuschließen, Schaffens. ist An eines mutig und kräftig vordringenden geistigen Ansätzen dazu fehlt es nicht, aber im allgemeinen zu sagen, daß unsere Literatur, die Literatur eines der größten Kulturvölker, nicht auf der Höhe der Zeit steht, daß sie dem modernen Menschen in dem Kampf um eine geistige Selbsterhaltung und um einen Sinn seines Lebens wenig Hilfe gewährt. dies offen auszusprechen. Es ist eine Pflicht, 5. Persönlichkeit Persönlichkeit. a) a. I und Charakter. Zur Geschichte des Ausdrucks. |en Ausdruck Person, einen der wenigen Termini lateinischen *-^ Ursprungs, von seinem Ursprung an in seine schon im Alter- tum reiche Verzweigung zu verfolgen, seine Bedeutung im römischen Recht wie in der christlichen Theologie darzulegen, das würde uns zu weit von unserer Aufgabe entfernen.^ So halten wir uns an die Philosophie und streben auch hier möglichst rasch zur Gegenwart. Die neuere Philosophie empfing den Ausdruck von der Scholastik, aber folgte der Definition des Boethius, diese nünftiges Einzelwesen.2 die Trinitätslehre Person sei ein ver- Aus der Anwendung dieser Definition auf entstanden ernste Verwicklungen (Roscellin), aber Über das Technische des Ausdrucks berichtet näher die RealenzykloÜber seine Anfänge und seine Schicksale bis ins Mittelschrieb einen anregenden Aufsatz Max Müller in Good Words, Juni ^ pädie von Pauly. alter 1866, bedeutender ist eine Untersuchung Trendelenburgs, die ich aus seinem Nachlaß in den Kant-Studien Bd. XIII, Heft 1 u. 2 veröffentlicht habe. ^ Näher lautet die Definition (in der Schrift de duabus naturis, ed. R. Peiper, pag. 193, 4): persona est rationalis naturae individua substantia. Im früheren Mittelalter ward Person wohl etymologisch erklärt als per se una. Über die verschiedenen Fassungen des Begriffes bei den wichtigsten Denkern s. Baumgartner, die Philosophie des Alanus de Indem Thomas, namentlich in seiner Untersuchung über die Dreieinigkeit (im 1. Buch der summa theologiae), die Lehre des Boethius weiter entwickelt, betont er, daß Personen non solum aguntur, sed per se agunt. Er verteidigt die Anwendung des Ausdrucks auf Gott, obwohl sie mittelalterlichen insulis, S. 45. Wie andere Scholastiker, so hat auch Thomas dem schon Eckhardt in personlicheit ein deutsches Gewand gab, sich nicht in der Bibel finde. personalitas, wie er auch personlich öfter verwendet. gebräuchlichste Definition In der späteren Scholastik von Person suppositum intelligens, war die suppositum aber bedeutete dabei eine substantia singularis viva. Zesen übersetzte persona mit Selbstand (s. Paul Piur, Studien zur sprachlichen Würdigung Christian Wulffs, S. 58), Clauberg (Wke. 1691, S. 321) mit „selbständig verständig Ding." Zu den Problemen des Menschenlebens. 344 haben ihren Gebrauch sie in der Scholastik nicht gehindert. Philo- sophische Probleme bedeutender Art werden dabei nicht aufgenommen. Erst die Neuzeit brachte die Sache in regeren Fluß. Der Be- dem Zuge nach einer gemeinsamen und gleichförmigen Weltordnung dem Menschen Person wird hier ein Hauptmittel, gegenüber griff eine ausgezeichnete übernommenen Stellung zu wahren. Begriffe Person Die von der Scholastik^ und Persönlichkeit werden dabei psychologisch genauer durchgebildet. Leibniz führt diese Bewegung, indem er das Wesen der Persönlichkeit in das Selbstbewußtsein, das Bewußtsein der Identität in den verschiedenen Zeitpunkten d. h. eignen Daseins, setzt und daraufhin die Unsterblichkeit des Menschen von der Unzerstörbarkeit der niederen Wesen abhebt^ des Wolff wie die Aufklärungsphilosophie nehmen diese Fassung Herbart führt sie bis ins auf, 19. Jahrhundert hinein.^ Das auszeichnende Merkmal der Persönlichkeit war bis dahin ^ Wie eng in der ersten Hälfte des 18. Jahrhunderts der Zusammenhang mit der Scholastik noch war, und wie sehr die Ausdrücke als bloße Termini der Schule galten, zeigt das weitverbreitete philosophische Lexikon von Walch. Es heißt dort (auch noch in der vierten, von Hennings besorgten Ausgabe von 1775) unter Person: «Person heißt in der Metaphysik eine besondere, vollkommene und vernünftige Substanz, welche ihr Wesen und ihre Subsistenz vor sich hat. Das Abstraktum davon oder die Subsistenz eines solchen Wesens ist Personalitas genannt worden." ^ Theodic^e I, § 89: l'immortalite, par laquelle on entend dans l'homme, non seulement que l'äme, mais encore que la personalitesubsiste: c'est-ä-dire, en disant que l'äme de l'homme est Immortelle, on fait subsister ce qui fait que c'est vant la la meme personne, laquelle garde ses qualites morales, en conser- conscience ou le sentiment reflexif interne de ce qu'elle est: ce qui Ja rend capable de chätiment et de recompense. Femer in dem Briefe an Wagner (de vi activa corporis et de anima brutorum) S. 466 b der Erdraannschen Ausgabe: Itaque non tantum vitam et animam, ut bruta, sed et conscientiam sui et memoriam pristini status, et, ut verbo dicam, personam servat. ' So sagt Wolff (psych, rationalis, §741): Persona dicitur ens, quod memoriam sui conservat, hoc est, meminit, se esse idem illud ens quod ante in hoc vel isto fuit statu. Femer in den »vemünftigen Gedanken von Gott, der Welt und der Seele des Menschen" § 924 (angeführt von Trendelenburg): »Da man nun eine Person nennet ein Ding, das sich bewußt ist, es sei eben dasjenige, was vorher in diesem oder jenem Zustande gewesen: "?o sind die Tiere auch keine Personen. daß sie Hingegen weil die Menschen sich bewußt sind, die vorher in diesem oder jenem Zustande eben diejenigen sind, gewesen: so sind sie Personen." Selbstbewußtsein, Herbart aber sagt: (Wke. III, 60): »Persön- worin das Ich lichkeit ist faltigen Zuständen als Eins und Dasselbe betrachtet." sich in allen seinen mannig- Persönlichkeit und Charakter. die Nun Intelligenz. 345 aber beginnt eine neue, eine ethische Phase. Nach mannigfacher Vorbereitung hat Kant mit seiner Voranstellung der praktischen Vernunft BPersönHchkeit« durchgesetzt sie ist einer der Hauptpunkte, welche die neue Denkweise zu greifbarem Ausdruck bringen, aus, in wächst bei es Kant weit über die bloße Intelligenz hin- ihr erscheint eine wesentlich höhere, Freiheit gegründete in Persönlichkeit bedeutet nämlich »die Freiheit Ordnung. und Unab- hängigkeit von dem Mechanismus der ganzen Natur", das »was den Menschen über sich selbst was ihn an eine Ordnung einen Teil (als der Sinnenwelt) erhebt, der Dinge knüpft, die nur der Verstand denken kann, und die zugleich die ganze Sinnenwelt, mit ihr das empirisch -bestimmbare Dasein des Menschen in der Zeit und das Ganze Zwecke, unter sich hat« (V, 91 Hart). aller Als Personen und dürfen nie als bloße Mittel gebraucht werden. Ja es wird in uns neben der Tierheit und Menschheit noch die Persönlichkeit unterschieden; der Mensch ist zunächst ein lebendes, dann ein lebendes und zugleich vernünftiges, sind Vemunftswesen Zwecke an sich als Persönlichkeit endlich ein vernünftiges nung fähiges Wesen 120). Denker, Spätere (VI, wie der ältere und zugleich der Zurechauch Schelling, H. Fichte, J. haben diese ethische Fassung des Persönlichkeitsbegriffes nach der metaphysischen Seite zu ergänzen und vertiefen gesucht,^ im großen und ganzen aber ist man seit ihm fest, daß das bei allen Kant verblieben. einzelnen Subjekt Zum mindesten steht Handlungen überlegene, auch mit praktischer als Vernunft Persönlichkeit bezeichnete auszustatten daß zu seinem Wesen nicht bloß das Selbstbewußt- sein, ist, sondern auch die Selbstbestimmung gehört ß. Zur Geschichte des Begriffs. Die letzterrungene Fassung des Begriffs als des selbstbewußten und selbsttätigen Subjekts ^ ist es, deren Geschichte wir nunmehr in Dne Geschichte des Persönlichkeitsbegriffes im 19. Jahrhundert wäre manchen Ertrag verspricht. Über den Gebrauch des Ausden verschiedenen Völkern bemerkt Alexander Chamberlain in eine Aufgabe, die drucks bei Harpers Monthly (1003, Juli, pag. 281): The word personality is not a natrve English term, but has been borrowed, ultiraately frora mediacN-al Latin and from the lau^-ers. The corresponding French term, was admitted to the Academy's dictionary so recendy as 1762. subsequently rescued personnalite, The German Persönlichkeit was once entirely in the possession of the mystics. Zu den Problemen des Menschenlebens. 346 Kürze aufrollen möchten. Die griechische Philosophie gelangte nicht zu einem deutlichen Begriff der Persönlichkeit, sowohl weil die Frage der Einheit des Seelenlebens nicht im Vordergrunde stand, das Denken dem vorwaltenden Intellektualismus das wahre Selbst des Menschen als Immerhin galt. großen Durchforschern des Menschenwesens, als weil der Kern und sich den arbeitet im Widerspruch fast mit ihren Hauptlehren, ein gewisser Begriff der Persönlichkeit her- So bei Plato, so mehr noch genug über einzelne Handdas sich selbst im Handeln er- aus und wirkt in ihrer Gedankenwelt. bei dessen Aristoteles, Ethik deutlich lungen zu einem Wesen vordringt, Das spätere Altertum lebt. hat mehr und mehr den Menschen auf und auch den eignes Innere gestellt sein Begriff des Selbstbewußt- seins entwickelt,^ aber zu einem vollen Begriff der Persönlichkeit ist es nicht Eine gelangt. unserem der Begriff Persönlichkeit ent- sprechende Fassung der Gottheit wurde von hervorragenden Denkern nachdrücklich abgelehnt.^ 1 S. Siebeck, Geschichte der Psychologie ausbildung des Bewußtseinsbegriffes." I, 2, S. 331—342: „Die Her- Trendelenburg hat in dem angeführten Aufsatz näher dargelegt, wie sehr die Stoiker die Entwicklung dieses Begriffs gefördert haben; er zeigt, wie »wir bei ihnen, die im Leben auf die Über- einstimmung mit sich selbst, Charakters gerichtet waren, das auf die Konsequenz des mit r.po'jujxo^j , die persona sich einigen zum Ausdruck des Ethischen werden sehen"; und weiter: »die gut gedichtete Rolle ist der Natur gemäß, wie der leben, die d. h. gut erste Grundsatz der Stoiker verlangt, der Natur gemäß zu der Vernunft, die der Natur gedichtete Rolle individualisiert zum Grunde liegt, zu folgen, und ferner das Allgemeine der eigen- tümlichen Natur des Einzelnen gemäß und gründet es in einem vernünftigen Mittelpunkt". * Zuerst tat das, so weit bekannt, der Akademiker Karneades (213/4 bis 129 V. Chr.), später mit größter Kraft und Schärfe Plotin. großem Werk S. darüber neben Grundriß der Gesch. d. griech. Philosophie. Karneades suchte zu zeigen (s. Grundr. 6. Aufl., S. 242 ff.): „daß man sich die Gottheit nicht als ein lebendes, vertiünftiges Wesen (Cwov Xoyizo'v) denken Zellers seinen könne, ohne ihr Eigenschaften und Zustände beizulegen, die ihrer Ewigkeit und Vollkommenheit widerstreiten." Besonders energisch aber kämpft Plotin dem Ganzen einer Weltanschauung dagegen, dem schlechthin Unendlichen und aller Besonderheit überlegenen Urwesen, wie er es faßt, Denken oder Wollen und weiter auch Selbstbewußtsein zuzuschreiben (s. Zeller a. a. O., S. 293 ff.). „So tritt die von Karneades vorbereitete Leugnung der Persön- aus lichkeit Gottes hier zuerst mit grundsätzlicher Entschiedenheit auf« (Zeller). Gründe sind für die spekulative Ablehnung einer Persönlichkeit Gottes maßgebend geblieben, auch Spinoza hat ihnen kaum etwas neues Plotins hinzugefügt. Persönlichkeit und Charakter. Die Belebung und größere seelische Nähe, welche kräftigere die Gottesidee im 347 Christentum gewann, alten gestattet hier weit eher, von einer Persönlichkeit Gottes und einem persönlichen Verhältnis des Menschen zu Gott zu eines Anthropomorphismus nicht unbemerkt: das zeigt schon dem höchsten Wesen ein Affekt der heftige Streit darüber, ob sich wie der Zorn zuschreiben Die darin liegende Gefahr sprechen. blieb Eine Lösung fanden die Probleme lasse. im Gottesbegriff unter Augustins Führung schließlich in der Weise, daß eine menschlich -persönliche Fassung auf die Grundlage einer Persönlichkeit Mittelalters absolutes empfand Widerspruch. ein und Gott aufgetragen wurde. spekulativ-mystischen Sein. Die zugleich sittliche ist mattere Denkweise des dem Nebeneinander beider Fassungen keinen in Der Neuzeit verwandelte Entweder -oder, was das Mittelalter sich auch hier alsbald als ein in Sowohl — als auch ruhig, ja willig ertragen hatte. So erfolgt denn in der Neuzeit eine energische Scheidung der Geister beim Gottesbegriff. folgt Was immer dem Zuge zur Immanenz und auf universalen Weltbegriffen besteht, das bekämpft die persönliche Fassung der Gottesidee als einen unerträglichen Anthro- pomorphismus. Der Widerstand und Widerspruch gegen den Pan- theismus dagegen stützt sich in seinem Verlangen nach einem lebendigen göttlichen Sein auf die Idee der Persönlichkeit und gibt auch dem Wort einen besonderen Nachdruck. Bis dahin hatte man viel von dem Verhältnis der drei Personen im göttlichen Wesen, aber wenig von der Persönlichkeit Gottes gesprochen.^ Nun aber wird Persönlichkeit ein Bekenntnis und Lieblingswort der Antipantheisten. So beteuert z. dem B. Jakobi in bekannten Gespräch mit Lessing seinen Glauben an eine «verständige persönliche Ursache der Welt" und vermißt an der Substanz Spinozas „eine eigne, besondere, indiVon da aus viduelle Wirklichkeit", „Persönlichkeit und Leben". zieht sich der Streit durch das 19. Jahrhundert bis zur Gegenwart. Wo immer der Lebensprozeß sich vorwiegend künstlerisch oder intellektuell und zu dagegen gestaltet, klein, da dünkt leicht die Idee der Persönlichkeit zu eng um das Ganze der Wirklichkeit zu beherrschen; wo der ethische Zug die Führung hat, da möchte man den * Wiederum sei hier Walch als Zeuge angeführt, der im Artikel Person wohl von Personen der Dreieinigkeit, nicht aber von einer Persönlichkeit Gottes spricht und in einer ausführlichen Erörterung des Wesens Gottes »Persönlichkeit" mit keinem Worte berührt. Zu den Problemen des Menschenlebens. 348 missen und strebt nach einer Fassung, die auch die Begriff nicht Gottesidee umspannen vermag zu Lotze und (so Ritschi). — Die neueste Zeit Spricht besonders viel von Persönlichkeit, die Wendung zu dieser erscheint oft als ein Heilmittel sicheres aller Schäden. Eine kräftigere Belebung der Persönlichkeit will die Kunst, die Redas allgemeine Leben; sie dünkt eine unentbehr- ligion, die Moral, liche Hilfe gegen die drohende Entseelung des Daseins, ein Mittel zur Abstoßung von Veraltetem und Überlebtem, der einzige Weg zur ersehnten Verjüngung und Vereinfachung des Lebens; mit der Persönlichkeit hofft man gegenüber der Erschütterung der Weltbegriffe einen Halt sicheren im eignen Innern gewinnen, zu auch einen Punkt, auf den sich die Menschen inmitten unerträglicher Spaltung und Zersplitterung einigen können. Wo so viel zusammentrifft, läßt sich viel Verworrenheit erwenn eine so leichte Wend- warten; es müßte wunderbar zugehen, ung, eine einfache Selbstbesinnung, uns den unermeßlichen Verwick- lungen entwinden könnte, die uns heute umfangen. Vermutlich ist entweder die Hilfe ein bloßer Schein, oder der Gedanke der Persönlichkeit enthält beilegt. mehr und fordert mehr, als die übliche Fassung ihm Sehen wie es damit wir, y. steht. Untersuchung des Problems. An dem Streit über Persönlichkeit ist ohne Zweifel viel Wortstreit; so lange der eine dem Ausdruck eine engere und niedrigere, der andere eine weitere und höhere Bedeutung ständigung ausgeschlossen. um das Wort nur Nicht deshalb Aber wie oft, so die Erscheinung schätzen ist gibt, ist eine Ver- auch hier der Streit eines sachlichen Gegensatzes. hervorragende Denker bis zur Gegenwart »Persönlichkeit", weil ihnen das Wort in die Ohren klingt, sondern weil sie zeichnet, in wenn auch unvollkommen, ihm, eine Tatsache verzichten zu dürfen. den Vorrang brachte, so sie Gedanken be- nicht glauben Wie Person und Persönlichkeit von alters her Menschen, des geistigen Wesens, zum Ausdruck desist einen behauptet finden, auf die es eine Grundüberzeugung vom Geistesleben, von seinem Inhalt und seiner Bedeutung, die in ihm sich ein, wenn auch unzulängliches Werkzeug schuf. Wer im Zusammenhang einer Welt- anschauung für eintritt, Persönlichkeit Geistesleben keinen bloßen Anhang behauptet damit, daß das der Natur, sondern eine eigen- tümliche Art des Seins besagt; er behauptet, daß es nicht in einzelne Persönlichkeit und Charakter. 349 Betätigungen und Vermögen aufgeht, sondern eine ihnen überlegene und umfassende Einheit enthält und damit zu einem Beisich- sie einem Selbstleben wird; selbstsein, Selbstleben bloßer kein sondern aktiver Art ist, Sammelpunkt ihm umwandelnde eine daß dies behauptet endlich, er zugeführter Elemente, Kraft an allem Empfan- Nur genen übt und das ganze Dasein auf eine höhere Stufe hebt. wenn dieses wesentlich Neues in sie von alles zutrifft, bringt Persönlichkeit etwas unser Dasein und rechtfertigt damit den Affekt, mit dem vielen ergriffen wird. Nun aber das ist Ganze Behauptungen nur im Recht, jener wenn im Gesamtbilde der Wirklichkeit und unserer Stellung in ihr sich eingreifende Wandlungen vollziehen. An der besonderen Stelle kann keine Wahrheit haben, was nicht im Ganzen wahr und Zusammenhängen begründet seinen in wäre jene Bewegung zur ist; Persönlichkeit eine bloße Privatangelegenheit des Menschen, so wäre sie mit ihrem Weltbilde eine bloße Illusion, so würde sie ins Leere Ein Durchdringen zur Wahrheit wird sie nur, wenn das Geistes- fallen. leben die Tiefe der Wirklichkeit bildet, in der sie ihr eignes Wesen Nur auf Grund einer neuen Weltstufe und im Zusammen- erreicht. Wendung zur Persönlichkeit hang mit ihr vollziehen und die Menschheit persönliches Leben kann der Einzelne die entfalten. Ja es muß jenes neue Leben, um den Menschen über die naturhafte Ordnung hinauszuheben, sein und in ihm wirken; er als muß an um der äußeren gewachsen So handelt es sich bei Persönlichkeit um ein neues Grund- inneren Unendlichkeit teilhaben, einer zu umfängt und beherrscht, die ihn zunächst Ganzes seiner Seele zugegen sein. verhältnis zur Welt, um eine neue Art des Lebens und Seins. Steht aber die Sache so, so keine fertige könnte, ließe, Tatsache, die er ist Persönlichkeit für den Menschen bequem und kein sicherer Ausgangspunkt, sondern Aufgabe, sie sie bedeutet für verlangt eine völlige hurtig sich aneignen der sich mühelos einnehmen ihn große eine und schwere Umwälzung der vorgefundenen Lage. Nicht Selbst, sondern der Gewinn eines neuen Selbst steht hier in Frage. Die die Entfaltung oder Ausschmückung des natürlichen Wendung wird nur da den vollen Ernst und Nachdruck erwo sie auch ein entschiedenes Nein umfaßt, eine Ver- langen, neinung der naturhaften Selbsterhaltung, ein Hinausstreben über die bloßmenschliche Form des Geisteslebens. Und es darf solches Nein keinen flüchtigen Durchgangspunkt bilden, es muß stets zugegen Zu den Problemen des Menschenlebens. 350 und energisch bleiben wenn nicht das Streben festgehalten werden, dem neuen Sein immer wieder zur nach zurücksinken Lebensform naturhaften soll. Ja auch innerhalb des Geisteslebens bildet Persönlichkeit einen Anstieg und eine Konzentration, die Erfahrungen und durch erst Entscheidungen des ganzen Menschen hindurch Das erreicht wird. Leben durchläuft die drei Stufen einer grundlegenden, kämpfenden, überwindenden Geistigkeit. Zuerst gilt es die Anerkennung einer Aufgabe überhaupt, eine Erhebung des Lebens über die geistigen eine Natur, Entwicklung geistiger Größen und Güter jenseit Das ergibt die Scheidung eines natürlichen Selbsterhaltung. der Idealis- mus vom Naturalismus, der alles Geistesleben als eine Weiterführung der bloßen Natur und damit behandelt, die Scheidung der erste Aber der Boden des Idealismus erzeugt alsbald Geister. ein neues Problem: das Reich d£r Erfahrung zeigt starke Widerstände gegen die vom Idealismus geforderte Ordnung; es fragt ob sich, Widerstände die Bewegung zum Stocken bringen, oder ob Preisgebung der Sache; hier muß irgendwelche Verstärkung, irgendDiese aber welche Weiterbildung des Geisteslebens erfolgen. der in von Dort entsteht der Pessimismus mit seiner ihr überwunden werden. die diese sie Wendung zum Persönlichsein behauptet ist wird. es, Das Persönlichsein erscheint hier als der Höhepunkt einer geistigen Be- wegung, und zwar der indem bindet, als ein solcher, sie zu einem Ganzen ver- Momente fest- er die früheren Stufen als beharrende Denn das Leben bleibt unablässig in Fluß, immer neu ist der Aufstieg von der Natur zum Geist zu vollziehen, immer neu der hält. Widerstand gegen die Vergeistigung des Daseins zu durchleben, immer neu eine innere Überwindung zu suchen. So bleibt das Ganze eine fortlaufende Tat, ein unablässiger Aufstieg; es läßt sich erwarten, daß nicht der ganze Umfang des Daseins darin eingeht, daß das Persönlichsein hält; es Kraft, Besitz, ist die nicht Seele sondern sein als ein bei uns selbst einen inneren Widerstand be- sowohl unser ganzes Dasein Augenscheinlich der Seele. das höchste Persönlichwerden. Ziel, Wie nicht als seine ist sowohl es so ein bewegende nicht ein Persönlich- dabei das Streben einem Ein- strömen des naturhaften Ich unablässig zu widerstehen hat, so haben die Begriffe immerfort ein Zurücksinken zur bloßmenschlichen Vorstellung abzuweisen, lässig bedroht. womit eine Ermattung des Denkens sie unab- Persönlichkeit und Charakter. 35I Wer solche Aufgaben und Verwicklungen im Persönlich>x'erden der wird auch die Kämpfe zu würdigen wissen, die anerkennt, Problem dies Persönlichkeit sich knüpfen. der Religion ward die Idee In zugunsten eines unpersönlichen Geisteslebens mit jener art der oft be- das natürliche Ich des Menschen starr kämpft, weil gelegt und zugleich das höchste Wesen zu menschenartig gefaßt schien; die Wendung zu einem unpersönlichen Sein derung einer völligen Auflösung des Einzelnen dünkte Unendlichkeit nehmere Denkart. demgegenüber eine fest- mit der For- den Ozean der in größere, reinere, vor- So die pantheistische Spekulation und die Mystik, so die Höhe der indischen Religionen, so Spinoza mit seinem Worte, wer Gott wahrhaft liebt, daß, wieder Solche liebe. nicht verlangen könne, Denkweise hat ihr Recht daß Gott ihn der Abweisung in der kleinmenschlichen Daseinsform, aber mit dieser Verneinung, Versinken dem, Geistesleben keine neue, wer in dem den Abgrund der Ewigkeit, kann nur abschließen, wer in selbständige zuerkennt, Wirklichkeit ihm wohl eine Befreiung von den Wirren und Mühen der menschlichen Existenz, von der Unruhe und Flucht der Zeit, von der Enge und Starrheit des kleinen Ich erblickt, nicht aber das Auf- und den Gewinn steigen Nein kann sich neuen eines nur eine Beruhigen beim und vorwiegend passive Lebens. kontemplative wo immer Lebensführung, eine weichere, mattere, schlaffere Denkart; das Geistesleben mehr Kraft und Zuversicht entwickelt, da wird es das scheinbar Unmögliche wagen einem Ja verlangen, da wird der Persönlichkeit angibt. Idee und über das Nein hinaus nach es die Bahnen einschlagen, welche die Nun von der Gefahr eines Zurücksinkens begleitet sein; in aber wird dies Streben stets die natürliche Lebensform in der Tat pflegt bei den Religionen eine höhere und eine niedere Art durcheinanderzulaufen: dort das Aufstreben zu einer neuen Welt, einem neuen Leben und auch einem neuen Gedankenfür reiche, bole liefert; Daseins das unser menschliches Dasein hier dagegen möglichst Gegenstück der gut alten, die Neigung, auszukommen, mehr nicht innerhalb des die neue Welt als Sym- gegebenen ein bloßes die höchsten Begriffe anthropomorph gestaltet, in dem allen weit mehr eine Festlegung des Kleinmenschlichen ein Aufklimmen zu neuen Höhen. ihrer Als Verdunklung des unentbehrlichen gegen das Persönlichsein der weltgeschichtlichen in Abweisung Nein ist als dieser Art mit der Widerstand gutem Recht und gewiß dem Ganzen Bewegung unentbehrlich; er gerät aber ins Zu den Problemen des Menschenlebens. 352 wenn er mit der niederen Art auch die höhere abweist und Unrecht, damit An dem Gewinn einer solchen Gestaltung liegt schließlich aufgibt. auch Hoffnung einer positiven Gestaltung des Geisteslebens alle Hoffnung einer gründlichen Überwindung des niederen alle Lebenstriebes. Denn letzthin ist positivem Streben nur ein anderes positive Streben gewachsen; alle Energie der Verneinung, alles Ver- schwimmenwollen die Unendlichkeit wird positive die So steht des Nein ist hier in Frage, Lebens- und Glücksdrang sich der bloßen Natur entwinden und der Stufe des Geistes zuführen Fall Selbstsucht nicht Bildung eines neuen geistigen Selbst als die und zwingenden Aufgaben. mit großen ob der in brechen so gründlich alle läßt oder Im nicht. Mühe schließlich verloren. unsägliche Ahnliche Probleme wie die Religion zeigt an dieser Stelle auch und Künstlerische die Kultur. der intellektuelle Weltbilder treffen in Abneigung gegen eine leitende Stellung der Persönlichkeit zusammen. Denn zum sehen darin ein Zurückziehen der geistigen Arbeit sie bloßen Menschen und eine widrige Störung ihres eignen Fortgangs durch eine Einmischung seiner kleinen Sorgen. Sich rein ständig entfalten zu können scheint das Geistesleben nur, wenn es vom Menschen und seinen Zwecken ledig- unter völliger Ablösung lich und voll- der eignen sachlichen Notwendigkeit folgt und sich im eignen Kreise zu einem selbständigen Lebensganzen mit eigentümlichen Gesetzen zusammenschließt Auch hier aber wird eine niedere und eine höhere Fassung der Persönlichkeitsidee zusammengeworfen und mit dem, was herabzieht, etwas aufgegeben, dessen die Kulturarbeit zur Erreichung ihrer vollen Höhe notwendig bedarf. Recht verstanden liegt das Sein und die dem Streben nach Persönlichkeit vorschwebt, nicht die Einheit, neben der Arbeit, sondern innerhalb zu bringen, das ein Selbstleben in ihrer. Diese selbst ist dahin ihr durchbricht, daß ein geistiges dem Denn es gibt schlechterdings keinen Inhalt ohne ein Selbst, das im Wirken und Geschehen Sein sich in ihr erfaßt, die Erfahrungen in Erlebnisse umsetzt, Geschehen allererst einen sich entfaltet. Inhalt verleiht. Nur ein solches innerhalb des Geisteslebens befindliches Selbst gewährt diesem eine Seele und einen Halt, behütet es vor der Gefahr, ein leerer Mechanismus oder ein seelenloser Kulturprozeß zu werden, gibt ihm die Kraft, seine eigne Arbeit zu bewältigen, von ihr einem bewältigt und erdrückt zu werden. solchen Selbst das Leben eine statt Auch kann nur mit volle Wirklichkeit gewinnen Persönlichkeit und Charakter. und sich einer Wirklichkeit sicher solchen Kern ein schatten- während und traumhaftes Dasein Empfangene verwandelt. alles fühlen, 353 es ohne einen führt und darin Die Inder zeigen uns solche Ver- flüchtigung in klassischem Bilde. - Augenscheinlich handelt es sich hier um eine eifrigere subjektive Aneignung einer gegebenen Wirksondern um eine reale Erhöhung und Umwandlung der gesamten Wirklichkeit, es handelt sich um den letzten Abschluß der Kultur, um die Möglichkeit eines neuen, wesenhafteren und seelennicht lichkeit, volleren Kulturideals. Das Entscheidende dabei ist für Menschen und Völker schließlich die Energie des Lebensaffekts, die kräftigere oder mattere Erfassung des Lebens; die sachliche Entscheidung aber Erwägungen, sondern liegt nicht bei begrifflichen keit der Entwicklung einer neuen Wirklichkeit hier stehen wir bei den letzten bei der Möglich- Nirgends mehr als Axiomen unserer geistigen Existenz. Von solchen Überzeugungen aus, die für ein Persönlichwerden das Aufsteigen einer neuen Welt und eine Umwälzung des natürlichen Seins verlangen, muß uns dje heutige Bewegung zum Persönlichsein als verworren, ja in mancher Hinsicht verfehlt erscheinen. Die übliche Behandlung der Sache erschöpft sich in das Verlangen einer kräftigeren Konzentration, einer Verstärkung des Subjekts, einer größeren Selbständigkeit gegenüber der Umgebung. das geschehen, Welt bleibt, Aber wie soll wenn der Mensch ein bloßes Stück der vorhandenen nicht von innen her an einer neuen Welt teil gewinnt? Ohne eine Umkehrung des nächsten Anblicks der Wirklichkeit und ohne den Gewinn eines neuen Lebensbodens könnte leicht die Sache mehr schaden als nützen, indem sie in eine bloße Verbrämung des Überspannung des Selbstgefühls, ein und Fürsichzurechtlegen aller Dinge auslaufen Und wenn gar die Bewegung zur Persönlichkeit als eine natürlichen Lebenstriebes, eine bloßes müßte. Genießen Abwendung von den Weltfragen und eine Zurückziehung auf einen Sonderkreis verstanden wird, so wäre das kaum etwas anderes als eine Verherrlichung eines engen und öden Spießbürgertums. Man macht dadurch nichts Neues aus dem Menschen, daß man ihm die Etiquette der Persönlichkeit anklebt. Ohne den Gewinn einer neuen Welt und eine Erhöhung des eignen Lebens bleibt das Ganze eines bequemen Surrogate, welche die Probleme des Menschenwesens und den Ernst der Zeitlage verdecken.^ jener Es wäre endlich wohl an der Zeit, das unablässige Zitieren des Qoethe„Höchstes Glück der Erdenkinder sei nur die Persönlichkeit« Eucken, Qrundbegriffe. 4, Aufl. 23 ^ schen Wortes: Zu den Problemen des Menschenlebens. 354 Nach unserer Überzeugung muß das persönliche Leben einen neuen Weltanblick entwickeln, aus seiner eignen Belebung, seinen seinen Weiterbildungen Erfahrungen, Lebenswahrheiten erzeugen. und Reich von Grund- ein Mögen sich diese nicht in angemessene Vorstellungsbilder umsetzen lassen, sie bleiben die Wahrheiten, wie schließlich geistige alles die Leben, so auch das Erkennen tragen, zentralen Wahrheiten, denen gegenüber alle anderen Einsichten nur periphere bedeuten. Nun behält unsere intellektuelle sie sind die und überhaupt unsere geistige Lage dadurch eine starke Spannung, daß ein Spalt zwischen den zentralen, persönlichen und den peri- pheren, unpersönlichen verbleibt, daß sich jene nicht unmittelbar in Aber trotzdem dürfen wir die Wirklichkeit diese überleiten lassen. zwei endgültig getrennte Gebiete zerlegen und das Reich nicht in Denn persönlichen Lebens gegen die große Welt starr abschließen. das wäre nichts anderes, und als das Leben zwischen leerer Subjektivität seelenloser Arbeit zerteilen, es wäre ein Verzicht auf seine innere und zugleich Einheit daher nach treibende einer Von beiden Seiten gilt, das Ziel seine volle Wahrheit. Einigung zu streben, es ist eine als und richtende Kraft tapfer festzuhalten, obschon wir keine Aussicht haben, es gänzlich zu erreichen und die beiden Ausgangs- punkte zu völliger Berührung zu bringen. Bei solcher Überzeugung treten eine persönliche jektive Gestaltung Die subjektive Art stellt sich der Wirklichkeit sobald sie über sich hinausgeht, hinein; die persönliche möchte dringen, und eine sub- von Arbeit und Kultur uns deutlich auseinander. nicht als zu etwas ihre gegenüber und Besonderheit in trägt, Dinge alle zum eignen Leben der Dinge vor- Fernem und Fremdem, sondern als zu dem, worin das geistige Wesen sich selber, die Wahrheit seines eignen Seins erreicht. Mit der Verwandlung der Dinge in ein Selbstleben vollzieht sich hier die Überwindung des Gegensatzes einer subjektivistischen und einer solchen, die souverän objektivistischen oder Behandlung zu gunsten einer eigenständlich heißen die Notwendigkeit der Sache unmittelbar ein und wird eigner Antrieb Menschen, hier erst wird die volle Einigung mit der Sache auf Denn könnte. hier allein erlangt das Schaffen eine volle Selbständigkeit des erreicht, Grund derer sie ihre eigne Natur rein und einfach aussprechen einzustellen. Die liebenswürdige und anmutige Stelle, an der es sich Westöstlichen Divan findet, will gar nicht so schwer genommen sein. im Persönlichkeit und Charakter, Erst die persönliche, die souveräne Art kann. 355 ist über das hinaus, was sich zwischen den Menschen und die Sache zu Der Mensch kann die Sache darf vielfacher Mittel, nicht ohne weiteres nischer Zurüstung, Übung, Gelehrsamkeit u. s. w. daß, was nur Ziele wird, daß es den Menschen bei Aber nun entsteht und Weg ist, zum Zwecke und Mittel Kaum Hauptsache ablenkt. sich festhält und von der dürfte ein Volk dieser Gefahr so aus- wie das deutsche sein pflegt. um an sie zu gelangen, er bedarf dazu tech- die Gefahr, gesetzt stellen ergreifen, er be- mit der Schwerfälligkeit seiner Art; besonders Gründlichkeit, aber auch mühsam gelangt es zu voller Überwindung der Technik durch das Schaffen, zu jenem Sichselbstin den Dingen, ohne das die Werke keine reinmenschliche Größe und schlichte Einfalt erlangen können. So besteht eben heute leben in unserem Leben ein arges Mißverhältnis zwischen dem Aufgebot und intellektueller Schöpfungen, die künstlerischer Arbeit und der Erzeugung von zum ganzen Menschen sprechen und den ganzen Wenn das Verlangen nach einer mehr persön- Menschen fördern. lichen Kultur dies heißen soll, daß es einfache Grundzüge aus der Verwirrung herauszusehen und damit zum Ganzen des Menschenwesens zu wirken dieses gilt, so ist dem nur freudig zuzustimmen. Aber keineswegs eine Sache raschen Entschlusses, sondern das ist Allerschwerste, etwas, aller Kraft, wozu es nicht nur der höchsten Anspannung sondern auch vielfacher Gunst des Schicksals bedarf. erfahren wir es auch heute: wie wenig hat alle So subjektive Beteuer- ung des Wertes der Persönlichkeit uns innerlich weitergeführt, wie wenig hat sie starke und selbstwüchsige Persönlichkeiten erzeugt! b) a. Geschichtliches Charakter, zum Ausdruck und Begriff. Charakter bedeutete bei den Griechen sowohl das Werkzeug zum Zeichnen und Prägen als das Gepräge selbst, das Merkzeichen; es schon hat im Altertum geistige Gebiet gefunden. als in die den Aristoteles, spätere Richtung. Theophrasts, tragen, naheliegenden Übergang auf das Es geschah das aber sowohl ästhetisch-literarischer Namen den des in ethischer Die ethischen Charaktere, Schülers und Nachfolgers des sind freilich aller Wahrscheinlichkeit nach eine Zusammenstellung aus größeren Werken des Mannes, aber 23* Zu den Problemen des Menschenlebens. 356 Neigung zu genauer Beobachtung und scharf umrissener Zeichnung verschiedener menschlicher Typen geht auf Aristoteles, den die ^ Kenner und großen Schule seiner Freund eigentümlich. zurück und blieb Nach derselben Richtung wirkten die Wirklichen, alles neuere Komödie und die Rhetoren; war dem späteren Altertum so der Blick für die Eigentümlichkeit verschiedener menschlicher Arten und Handlungen geschärft.^ Zugleich aber bezeichnet Charakter auch die Eigentümlichkeit schriftstellerischer und künstlerischer Dar Stellung, das individuelle Gepräge Im kirchlichen Sprach- •gebrauch wurde es Augustin der technische Ausdruck für ein seit w. u. s. der Seele durch gewisse Sakramente (im Mittelalter Taufe, Firmung, Priesterweihe) unverlierbar eingeprägtes geistiges character sacramentalis, auch spiritualis genannt). vereinzelt auch So (später findet es sich im Mittelhochdeutschen, ebenso vereinzelt ist hier die Bedeutung Schriftzeichen (der karakter a b c). Gegenwart Zeichen hineinreicht, so erinnert es Wie das bis in die auch an jenen älteren Ge- wenn der amtliche Stil von der Beilegung eines »Charakters" (Titels und Ranges) spricht. Zu allgemeinerer Verwendung im psychologischen und ethischen Sinn ist bei uns das Wort wohl von Theophrast her und durch die brauch, Vermittlung der Franzosen gelangt.^ La Bruyere: ies Typus und ^ caracteres Im Jahre 1687 erschien von de Theophraste, avec les caracteres ou typisch im jetzt üblichen Sinne als Bezeichnung gemein- samer Seins- und Lebensformen dürfte aus der Medizin stammen. Es bemerkt darüber Dilthey (Sitzungsberichte der K. Preuß. Akademie der Wissenschaften 1896, XIII, S. 18): „In diesem Sinne finden wir auch den Ausdruck zunächst technisch gebraucht, wenn der Arzt Coelius (wahrscheinlich im 2. Jahrh. n. Chr.) vom Typus des Wechselfiebers spricht und darunter die Regel seines Ablaufs So sprechen wir überhaupt von einem typischen Verlauf." über das Ganze Sauppe, Philodemi de vitiis 1. X, pag. 7: Peripatetici disciplinae suae principis et auctoris exemplum nulla in re magis secuti sunt, quam ut omnia quae vel in natura rerum existerent vel in vita hominum versteht. * et S. publica et privata usu venirent accuratissime observarent et observata sive libris singularibus explicarent sive ad sententias suas firmandas et illustran- das adhiberent. pag. 8: neque vita ipsa tantum exempla suppeditabat, sed maximam notationum copiam nova comoedia habebat. Quae ut eidem saeculi ingenio originem debebat, atque aristoteleum illud Studium vitam quotidianam moresque hominum observandi, phrasti libris desumta in ita quaedam fortasse ex Aristotelis vel Theousum suum converterat, sed multa plura certe, quam acceperat, deinde philosophis et rhetoribus suppeditavisse censenda est. ^ Wir besitzen darüber eine ebenso feinsinnige wie tiefdringende Untersuchung von R. Hildebrand: „Charakter in der Sprache des vorigen Jahr- Persönlichkeit und Charakter. les moeurs de viel Beachtung siede, ce ein Buch, das auch und Einfluß gewann. 357 Völkern bei anderen Sicherlich stehen damit im Zusammenhang wie andere deutsche Werke zur Charakterschilderung, so auch Qellerts «Moralische Charaktere", eine Zugabe zu seinen Hier wie sonst bedeutet Charakter soviel moralischen Vorlesungen. wie Bildnis, welches Wort bisweilen zu seiner Übersetzung dient, Zeichnung, Porträt (so spricht terisieren" Rabener B. das fort lebt in seinen Satiren von dem Ausdruck „charakbis zur Gegenwart. Von dem Bild überIn der Ausdruck auf die Sache und wird zur Bezeichnung sich trägt z. zu meinen Charakteren"). w Originalen der seelischen, namentlich der moralischen Art, der Orundbeschaffenheit des Menschen. diesem Sinne kann es eine Fülle verschiedener In Charaktere geben, guter und böser Art; keinen Charakter haben, Woher der Gabe der Natur oder als Werk das heißt hier einer scharfen Ausprägung entbehren. ob Charakter entstanden freier Tat, das bleibt dabei unentschieden. sei, Kant hebt den Erst wichtigen die Höhe, ihn zu einer die These moralischen Charakter. Naturell Begriff auf und einem schweren Probleme macht. scharfe Grenze zwischen einem physischen und einem ethischen Er zieht eine als Charakter schlechthin und Temperament umfassend, Menschen machen läßt, ist zeigt an, nur dieser; jener, was sich aus dem der eigentliche Charakter hingegen, was er aus sich selbst zu machen bereit ist. „Einen Charakter schlechthin zu haben, bedeutet diejenige Eigenschaft des Willens, nach welcher das Subjekt sich selbst an bestimmte praktische Prinzipien bindet, die es sich durch seine eigne Vernunft unabänderlich vorgeschrieben hat" 614). (VII, Natur aus „Es kommt hierbei auf das an, nicht was die dem Menschen, sondern was dieser aus sich selbst macht." „Die Gründung eines Charakters Prinzipes des ist absolute Einheit des inneren Lebenswandels überhaupt" (617). In diesem Sinne wollte Kant nicht sagen, der Mensch habe diesen oder jenen Charakter, sondern er habe überhaupt einen Charakter, „der nur ein einziger oder gar keiner sein kann." Diese kantische Fassung mit ihrer Erhebung des die Stufe geistiger Hunderts« (Zeitschr. Selbsttätigkeit f. d. ist rasch deutschen Unterricht, Lebens auf durchgedrungen; ^ 6. Jahrgang, 7. Heft.) der Ihr folgt unsere Darstellung jener Zeit. ' Wie Charakter (in zeigt u. a. eine Abhandlung von den Abh. d. K. Pr. Akad. d. Wiss. 1803). rasch, E. Biester über den Zu den Problemen des Menschenlebens. 358 hohe Ton, dem in Wertschätzung, die Folgezeit von die sie Aber neben Kant zurück. und Charakter spricht, der Charakterbildung beilegt, sie die weisen auf seiner ethischen Fassung erhält sich auch man nicht so viel einem durch Anpassung und Gewöhnung er- die ältere, empirisch-psychologische; sonst könnte von einem ererbten, So laufen wiederum in einem Wirkungen verschiedener Zeiten und Welt- worbenen u. s. w. Charakter sprechen. Wort alltäglichen die anschauungen bunt durcheinander. ß. Die Lage der Zeit. Der Begriff des Charakters im ethischen Sinne steht in engem Zusammenhange mit dem der Persönlichkeit, nur hebt er die Selbstdes Menschen tätigkeit mit dieser Begriff eine präzise besonderer Stärke hervor. So gewiß Bezeichnung erst spät gefunden hat, der Gedanke einer durch eigene Willensenergie erreichbaren Selbständigkeit und Weltüberlegenheit ihn hielten Zeiten, wo Zusammenhänge licher suchen zwang. Stoikern gefunden, die weit zurück; hoch besonders Auflösung überkommener gesellschaftihren Halt in sich selbst zu die Individuen Seinen ein reicht Ausdruck klassischen hat das bei den eigentümlicher Lebenstypus zieht sich von dort durch die ganze Geschichte, er ist namentlich von hervorragen- den Persönlichkeiten der Aufklärung aufs neue bekräftigt worden. Auch Kants Erörterungen über den Charakter haben viel Verwandtschaft damit und benutzen mit Vorliebe Wendungen aus dem stoischen Die Entwicklung nach dieser Richtung Gedankenkreise. die enthält Gefahr einer schroffen Isolierung genügsamkeit des Individuums, einer und stolzen Selbst- Verkennung der Bedingtheit des Einzelnen, wenn nicht durch sichtbare, so doch durch unsicht- bare Zusammenhänge, aber bei solcher Gefahr blieb sie in beson- deren Zeitlagen das einzige Mittel zur geistigen Selbsterhaltung des Menschen. Der Begriff des Charakters reicht aber über solche Zuspitzung hinaus; als Bezeichnung des Selbstwertes und der Selbständigkeit des Innenlebens gegenüber aller bloßen Außenwelt, als Bekenntnis zur Überlegenheit auch da in worfen wird. dem der inneren Ehren stehen, Güter über alle äußeren kann er wo jene Isolierung des Individuums ver- Es berührt sich dann aber der Begriff so eng mit der Persönlichkeit, Erörterung bedarf. So daß er hier nicht noch einer besonderen sei nur mit einigen Worten darauf hinge- Persönlichkeit und Charakter. 359 wie das Problem des Charakters und der Charakterbildung wiesen, vom Standort unserer eignen Zeit sich ausnimmt Diese Zeit beschäftigt sich viel mit dem Problem des Charakters, sie klagt zugleich viel und ausgeprägten Persönlichkeiten, sie fordert von der Kultur- teren mehr Fürsorge für die Bildung namentlich von der Erziehung, arbeit, Aber von Charakteren. und Halbheit, heit über den heutigen Mangel an festen Charak- in dem allen erscheint wieder die Unklar- die solchen Durchschnittsbestrebungen eigen ist Nicht selten sieht es aus, als ob unversehens ein moralisches Sinken und es nun nur einer eindringlichen Zuspräche oder ge- erfolgt sei, schickten Einrichtung So einfach bringen. bedürfe, um die Sache wieder in Stand zu steht doch es nicht. Der Mangel an selbst- wüchsigen und selbständigen Menschen, den wir heute schmerzlich empfinden, hat sicherlich tiefere Gründe, Immer mehr ist in den Wandlungen der Jahrhunderte der Menschheit die mühsam erkämpfte Innenwelt erschüttert oder verdunkelt, üben kraft daher Güter, ihre immer weniger Anziehungs- immer leerer werden zugleich die Dazu die stürmische Okkupation des modernen Menschen Seelen. durch die Außenwelt, die Streberei nach sichtbaren Erfolgen, der wachsende Kampf ums Dasein und die unheimliche Beschleunigung des Lebens, die Zerstückelung des Menschen durch die immer technischer, immer komplizierter gestaltete Arbeit, die abschleifende Wirkung einer Durchschnitts- und Massenkultur. Kann ein solches Getriebe einen Sinn und einen Platz für die Ausbildung selbstän- diger Charaktere haben? Wer im Vertrauen auf die innere Notwendigkeit der Sache jenes Ziel festhält, der wird seine Erreichung sich keinenfalls so leicht denken dürfen, wie manche Strömungen der Zeit, die ihre Schätzung von Persönlichkeit und Charakter mit möglichstem Nachdruck beteuern, zugleich aber die Bedingungen zerstören, unter für jene Größen ein Platz ist ralismus, Liebe gültiges, läßt, indem alles er bei Dies tut z. allein den Weltfragen mit besonderer Lust und begrüßt, was den Menschen als klein unselbständiges denen B. oft ein radikaler Libe- Stück und als ein gleich- einer seelenlosen Natur erscheinen zugleich aber auf praktischem Gebiet für die Größe und Würde des Menschen schwärmt, sich für Humanität begeistert und entrüstet ist, wenn er einen Mangel an selbständigen Charakteren, wuchern der Streberei gewahrt es heute in den ein Über- Ein solches Überwuchern, wie wir mannigfachsten Formen gewahren, ist schlimm Zu den Problemen des Menschenlebens. 360 genug, aber wird ihm kräftig zu begegnen sein, wenn dem Menschen alle selbständige Innenwelt höher ein etwas wenn er gar nichts anderes ist als fehlt, geartetes Tier und daher keine anderen Ziele kennt der natürlichen Lebenserhaltung? als die Wir werden an dieser Stelle in einen sicheren Fortgang nur kommen, wenn die Probleme des inneren Menschen wieder zur Hauptsache werden, und wenn sie sich zum Ganzen einer Lebensüberzeugung zusammenfassen, das mit aufrüttelnder, richtender, Davon höhender Macht die Gemüter zu ergreifen vermag. Aber so sehr es abzulehnen einstweilen noch weit entfernt. die Bildung von Persönlichkeit und Charakter als handelt werde, die sich so nebenbei abmachen wirken dafür läßt unmittelbar auch sich erwägen eignen Zeit; wir also auf Kürze, in ist, daß Sache be- eine läßt,^ er- sind wir einigermaßen dem Boden unserer welchen Richtungen in vornehmlich das geschehen kann und geschehen muß. Zunächst ist notwendig mehr Anerkennung und Hochhaltung der bloßen mehr Zurückstellen und Durchschauen Aufmachung und des bloßen Scheins. Was uns daran hindert, namentlich der Epikureismus einer reifen, der echten Werte des Lebens, ist überreifen ja Kultur, der jeden Einzelnen möglichst seinem individuellen Behagen nachgehen, ihn ängstlich allen Zusammenstoß scheuen und willfährig vor sozialen aller alsdann Wert, Konvention sich ducken Der Mensch stellt läßt. weniger auf sich selbst und gibt sich selbst seinen sich daß er das Gelingen seines Lebens an die Anerkennung als durch andere setzt, damit aber sich unvermeidlich zu ihrem Diener Jedes Volk hat hier seine besonderen Gefahren, bei uns erniedrigt. Deutschen nehmen unverkennbar künstliche Abstufungen, Fragen des Ranges, Dekorationen wie Titel und Orden, werk des Lebens, damit einen viel das Aufsichselbststehen zu alles dieses bloße Bei- und schädigen breiten Platz des Lebens und seine Nebensachen wie Hauptsachen Nebensachen herabgesetzt haftigkeit. Unmöglich lassen behandeln, ohne daß Hauptsachen sich zu ein volle Mann- werden. Jeder Beruf und auch jeder Mensch hat ein Recht auf Achtung und Anerkennung; er muß es sich erstreiten, wenn es ihm versagt wird. 1 Es sei Aber er es „Wohl wachsen Schwämme nur regnet, aber Menschenwürde, wächst nicht daß von außen her hier an das derbe, aber nicht unberechtigte Wort Pestalozzis erinnert (Wke. XII, 217): wenn erringt es nicht dadurch, aus der Routine hervor, wenn ihr leicht aus dem Mist, und Charaktergröße auch die Sonne scheint." Geistestiefe Persönlichkeit und Charakter. ihm Rangklassen oder Dekorationen bewilligt, 361 sondern dadurch, daß seine Arbeit bei sich selbst gestärkt, unabhängig gestaltet, zu vollem Ganze des Lebens gebracht wird. Einfluß auf das führt schon auf den zweiten Das Das bildung bedarf. ist Wir die eigne Verantwortung innerhalb unseres Lebenskreises. und über Vielregiererei und pflegen zu durch die Bureaukratie jener die Tendenz innewohnt, Selbständigkeit zu alle Befugnis hätte die Bureaukratie von als den Entfaltung freier gewiß insofern mit Recht, zu als nur eine alles andere von ihr abhängig zu einzige Stelle voller Aber übertragen zu verstehen. ihr nimmer langt, entspräche sie nicht einer nicht über Schädigung klagen, berufen und machen^ es Punkt, dessen die Charakter- die Selbständigkeit, die freie Entscheidung solche eine Macht bei uns er- uns innewohnenden Neigung, stäcke uns eine Lust zu regulieren und zu schabionisieren, über in unsere Denkweise eigensinnig anderen eine Polizei zu üben, auch den anderen aufzudrängen. Es fehlt uns vielfach die Bereitschaft, den anderen lassen, seiner Art gewähren, in wo er uns schroff widerspricht; uns erscheint leicht als Verleugnen der eignen dem Gedanken eine Überzeugung. ein frei der eignen Gesinnung, möglichst als erhalten mög- um ihnen vorzubeugen, drücken wir von der Ausnahme So ein. ein Auch stehen uns leicht bei den Gesamtstand des Lebens herab, gestalten von der Regel schalten zu solches Gewährenlassen der Freiheit vorwiegend die Gefahren eines lichen Mißbrauclis vor Augen; lieber Mattheit ihn auch da wir schnüren und engen es her, leicht es nicht sowohl konventionelle Gestalten, Menschen, Exemplare einer bloßen Gattung, während die t)'pische Ausbildung individueller Art unterdrückt wird und damit etwas verloren geht, dessen dringend bedarf. Aufrechterhaltung die Wie vieles wirkt innerer Selbständigkeit im Leben der Gegenwart zu- sammen, um den Menschen abzuschleifen und ihn gleichförmig zu gestalten, wie sehr bedrohen Massenwirkungen die Entfaltung unserer Individualität, Massenwirkungen namentlich auch der Individualität mit besonderem unsere Individualisten sind oft da, wo das Recht Nachdruck betont wird! gar nichts anderes als eine Denn besondere Art von Gattungsmenschen mit durchaus uniformen Zügen. Zur freien Entwicklung der Individualität bedarf es aber mehr Muße, mehr innerer Lebens zu gestatten Sammlung, pflegt. nur zahllose Individuen, als die Hast des gegenwärtigen Die Überbürdung mit Arbeit, die nicht sondern ganze Stände bedrückt, wird zu Zu den Problemen des Menschenlebens. 362 Gefahr für eine innere Bildung, ernstlichen einer ruhige Selbstbesinnung, alle beharrende sie alle zu- Wir haben sammenhängenden Aufbau des Lebens hemmt. ausgezeichneten Lehrstand, Vertreter ist ihm Aber dieser Lehrstand schwer überbürdet, zürn Teil mit mechanischen Geschäften, ganz abzunehmen wohl daß wären; frische Menschen einer ganz anderen Wirkung fähig sind abgestumpfte, das wenn erwogen, wird keine nicht und als Raum für Beisichselbstsein des Lebens schafft, Wer freudige hier es findet, wie auf eine innere Bildung, der hilft die müde und genügend erwogen, oder durchgreifende Abhilfe. anderen Gebieten mehr freien ein einen den besten auf der Erde, den gebornen und Wesenskultur. Innen- einer indem Konzentration, allen für auch zur Förder- ung jener Ziele, die das Problem des Charakters vorhält. Wo so Großes auf dem Spiele steht, da gewinnt auch das Bedeutung, was, für sich selbst betrachtet, klein scheinen mag. Freiheit des Willens. 6. Einleitung. a) \/\ an kommt nie zu Ende, wenn man sich auf die Fragen der "*^* Freiheit einläßt; jede Partei hat unbegrenzte Hilfsmittel", so sagte der große Kritiker Bayle bei Erörterung des Willensproblems.* Ein angesehener gegen die Akten nismus für die letzten deutscher Jahrhunderte so daß für uns der Gegenwart erklärt da- des Determinismus gegen den Indetermi- in Sachen M geschlossen ".2 aufgeklärt, Gelehrter Wer hat nun Recht? viel erledigt völlig Haben wirklich Neues gebracht und die Sache so ist, was früher die Geister Oder erscheint uns vielleicht nur deshalb unversöhnlich entzweite? die Sache als ausgemacht, weil wir sie unter dem Einfluß besonderer Gedankenmassen nur von einer besonderen Seite zu betrachten pflegen? Sehen wir, schon als wie es damit steht, und ob der Sieg des Determinismus entschieden gelten darf. Der Determinismus seinem Grundgedanken nach alt, ver- ändert ward nur das Nähere seiner Fassung und Begründung. Bei den Stoikern, dürfen, 3 ist die es der als ist die bewußten Deterministen gelten Gedanke eines durchgängigen Kausalzusammen- Oeuv. div. (Haag 1727) ^ ersten III, 794a: On ne finit point quand on s'en- gage aux questions de la liberte, chaque parti a des ressources infinies. ' S. Meinong, Psychologisch-ethische UntersuchuTigen zur Werttheorie, S. 209. Es heißt dort: »Es ist aber nicht die Kontroverse des Determinismus, die hier aufgenommen werden soll : in ihr sind die Akten längst geschlossen oder sollten es doch meines Erachtens sein, indem, wer an das Kausalgesetz glaubt, konsequenter Weise auch nicht Indeterminist sein kann." meint bei Anführung dieser Stelle (Ethik, bei 2. Höffding deutsche Aufl., 102), daß Betrachtung der dänischen Literatur über diese Frage einen man anderen Eindruck erhalte. ^ Daß Aristoteles keineswegs Indeterminist war, aber das Problem noch nicht zu voller Klarheit brachte, hat in sorgfältigster Untersuchung R. Löning gezeigt (in dem Werk »Die Zurechnungslehre des Aristoteles", 1903). Zu den Problemen des Menschenlebens. 364 hanges der Welt, der es alle Willkür an den einzelnen Stellen aufhebt; drängt hier weniger die psychologische Analyse als das Ganze Nachdem die praktisch- Weltansicht der zu jener moralische Richtung des Behauptung. ältesten Christentums der Willensfreiheit das entschiedene Übergewicht gegeben hatte, ohne sie aber wissenschaftlich irgend zu begründen, verficht Augustins theozentrische Be- trachtung der Wirklichkeit einen völligen Determinismus; jede eigne Entscheidung des Menschen erscheint hier Allmacht und der Allwissenheit Gottes. als Aufhebung der eine Die Milderung, welche die Kirche und das Mittelalter daran vollzogen, wurde von der Reformation, namentlich ihren Anfängen, aufs schroffste abgewiesen und der religiöse Determinismus wieder in voller Stärke aufgenommen. Die Höhe der Aufklärung bringt einen kosmischen Determinismus und gibt ihm bei Ein scheinbarer Spinoza seine klassische Gestalt. Gegner des Determinismus wie Leibniz bildet diesen in Wahrheit nur feiner durch, und gibles Reich gewährt unserm inmitten der Zeit befindlichen Leben die kantische Rettung der Freiheit in ein intelli- und Handeln keine genügende Hilfe.^ So überwog schon vor dem 19. Jahrhundert in der geistigen Arbeit entschieden der Determinismus, gerade an ihren Höhepunkten gewann er besondere Klarheit und Kraft, er scheint hier die Lebens- energie nicht zu mindern, sondern zu steigern. Paulus stand dem Determinismus näher als irgend ein anderer zu Beginn des Christen- tums und durfte zugleich von habe als die anderen Natur und von alle; sich sagen, daß er mehr gearbeitet auch Augustin war eine unablässig tätige gewaltigem Organisationsvermögen. Und in den Kämpfen der Reformationszeit war die Überzeugung, im Tun und und gar, aber auch lediglich und allein von Gott, Ergehen ganz nicht von irgendwelcher menschlichen Macht, abhängig zu sein, die Hauptquelle festen Vertrauens und unbeugsamer Willenskraft. Zu diesem allen kommt die neueste Phase des Determinismus im 19. Jahrhundert. Ging er früher aus religiösen oder spekulativen Überzeugungen hervor, so ist es jetzt die gründlichere Durchforsch- ung der Erfahrung, welche von den verschiedensten Punkten her zu ihm drängt und ihn sowohl anschaulicher als eindringlicher macht Immer dichter sehen wir jetzt das Netz der Kausalität den * Streng genommen, müßte jene intelligible Freiheit das ganze Leben in der Zeit zur Untätigkeit wegung rauben. verdammen, ihm alle Möglichkeit eigner Be- Freiheit des Willens. Menschen umschlingen, stock seiner Natur Erfahrungen gelangen dabei durch die alte präzisere Fassung zu neuer 365 und Wirkung. gesteigerter Den Grund- der Mensch augenscheinlich ererbt, hat weitere Entwicklung hängt an der gesellschaftlichen seine Umgebung und an der Erziehung; wenn er zu klarer Bewußtheit erwacht, findet er im wesentlichen schon sich fertig, das Schicksal, nicht sein eigenes daß Wollen, hat ihn gebildet. Die neuere Gesellschaftslehre unser Handeln bis Wurzeln hinein dem Einfluß der Gedie geschichtliche Betrachtung, daß wir in samtverhältnisse unterliegt, ganz und gar Kinder der Zeit sind, selbst da, Die moderne Psychologie aber sprechen. Gewebe des Innenlebens blicken, verkettet ein und zeigt, seine sie zeigt läßt uns genauer jede einzelne bedingt, ja ringsumher festgelegt, Geschehen unvermittelter Art. wo wir ihr wider- sie in das Handlung duldet nirgends Dabei aber hofft man der moral- Man ischen Seite des Lebens ihr volles Recht gewähren zu können. sucht darzutun, antwortlichkeit, daß, was ihr notwendig ist, wie z. B. Ver- die auch nach Aufhebung der Freiheit verbleibt; ja die moralische Aufgabe scheint dadurch erheblich zu gewinnen, daß die einzelne Handlung einem Ganzen des Lebens und dies Ganze wieder geschichtlich-gesellschaftlichen Denn wenn damit die Zusammenhängen fester eingefügt wird. dieser Zusammenhänge die Verbesserung Hauptaufgabe des Handelns wird, so erhält es eine breitere Basis sowie festere Angriffspunkte. Zugleich entfaltet sich stärker das Ge- lühl einer moralischen Solidarität und läßt die Verfehlung des Indi- viduums milder beurteilen. So zeigt namentlich das moderne Strafrecht bedeutende Bewegungen zu größerer Humanität unter dem Einfluß des Determinismus. Wenn in dieser Weise ^Ue Interessen zusammen- streben und die engere Verflechtung mit dem unmittelbaren Dasein dem Gedanken der Jahrtausende eine gesteigerte Wirkung gibt, so verstummen zu müssen und an dem endsich nicht mehr zweifeln zu lassen. So Determinismus gültigen Siege des alten scheint aller Widerspruch reicht denn das Bekenntnis zum Determinismus weit über die Kreise der Wissenschaft hinaus und gehört, wenigstens in Deutschland, zu den Ausrüstungsstücken des Menschen, der sich »auf der Höhe der Bildung" fühlt Von hier aus glaubt der Adept moderner Weisheit auf alle, die gegen den Determinismus noch irgend welche Bedenken haben, als auf arg Zurückgebliebene stolz herabsehen zu dürfen. Zu einiger Behutsamkeit demgegenüber könnte wohl die Wahrnehmung stimmen, daß es noch immer eine ganze Anzahl von Zu den Problemen des Menschenlebens. 366 Forschern gibt, tüchtigen welche dem Determinismus widerstehen, und daß sie eher zu wachsen als abzunehmen scheint, ^ sowie die andere Wahrnehmung, daß bei anderen. Kulturvölkern der Determinismus keineswegs so argem Verruf steht wie bei uns Deutschen. in Das gilt namentlich von Frankreich, wo die «Diskontinuitätsphilosophie" bewußt und energisch den Determinismus ablehnt, wo selbst die in lebensvolles Gemälde des Seelenlebens die Freiheit als wesent- ein lich einschließt. Das bezeugt doch schon endgültig erledigt deutschen Lebens b) Boutroux ein „Kontingenz" der Naturgesetze verteidigt,^ wo ein Bergson ist als vielleicht, unter daß die Sache weniger besonderen Strömungen des erledigt erscheint.^ als Erwägungen zur Behauptung des Determinismus. Ein Problem, das so viel Verwicklung in sich trägt, und das so sehr die Zeiten wie die Geister entzweit, in raschem Fluge nebenbei behandeln zu wollen, das müßte als unbescheiden, ja vermessen Aber wer sich mit den geistigen Strömungen der Gegenwart gelten. befaßt, der muß irgend auch der Bewegungen gedenken, welche in der Behandlung jenes Problems zutage treten. Die Art, wie sich heute der Determinismus in der Breite des Lebens ausnimmt, scheint uns an einem starken Dogmatismus zu leiden und das alte Problem viel zu aus dem Gesichtswinkel der besonderen Zeit zu Wer auf die Geschichte einseitig behandeln. blickt, der erhält nicht den Eindruck, daß der Determinismus sich zu seinem Gegner wie Höheres zu Niederem verhält, und daß bei wachsender Aufklärung der Wider- mehr und mehr verschwunden stand ist. Denn schon vor Jahr- tausenden lag die deterministische These deutlich vor Augen, es sind dann immer wieder Gegenströmungen gekommen, und zwar nicht nur in den Niederungen des Durchschnittslebens, sondern auch auf ^ Es seien u. a. von Werken der letzten Jahre erwähnt: von Rohland »Die Willensfreiheit und ihre Gegner" (1905), Froehlich «Freiheit und Notwendigkeit" (1908), Joel „Der freie Wille" (1908). "^ und in E. Boutroux „Über den Begriff des Naturgesetzes in der Wissenschaft der Philosophie Boelitz „Die Lehre der Gegenwart" (deutsche Übers. 1907), s. auch vom Zufall bei Emile Boutroux" (1907). ^ In vortrefflicher Weise hat zur Klärung des Problems neuerdings Windelband gewirkt („Über Willensfreiheit", 2. Aufl. 1905), indem er die Notwendigkeit einer Scheidung verschiedener Formen zeigte, „die in dem Worte ,Willensfreiheit' kritiklos zusammengefaßt zu werden pflegen" (s. S. 222). Freiheit des Willens. der Höhe der geistigen Arbeit, den leitenden Deterministen Durchführung ja, 367 was am schwersten wiegt, Zur selbst. vollen bei Konsequenz der der Determinismus wohl an keiner Stelle gelangt. ist Wenn die Stoiker das All in ein kausales Gefüge verwandeln und dieses auch das Schicksal des Menschen gänzlich bestimmen lassen, so bleibt seiner eignen Entscheidung vorbehalten, ob er den Weltlauf an- erkennt und in die eigne Überzeugung aufnimmt, oder ob er sich ihm fortschleppen läßt; widerwillig von die Möglichkeit einer solchen Entscheidung, dieser Kern der stoischen Moral, durchbricht aber augen- Augustin ist strenger Determinist nur scheinlich den Determinismus. so lange, als ihn gänzlich die theozentrische Betrachtung erfüllt; so- bald er das menschliche Handeln, im besondern das praktisch-kirchliche Leben, ins Auge faßt, gilt ihm der Mensch als zu selbstständiger und zu eigner Entscheidung berufen. Auch Luther hat die anfängliche Strenge seines Determinismus später beträchtlich ge- Mitarbeit mag den Menschen noch Spinoza endlich mildert. so sehr einer lückenlosen Weltverkettung angehörig zeigen, für ihre Anerkennung ist er erst gewinnen; zu Wendung des Lebens, zu mit eine einem licher Illusionen nicht endlich auch bei einer minismus wie ihr ergibt sich aber eine völlige Wendung von einem Gewebe menschMacht es lauterer Wahrheit mehr empirischen Fassung des Deter- Reiche der Gegenwart einen großen Unterschied, ob die in Verkettung uns gänzlich unbewußt wirkt, oder ob sie von uns durch- und schaut in unser Handeln aufgenommen wird? Durchgängig Ordnung nicht schon ihre Anerkennung mit sich bringen, nach dem Ja oder Nein an dieser Stelle aber das Leben sich grundverschieden gestalten. Ganz gleichwir die Tatsache einer kausalen sehen gültig scheint die Entscheidung des Auch die Kants Stellung zu Menschen also nicht zu sein. unserem Problem könnte die heutigen Deterministen vor einem allzu sicheren Auftreten warnen. Kant pflegt auch ihnen System aber ist ein entfernen heißt seinen Man großer Denker zu gelten und sein philosophische Leistung der Neuzeit Die unentbehrlicher Eckstein dieses Systems, ihn als die gewaltigste Freiheit ^ als ein Gesamtbau zerstören.^ Hat doch Kant die sehe nur die Äußerungen Kants in der Vorrede zur Kritik der praktischen Vernunft: »Der Begriff der Freiheit, sofern dessen Realität durch apodiktisches Gesetz der praktischen Vernunft bewiesen ist, macht nun den Schlußstein von dem ganzen Gebäude eines Systems der reinen, selbst der spekulativen Vernunft aus« (V, 3, Hart.). Femer: „Freiheit ist aber auch ein Zu den Problemen des Menschenlebens. 368 Raum und Zeit und die Realität des Freiheitsbegriffes um welche sich die Vernunftkritik drehe, von Idealität die beiden Angeln genannt, und ist auch seiner Erkenntnislehre der Freiheitsgedanke von Anfang an gegenwärtig. Mag man der besonderen Art, wie Kant das Frei- heitsproblem löste, noch so kritisch gegenüberstehen, es gibt doch zu denken, daß dieser große Philosoph auf Freiheit glaubte unmögverzichten zu können. lich Was ist es wohl, das trotz aller Häufung anscheinend unwiderleglicher Gründe immer wieder über den Determinismus hinaustreibt? daß seine konsequente Durchführung alles zerstören Dieses wohl, müßte, was dem geistigen Leben des Menschen eigentümlich ist. Dem Determinismus ist, wie die Dinge draußen, so auch die Seele aus dem Zusammentreffen ein gewisses Ergebnis mit zwingender Notwendig- Menschen eine gegebene des ihm beider geht hervor. keit Aber läßt sich Größe; im Grunde dann noch von eignem Handeln reden, und können wir uns innerlich irgend dafür verant- Wird die Sache in ihren eignen Grenzen gehalten überkommene Bild unseres Lebens und Wesens verändert oder ergänzt, so müßten wir bloße Zuschauer wortlich fühlen? und nicht unvermerkt durch das dessen werden, was an uns vorgeht, an der Seele genau wie an dem Körper; was je aus uns werden wird, das liegt mit sicherem Zuge schon vorgebildet, wir spielen eine zugewiesene Rolle, wir müssen eine vorgezeichnete Landstraße geduldig weiter als völlige Sklaven des Schicksals. und weiter wandeln Verschwinden würde damit alle wo es keinen Aufruf zur Entscheidung, keine Spannung, kein ursprüngliches Handeln gibt, wo die Zukunft wahrhaftige Gegenwart, denn aus der Vergangenheit wie die Frucht aus dem Samen da gibt es nur den Schein einer Gegenwart. zugleich aller innere Zusammenhang und alle hervorgeht, Auch verschwindet beherrschende Einheit des Lebens, denn eine solche läßt sich nicht überliefern, nur aus eigner, ursprünglicher Tätigkeit hervorgehen, sie sie kann muß immer von neuem geschaffen werden; so verwandelt sich unsere Seele dort in ein Nebeneinander einzelner Elemente, das nach außen hin ein Ganzes scheinen mag, das entbehrt. lichkeit, die In in Wahrheit aller inneren Verbindung Summa ist es namentlich die Leugnung aller Ursprüng- welche jener Denkweise anhaftet, und dieser Verzicht auf einzige Möglichkeit unter a allen priori Ideen wissen, der spekulativen Vernunft, ohne sie wovon wir doch einzusehen, weil dingung des moralischen Gesetzes ist, welches wir wissen" sie (V, 4). die die Be- Ursprünglichkeit, dies Freiheit des Willens. 35g gänzliche Geschoben- und Getriebenwerden durch ein dunkles Geschick, näher erwogen, etwas ganz Ent- Das Unerträgliches. schlechterdings setzliches, ist, Entsetzliche Verstrickung in ein allgewaltiges, unentrinnbares Schicksal, einer das uns nun und nimmer losläßt, haben namentlich die tieferen Geister unter den Indern mit erschütternder Stärke empfunden; so ward ihnen die Befreiung von solchem Schicksal, von dem Rad der Wiedergeburt, zum dringendsten Anliegen, zur sehnlichsten Hoffnung. Aber, so wird uns eingewandt, was hilft Sträuben gegen alles eine eherne Notwendigkeit? Ergebung und Resignation ist das einzige, dem Menschen bleibt und das ziemt. Erhält er nicht in Wahrheit seine Natur als ein unabweisbares Erbe mitgeteilt, haben ihn zusammen mit diesem nicht seine nähere und seine weitere Umgebung zu dem Und ist es nicht das Schicksal, das den gemacht, was er heute ist? Fertigen Motive, wenn oder dorthin hieher Bedarf läßt? ferner nicht stellt, ihn und würde nicht das Leben oder jenes wirken dieses menschliche das Handeln bestimmter Chaos in ein wirres unter diesen Motiven willkürlich zu wählen wäre, zerfallen, wenn sich ohne allen Zusammenhang mit dem bisherigen Tun der Gute zum zum Guten entschließen könnte? Bösen, der Böse verlangen Sicherlich Würdigung; ob sie solche Wahrheiten gewissenhafte eine aber die Sache erschöpfen, ob sie das Eigen- tümliche des Menschen mit seiner geistigen Art vollauf zur Geltung bringen, das ist nicht so leicht zu sagen. Es ist eine unbestreitbare Tatsache, daß der Mensch, zunächst mit seinem Denken, nicht einfach innerhalb der sondern daß Verkettungen des Daseins verbleibt, wie das Tier, sich ihnen gegenüberzustellen und sie als Ganzes zu überschauen vermag. Ohne er aus diesen Verkettungen heraustritt, das gäbe es kein Fragen nach Wahrheit, und schon daß eine solche Frage überhaupt des Lebens. gestellt wird, Steht es mit enthält eine erhebliche Weiterbildung dem Tun nicht ähnlich? Wir gehen nicht auf in ein Nebeneinander einzelner Antriebe, einer eine neue Stufe des Lebens; als erheben uns zu wir überlegenen Einheit und gewinnen damit eine Selbsttätigkeit heit überblicken von hier aus können wir jene Viel- und jedes Stück nach seinem Werte schätzen; dieser Wert ist nicht schlechthin gegeben und ausgemacht, er gestaltet sich von jener Einheit aus, und ihre Umgestaltung muß auch ihn erst verändern. Fragt man uns, wie jene Selbsttätigkeit, wie jenes Hervor- brechen ursprünglichen Geisteslebens im Menschen möglich Eucken, Grundbegriffe. 4. Aufl. 24 sei, wie Zu den Problemen des Menschenlebens. 370 sie sich aus dem Ganzen des Alls erkläre, so bekennen wir offen Unvermögen einer Antwort darauf. Aber wie arm würden wir werden, wenn wir alles leugnen wollten, was wir nicht zu erklären vermögen ? Wir sehen um uns in unerschöpflicher Fülle bewußte und fühlende Wesen, eigentümliche Lebenseinheiten, immer Ist uns das irgend erklärlich, könnte man von neuem entstehen. nicht, wenn es nicht eine unbestreitbare Tatsache wäre, es ebenso und ehrlich das als unmöglich verwerfen wie Erwachen ein von Selbsttätigkeit? Denn eine Lebenseinheit scheint weder durch eine Zusammensetzung von Leblosem noch durch eine Teilung von Lebendigem entstehen zu können. kann es neue Lebenseinheiten So müssen wir doch wohl unsere leugnen. keit Also in keiner Weise Aber sie quellen unablässig auf, wir können sie unmöglich geben. Begriffe von Möglich- der Wirklichkeit der Dinge unterordnen, nicht diese in ein von unserm engen und kleinen Verstände gebildetes Maß hineinzwängen. Im Grunde ist es der Intellektualismus, der dem Determinismus die Wurzeln liefert Dazu sei bei diesem Problem auch die besondere Lage des Menschen mit ihrer Verwicklung nicht vergessen. Daß er einerseits zur Natur gehört und andererseits den Beginn einer neuen Stufe stärksten der Wirklichkeit, eines Reiches des Beisichselbstseins, wandelt sein bildet, das ver- ganzes Leben in eine Aufgabe, zu deren Lösung es unbedingt seiner eignen Entscheidung bedarf. entgegengesetzte Antriebe, das schlechterdings unvergleichlich. gesellschaftliche Dasein macht die Das stellt es unter Motive hie und da Dort das« natürliche oder auch das mit seiner Lust, hier die geistige Ordnung mit ihrem neuen Selbst und seiner Unendlichkeit, Läßt sich direkt miteinander vergleichen, und aneinander messen, was ein Handeln an selbstischem Glück, und was es an Erhöhung des Wesens in Erfüllung von Pflicht und Entwicklung von Liebe bringt?^ scheinlich stehen die Hauptrichtung wohl um * das, hier einzelne Leistungen, nicht des Lebens in Frage, was wir tun, als es Augen- sondern es steht handelt sich nicht so- um das, was wir sind, oder vielmehr So müssen wir auch die Vorstellung ablehnen, als ob die Motive und gegebene Größen wären, die in der Seele wie Gewichte auf einer Wagschale zusammenträfen und eine Entscheidung bewirkten. Muß denn alles Handeln aus gegebenen Motiven erfolgen, können bei innerer Wandlung feste des Lebens nicht neue entstehen? Und muß die Seele den Motiven ihren Wert nicht immer von neuem erteilen? Freiheit des Willens. um 371 worin wir unser Wesen setzen. Die Seele des Menschen bildet nicht einen bloßen Schauplatz, an dem zwei Stufen der Wirk- das, begegnen, sondern lichkeit sich wird selbst zur Mitwirkung auf- sie gerufen, nur durch selbsttätige Aneignung hindurch kann das Geistes- leben an dieser darin an zur vollen Wirklichkeit Stelle Entscheidung liegt, das kommen. Was aber ein besonderer nicht vollzieht Augenblick, sondern das vollzieht unser ganzes Leben, das will unablässig bejaht und bekräftigt sein. Geistiges Leben, so sahen wir, verbleibt nicht, wie es einmal sich findet, es muß immer von neuem So behält unser Leben entstehen, soll es nicht aufs rascheste sinken. stets eine Spannung, es wird, geistig angesehen, nie zu ruhigem Besitz. Damit erscheint Wirken freies Wendung Augenblicks, die Sache des bloßen nicht als ein plötzlicher Einfall. Denn nicht eine als auch der Augenblick eine hervorragende Bedeutung ge- so gewiß winnen kann, er kann das nur in weiteren Zusammenhängen, nur als der Höhepunkt eines durchgehenden Strebens. An erster Stelle geht die Frage auf das Ganze, auf die Hauptrichtung des Lebens, nicht auf einzelne Punkte. Wie in unserem Leben Freiheit und Schicksal zusammenwirken, wie das eine auf das andere angewiesen ist, das zeigt mit beson- derer Anschaulichkeit die Bildung einer geistigen Individualität bloßem Entschlüsse Un- möglich kann Schicksal geht hier voran und weist uns die Richtung des Weges. Aber sofern erst eine dieses Individuelle geistiger Art errungen, geschieden, Stärke solche aus muß eigne in Tätigkeit Mittelpunkt als erst freigelegt muß aufgenommen, anerkannt und ist, werden. der Seele der Seele, kann harte des eignen Wesens, von uns es von das Fremdem Der Punkt unserer sein. ergriffen entstehen, Das Suchen Mühen und schwere Irrungen kosten, wir können uns weit verlaufen, ehe wir Und wenn wir ihn gefunden haben, Mühe und Arbeit, um ihn festzuhalten zu jenem Punkte gelangen. so bedarf es weiterer und durchzusetzen; so verwandelt der Verlauf des Lebens mehr und mehr die Gabe des Schicksals in ein eignes Werk des Menschen, so erhebt sich unser Leben immer mehr zur Selbst- tätigkeit Ähnliches gilt gebenem vorliegt, von ganzen Völkern und ist, geistig angesehen, Zeiten. Was an Ge- nur eine Möglichkeit, zur Verwirklichung und zugleich zur konkreten Gestaltung nur durch unsere eigne Tat gelangt. die immer Wir können uns willenlos 24« Zu den Problemen des Menschenlebens. 372 von dem, was wir empfangen und was uns umgibt, treiben lassen wir können ihm gegenüber eine Selbständigkeit gewinnen und von aus erst ihm entringen, was an Geistigem in ihm angelegt ihr ist. Die Geschichte baut sich ihrem Geistesgehalt nach nicht auf einer und gesicherten Grundlage auf, sondern immer von neuem mag über ihr Ganzes Zweifel entstehen, immer von neuem gilt es, ihr eine Grundlage eret zu sichern, immer neu ist sie in ein gegebenen Ganzes zusammenzuschließen. Solche Überzeugungen ergeben ein wesentlich anderes Bild der Wirklichkeit, als es dem Determinismus vorschwebt. ihm scheint bei alles in einer einzigen sein ein Fläche zu liegen oder doch aus einem gegebenen Grundstock hervorzugehen, Leben nicht so einfach und Vor allem Dem Determinismus anderes Bild von unserem eignen Seelenleben. Wahrheit ist unser Inhalt nicht so gleichartig. Ver- in schiedene Möglichkeiten und verschiedene Höhenlagen durchkreuzen einander und ziehen bald hierher bald dorthin. Eine von ihnen ist durch den Verlauf des Lebens zur Herrschaft gelangt und das Ganze Lebens und Seins. Aber gilt leicht es brauchen nur wesentlich neue Aufgaben, nur große Erschütterungen und Umwälz- als unseres ungen zu kommen, und wartetes uns in es steigt während auf, etwas altes völlig neues, etwas uner- und versinkt, verblaßt anderes in uns belebt, und es verändern sich alle es wird Werte; es kann uns unsäglich klein und nichtig erscheinen, was früher unsere Seele erfüllte. Das an uns, sondern durch unsere eigne Erregung und Bewegung hin- alles aber nicht durch daß ein was mechanisches Vorgehen Ganze durch. Alsdann dünkte, nur eine gewisse Schicht, nur eine besondere Möglichkeit war, wird deutlich, uns daß wir nur ein Stück unseres Wesens schaftliche Dasein und der vorher erlebten. das Das gesell- Zwang der Lebenserhaltung drängen zu einer Festlegung auf eine solche Besonderheit, hier gilt der Mensch nur als Träger eines eigentümlichen Berufes, hier darin aufgehen, hier wird gezogene Linie einhält. ihm alles verübelt, soll er möglichst was nicht die dadurch Wer sich aber ganz einer solchen Vereng- nicht eine größere Weite mit offnen Mögdem muß es erstarren und verknöchern, der handelt ung ergibt, seinem Leben lichkeiten wahrt, weniger als durch ihn gewirkt wird, abgesteckte Chaussee, verwandeln möchte. in die der geht in Wahrheit jene der Determinismus unser aller Leben Das ist der Segen des Leides und aller großen Erschütterung, daß es den Menschen solcher Erstarrung zu entreißen Freiheit des Willens. 373 Und darin hat und ihm neue Lebensquellen zu eröffnen vermag. auch die Kunst eine hervorragende Aufgabe, der üblichen Einpferch- ung des Lebens den weiteren Kreis mit seinen offnen Möglichkeiten entgegenzuhalten und dadurch der Bindung der Verhältnisse gegenüber zur Befreiung der Seele zu wirken. Immer ist dies der Scheidepunkt, ob unser Leben sich aus fertigen Daten zusammensetzt, oder ob es noch mitten im Fluß Was vom Einzelnen, ist. das von der Menschheit im auch gilt Ganzen. Wie jenen die Besonderheit seines Berufes und seines Ge- schickes festlegt und einspinnt, so ist Menschheit eine bei der es besondere Art der Kultur, auf welche die gewöhnliche Denkweise sie und festlegt, in die zur Verengung auch man sie einspinnen möchte. Das aber bringt Die Menschheit eine Erstarrung des Lebens. dann nur eine gewiesene Bahn zu verfolgen, hätte einer, irgendwelcher zu bloßes Mittel solchen Kultur fühlt, der wer Leistung; mag glauben, sich würde ein sie Höhe auf der mit voller Sicherheit nachweisen zu können, wie alles gekommen ist, wie alles so kommen dem stellt sich die ganze Geschichte als eine Kette der Not- mußte, Aber auch Kulturen leben wendigkeit dar. altem; es wäre schlimm, zum All und sie von dem Alten Altertum ergreifen wie die Gestaltung welken und durchlaufenen in sich die als ausbilden irgendwie her aus, wenn die Menschheit in ihrem Verhältnis nicht andere Möglichkeiten trüge sich könnte. ableitbar? voraussehen, irgend Ist nun dies Neue Konnte das griechische die durch das sich Oder Christentum und den Aufstieg neuer Völker vollzogen hat? war vom Neuzeit Mittelalter brachte? modernen Kultur, aus Wendung die Und wenn ein wir zu jetzt Greisenhaftwerden welche die Schranken der erwarten, innere ihres inneren Bestandes immer stärker empfinden, was anderes läßt uns freudig am Streben und Wirken festhalten, als die Hoffnung, daß die Menschheit sich auf den bisherigen Bahnen nicht schon ausgelebt hat, daß noch Aber ohne unsere Selbsttätigoffne Möglichkeiten vor ihr liegen? keit werden solche Möglichkeiten sich schwerlich beleben lassen; wir dürfen dabei nicht bloße Zuschauer sein, wir müssen Mitarbeiter werden. Sollte eine derartige Denkweise mit ihrer Erweiterung und Be- lebung des Wirklichkeitsbildes nicht auch für das Ganze des Weltalls gelten? Viel zu sehr neigen wir Modernen und den Weltstand, der uns umfängt, als die dahin, die Welt einzige Möglichkeit, Zu den Problemen des Menschenlebens. 374 als den Inbegriff Wirklichen anzusehen. alles nur eine besondere leicht müssen? ja bestehen Ist jene nicht viel- neben der andere bestehen können, Art, Als ein Anzeichen dessen könnten die mannig- fachen Unfertigkeiten, Verwicklungen, Widersprüche gelten, in denen Gemenge von Vernunft und UnverVon hier aus muß es als ein starrer und unsere Welt sich befindet, das das sie aufweist. nunft, einengender Dogmatismus erscheinen, alle Wirklichkeitsbildung an das »Gegebene" zu binden. und licher selbstverständlich gibt, Ursprünglichkeit ihrer ein höchst unglück- und da es alle Selbsttätigkeit Eine leugnet. kaum, empfindet heute weise ist da es eine höchst problematische irreleitender Begriff, Behauptung als mit Dies «Gegebene" eingeschüchterte Herabsetzung welche der Denk- geistigen »Der Energie in der völligen Bindung an die Gegebenheit Geist geht auf alle Arten an die Kost und schmiegt sich an das Ge- gebene" (J. Wie liegt. Burckhardt). sich von unseren Überzeugungen aus das Problem der und Gebundenheit des Handelns darstellt, das läßt sich Freiheit in der hier gebotenen Kürze unmöglich erörtern, das hoffen wir bald in einer Grundlegung der Ethik näher darlegen es zu können. Hier galt nur bemerklich zu machen, daß der Determinismus auf ganz be- stimmten Voraussetzungen von der Wirklichkeit ruht, und daß die Erkenntnis dessen seine Selbstverständlichkeit sofort zerstört. handelt die Welt als gegeben bloßes Stück dieser Welt; als ein zu einer kaum verständlichen irgend zu zweifeln; Er be- und geschlossen und uns Menschen hat er damit Recht, Torheit, an seiner so wird es Wahrheit noch aber die Welt noch im Fluß und können ist wir selbst an vordringendem Schaffen teilnehmen, so wird jemand, der andere Wege versucht, nicht schon als ein intellektuell Verlorener anzusehen Schlimmsten Falles sein. freilich müßte er sich dessen in Gemeinschaft mit einem Plato und einem Kant vertrösten. Die große Klärung und Förderung, welche der moderne Determinismus dem Freiheitsprobleme gebracht hat, wird damit keines- wegs verkannt, er hat das ganze Problem der Freiheit aufs wesentlichste vertieft und Daß viel gemacht. zum guten Teil nur das bleibt auch die ihre sammenstoß von in unserem Leben das Schicksal es bereitet, in Frage, Freiheit naive Bejahung schlechterdings unmöglich Notwendigkeit ein Recht Freiheit ist ob dieses das Ganze behält, und ob waltet, daß nicht zu verkennen; ist, ob nicht zugleich der Zu- nicht eben und Notwendigkeit unserem Leben seinen Freiheit des Willens. eigentümlichen Sinne erst Charakter möglich macht verleiht, ob er nicht 375 Leben in vollem Wir stehen zu Schelling, wenn er sagt: »Ohne den Widerspruch von Notwendigkeit und Freiheit würde nicht Philosophie allein, Tod versinken." sondern jedes höhere Wollen des Geistes in den E. Letzte Probleme. 1. Der Wert des Lebens. Einleitendes. a) D ein en Wert des Lebens nach der individuellen Empfindung mit ihrer Zufälligkeit und ihren Ding der Unmöglichkeit; Schwankungen abzuschätzen, hat die Frage Pessimismus* keinen anderen Sinn, so weisen. Aber unmöglich stellen, schon deshalb unwiderstehlicher nicht, sie von vornherein abzuüber das Leben ein- weil dies uns nicht mit schlichter Tatsächlichkeit ist oder Urteil ist läßt sich alles des Optimismus einnimmt, sondern weil es und eine Entscheidung unsererseits verlangt, diese aber entgegengesetzt auskann. fallen anschließen aber wir ^ Denn entweder mögen wir uns seinem Strome freudig und ihn möglichst durch unsere oder Kraft verstärken, mögen uns ihm entgegenstemmen und ihn bei uns zum Die Ausdrücke Optimismus und Pessimismus sind neueren Ursprungs, zuerst zur Bezeichnung der Leibnizischen Lehre von der besten Welt verwandt worden. Brucker (IV, 2, S. 385) bemerkt darüber: Non tacendum vero, ipsos Jesuitas Trivaltinos, magnos cetera Leibnizii admiratores, jenes ist cum recensione Theodiceae facta sententiam dicerent (wie eine hinzufügt 1737, Febr. art. 1), Anmerkung laudata ingenti lectionis et judicii copia, tractationis ordine, accuratione et concinnitate systematica, fateri tamen, errores philosophum summum admisisse, maxime vero optimi mundi tionem (optimismum vocant) non nisi larvatum materialismum et multos asser- et spiritualem Spinozismum involvere, s. auch S. 415. Zu seiner Verbreitung hat wohl namentlich Voltaire mit seinem Candide ou l'optimisme beigetragen. Bei Pessimismus pflegen wir zuerst an Schopenhauer zu denken, aber dieser selbst gebraucht das Wort nur spärlich; Caldwell in seinem vortrefflichen Werke über Schopenhauer bemerkt darüber (S. 522): He rarely uses the word pessimism perhaps three or four times at all — and than only about the philosophy of others, and generally in the adjective form as opposed to an optimistic view of — — things. Der Wert des Lebens. uns stellen bringen suchen. Große beide Antworten deutlich zu Stillstand 377 geschichtliche Entwicklungen Die Höhe des vor Augen. indischen Lebens war von der Überzeugung beseelt, daß das Leben mit seiner endlosen Sorge, Mühe und Not vornehmlich ein Leiden und daß das Streben nach Befreiung von ihm oder doch nach sei, Herabsetzung Summe die Stärke seiner Entgegen solcher Lebensverneinung kreis einer Gut, das anderen Schätzung: man festhalten eifrig folgt Lebensweisheit unser westlicher Kultur- zu erweisen. dünkt und steigern möchte; das allumfassende soweit das Problem in sie Gerechtigkeit und sittliche zu Die griechischen und Widersprüchen der Welt erweisen; beschäftigte, Liebe bemühen diese als ein vollendetes Kunstwerk, sie Harmonie der Wirklichkeit eine hohes ein so Die geschichtliche Bewegung nach Dunkelheiten überlegen zu werden, indem eine Leben hier dieser Richtung zerfällt aber in drei Hauptphasen. Denker suchten den bilde. Bejahung zu begründen und die Wirk- sich auch die Denker, jene lichkeit als wertvoll der wie die z. Ordnung, voller christlichen Denker, B. einen Augustin, sahen von die den Gegensatz Ausgleichung den bringe; modernen Denkern endlich ward die Welt ein vordringender Lebensstrom, ein unaufhörliches Anschwellen der Kraft; als ein Reiz und Antrieb als dafür schien- sich auch das zu rechtfertigen, was zunächst Störung und Widerspruch erscheint Diese Versuche der Denker sind oft hart angegriffen, ja arg ver- spottet worden; sie möchten eine solche Behandlung verdienen, wenn nur spielende Reflexion sie hervorgebracht hätte, nicht tiefere Beweg- ungen hinter ihnen stünden. Dieses aber war in der Tat der Fall. Denn jene Versuche einer Rechtfertigung des Lebens wurzelten in einer tatsächlichen Gestaltung seiner, in einer Selbstkonzentration, die einen Kern vom übrigen Dasein schied und von ihm aus eine Weiterbildung des So wären die griechischen Versuche, die Welt als Kunstwerk darzustellen, ohne Saft und Kraft gewesen, hätte nicht Ganzen erstrebte. ein die großartige künstlerisch-plastische Gestaltung des Lebens ganzen Wirklichkeit, die wir an den Griechen bewundern, tragen und getrieben; und der sie ge- dies künstlerische Schaffen mit der ihm eignen und Freude war es, was die griechische Welt gegen die Unvernunft des Daseins wappnete, die sie keineswegs gering anschlug, und Kraft was ihr die Zuversicht gab, dem Dunkel und Leid gewachsen zu sein. Das Leben zerlegte sich damit in eine höhere und eine niedere Stufe, in Gestalt und Ungestalt; der Mensch aber vermochte sich auf die Seite Letzte Probleme. 378 des Höheren zu und stellen seinem Bereiche dafür zu wirken. in An stärkster Nicht anders stand es auf dem Boden des Christentums. Empfindung des Bösen wahrhaftig nicht, hier es fehlte aber das Bewußtsein, ein unverlierbares Glied einer weltbeherrschenden Ordnung zu lichen Zuversicht, es sein, gab ihm dem Menschen verlieh viel zu tun, es stählte ihn, sitt- Größe und eine den schweren Kampf gegen die überwuchernde Unvernunft getrost zu beginnen. Daß es sich in der Neuzeit ähnlich verhält, daß hinter dem Glauben an das Vermögen der Entwicklung eine tatsächliche Steigerung des Lebens, eine rastlose Daseins steht, das haben wir Arbeit Verbesserung des zur menschlichen vor Augen; ohne solche alle deutlich Erfahrung des Fortschritts hätte der Glaube an die Entwicklung die Gemüter wohl nur flüchtig berührt. So waren waren es durchgängig eigentümliche Lebensgestaltungen, es Konzentrationen des Lebens bei sich selbst, Tatsynthesen, nicht bloße Begriffsklaubereien, Tatsynthesen, Lebensenergien, welche dem Menschen die Überzeugung einflößten, den Gründen der Wirklichkeit ihn verbunden zu sein und aus ihnen Kraft zu empfangen, die dem aus in den und ihn zugleich mit freudigem Mute er- Stande eines bloßen der Aktivität versetzten Übersichergehenlassens Die Unvernunft verschwand damit keineswegs, füllten. mochte sie eher noch gesteigert scheinen. nicht mehr einsam und wehrlos gegenüber, nun durfte er mitarbeiten Aber nun stand der Mensch ihr am Bau der Wirklichkeit, nun hatte sein Leben einen Sinn und zugleich einen Wert gewonnen. Wer solche Zusammenhänge vor Augen hat, schätzig der wird jene Versuche ansehen, wie führung finden mag. unzulänglich Denker minder gering- der er das Nähere ihrer Beweis- Aus Beweisen hat das Leben nie seine Kraft gezogen. b) Die Verwicklung der Gegenwart. Heute ergeht es uns bei diesem Problem wie bei vielen an- deren; aus vermeintlichem Besitz sind wir wieder in ein tastendes Suchen geworfen. Alle jene Lebenskonzentrationen sind in ihrem Bestände und in ihrer Herrschaft aufs schwerste erschüttert: von der künstlerischen und der ethischen ist es augenscheinlich, der Fortschrittsgedanke hat nicht mehr die Zauber; wir wissen nicht recht, alte Kraft aber auch und den alten wohin uns der Fortschritt führt und Der Wert des Lebens. wem er zugute kommt, tieferen 379 auch seine Tatsächlichkeit ist uns bei den Lebensfragen stark ins Wanken geraten; ein hohles Phrasen- tum umspinnt hier oft den Kern der Behauptung und zieht das Ganze herab. Fehlt demnach dem heutigen Leben überhaupt ein fester Zusammenschluß und mit ihm ein allbeherrschendes Ziel, so zugleich fehlt dem Zustrom die Kraft, der Wirklichkeit gewachsen zu werden, ihn innerlich zu bewältigen und in solcher Verwandlung des das Bewußtsein Daseins in Aktivität Dieser Mangel wegung der Neuzeit sie läßt, Größe zu einer finden. muß um so empfindlicher werden, als eben die Bedie Außenwelt aufs gewaltigste anschwellen uns unvergleichlich größer vor Augen und stellt stärker Indem so immer mehr die auf uns eindringen läßt als je zuvor. Welt uns besiegt und uns zu ihrem bloßen Anhange herabsetzt, verfallen wir bei aller Ausdehnung des Lebens einer inneren Schwäche und Kleinmütigkeit; immer mehr greift die Neigung um sich, in den mannigfachen Erfahrungen vor allem das zu sehen, was den Menschen als klein durchgängig scheint ziehe, als Verhältnis darstellt es, als und ihm alle Besonderheit raubt; ob sich mehr ein Schicksal an uns voll- daß wir zu Herren der Dinge werden und ein inneres zur Wirklichkeit Außenwelt größer geworden setzen haben, ist es, ausbilden ist, als können. Weniger daß die daß wir ihr nichts entgegenzu- was unseren Lebensmut niederdrückt und uns am Inhalt der Wirklichkeit vor allem die Verneinung gewahren läßt So erfahren wir es zunächst im Verhältnis zur großen Natur. Auge fällt uns vor allem ihre Unermeßlichkeit, ihre UnendlichRaum und Zeit, ihre Grenzenlosigkeit im Großen wie auch im Kleinen. Dieser Gedanke der Unendlichkeit hat frühere Zeiten freudig ergriffen und innerlich erhöht, indem er ihnen ein AusIns keit in druck der unbegrenzten Lebensfülle fanden sie darin einen besonderen der Wirklichkeit war;i auch Vorzug des Menschen, daß sein Denken ihn über alle Begrenzung hinaus zum Unendlichen und Ewigen führe und ihn daran teilnehmen lasse. Heute aber denken wir weniger an die innere Gegenwart der Unendlichkeit, sie ausgebreitet in als daß Raum und Zeit um uns liegt und zugleich unser Zu voller ganzes Dasein zu verschwindender Kleinheit herabsetzt Gleichgültigkeit scheint ^ es damit zu sinken. Denn was Die klassische Zeit des Altertums scheut das Unendliche bedeutet. als etwas Grenzenloses und damit einer künstlerischen Gestaltung Entzogenes, zu positiver Schätzung hat den Begriff erst Plotin gebracht. Letzte Probleme. 380 so hören wir, was auf dieser winzigen Erde vorgeht, gegen- alles, über der unermeßlichen Fülle der Welten, die das geschärfte Auge der Neuzeit uns wahrnehmen das Maß, ein anderes Die äußere Größe gibt hier läßt? — Dazu kennen wir nicht Natur bleibt die uns innerlich fremd und verschlossen, sie scheint, indem die Forsch- ung ihr immer mehr Boden abgewinnt, ihrem Grunde nach ferner und ferner zu rücken. Denn es fehlt uns ein inneres Verhältnis zu wie es früheren Zeiten eine religiöse oder eine künstlerische ihr, Überzeugung gab. Uns erfüHt ganz und gar der Gedanke der Be- grenztheit des Menschen, wir scheinen einem besonderen Kreise an- zugehören und ihn wir keiner Weise überschreiten zu können. in den von derart Zusammenhängen großen Sind abgeschnitten, so wird es zu einem vermessenen Unterfangen, zu einem eingebildeten Anthropomorphismus, Natur die deuten und verstehen zu wollen. sie ganz ein nis, tätigen irgendwie tiefes Geheimerzeugt wir uns nur durch die Analogie eines zweck- Sie bleibt Rätsel. In uns ein Handelns irgend vorstellbar machen können, aber wenn sich von Zwecken überhaupt einander dem die Gestalten unermeßlicher Fülle unlösbares Bildungen, ihrem mit der Natur reden widersprechen und sich zu nämlich die Natur mit so scheinen diese läßt, gegenseitig aufzuheben. In- bewunderungswürdiger Sorgfalt eine Art von Wesen bereitet, bereitet sie mit gleicher Sorgfalt eine andere, was die jene zu zerstören vermag, so scheint sie hier zu verneinen, sie dort bejaht, treibt sie Individuen, oft Geschöpfe gegeneinander und aufsteigender mühsamen Umwegen, auf massenhaft werden zierter hetzt ihre eignen sie einen unerbittlichen Daseinskampf. in Zug Inmitten alles aufgeopfert. sie Massenhaft werden gebildet, des Lebens nicht zu verkennen, ebenso aber Kampfes ist ein immer kompli- wird der Bau der Organismen, immer abgewogener die Diffe- renzierung der Teile, immer größer die seelische Leistung. Aber dem eignen Gebiet der Natur sehen wir keinen rechten Gewinn dieses Forttriebs. Denn wenn auch die höchste Stufe über die Lebenserhaltung Im Kampf ums Dasein nicht hinauskommt, so wird im Grunde nur dasselbe Ziel, was auch schon die niedere Stufe auf hat, in weit umständlicherer Weise erreicht; Rückschritt als ein Fortschritt zu nennen? nach Leben und die völlige Leere des bilden den härtesten Widerspruch. viduen an das Dasein, zu seiner ist das nicht eher ein Der ungeheure Drang mühsam errungenen Lebens Gierig klammern sich die IndiFesthaltung bieten sie höchste Der Wert des Lebens. Kraft und Leidenschaft auf. wesen selbst, damit, welchen Sinn hat dies ganze sie zu walten, aber sie scheint unablässig selbst sobald Irgendwelche Vernunft scheint gehemmt und gebunden, zu durchkreuzen, sie ja sie scheint scheint in eine Breite Dazu die sich innerlich nicht zusammenfindet. aufgelöst, wir keine weil wir uns verwirrt und niedergeschlagen, wir die Frage aufs Ganze richten.^ sich und darauf keine Antwort, finden fühlen so finden, Aber was liefert dieses Dasein den Lebe- was gewinnen Wir Getriebe? 381 zeigt die Entwicklungslehre uns diesem dunklen Getriebe weit enger verkettet, als gegenwärtig war, immer enger der Vorstellung früherer Zeiten ziehen sich die Fäden zwischen uns und nur nicht körperlich, auch dem was unter uns liegt; scheinen seelisch ganz dieselben uns Kräfte zu beherrschen, die dort das Leben bestimmen; so wird das Dunkel der Welt auch zum Dunkel für unser eignes Leben, eine Notwendigkeit drängt und nunft dient, Nun ist treibt uns, aber wie weit sie einer Ver- nicht zu ersehen. verbleibt dem Menschen die Wendung von der Natur zur Kultur, er kann sich ein eignes Reich aufbauen und in ihm sich eine Größe, sowie seinem Leben einen Wert bereiten. hier sieht heute, als wer die Sache reinen Gewinn. befriedigt Aber auch Ganze faßt, mehr Verwicklung ins Daß die Kultur den Einzelnen nicht unmittelbar und glücklich macht, daran zweifeln wir heute nicht; so muß sie ihm etwas über das Glück hinaus gewähren; was aber dies sei, das wissen wir nicht. Gewiß wächst unaufhörlich unsere Macht über die Umgebung, sicherlich verbessern sich unablässig die Be- dingungen unseres Daseins, Schmerz und Not werden siegreich bekämpft, Genüsse gewinnt unser eröffnen Leben. in reichster Fülle, alles zusammen sich Aber selbst gibt an Dauer ihm als einem Ganzen noch keinen Gehalt und Sinn; nach einem solchen zu fragen, kann aber ein denkendes und überschauendes Wesen nicht lassen. Größe der Leistung jene Konzentration des Lebens bei sich selbst, die, wie wir sahen, dem Menschen einen festen Halt und das Bewußtsein eines inneren Zusammenhanges mit dem Ganzen der Wirklichkeit gab, die ihm das Leben Damit zu einer großen und aussichtsreichen Aufgabe gestaltete. Es fehlt unserer Kultur bei aller aber fehlt uns die Möglichkeit, uns über die Verwicklungen hinaus- ^ So mag sich wohl bei Erwägung des Ganzen das aristotelische Wort aufdrängen: r «püm? SatjjLovia, aXX' ou ö-£(« (463b, 14). Letzte Probleme. 382 zuheben und ihnen Komplexe zu begegnen, die jeder Kultur anhaften, kräftig moderne Kultur in besonderem Maße zeigt die aber unsere verbinden und verweben die Kräfte entstehen, Große mit sich, solchem Zusammenschluß befreit sich die Arbeit von der Zufälligkeit der bloßen Individuen und gewinnt eine Selbständigkeit; kraft solcher kann Wege eigne sie Aber zugleich und glänzende Triumphe feiern. der Einzelne immer mehr zu einem bloßen sinkt verfolgen und Werkzeug, und Mittel mehr je er das tut, mehr wird desto das Ganze der Kultur seiner Seele fremd, desto weniger kann im Wirken für ein sie nach Betriebsamkeit größter Selbst geistiges außen behaupten. mit hin, atemloser Lebens eine innere Gleichgültigkeit verbunden sein und die und Freude rechte Kraft fehlen. es doch Hast des dem Leben in lauter ein- Erscheinungen aufgelöst und beinahe sich selbst entfremdet. zelne Auch das schwer zu vermeiden, ist herrschende und bewegende Seele alle Wird er So kann mit Kulturentwicklung stark zur daß, fehlt, wo der Kultur eine be- das Kleinmenschliche, das besonders aufwuchert und besonders begleitet, Empfindung kommt: die Verquickung alles Strebens mit kleinmenschlichen Zwecken, die durchgängige Unwahrhaftigkeit des gewöhnlichen Betriebes, der hohe Ziele verkündet, zugleich aber Wohl verfolgen lehrt; dazu die vielfache Eitelkeit, die allen Erfolg zum Preise des kleinen die Handelnden Ich verkehrt; und es erster in Linie ihr eignes das braucht nur ins Ganze gefaßt zu werden, alles kann uns eine starke Unlust an diesem Ganzen befallen; wir haben das Gefühl, hier Mächten gegenüberzustehen, denen wir das Feld unmöglich überlassen dürfen, und denen gegenüber wir doch nichts rechtes vermögen. zeitweilig eine Hülfe Auch der Fortschrittsgedanke, der zu bieten schien, verblaßt uns bei diesen Pro- blemen mehr und mehr; denn augenscheinlich reicht der Fortschritt nicht in diese elementaren Verhältnisse hinein; allem Aufstreben scheinen hier natürliche Triebe und Leidenschaften eine Grenze zu setzen, die wir hören als So läßt können. Gegenwart Lebens doch Schranke peinlich zu empfinden nicht aufsich in der Kultur einen findet, nicht sagen, daß der Mensch der genügenden Sinn und Wert seines und daß ihn die Arbeit für sie über die Zweifel und Nöte des Daseins sicher hinaushebt Die Strebens. Kultur In bildet nicht die äußerste Grenze menschlichen kühnem Aufschwung kann der Mensch sich über ihr ganzes Gebiet erheben, sich auf seine eigne Innerlichkeit stellen, in Der Wert des Lebens. 383 Ausbildung weltumspannender Persönlichkeit jenem ganzen Gewirr und Schein überlegen werden, von dort aus ein unmittelbares Verhältnis Das haben mit bewußtem Streben zuerst die zur Wirklichkeit suchen. Stoiker unternommen, von ihnen zieht diese Denkweise sich als ein bleibender Typus durch die weitere Geschichte, in der Aufklärungszeit auf stark Weise sucht die Religion durch nächsten Daseins ob dabei ziehung die in Weg zu fallen, nicht erörtern, und Zurück- ob uns heute dieser wir fragen nur, hat, Jene Ablösung von der sichtbaren ist Welt und vom Kreise des Menschen zu hier der Welt mit ihrer Spaltung des Daseins nicht Seele möglich betreten anderer Verwicklungen des alle Wir wollen hinauszuheben. eigne In die Entwicklung eines unmittelbaren empfohlene Ablösung von Gefahren und Schranken hat besonders sie Seelen gewirkt. Gott den Menschen über Verhältnisses zu die die um nicht ins Leere verlangt, den sicheren Besitz einer Innenwelt, und ein solcher kann nur aus einem unmittelbaren Verhältnis zu einer überlegenen Macht, möge sie als Gottheit, möge sie als Weltvemunft vorgestellt werden, Dem modernen Menschen aber ist alle Wirklichkeit einer hervorgehen. solchen Macht unsicher geworden. So hat bei ihm auch eine selb- und er verliert damit die Möglichkeit eines Selbständigwerdens gegen die sichtbare Welt ständige Innenwelt keinen festen und das menschliche Getriebe. keit wird mit solchem Grund, Das Sichgroßfühlen der Persönlich- Schwinden des Grundes zu einem eitlen Dünkel und zu einer leeren Phrase; was hat denn der Mensch ohne ein Reich selbstwüchsiger Innerlichkeit der Welt entgegenzusetzen, die mit überwältigender Macht ihn umklammert? Art der modernen Kultur steigert Ja die besondere noch das Gefühl der Abhängig- Maße immer mehr auf ein Wirken und Schaffen in Reih und Glied verweist Wo aber das Vermögen des Einzelnen als engbegrenzt und gering erscheint, da muß zugleich keit, indem an seine sie der Antrieb zu ihn eignem Beginnen sinken; Seelenstande unsere eigne als in seiner Arbeit in wachsendem den Menschen Umgebung bindet, Meinung uns dazu oft bei stellt mattem noch abhängiger dar, wir es in Wahrheit sind, wir neigen dahin, überall Anschluß an andere zu suchen, unser Wollen dadurch bestätigen zu lassen, nur das gibt uns ein Gefühl der Sicherheit; auch erwarten wir vieles vom Vermögen gemeinsamer bei der Einrichtungen, Gesinnung des Einzelnen liegt, wo kurz Energie des Lebens ohne zwingende Not herab. die wir Ist Hauptsache drücken die bei Überwiegen Letzte Probleme. 384 solcher Gesinnung hoffen ?i So sichtiges ist von einem Aufruf zur Selbständigkeit der Anblick des Ganzen wenig erfreulich. viel zu Ein undurch- Reich der Natur umschließt und beherrscht uns, ein Reich der Kultur strebt darüber hinaus, aber es seelenloser Arbeit daher unser Glücksverlangen Kraft möchte unter den Gegensatz fällt und kleinmenschlicher Subjektivität und nicht, befriedigt gesteigerte Anspannung geistiger uns darüber erheben und uns einen sicheren Grund im eignen Wesen geben, aber dem Wunsche entspricht nicht das Vermögen, und der Versuch der Befreiung läßt noch Mühe und stärker empfinden. wandelt sich uns nicht in Sinnes zu entbehren; was Die unsägliche einen reinen Ertrag, unsere Bindung nur Arbeit ver- scheint alles ja sie der Erfahrung des Lebens besonders in schmerzlich berührt: innerlich Überlegenes von Niederem abhängig zu sehen, das scheint die Gesamtgestaltung unseres Daseins uns auf- Denn wohl zuerlegen. regt sich in ihm aufsteigende Leben dringt vor und neue Ausblicke tun sich auf. und Aufstrebende gelangt nicht das Kraft, Aber das Neue zur Selbständigkeit, es bleibt an eben das gebunden, worüber es hinaus will, oft wird es dadurch zurück- gezogen und in seinem Wirken gelähmt. bei Erfahrung dessen, lich gilt, ein trüber eben den tieferen Pessimismus Lebensbejahung reden, * Ist Wunder, wenn es ein namentlich v/enn diese Lage als unveränderSeelen der Lebensmut schwindet, und um sich greift? Wir hören ja heute viel von Hymnen ja auf das Leben singen, aber es Jenen Mangel an Selbstvertrauen, jenes Hoffen und Karren auf andere schildert vorirefflich ein Artikel im Spectator über English Pessimism (11. Aug. 1906); es heißt dort S. 190: we try to correct If we were to suggest the spirit which, our pessimism, would be most efficacious; increase in individual self-reliance. it when would be an We are not beaten in public affairs as we imagine we are, and there is no necessity in carrying out our works of philanthropy for relying so entirely upon associations. We establish far too many societies. Everybody seems too feel that before he can do anything he needs the protection of a crowd. He cannot even denounce or defend motor-cars unless hundreds will join him to protect him from the consequences of thinking independently. The result is that every one who wants to do something good devotes to it some fraction of Chip of his energy, and that the slrength which his mind, some little derive from the we would We develop some new is seldom or nfever present. and small group, not a Loyola or a Wesley. This, always the danger of democracy, is the danger also of the mental processes of our time, and deprives us first and foremost of all help from mdividual genius. strong will of a leader Der Wert des Lebens. gehört das zu den vielen, 385 innerlich hohlen Schaustücken der Zeit- oberfläche, es ist das eine erkünstelte Lebensbejahung, die dem Grunde der Seele fremd bleibt, es ist wie ein Rausch, der ein unbefriedigen- des Dasein zeitweilig vergessen machen soll. Ein derartiges Schein- wesen kann dem Pessimismus kaum etwas anhaben. Aber jede eingehendere Erwägung des Pessimismus zeigt, daß daß sich daher unmöglich er in sich selbst einen Widerspruch trägt, Ein wahrhaftiger Schmerz kann nur ent- mit ihm abschließen läßt wo es etwas wertvolles zu verlieren gibt; wo alles nichtig und stehen, gleichgültig wäre, da könnte auch die Versagung oder der Verlust in keiner Weise erregen. Christentum alte Das ausgehende Altertum und mit ihm das verfochten die wie z. sie schöpften B. erblinden daß das Böse keine Lehre, ständige Wirklichkeit, sondern nur die selb- Wegnahme eines Guten sei, nur könne, wer von Natur das Augenlicht besitzt; daraus die Überzeugung von einem sicheren Über- freilich über die Klippe ist nun hinwegzukommen, das Böse bedeutet wohl mehr als einen bloßen Mangel. Aber richtig ist, daß eine Empfindung, und gar eine starke Empfindung des Bösen ohne irgendwelches Gegengewicht wiegen des Guten. ^ So einfach nicht schlechterdings undenkbar wäre. »Wer anders findet sich darüber, nicht König zu sein, lich als ein meint Pascal gewiß mit Recht; würden gänglichkeit der z. unglück- entthronter König", so B. die Menschen die Ver- Dinge und den raschen Flug des Lebens so un- ablässig beklagen, wenn sie bloße Eintagsfliegen wären, nicht in ihnen irgend etwas wirkte, was in sich die Forderung ewiger Dauer trägt? So ist inmitten aller Bedrängnisse der Zeit die tiefe Empfindung dieser Bedrängnisse ein vollgültiges Zeugnis dafür, in jene Lage enthält, nicht ganz und gar was ihr Widerstand Befreiung von leistet. dem bloßen aufgeht, sein Wesen etwas Könnten wir so sehnlich eine Kulturgetriebe etwas an uns ihm überlegen wäre? daß daß der Mensch erstreben, Könnte uns in wenn nicht der Kultur der Mangel innerer Zusammenhänge und reiner Sachlichkeit so sehr schmerzen, wenn unsere Natur nicht solche verlangte? Könnte das tiefe Dunkel der Welt unserem Leben zur Einengung werden, wenn wir nicht müßten? * auf irgendwelchem Unmittelbar inneren Verhältnis zu ihr bestehen mag uns das alles an Positivem kaum etwas Mit besonderem Nachdruck hat Augustin jene Lehre vertreten, nament- dem enchiridion ad Laurentium de fide, spe et caritate. Nach seiner Bezeichnung ist das Böse nicht causa efficiens, sondern nur causa deficiens. Eucken, Grundbeg^riffe. 4. Aufl. 25 lich in Letzte Probleme. 386 wohl aber überzeugt liefern, daß die es uns, glatte Verneinung die Sache nicht erschöpft, daß jenseit ihrer manches zu fragen bleibt. wenn wir uns das Ganze des gegenwärtigen Lebens vor Augen stellen. Auch Aber wir dürfen noch einen wie es vorliegt, geht auf in das Bild, nicht es Schritt weiter grenzenlosen Ausdehnung des modernen Lebens gehen, das eine von der streben, enthält herrscht. Ein mehr, als Ge- überwältigte sinnung von ihm entwirft; schon das, was wir haben, nicht erst er- den Blick und die Schätzung be- dort Leben aus weltumspannender Persönlichkeit uns ist frommer und matter Wunsch; wohl können wir es erreichen, aber wir bemühen uns eifrig darum, wir suchen auf, was solches Streben verstärken könnte, wir möchten im besondern die großen Persönlichkeiten, welche das geschichtliche Leben bietet, uns näher rücken und mit ihnen das eigne Leben verknüpfen; mag das alles unvollkommen und unfertig sein, mehr als ein Einem Schlage nicht mit eine Bewegung zu jenem Ziele ist zweifellos im Gange. Noch deutlicher sind die Schranken der pessimistischen Be- trachtung gegenüber der Kultur. Es ist nicht richtig, daß uns heute nur der Mechanismus der Arbeit verbindet und uns ganz zu Stücken seines Räderwerkes macht; Streites und Werten halten wir besitzen inmitten auch eine gemeinsame Gedankenwelt wäre überhaupt nicht zu — ohne alles eine solche — uns umfängt mit geistigen In- gemeinsame Atmosphäre, wir sehen bei streiten eine und gar , näherer Betrachtung dessen eine innere Weiterbildung und Erhöhung des Menschen durch die Kultur; wir überzeugen uns, daß eine neue Stufe der Wirklichkeit aufsteigt, in ihr daß hier die Welt aus einem bloßen Neben- und Gegeneinander ein innerer Zusammenhang wird, und daß, was hier geschieht, über die Zwecke Menschen weit hinausreicht. So läßt sich des bloßen auch die Leistung der Gegenwart unmöglich verstehen vom kleinen Ich des Menschen her. Das gewaltige Vordringen der Wissenschaft und das unermüdliche Gestalten der Kunst ist nur begreiflich als das Werk von inneren Notwendigkeiten, die den Menschen packen und ins Schaffen treiben; mag kleinmenschliche Art dies Schaffen noch so umspinnen, seine Überlegenheit wird dadurch es mit dem praktischen nicht gebrochen. Wirken der Gegenwart. Nicht anders steht Eine Zeit, die an Erweisung humaner Gesinnung alle früheren Zeiten weit überflügelt, die zugleich das Recht jedes Einzelnen auf Entwicklung seiner geistigen Kraft und auf Teilnahme an den Lebensgütern bereitwillig anerkennt, Der Wert des Lebens. 387 Macht über die Gemüter gfewährt, die der sozialen Idee so viel Wir sehen dieses Große nicht klar, weil wir Egoismus beherrscht. die Erscheinungen einzelnen fassen, aber es nicht genug kräftig Ganzes ein in wir müssen es gewahren, sobald uns durch da, ist ist vom bloßen keineswegs ganz und gar odec auch nur überwiegend die trübe Oberfläche des Alltags hindurch die richtenden Grundlinien hervorscheinen. Die Anerkennung einer in der Menschheit aufsteigenden Geisteswelt verändert aber auch den Gesamtanblick des Alls und unsere Aufgabe ihm gegenüber. Nun bildet die Natur nicht mehr das Ganze Denn dar- der Wirklichkeit, nun gewinnt diese eine größere Tiefe. über kann kein Zweifel sein, daß, wenn eine solche Wendung zur Ganze von Haus aus mehr sein muß, als es dem ersten Anblick erscheint. Auch die Entwicklung nimmt sich Innerlichkeit statthat, das anders wenn aus, Natur ist, sondern das Geistesleben Erzeugnis kein bloßen der aus der Natur nur hervorgehen kann, weil es diese eine tiefere Wirklichkeit hinter sich hat; die engere Verbindung mit der Natur wird dann des Menschen nicht sowohl ihn herab- Wandlung der Grund- als die Natur erhöhen. anschauung muß auch der Arbeit neue Aufgaben Mensch mit seiner Geistigkeit drücken beschränktes Einzelwesen, tritt auch Solche kein sondern wirkt ein Weltleben Erkenntnisstreben sein stellen. auf einen bloßes, günstigere unter Ist der Sonderkreis in ihm, so Bedingungen. Denn nun läßt sich fragen, ob sich bei ihm nicht zwischen Echtgeistigem und Kleinmenschlichem scheiden läßt, und ob sich von jenem aus Brücke finden mag, die uns enger mit der nicht eine sie uns mehr zur Heimat macht. Doch solche Gedankengänge lassen sich hier nicht weiter ver- Welt verbindet und folgen. Hier galt es nur festzustellen, daß das des Weltbild Pessimismus nicht das Ganze der Wirklichkeit aufnimmt, sondern nur eine gewisse Durchsicht lage entspricht, bindet. Es einer Seelenlage, Freilich ist die einer eigentümlichen Seelen- die uns keineswegs endgültig mehr in unserer Wirklichkeit, als der Durch- steckt weit schnitt des Zeitlebens bietet, uns sehen nicht zu läßt. bestreiten, daß solches sammengefaßt und vollauf angeeignet werden muß, ständen gewachsen zu sein; Mehr um erst zu- den Wider- jenes aber kann nur geschehen, wenn es gelingt, wieder zu einer Selbstkonzentration des Lebens, damit zu einem ausgeprägteren Charakter und zugleich zu einem 25' aktiveren Letzte Probleme. 388 Verhalten zur Wirklichkeit zu gelangen.* Mit steckt in der Wirklichkeit; wir können Wendung einer bekannten Dürerschen Wortes könnten wir sagen: des «Die Vernunft wer sie herausreißt, der hat sie." Aber sie nicht herausreißen, bevor sich uns selbst das Leben zusammenschließt und sich damit unsere Organe inneren weiter- bilden. Es kann sich uns aber unmöglich das Positive in Leben und Wirklichkeit zusammenschließen, ohne daß sich uns das Dasein zerlegt, und Dunkel sich Licht des Menschen wie der schärfer scheiden, sich das ganze Leben Menschheit in eine durchgehende Aufgabe So verschwindet mit dieser Wendung die Unvernunft verwandelt. keineswegs, wohl aber gewinnen wir die Möglichkeit, zu entrinnen. Woher der ziehung des Höheren gültigkeit und werden überlegen ihrem damit Widerstand lähmenden was das, woher stamme, zum Niedern, woher des Weltlaufs gegen ihr innerlich Druck die zu Herab- die scheinbare Gleich- er selbst als Ziel hervor- zubringen scheint, das können wir Menschen unmöglich enträtseln; die Philosophie wie die Religion haben das Problem beim Versuch einer Lösung nur immer weiter verwickelt. So muß uns genügen und kann uns genügen, daß bedeutendes uns vorgeht, und daß wir nicht als tatenlose Zuschauer lassen müssen, sondern stellen und zu kommt das ihrer das bei Weltgeschick über uns ergehen daß wir uns auf die Seite der Vernunft Förderung zu wirken vermögen. zu Damit be- Wort von Vauvenargues ein gewisses Recht: «Die Welt ist, was sie für ein tätiges Wesen sein muß, voll von Hindernissen." Je mehr wir uns wieder einer vollen Lebenssynthese nähern, desto mehr läßt sich auch der Lebensmut wiedergewinnen, desto mehr wird uns das innere Gefüge des Lebens selbst einen sicheren Halt gegen die Unvernunft des Daseins gewähren. Wenn so unsere Zeit mit der Ergreifung tieferer Zusammenhänge der Wirklichkeit wieder zu einer positiven Schätzung des Daseins zurückkehren muß, so bleibt dabei aller Optimismus, alle Abschwächung des Dunkels fern; wir müssen im besondern eine nicht geringe Differenz zwischen der hier gewonnenen Lebensstimmung und derjenigen anerkennen, welche Literatur beherrschte. ^ die Höhe unserer klassischen Dort schien die Welt ein Reich ungetrübter Daß der von uns geforderte Aktivismus keineswegs mit der Wendung zur praktischen Vernunft oder gar zur moralischen Betätigung zusammenfällt, kam schon oben zur Sprache. Der Wert des Lebens. 339 Vernunft zu bilden, der Mensch aber die Höhe seines Lebens in der künstlerischen Anschauung oder im denkenden Begreifen der Welt- harmonie zu finden; die Hauptaufgabe der Menschheit war voller Bewußtheit zu bringen, allen Seiten umfängt. Uns Neueren haben sich im menschlichen Leben die Probleme daß wir so rasch heraustreten dürften. hier, viel in der Natur wie zu sehr verschärft, mehr als wagen und aus dem Kampf Aber wenn wir mit solcher Erschwerung des einen Abschluß Daseins vieles verloren haben, wir haben eines gewonnen, das Verluste zu was uns mit unbewußtem Wirken von als aufwiegt: wir alle dürfen selbst zur Förderung des Ganzen wirken, weit mehr sind wir aus Zuschauern zu Mitarbeitern am großen All geworden. Das Problem der Religion. 2. (Immanenz — Transzendenz.) Immanenz und Transzen- |ie Behandlung des Gegensatzes von I *-^ denz könnte zur Aufrollung des gesamten religiösen Problemes um so weniger, als wir uns über dies neuerdings führen; sie soll es wiederholt geäußert haben ;i so tümliche Verhalten hier sei nur das der Gegenwart eigen- diesem Problem zu Die jetzt Auch übliche Zusammenstellung von immanent und transBis dahin zendent reicht nicht hinter Kant zurück.^ ander immanens 13. (auch sofern sie transeunt, sofern sie das berühmte ^ S. standen ein- permanens) und transiens gegenüber; Jahrhundert nannte immanent, ist erörtert. Zur Geschichte der Ausdrücke. a) dem Kürze in mag das Wort uns zur Sache leiten. man seit eine Handlung oder eine Ursache des wirkenden Subjekts verbleibt, innerhalb darüber hinaus auf etwas anderes geht.^ So Wort Spinozas zu verstehen, daß Gott die imma- „Der Wahrheitsgehalt der Religion", 2. Aufl. 1905, »Hauptprobleme der Religionsphilosophie der Gegenwart", 1907. S. z. B. III, 245 (Hart.): „Wir wollen die Grundsätze, deren Anwendung sich ganz und gar in den Schranken möglicher Erfahrung hält, imma"^ nente, diejenigen aber, welche diese Grenzen überfliegen sollten, transzendente Grundsätze nennen." So unterscheidet z. B. Thomas v. Aquino eine actio manens und actio s. Thomaslexikon von Schütz unter actio: duplex est actio, una quae transit in exteriorem materiam, ut calefacere et secare, alia, quae manet in agente, ut intelligere, sentire et velle. Das erstreckt sich bis in die Neuzeit. Clauberg (op. omn. 1691, S. 322) gibt der Sache die Fassung: si ipsius rei, quae dicitur agere, Status mutetur, est actio immanens, sin alterius, est actio transiens. Es stammt aber diese Unterscheidung, wie der gesamte Grundstock der scholastischen Terminologie, von Aristoteles. S. z. B. Met. 1050a, 24: ^ transiens; TtTv [i£v eoyaTov £T£pov ot/.t'a aKo T^; napa x;^v r\ y^r'jii, o'ieo)«; oTov epyov, oiy.oo6[xri^v/. oJ/ew? aTi'Evi'wv ri opaat;, 8e xa\ Yt^vexai ouO'kv Tt, o!ov yiYvexat axo xf? jrapa Tau"n)v o?xo5o[itxf? In soIcher Unterscheidung eines Tuns, das gegen Das Problem der Religion. aber nicht nente, die daß Gott nicht aus Dingen wirkt, Welt also in der Welt als aller sich selbst heraustritt, sondern daß er dabei bei sich Dinge Es sei.^ wenn er zu den selbst verbleibt, die Demnach ist hier nicht sowohl Gott in Neu .gegenüber der Scholastik ist die Welt in Gott. sich nur die dabei Wirken Ursache transeunte bedeutet, 39I neben trägt. Ausschließiichkeit der Immanenz, dem hat auch jene bereitwillig transeunten ein immanentes aner- - Einen anderen Ausgangspunkt hat transzendent und trans- kannt zendental. Transzendent hießen (transzendentia) des Mittelalters die allgemeinsten Hälfte in Eigenschaften der zweiten der Dinge, welche nach neuplatonischer Lehre jenseit der einzelnen Kategorien liegen. 2 als Von aus ergab sich hier leicht eine Beziehung auf Gott das allen menschlichen Begriffen überlegene Sein; so wirkt der Sprachgebrauch auch bis in die Neuzeit.^ Kant hat dann transzendent und transzendental geschieden und unter Umprägung der Bedeutung zu "V^erkzeugen seiner eigentümlichen Denkweise gemacht.* sich selbst und richtet begründet sich das deutliche Auseinandertreten von praktischer ist, eines anderen, das auf die Hervorbringung eines Werkes ge- und künstlerischer Wirksamkeit. Für den Ausdruck Immanenz sei noch die von Heman (Kantstudien VIII, 1, S. 58) angeführte Stelle des Augustin erwähnt (epist. 268 ad Nebr.): In se habeat haec tria et prae se gerat, primo ut sit, deinde ut hoc vel illud sit, tertio, ut in eo quod est maneat, quantum potest. Primum illud causam ipsam naturae ostentat, ex qua sunt omnia. Alterum speciem, per quam fabricantur et quodammodo formantur omnia. Tertium manentiam quandam, ut ita dicam, in qua omnia sunt. ^ Ethic. pars non vero transiens. prop. XVIII: deus est I, omnium rerum causa immanens, Begründung heißt es: om.nia quae sunt in deo sunt et per deum concipi debent, adeoque deus rerum, quae in ipso sunt, In der est causa. ' Grund der Schrift de causis zuerst die vier Beunum, verum, bonum, später außerdem res und aliquid; so sprach Als solche gelten auf griffe ens, man von einer unitas, veritas transcendentalis u. s. w. ' S. z. B. Bayle oeuv div. (Haag 1727) III, 871a: Si l'Origeniste repond que les vertus de Dieu sont transcendentelles, qu'elles ne peuvent point etre dans la meme categorie que Celles de l'homme. Transzendental im älteren Sinne verwenden noch Ch. Wolff und Lessing. Lambert nennt transzendente Begriffe solche, die „das Allgemeine der Körper- und Geisterwelt zusammen- nehmen". * Über transzendent s. 0., über transzendental sagt er (Kritik d. r. V. III, 49) »Ich nenne alle Erkenntnis transzendental, die sich nicht sowohl mit Gegen- ständen, sondern mit a priori möglich sein unserer soll, Erkenntnis von Gegenständen überhaupt beschäftigt." sofern diese Letzte Probleme, 392 Die b) Bewegung der Neuzeit zur Immanenz. Der Gesamtverlauf der Neuzeit zeigt einen Zug zur Immanenz, Eigentümlichkeit dessen durch eine Vergleichung der mit Haupt- bewegung der griechischen Kultur besonders deutlich erhellt. Das Griechentum wurde durch die Erfahrungen seiner Arbeit immer Von der Außenwelt, weiter über die sinnliche Welt hinausgetrieben. von der die Forschung begann, schrittweise religiösen zurück in verlegte sich ihr der Innenwelt, die bis der in Gestaltung der Wirklichkeit bei Plotin die nächste Welt zum bloßen Gleichnis einer unsichtbaren wurde. religiösen Überzeugung das Jenseits als Die Neuzeit ver- Galt im Mittelalter der gerade entgegengesetzte Richtung. folgt die Schwerpunkt abschließenden das wahre Vaterland und gab nur die Beziehung darauf dem Diesseits einen Wert, so beginnt dem Verlangen, Neuzeit mit die das Wirken des Göttlichen mehr und innerhalb der Welt aufzusuchen, ja diese als einen Ausdruck Abglanz des göttlichen Wesens zu verstehen. Das ergibt zunächst einen Panentheismus, naissance. das Bekenntnis der edelsten Geister der Re- Bald aber verschiebt sich das weiter dahin, daß mehr und mehr die Welt zur Hauptsache wird, daß die Gottesidee mehr dahin wirkt, ihr eine größere Tiefe zu geben lichkeit So der Pantheismus zu eröffnen. und eines Spinoza. ratur überwältigend angezogen, indem er als eine eines neue Wirk- Giordano Bruno Er hat die klassische Zeit der deutschen brücken, im besondern die weiteste und alle Lite- Gegensätze zu über- freieste Behandlung der sichtbaren Welt mit aufrichtiger Anerkennung einer unsichtbaren zu verbinden versprach. Solche pantheistische Denkweise ist auch im wo dies seine eigentümda neigt es weit mehr, wenn nicht zum 19. Jahrhundert keineswegs erloschen; aber Art vollauf liche entfaltet, Atheismus, so doch zu einem Agnostizismus,^ einer Ablehnung transzendenten Fragen als schlechterdings unlösbarer aller Probleme. Über den Ursprung des Ausdrucks berichtet genau R. Flint in seinem Der Schöpfer des Wortes agnostic, aus dem dann bald agnosticism hervorging, ist Huxley. „According to Mr. R. S. Hutton this latter word (d. h. agnostic) was suggested by Professor * hervorragenden Werke Agnosticism (1903). Huxley, at a party held previous to the at in now defunct Metaphysical Society, James Knowles's house on Clapham Common, one evening in 1869, my Hearing. He took it from St. Paul 's mention of the altar to ,the iVlr. unknown God'" (s. Hint, S. Iff.). Das Problem der Religion. Praktisch ergibt das Religion aus dem wie eine eine Ausscheidung das andere der War also das Göttliche zuerst unserem Leben. Dasein angenähert, dann 393 beseelende Kraft ihm aufs engste ver- als bunden, so verschwindet es schließlich gänzlich oder wird zu einer unzugänglichen Ferne; so aus ist die Religion dem modernen Menschen Macht zu einer allesbeherrschenden der Nebensache, ja zu bloßen Illusion gesunken, und es hat die unmittelbar gegen- einer immer wärtige Welt Sinnen und Denken an ausschließlicher alles und fehlt es nicht an Widerständen gegen eine solche Bewegung, schon deshalb nicht, weil jede frühere Phase sich gegen die späteren behauptet; aufgehalten aber haben Natürlich fehlte sich gezogen. sie jene nicht. So tiefgehende Wandlungen bloß dem Unglauben und bösen Willen der Individuen schuldgeben kann nur eine flache Denkweise; sicherlich hatte die Sache tiefere Gründe in den allgemeinen Verhält- Gründe, die unbefangen gewürdigt sein wollen. — Die ältere nissen, Art der Religion stieß zunächst mit einem wesentlich veränderten Lebensgefühle der Menschheit hart zusammen. wo Lebensmut, aller Glaube an aller Sie entsprach einer Zeit, irdische eine Zukunft ge- wo man zur Religion seine Zuflucht nahm, um Nun aber hatten Frieden und Sicherheit zu finden. brochen war, und Ruhe, dort lange Jahrhunderte bei jugendfrischen Völkern einen neuen Lebens- mut erzeugt; ein solcher wollte nicht sowohl nicht sowohl ein sicheres Geborgensein Ruhe als Betätigung, Gefahr und Wagnis, als Kampf; er konnte die Welt nicht verschmähen, ihn in sie hinein, Zu steigern. solcher Wandlung der Grundstimmung kamen entsprach und zu einer Arbeit, wirkte. Nach den verschiedensten Richtungen hin tiefer sich die ihr die ihrer Verstärkung Erfolge nächste Welt trieb es mächtig um seine Kraft an ihr zu erproben und durch sie zu ist die sinnlich- dem Menschen bedeutender geworden, hat sie ihm sich und zugleich einen engeren Zusammenhang bei erschlossen selbst gefunden, hat sie sein größeren Leistungen geführt. allgemeinen Gesetzen und in Handeln stärker bewegt und zu Die Wissenschaft zeigt die Natur unter festen Zusammenhängen, sie entfernt auch aus der Geschichte das Wunderbare und versteht sie aus ihren eignen Verkettungen. Das gesellschaftliche Zusammensein der Menschheit nimmt mehr geistige Aufgaben an sich und sucht mit Ein- setzung gewaltiger Kraft aus unserm Dasein ein Reich der Vernunft zu machen. Alles zusammen hat diese Welt mehr als je zuvor dem Letzte Probleme. 394 Menschen auch zur Heimat gemacht. geistig Sonderstellung seine aufs ärgste nämlich die Welt wird, ständiger und Walten eigne Gesetze Zugleich aber wird bedroht. Je mehr sie je erweist, desto kleiner, der wird ihr gegenüber der Mensch. größer durch und selb- Wirken alles desto verschwinden- Bei solcher Kleinheit können unmöglich die ihm eigentümlichen Größen die Wirklichkeit fassen und seelisch Annäherung nahebringen. innerlich hältnis zu ihren Rückt Welt die bei äußeren aller so entfällt alles innere Ver- Gründen, so droht alle Religion ein bloßer Anthro- pomorphismus zu werden wo so Ferne, weite in die Religion und zur Mythologie zu sinken. rückt sie leicht aus behauptet, sich Auch dem Zentrum des Lebens in seine Peripherie, und wird sie aus einer natürlichen, beinahe selbstverständlichen Überzeugung zu einer kühnen, So haltbaren Behauptung. ist kein es Wunder, daß derer, die alles Überschreiten der Erfahrung verwerfen «immanent" fassen wollen, gaben sich immer lauter die mühsam Stimmen und alle Auferheben und wachsendes Echo finden; nie dürfte die Verneinung der Religion ein so in die Massen gedrungen sein, gewaltet haben, mehr eine wie ist Mag der eine in ihr Hemmung klarer Einsicht, der andere eine Lähmung der Unterdrückung freudigen Lebensgefühls Tatkraft, der dritte eine blicken: nie so viel Antipathie gegen sie der Fall heute es er- dünkt sie ein verderblicher Wahn, der mit hier wie dort das der endgültige Abschluß des allen Kräften auszutreiben alten Problems, oder ist es eine bloße Woge der Zeit, die vorbeigehen sei. Ist und vielleicht das gerade Gegenteil der eignen Absicht bewirken wird? c) Die Verwicklungen im Begriffe der Immanenz. Ihre Stärke hat die im Angriff; sobald aus das Leben gestalten wicklungen. Bewegung gegen die Religion vornehmlich eignes Vermögen zeigen und von sich ihr sie soll, erscheinen Verwicklungen Erschreckend dürftig pflegt zu sein, über Ver- was als Ersatz der und selbst dies Dürftige ist zum guten Teil fremdem Boden gewachsen und von dort aus zugeführt. Die Religion geboten wird, auf «immanente" Lebensführung und Weltanschauung pflegt keineswegs aus reiner Erfahrung zu schöpfen, sondern sie un- idealisiert diese vermerkt, sie mischt ihr etwas bei, was einer ganz anderen, nämlich der pantheistischen Denkweise angehört; ein Pantheismus verblaßter Gestalt als hat sich der selbstverständlich. einzelnen Gebiete bemächtigt und Er pflegt dabei kein gilt dort offenes Bekenntnis zu Das Problem der Religion. 395 wagen, sondern seine Erhöhung der Dinge eher zu verstecken, er ist mit solcher Unklarheit gegenüber dem Pantheismus eines Spinoza und eines Goethe und ein unechter schlechter Pantheismus. Pantheismus erscheint schlechter in sophie, welche unbedenklich die Natur beseelt Wertbegriff behandelt; erscheint er Ein solcher monistischen Naturphilo- einer und als einen hohen einer Geschichtsphilosophie, in welche bloße Massenbewegungen Vernunft erzeugen und eine läßt Evolution zur Vernunft verkündet, obwohl dieser Begriff in ihrer Ge- dankenwelt keinerlei Grundlage hat; er erscheint groß behandeln. politischsozialen in Bewegungen, die den Menschen wie er leibt und und lebt als edel Überall ein verstecktes Idealisieren der Erfahrung, zugleich aber ein Abschleifen der Gegensätze, ein Verkümmern der eigentümlich geistigen Art, ein Einschläfern aller Selbsttätigkeit. Auch nicht so wissenschaftlich angesehen wie er sich zu geben einfach nächste Wirklichkeit, die uns ganz wirklich an uns selbst? einander, wenn Ist völlig er Was pflegt. soll, die was ist Befund des Neben- wird? herausgestellt denn ist und gar einnehmen es der unmittelbare rein Immanenz der Begriff der ist Dann müßten wir uns in einen Haufen, ein Bündel einzelner Empfindungen auflösen; das aber geht aus dem einfachen Grunde nicht, weil es freischwebende Empfindungen überhaupt ungen eines Ich, nicht gibt, sondern immer nur Empfind- meine und deine Empfindungen, nicht Empfind- ungen an sich. So werden wir von der Empfindung immer wieder auf eine zusammenhaltende Einheit gewiesen, erscheint es eignen Bereich ein Gegensatz, und es wird zur Frage, des Lebens erscheint liegt. bei uns Reicht aber selbst eine in unserem wo der Kern das Problem so weit zurück, Abstufung, so erhellt deutlich, und wie wenig mit dem Schlagwort der Immanenz gewonnen wird. Beim Recht der religiösen Problem im besondern geht gewiß mit gutem Zug der Neuzeit gegen die mittelalterliche Transzendenz mit ihrer Verdopplung der nächsten Welt, aber damit ist keineswegs entschieden, daß unser ganzes Leben eine einzige Fläche bildet. Es notwendig werden, Um- könnten Abstufungen kehrung dahin erfolgen sichere in müssen, daß, es ja was Boden unseres Lebens und Wirkens könnte eine als der selbst einen Halt uns zunächst gilt, einer tiefer gegründeten Welt erst zu suchen hätte. die Wirklichkeit, die unser ganzes Leben Was ist denn und Streben umfassen soll? Die Welt des unmittelbaren Sinneseindrucks dafür zu erklären, das hieße den großen Vorkämpfern der Im.manenz, einem Spinoza und Letzte Probleme. 396 einem Goethe, schroff widersprechen, das hieße die seeHsche Tiefe der gesamten modernen Kultur verkennen. Die Anerkennung einer von geistigem Leben getragenen WirkHchkeit aber erzeugt sofort die Frage, ob jenes den ganzen Umkreis unmittelbar an es nicht windung, ja Bekämpfung es erst nach Hilfe weiterer Zusammenhänge ob sich zieht, Hemmungen stößt, deren Über- draußen und drinnen auf weiterer Kräftigung unternehmen kann. und mit Namentlich ist es die Tatsache der vielfachen Unvernunft im Natur- und Menschenleben, an der jedes System ausschließlich immanenter Vernunft mit seinem Denn hier bleibt die Unvernunft wegzudeuten und abzuschwächen, aus den Augen zu oder sie Pantheismus scheitert. rücken, Wirklichkeit anzuerkennen als nur die Wahl, ein sie entweder möglichst Grundelement der und damit für unangreifbar zu erklären. Entweder also eine Tendenz zum Optimismus mit seiner verflachen- den Art, oder zum Pessimismus mit seiner Verneinung und schließ- demnach die Dinge nicht, wie das Verlangen nach Immanenz es darstellt. Hüten wir uns, ein Weltbild deshalb für wahr zu erklären, weil es sich unserer Vorstellung als das glatteste und bequemste empfiehlt. Denn was anderes So einfach lichen Verzweiflung. liegen wäre das als eine neue Art von Anthropomorphismus, die das menschliche d) Wollen und Wünschen zum Maßstab der Wirklichkeit macht? Das Wiedererwachen des religiösen Problems. So erscheinen arge Verwicklungen beim Versuch eines Aufbaues des Lebens ohne alle Religion. Aber das würde eine derartige Be- wegung noch keineswegs hemmen; sehr viel Unklarheit und Widerspruch läßt sich ertragen, wenn der Zug des Lebens kräftig und selbstbewußt Problems Nun aber ist ein Wiederaufsteigen des religiösen ist. inmitten aller leidenschaftlichen Befehdung der Religion heute nicht zu verkennen; das Vordringen der Verneinung in immer breitere Massen hindert nicht, daß auf der Höhe des Geisteslebens die Religion wieder weit schaften erregt; es ist mehr das Denken einmal so, daß beschäftigt und Leiden- in derselben Zeit verschiedene Strömungen durch- und gegeneinandergehen können, und daß dabei der Unterstrom dem Zuge der Oberfläche direkt widersprechen mag. Um aber der Tatsache eines Wiederaufsteigens der Religion gewiß zu werden, brauchen wir nur unsere Zeit mit der unserer Klassiker zu vergleichen; dort war die Religion mehr eine freundliche säumung des Lebens, heute ist sie in Um- seinen Mittelpunkt getreten, Das Problem der Religion. 397 entzweit sie die Menschen bis zu härtestem Kampf, fließt sie in die Behandlung aller gewaltige eine Angelegenheiten ein, übt Denn auch die Verneinung Kraft. der Art, daß die Religion als heute nicht ist etwas welkes und greisenhaftes ruhig geschoben würde, sondern die stürmische Leidenschaft des bei Seite genug als etwas noch sehr Angriffs zeigt sie deutlich und Wirksames. tiges im Ja wie im Nein sie Vielleicht besagt sogar die Reales, Kräf- Verneinung selbst oft weniger eine völlige Ablehnung der Religion, als sie ein Verlangen nach einer anderen, einfacheren, den Bedürfnissen der Gegenwart an- gemesseneren Art der Religion bekundet. Jedenfalls erscheint danach die Religion nicht wie ein mattes Licht, das langsam und still verglimmt. Was mag diese es Umschlag herbeigeführt hat? das diesen sein, war er die Schwerlich von Frucht apologetischer vorwiegend auf solche zu wirken, pflegt Denn Arbelt. welche schon ge- wonnen sind; sie mag befestigen und zusammenhalten, vorzudringen ist nicht ihre Art. In Wahrheit ist es ein Rückschlag im modernen Lebeh selbst, in dem die Bewegung wurzelt; eben indem jenes Leben mit seiner Weltfreudigkeit sich frei entfalten und sein volles Vermögen einsetzen konnte, sind seine Schranken, ja ist seine Ohnmacht gegenüber den erfahren wir welche durch Sichausleben zwingend Dasein letzten die Verneinung des Gegenteils, und geleistet wurde, dingungen vertieft; einmal das ungehemmte einer Behauptung Die Richtung des Lebens auf das unmittelbare Aberglauben ausgetrieben, das ist in viel sonst mannigfachster Weise Aber was immer in dieser Richtung weitergebildet. vorwiegend peripherer Art, es hat die Be- unseres Lebens verbessert, nicht aber das Leben selbst aus aller unsäglichen Arbeit steigt daher erscheinen. Zusammenhänge muß und damit innere Leere auf, länglich wieder Weltgeschichte, durch schlummernde Kraft erweckt, jenes Dasein gefördert der Unerläßlichkeit hindurch, die Wahn und viel geworden; deutlich Beweisführung indirekte erweist. hat Fragen jene Die Ablehnung gestaltete die alles jenes aller Kultur schließlich eine Bemühen als unzu- und jeder unsichtbaren mehr und mehr zu einer bloßen Menschenkultur; das mochte so lange keinen Anstoß erregen, als das Menschsein selbst klärter Gestalt ^ So machte umfassenden Ideal. als als ein gesehen wurde.^ Er selbst ist." hoher Idealbegriff galt und in verDas aber geschah unter dem Ein- vornehmster Weise Herder die „Humanität" zum all„Der Mensch hat kein edleres Wort für seine Bestimmung, in Letzte Probleme. 398 Denkweise, die fluß derselben lichkeit abgelehnt Verklärung wird; jetzt als muß so der Mensch fallen, eine Verfälschung der Wirk- mit ihrem Schwinden auch jene seiner natürlichen in ohne Hülle erscheinen und zum einzigen Maß Beschaffenheit Wahren und Nun hat aber eben das moderne Leben mit seiner Guten werden. Entfesselung alles so Kräfte aller Niederes aufgewühlt, es stellt Unerquickliches, viel Trübes und uns das Kleine und Scheinhafte einer vor Augen, daß die Hoffnung immer geringer wird, von hier aus einen zusagenden Lebensstand erreichen und dem menschlichen Dasein einen Sinn und Wert verleihen zu können. Immer mehr greift die Empfindung um sich, daß im Menschen etwas steckt, was durch jenes immanente Kulturleben nicht belebt wird, und daß jenes Verkümmerte etwas Unentbehr- bloßen liches, Menschenkultur so das Beste von allem vielleicht So erwächst Menschen, nach festhält und einmal wieder sichtlich von von Befreiung einer niederdrückt; ist. nach einer inneren Wandlung des ein Verlangen der neue Zeit eine kleinen Art, gekommen, ist die ihn. es treibt bloßen Menschenkultur zu einer wesen- einer erhöhenden und umbildenden Geisteskultur; das aber führt mit Not- Forderung einer neuen Wirklichkeit und damit auf wendigkeit zur Weg der Religion. den Zunächst freilich ergibt es eine höchst verworrene Lage. Während von innen her eine neue Art des Lebens und Seins verlangt wird, hält unser Verstand und unsere Arbeit uns beim unmittelbaren Da- sein fest; wir möchten etwas Höheres, aber wir finden keinen Weg dahin und können doch vom Ziele nicht lassen. vom hin- einen zum andern So werden wir und hergeworfen und widersprechen unablässig uns selbst. Aber bei aller Unfertigkeit und allem Unbehagen ist wenigstens das eine gewonnen, daß wir aus Besitz wieder gekommen in ein Suchen, sind, daß die ein alten redliches und ewigen dem vermeintlichen und eifriges Fragen mit Suchen frischer Kraft wieder aufsteigen. Wie sich das freilich weitergestalten wird, an Bedingungen des Menschen und des Ge- das liegt vielfachsten schicks; darüber e) kann erst die Zukunft entscheiden. Forderungen für die gegenwärtige Lage der Religion. Beim Überblick der gegenwärtigen Lage der Religion bemerkenswert besonders die Tatsache ins fällt als Auge, daß zwischen der Das Problem der Religion. 399 überkommenen kirchlichen Form der Religion und einer univerBewegung zur Religion aus dem eignen Streben der Zeit eine schroffe Spannung besteht; es gibt heute viele, welche religiös, saleren aber durchaus nicht kirchlich sein wollen, von der Kirche abgestoßen Den als welche sich ebenso sehr von der Religion angezogen fühlen. Grund dessen mag man darin nächsten suchen, daß zwischen der überkommenen Form des Christentums und dem Kulturleben der Gegenwart eine weite Kluft besteht, aufs Äußerste die ein gegenseitiges Verständnis Das Weltbild erschwert. hat wesentlich sich ver- und Außermenschliche verschoben, die ältere Gefühlslage erscheint dem modernen Menschen als zu weich und zu matt, die Zeit stellt ihm neue praktische Aufgaben, die seine ändert, namentlich ins Große ganze Kraft verlangen; während das alte Christentum einer ermüdeten und eingeschüchterten Menschheit neue mut zuzuführen suchte, und starken Lebensdranges dieses, ist das Leben in Die Haupt- tun. dem Widerspruch seine Schärfe daß sich der Zeit nicht mehr aus eigner Erfahrung eine Frage zusammenfaßt, deren Beantwortung bildet: Vor all dem Wirken und Schaffen frischem Menschheit verblaßt; wo sprünglichkeit da fehlt, in Kraftgefühl der moralischen Frage die in Befreiung und der inneren vor jugendlich frischen Lebens- die den Kern des Christentums Rettung, und Wirkens zu rastlosen sache aber und was vornehmlich gibt, Kraft hat jetzt die Religion mit einer Menschheit Erneuerung der Menschheit. die sichtbare Welt hinein und ist jene Frage der modernen aber der Frage die volle Kraft und Ur- muß auch die Antwort die Herzen gleich- gültig finden, da verdunkelt sich das Recht und die Notwendigkeit der Sache, da wird hingegen die Schätzung voUkommnem enthält. die Abhängigkeit gewährte Hilfe der viel zu entfremden. die alles grell was beherrschen, die in die Augen fallen und überlieferte Endlich hat wohl auch bei Kirche vom Staate und leicht Fassung an Ununs Deutschen die ihr vom Staate dazu beigetragen, jener die Gemüter innerlich Denn den anderen germanischen Völkern bei ist Entfremdung anscheinend nicht so groß. So wegung ist es ihre älteren Art begreiflich, eignen Bahnen Vor allem sucht. religiöse erstrebt sie Beder gegenüber, die ihr zu eng und gebunden dünkt, eine größere Weite, für Weltumgebung; die daß die neu aufsteigende eine möglichste sie sorgt Universalität, sich lungen im eignen Innern des Menschen weniger als mehr um die Offenheit Verwick- um sein Verhältnis zum Letzte Probleme. 400 All; dieses keit ihn möchte ihm sie innerlich naherücken, seine Unendlich- miterleben, seine Schönheit ihn genießen lassen; in solcher Stimmung künstlerischen scheint von Befreiung eine sich allem Kleinmenschlichen zu vollziehen und die Seele im reinen Äther des Alls sicher und zu schweben. selig Solches Streben gegen ein Sicheinspinnen des Menschen in die bloße Menschlichkeit, solches Verlangen zum der Religion und in wesentliche Seite deutende Wirkungen hier aufstrebt, die hat bedeutet eine Geschichte be- All ihrer Aber eine andere Frage geübt. ist, ob, was Gesamtaufgabe der Religion zu lösen und damit alle geschichtliche Gestalt zu verdrängen und ersetzen vermag. Wird nämlich das Neue streng auf sein eignes Vermögen beschränkt und nicht stillschweigend durch das von der geschichtlichen Religion ge- botene Leben in mannigfachster Weise ergänzt, so kann und Freiheit Diese Art und Leere starke Vagheit eine dringt über feine der Religion nicht vor zu einer echten Tatsächlichkeit; neue Welt zu eröffnen, eine stellt sie Weite zarte Stimmungen dem Menschen eine ihm nur die vorhandene Welt in oder Beleuchtung, liebenswürdigere und statt alle wohl verdecken. nicht umsäumt sein sie Leben mit gefälligen Stimmungen, welche Mußestunden angenehm ausfüllen mögen, die aber gegenüber dem Ernst des Lebens in kläglicher Weise versagen. Denn nun und nimmer wird damit der erreicht, Seele werden Kräfte Schuld befreien, wird Menschheit durch sammengeführt. entbunden, eine Weiterbildung die von Not und dem Leben ein fester Halt geboten, wird die eine bei Schöne sich Bilder, selbst befindliche Innenwelt zu- schöne Aussichten, aber Bilder, die den bloßen Entwurf nicht überschreiten können ! Die hier erwachsende ästhetisch -pantheistische Stimmung mag Vorbereitungen liefern, der Hauptaufgabe der Religion gewachsen; was sie an Wahrheit und festerem verbinden, enthält, wertvolle das Anregungen und ist sie nicht muß sich mit anderem um zur Förderung zu wirken. Aber so wenig jene neue Art der Religion befriedigen kann, gegen die kirchliche Form, es bleibt die Tatsache des Widerspruchs es bleibt ein Spalt im unabweisbar wird damit Überwindung religiösen die arbeiten läßt. wieder zusammenfinden, so entgegenbringen, so Leben und Streben der Gegenwart; Frage, ob und wie Sollen sich die Zeit sich und zu seiner die Religion muß die Zeit der Religion eine Frage muß diese sie aber in einer Weise beantworten, welche anzunehmen der Zeit nicht unmöglich ist; dazu aber wird Das Problem der Religion. beiden auf es 401 Wandlungen oder doch Weiter- erheblicher Seiten Nur dann wird das Verlangen der Zeit nach Religion wieder stark und überwältigend werden, wenn jene im menschlichen Leben große innere Verwicklungen erkennt und zum eignen Erlebnis macht, und wenn sie zugleich die Spitze dieser bildungen bedürfen. Verwicklungen im moralischen Probleme Aber andererseits findet. muß auch die Religion das moralische Problem nicht nach der Enge des unmittelbaren Eindrucks, sondern den Gipfel einer allum- als fassenden Bewegung verstehen und behandeln; damit wird sie selbst Grundlage gewinnen und der eine breitere sonst unvermeidlich die ihr die Religion einen sicheren der Zeit gewonnen, und und klar ist sicher geworden, kommenen Bestand Kontakt sie so mit dabei kann dem ihrer sie innersten Streben eignen Grundtatsache ohne Gefahr ihren über- Prüfung daraufhin einer entgehen, Partikularität Hat an diesem Zentralpunkt anhaftet. was unterziehen, in ihm wesentlich und unwandelbar, was aber nebensächlich und den Wa'ndlungen der Zeit unterworfen ist. Vor allem muß die Religion verschanzen. nicht Wesen besinnen und auf ihr eignes kräftig sich Ihrer intellektuelle letzten Absicht nach Aufklärungen sich fest in bringt sie über die Welt, ihm dem Menschen erweckt auch sie nicht bloß neue Gefühle oder stellt neue praktische Aufgaben, sondern eröffnet aus sie Seins, ja zur einem direkten Verhältnis zum beherrschenden neue Welt; eine erster Stelle sie Urkraft führt den des tiefsten Lebens ein Grunde des neues Leben, Beweis dieses neuen Lebens an durch die Tatsächlichkeit seiner weltgeschichtlichen Ent- wicklung, durch die Umgestaltung der Wirklichkeit, die unablässig von ihm ausgeht; darauf freilich muß botene Leben als die sie beherrschende Seele dringen, daß das hier gealles Lebens, als die un- erläßliche Bedingung aller Geistigkeit anerkannt wird. dies Leben faltung auf kann seine Ent- ist, dem Boden der Menschheit steht unter den Bedingungen der Zeit und Geschichte; es es Aber so sehr seinem Grunde nach überzeitlicher Art das, ohne sich muß sich mit ihnen auseinandersetzen, selbst zu verlieren, nur, wenn bei ihm zwischen Substanz und Daseinsform deutlich geschieden wird; denn dann wird eine Unwandelbarkeit der Substanz mit einer geschichtlichen Bewegung der Daseinsform Hier nun liegt eine besonders wichtige und schwierige Aufgabe der Gegenwart, es gilt eine der weltgeschichtlichen vereinbar. Lage des Geisteslebens bloßen Strömungen der Zeitoberfläche Eucken, Grundbegriffe. 4. Aufl. — nicht den - entsprechende Daseinsform 26 Letzte Probleme. 402 der Religion zu gewinnen, ohne darüber ihre Substanz zu verlieren oder auch nur irgend abzuschwächen.^ Diese Verständigung zwischen Christentum und Neuzeit manchen Vor allem ist aber dazu nötig, Anerkennung und Würdigung finde. erscheint. minder daß der große Wandel der Zeiten mit allem, was in ihm an inneren leicht, als Notwendigkeiten In der sie liegt, volle gewöhnlichen Apologetik geschieht das nicht ist Sie faßlr nicht die Sache ins Ganze, sondern sje verbleibt bei zerstreuten Punkten, sie sich versetzt nicht in den anderen hinein, sondern behandelt ihn gänzlich von außen her, sie operiert mit bloßen Möglichkeiten, indem sie Wege offen zeigt, daß die moderne Bewegung noch immer gewisse lasse, die gutem Willen wohl bei mit dem kirch- immer mehr ins die Gefahr einer inneren Unwahrsie kommt in hinein, ja Künstliche lichen Glauben vereinbar haftigkeit; sie gerät damit man mag hier des Hume'schen Wortes von der Vergeb- lichkeit eines mit seien, Unternehmens gedenken, einen Durchbruch des Ozeans Strohwischen verstopfen zu Auf diesem Wege wird wollen. nun und nimmer die Religion die erstrebte Stellung im Ganzen desLebens wiedererringen, nun und nimmer kann sie so die schlichte Einfalt, die seelische Nähe, die sichere Überzeugungskraft erlangen, ohne die ihre Aufgabe sich nicht Religion in der Gegenwart ist lösen läßt. Ein Unsicherwerden der nicht zu verkennen und sei nicht ver- Der christliche Lebenstypus ist heute der Menschheit keineswegs mit solcher überzeugenden Nähe gegenwärtig, daß ihn sowohl jeder einzelne unmittelbar ergreifen und erfahren, als von ihm die dunkelt. Gesamtgestaltung des Lebens mächtige Einflüsse empfangen könnte. Wo die Religion so viel Fremdes und Veraltetes mit sich schleppt, wo vielfach der Schutt von Jahrtausenden die ewigen Wahr- heiten verdeckt, da kann die Religion nicht ihre volle Kraft mit siegesgewisser Zuversicht entfalten, Gewißheit haben, da wird da kann sie selbst sie keine axiomatische durch Angriffe von kläglicher Weshalb uns das Christentum bei allem, was an ihm vergänglich ist, und es daher keines Bruches mit ihm zu bedürfen scheint, darüber haben wir uns in den religionsphilosophischen Werken näher geäußert. Hier sei nur noch gesagt, daß uns kaum ein Unterfangen so töricht erscheint als das, eine Religion durch bewußte In allen anderen Gebieten haben wir eine Reflexion bereiten zu wollen. derartige Denkweise der sich verflachenden Aufklärung glücklich überwunden, gerade das allerinnerlichste Gebiet aber, das sie am wenigsten verträgt, muß sich ihrer noch immer erwehren. ^ einen unvergänglichen Kern zu enthalten, Das Problem der Religion. 403 Flachheit beunruhigt, die an einer gefestigten und selbsterlebten Über- So bedarf zeugung ohne weiteres abprallen müßten. sie dringend durchgreifenden Revision, einer energischen Freilegung ihrer einer beherrschenden Grundlinien, einer Abstoßung alles dessen, was welk und morsch ward, Eine Interesse. sie bedarf dessen solche Aufgabe aber besonders in ihrem eignen kann nur im Elemente voller Freiheit gelingen. Aber zugleich muß auch die Substanz der Religion energisch gewahrt, zu kräftiger Betätigung aufgerufen, zur Scheidung von Echtem und Unechtem, von Wesen und Schein im Befunde der Zeit verwandt werden. Bedeutendes nichts Religion die Ohne eine Selbständigkeit gegen die Zeitlage kann schlechthin, wohl aber eines im besonderen leisten; Christentum mit seiner Verneinung nicht letzten freilich steht das der nächsten Welt Abschließens bei dieser Welt in unversöhnlichem Gegensatze zu einer bloßen Daseins- und Menschenkultur, wie sie ist "keinerlei gewiß den Hauptzug des modernen Lebens beherrscht. Hier Ausgleichung möglich, sondern nur ein ehrlicher Kampf; muß auch er einen schließlichen Frieden ins Auge fassen, aber es macht für das Ergebnis einen gewaltigen Unterschied, ob der Gegen- vor allem zunächst mit voller Klarheit herausgestellt, oder ob er satz von vornherein abgeschwächt wird. Hier findet sich nun auf dem Boden des Protestantismus viel schwächliche Konnivenz und viel ängstliche Reverenz gegen die moderne Kultur, als sei sie im Ganzen ihres Seins schon eine ausgemachte Wahrheit und nicht selbst voll schwerer Probleme; man scheut alles kräftige Nein, als aus 'dem nicht ein Nein hervorspringt; sein, könne ein Ja von Wert man denkt zu klein von der eignen Sache und kommt daher natürlich nicht weiter. Bescheidenheit mag den Menschen zieren, für die Sache kann sie zum Unrecht Eine Religion von Gnaden der bloßen Kultur, eine Religion, werden. die gar allen Wandlungen der Zeitoberfläche folgeleistet, liches und Freiheit haltloses binden; in der Sache zu erreichen. Gegenwart So voller ist zeigt muß So Ding. und Ursprünglichkeit sich mit das ist ein kläg- nach Verlangen mehr dem nach mehr Tiefe ver- das möglich, es liegt nur am Menschen, es auch daß freilich sich Verwicklungen hier, und Widersprüche die Lage der ist, daß wir aber diesen keineswegs wehrlos ausgeliefert sind, sondern bei Auf- bietung geistiger Kraft über sie hinauskommen können. 26* Schlußwort \ V/ir haben verschiedene Gebiete durchwandert und verschiedene ^^ Probleme behandelt, soweit es im Plan unserer Arbeit lag. Wir überzeugten uns dabei von der überströmenden Fülle des Lebens, das unsere Zeit durchdringt; eine greisenhafte Zeit die Arbeit leistet. namentlich Stoffes eine deshalb, geistigen Gehalt und die Hauptist das dem unermeßlichen Zustrom des unfertige des Zusammenfassens die Kraft nicht Expansion des Lebens seine Konzentration Aber wir sahen zugleich, es wahrlich h-ervorragende hat durchaus weil ist und so Probleme Aber es ist, auf den angesehen, richtung aber bedeutende so nicht, Zeit; sie entspricht, weil die weitem überwiegt. bei daß das nicht wie ein starres Schicksal hingenommen zu werden braucht, daß vielmehr das Leben der Gegenwart auch voller Möglichkeiten ist, und fördern können; nur muß ein die eine Synthese vordringendes Schaffen diese Wir sahen dabei weiter, Möglichkeiten ergreifen und weiterführen. daß das nicht «gegebenen" unmittelbar aus der vorbereiten Lage heraus ge- schehen kann, daß es vielmehr dazu eines Überlegenwerdens gegen diese Lage, der Erreichung eines neuen Standorts des Lebens, einer Umkehrung des nächsten Daseins bedarf; eine solche Umkehrung aber geigte sich möglich und nicht anders möglich als durch ein Selbführten alle einzelnen Punkte ständigwerden des Geisteslebens; so darauf zurück, und an jeder Stelle kam zur Sprache, wie die Über- zeugung von solcher Selbständigkeit die Aufgabe verändert und ihre Lösung anbahnt. Bei solchem Gedankengange drängt die Betrachtung der Zeit über den eignen Befund der Zeit hinaus zum Ausblick in die Zu- kunft; daß wir aber aus der Zerstreuung in vielfache Aufgaben zu einer allumfassenden Aufgabe, von betrachtender und zerlegender Reflexion zu mehr schöpferischer Synthese, von überwiegender Hingabe an die Außenwelt zu mehr Leben Selbständigkeit gelangen, ist bei uns selbst und mehr innerer dazu nach bestem Vermögen mitzuwirken auch die Philosophie berufen. Sachregister Abstrakt, Geschichte des Begriffes Macht abstrakter Begriffe in 55 der Gegenwart 55. Agnostizismus, Ursprung des Aus; — drucks 392. Aktivismus, sein Unterschied vom Pragmatismus 51. Aktuell, Geschichte des Wortes 220 Zurückweisung der landläufigen — Aktualität 220. Beharrungslehre Altertum (im und Mittelalter) 194 ff. Beharrungs- und Bewegungslehre, ihr Kampf 216; — drei verschiedene Lebenstypen dabei 223 ff. Bevölkerungszunahme, ihre Bedeutung die für Auffassung der Geschichte 214. Allegorische Deutung, ihre Wirkung auf das logische Denken 58. Allgemeinbegriffe, ihre eingewurzelte Überschätzung 54 ff. Allgemeines Stimmrecht, wann zuerst gefordert 290. Altertum (s. Griechentum) sein Intellektualismus 39; Ausgangspunkt der Erkenntnisarbeit, wo gesucht 64ff., 222. — was an ihm zu verschiedenen Zeiten geschätzt Bildung, Geschichte des Begriffs und Ausdrucks 232 ff. Charakter, Geschichte des Aus- — Erörterung aus drucks 355 ff.; der Lage der Zeit 358 ff. Christentum, seine Stellung Intellektualismus 39 ff.; — zum seine Steigerung der Geschichte 197. Demokratismus, seineEntwicklung 263 ff. — a posteriori, Aus— modruck und Begriff 84 A priori ff.; derne Verflachung abgewiesen 86. Arbeit, Eigentümlichkeit und Probleme der modernen Arbeit 18 ff., 313ff.; — seine Bedeutung und seine Schranke 317 ff. Denken, Verschiedenheit seiner geschichtlichen Stniktur 58. Denknotwendigkeit, Kritik ihres Vermögens 94 ff. 73 ff. Arbeitswelt (des Menschen) 113ff. Ästhetizismus 336ff. Aufklärung, ihr Intellektualismus — ihre Scheidung von Natur und Seele 173; — ihre Stellung zur Geschichte 254; — Folgen ihrer — ihre Ungeschichtlichkeit 261 41; ff.; Fassung des Geisteslebens 271. Determinismus, seine verschiede- nen geschichtlichen Formen 363 ff. — sein Wachstum im Jahr— seine Voraushundert 364 setzungen 368; — sein Bild der 19. ff.; Wirklichkeit 372. Diskontinuitätsphilosophie (in Frankreich) 146. Sachregister. 406 Dualismus, das Wort 170; — sein Hervortreten der Neuzeit 173; in — Dualismus innerhalb des Monismus der Gegenwart 190. Empirismus, sein Erkenntnisbild — sein Recht und sein Unrecht 117ff.; — sein Ungenügen 88, 90; für die Gegenwart Energie, Definition 141 ; 124. Ostwald von — geistige Energie = Lebens- Entwicklung, Ausdruck 192ff.; — ihr Aufkommen und Vordringen — drei Hauptphasen der Entwicklungslehre (religiöse, künst- empirische) lerische, — — Schei- 198 ff.; neuer Lebenstypus 204 ff.; dung von Entwicklung und Evolutionismus 204 ff., 226; — Gefahren des Evolutionismus 210, 215. Erfahrung, Ausdrücke 84; — ihre zwiefache Bedeutung für das Er- kennen 114 ff.; keit auf dem — ihre SchwierigGebiet des Geistes- lebens 120. Denken, irre- leitender Sprachgebrauch 119. Lebensprozesse 56 ff. ; 100 ff .; 1 1 1 ff. — Kampf über seinen Ursprung 87; — Erkennen unterschieden vom Kennen 99; — inwiefern immanenter Art 113; — inwiefern auf Erfahrung angewiesen 114, — verschiedene Stufen 116; — wo die Entscheidung darüber 121 ff. Erkenntnislehre (moderne), ihr (s. 192ff.; Ästhetik, Entwicklung), Aus- — evolutionistische Ethik, Rechtslehre 212 ff. — AufFortschritt, Ausdruck 200; kommen und Durchdringen der Idee 199ff.; — seine Verschieden- heit in verschiedenen Lebensgebieten — vager Fortschrittsglaube 213; — Schranken 382. 215; 111; 30ff., sein Grundbegriff — sein Verhältnis zum geschichtlich -gesellschaftlichen Da- sein lOOff., 103ff. Geistigkeit, ihre drei Stufen (grundlegende, kämpfende, überwindende) 350 ff. Genie, zur Geschichte d^s Ausdrucks 307. Geschichte, Zusammentreffen von Zeitlichem und Zeitlosem in ihr 5, 103; — ihre Bedeutung für die Philosophie 65; — Bedingungen der menschlichen Geschichte 21 ff.; — Bedeutung und Schranke der — Gründe der Geschichte 253 — Bewegung zur Geschichte 254 ff.; ff ; 260ff.; — Bedeutung der Geschichte Geschichte der Philosophie, Forderungen für ihre Behandlung 66 ff. Geschichtliche Gesetze 166ff. Geschichtsforschung und Geschichtsphilosophie 256. Geschlechtliches, seine Behandlung im modernen Subjektivismus in der modernen Kunst 342. 338; — Gesellschaft und Individuum, Einfluß auf das Weltbild 187. druck Begriffes 374. für das Geistesleben 265 ff. Erkennen, seine Abhängigkeit vom Evolution auf das Gefühl 20 ff. Gegebenes, das Bedenkliche dieses der Bewegung gegen die Geschichte Erfahrung und letzte Freiheit und Schicksal 371 ff. Kritik des Zurückgehens Gefühl, Geistesleben, konzentrationen 60, 62. 197 ff.; Freiheit und Gleichheit, ihre Verbindung 290. 283 ff.; — Art der gesellschaftlichen Kultur 288. Gesetz, 154 ff.; Geschichte und Problem — Kampf darum in den verschiedenen Gebieten 159ff. Gleichheitsidee, ihr Ursprung und ihre Geschichte 289. Gleichmacherei, ihre Gefahren in der Gegenwart 299 ff. Sachregister. Gottesidee, der über Streit ihre — zweierlei Fass- Fassung 346ff.; ungen innerhalb des Christentums Griechische Denkweise (s. Altertum), ihr Verhältnis zum Wahrheits- 12; — ihr Intellektualismus 39; — Eigentümlichkeit ihres logischen Verfahrens 58; — ihr Vor- problem Problem auf geistigem Gebiet 31 Off. große Persönihr — ; zur Zeitum- lichkeit, ihr Verhältnis gebung 297 ff. Hierarchische Ordnung 285. Historismus, seine Gefahr 258ff.; — Abwägung von Historismus und Rationalismus 271 ff. Humanismus, als moderner Terminus 48. Höher, Ausdruck und Begriff 78. Idealismus, Kritik der Ausdruck 68ff.; — überkommenen Formen Idealismus ungen 76 ff.; für einen — Forder- neuen Idealismus — kritischer und positiver 81 ff.; Idealismus 124 ff. Immanenz, Ausdruck 390; — Bewegung der Neuzeit zur Immanenz — VePÄ'icklungen im Begriff Immanenz 394 ff. Individualkultur 302ff. der Ausdruck 283 — Begriff 284 — verschiedene geschichtliche Geff.; ff . Kultur, Geschichte des Ausdrucks und Begriffs 228ff.; unterschieden von Zivilisation 231 ff.; ihr Wesen und Wert 235 ff.; ihre — — Arten das von Mensch Verhälthis mikrokosmische) 284 ff.; der geistigen — Bild- Individualität — Bedeutung der Individuen — moGeschichte 297 in der ff.; derner Individualismus 306 ff. Intellekt, seiner Geringschätzung widersprochen 52 ff. — — seine und und Kultur — Teilkulturen — Probleme und Gesamtkultur 249; gegenwärtigen der Kultur 250 ff., 381 ff. Kulturstaat, bei Fichte 231. Kunst, ihr Verhältnis zur Moral 329 ff.; — ihre Stellung im modernen Leben 339; Verhältnis von Inhalt und Form 340; Bedeutung der Kunst für den nordischen Menschen 341. Künstlerischer Individualis- — — mus 306ff. pour L'art Geschichte l'art, des Ausdrucks 339 ff. Leben, wissenschaftliche einer näheren Versuche Bestimmung 145 ff. Lebensbejahung und Lebensverneinung 376 ff.; — gegen erkünstelte Lebensbejahung 384 ff. Lebensenergien 60. Lebensprozeß als Ausgangspunkt Stell- ung zum Geistesproblem 342. Logik, Überschätzung ihrer Form — 54ff. ihre Abhängigkeit Lebensprozeß 58 ff. Materialismus, Ausdruck vom und Lehre 175 ff. Mechanismus, zur Geschichte des — Ausdrucks 126; zur Geschichte des Problems 133 ff.; unzulängr — lieh als philosophische Weltanschau- ung Intellektualismus, Wort und Sache 37 ff.; Überflutung des modernen Lebens durch ihn 53 ff.; — 240 ff.; Kultur — 237 ff.; geschichtlichen ; staltungen(organische, hierarchische, 371; Kant 41 ff.; Lebenstypus 39. der Untersuchung 31. Literatur, ihre gegenwärtige Individuum und Individualität, ung — durch Geistesleben 243 ff.; anstellen des Beharrens 194 ff. Größe, 392; Brechung intellektualistischer — 347. des 407 142. Mechanismus und Teleologie, ihre Ausgleichung bei Leibniz 134; — bei Lotze 138. Sachregister. 408 156 ff.; Metaphysik, der Ausdruck 108; — Metaphysik das Problem 104 — Metaphysik 108; und Kultur ff.; und Moral 324 ff. des Ausdrucks Art logischen seines — Hauptstätte der — sein Ord— Bekämpfnungssystem 196 Verfahrens 58; organischen Lehre 151 ; ff.; ung dieses Begriffes 277 ff. — Problem — Bedeutung bei Kant 16, Modern, Verschärfung des Begriffes durch die Entwicklungslehre 205; Ausdruck und Begriff 273 ff.; — — echtes und falsches Moderne 280ff. Monismus, Wort 170ff.; — Lehre _ Monismus der Gegen- wart 183 ff. Moral, gegenwärtiger Mangel einer genügenden Begründung 322ff.; überkommene Typen 323 ff.; — — — Moral und Metaphysik 324 ff.; AbMoral und Kunst 329 ff.; lehnung der ;neuen Ethik" 336ff. Natur, der Ausdruck angegriffen — — Definition des Aristoteles 172; — Naturbild der von Boyle 127; Gegenwart 380ff. Naturgesetz, zur Geschichte 154 ff.; Naturgesetz und Sittengesetz 163. Naturkultus, seit Giordano Bruno 158. Naturwissenschaft Ge- und Neunzehntes jahrhundert, seine Bewegung^ gegen tualismus 43 ff.; 18ff.; — — seine den Intellek- sein Realismus ihre Goethe 28. Ökonomismus 311ff., 318ff. blem 376ff. zwiefache zum Geistesproblem 13ff.; Organismus, und Organisch Ausdrucks 128ff., Organische des Problems 130 ff.; des Geschichte — Natur- 136 ff.; Geschichtsauffassung und — Unterschied der alten und der neuen Fassung 139; — organische Gesellschaftslehre 149 ff. Panentheismus 392. Pantheismus, der Neuzeit 392; sein Verblassen in der — Gegenwart 394 ff. Persönlichkeit, Geschichte des — des Begriffes Ausdrucks 343 ff.; Problem der Persönlich345 ff.; Motive des keit Gottes 346; — — Kampfes gegen die PersönlichkeitsPersönlichkeit und idee 347 ff.; — Metaphysik 349 ff.; — Kritik der h^tigen Bewegung zur Persönlichkeit 353; — persönliche Gestaltung der Arbeit unterschieden von subBedeutung der jektiver 354 ff.; — im geschichtlichen Leben 297 ff. Pessimismus, Ausdruck und Pro- blem 376ff. Phantasie, ihre Unentbehriichkeit für die Philosophie 98, 106 ff. 72 ff. Neuzeit, bei Öffentliche Meinung 297. Persönlichkeiten schichte 168. Bewegung 12 ff.; Ontologie, Ausdruck 109. Optimismus, Ausdruck und Pro- Mittlerer Mensch 289. innere Normen 24, 163. Objektiv, Wort lOff.; 289. Mittelalter, 179ff.; ff.; ung des Individuums 151, 286 ff. Noologische Betrachtung, unterschieden von psychologischer 33. Mikrokosmos, Ausdruck 285. Milieu, Geschichte — ihr Eintreten für Ent— ihre Befrei197 wicklung Stellung — ihre — Behandlung des Denkens 41ff.; ihr wissenschaftliches Verfahren 59, Philosophie, ihre Aufgabe und Methode 63; — das Recht einer selb- ständigen Philosophie 63 ff.; 93 ff.; der ihr Zusammenhang mit — ^9 Sachre gister. Bewegung der Weltgeschichte 100 ff.; — philosophia 62, perennis 278. Pragmatismus, und Darstellung Kritik 47. — Geschichte der — verschiedene Aristoteles 37; Begriffe 37 ff.; Lebenstypen 39 ff. Psychologismus, abgelehnt 19. Rationalismus, sein Recht und Unrecht — sein Er- 11 7 ff.; kenntnisbild 88; — sein Geschichts- — im 19. Jahrhundert 72ff.; — seine Schranken 74 69; ff. Reformation, ihre zum Stellung Intellektualismus und zum Volun- 40 ff.; — ihre Stellung tarismus Staatsidee, ihr stärkeres Hervortreten im 19. Jahrhundert 290, 301, Stufen und des Lebens kennens 102, 350. des — Summierungder Vernunft 296 Verstand, Ver- reine nunft) 86. Religion, ff. System, Bedeutung und Grenze 67. Teleologie 135ff.; empirische — Teleologie 141. tisch) prak- (s. 37 ff. Transzendenz, Ausdruck 391. Typus und typisch, Ursprung des Ausdrucks 356, Unendlichkeitsgedanke, verschiedene Schätzung seine und Be- handlung 379. zur Kunst 332. (reiner Er- Subjektiv, Wort 10; Wendung der Gegenwart zum Subjekt 19 ff. Theoretische Vernunft 118. Realismus (s. Idealismus) Ausdruck Rein Soziale Lebensführung 292ff. 318. Praktische und theoretii^che Vernunft, ihre Scheidung durch bild 164. Spiritualismus 178ff. Politismus, 301 ff., 305 ff. sein Soziale Gesetze Vitalismus 144ff. Volltätigkeit 31. Bewegung gegen der Neuzeit 392 ff.; aufsteigen 395ff.; sie in — ihr Wieder— Forderungen für ihre gegenwärtige Lage 398 ff. Renaissance, ihre Stellung Kunst und zur Moral 331 ff. zur Romantik, ihre Schätzung des Or- — ihre Entwicklungsidee 204, 215; — ihre Ge- ganischen 136 ff.; Voluntarismus, Wort und Problem 37 ff.; — seine Entwicklung 43. Von vornherein 86. Wahrheitsbegriff und Wahrheitsproblem 12ff.; — nicht bloß intellektuell 64, 122; ff. Weltgeschichtlicher Stand der Arbeit, schichtsauffassung 255. 35ff., — unsere Fassung 35 als Maßstab 267. Schöne Seele 307. Wert, Erörterung 22ff. Scholastik, ihre Fassung der Wahr- Wert des Lebens 376ff. Wertschätzungen in der Ge- heit 12 ff.; — ihre Stellung zum — Grenze ihrer Leistung 57; — ihr Mangel schichte 257. Werturteile 20ff. an disjunktiver Kraft 58ff. Willensfreiheit, Intellektualismus 40; Souverän, souveräne Art der geistigen Arbeit 354 ff. Sozialdemokratische Bewegung 313ff. Erörterung des Problems 363 ff. Selektionslehre 208ff. Wirklichkeitsproblem 79ff. Wirtschaftliche Frage, ihre ge- schichtliche Entwicklung 314 ff. Wirtschaftliche Gesetze 104ff. Sachregister. 410 Zeitgeist, Aufkommen des Begriffs und Problems 273. Zivilisation, drucks 229; Kultur -231 ff. Urspnmg Zufall, Grenze seines Vermögens nach Aristoteles 132. des Aus- — unterschieden von Zweck, sein Recht schauung 143 ff. in der Weltan- VEIT & COMP, in Leipzig Verlag von GESCHICHTE DER MEÜEREH PHILOSOPHIE VON NIKOLAUS VON KUES BIS ZUR GEGENWART. Im Grundriß dargestellt « von Richard Faickenberg, an der Universität Erlangen. o. Professor Sechste, verbesserte und ergänzte Auflage, gr. 8, geh. 9 M., geb. in Ganzleinen 10 M. 1908. Großartig durch die ,,Ein Buch von mäßigem Umfange und doch ein großartiges Werk. Fülle des verarbeiteten Stoffes, da nicht allein alle Philosophen von Fach, sondern alle Männer, deren Denkarbeit bestimmend auf das Geistesleben der modernen Völker eingewirkt hat, Berücksichtigung gefunden haben." Durch die Gediegenheit des Inhaltes, die geschickte Anordnung und die Klarheit der Darstellung, durch vorzügliche bibliographische Nachweise hat sich Falckenbergs Geschichte der neueren Philosophie allgemeine Anerkennung erworben, nicht nur in Deutschland, sondern auch im Auslande. Den Schluß des Werkes bildet eine Erläuterung der wichtigsten philosophischen Kunstausdrücke. — GRIECHISCHE^ DENKER, Eine Geschichte der antiken Philosophie. Von Theodor Gomperz. Vollständig in drei Bänden. » Erster und zweiter Band. Zweite Auflage. Lex. 8. 1903. geh. 23 M, geb. in Halbfranz 28 M. diesem einzig dastehenden, die Summe seiner durch zahllose Einzelforschungen ausgefüllten Lebensarbeit ziehenden Werke behandelt der große Gelehrte nicht die Gedanken oder die Systeme oder die Geschichte der griechischen Philosophie, sondern die „Denker", die Individuen. Der Werdegang ihrer Ideen wird auf dem Hintergrund ihrer Zeit geschildert, und die geistige, Jahrtausende überspannende Brücke, die sie mit der Gegenwart verbindet, wird geschlagen. Der dritte Band, dessen erste bis dritte Lieferung erschienen sind, wird Aristoteles behandeln. In GESCHICHTE DES GELEHRTEN UNTERRICHTS auf den deutschen Schulen und Universitäten vom Ausgang des Mittelalters bis zur Gegenv^^art. Mit besonderer Rücksicht auf den klassischen Unterricht. Von Dr. Friedrich Paulsen, o. ö. Professor an der Universität Berlin. Zweite, umgearbeitete und sehr erweiterte Auflage. Zwei Bände. 1896 u. 1897. gr. 8. Erster Band: Zweiter Band : geh. 30 M, geb. in Halbfranz 34 M. Der gelehrte Unterricht im Zeichen des alten Humaniimus. 1450—1740. Der gelehrte Unterricht Im Zeichen des Neuhumanismus. 1740—1892. „Wenn diese Deutung der historischen Tatsachen nicht gänzlich fehlgeht, so wäre hieraus für die Zukunft zu folgern, daß der gelehrte Unterricht bei den modernen Völkern sich immer mehr einem Zustande annähern wird, in welchem er aus den Mitteln der eigenen Erkenntnis und Bildung dieser Völker bestritten wird." VEIT & COMP, in Leipzig Verlag von JOHANN HEINRICH PESTALOZZI. Seine Ideen in systematischer Würdigung. Von Dr. Hermann Leser, Professor an der Universität Erlangen, gr. 8. geh. 3 1908. M 50 Pf. VORLESünQEM ÜBER HRTÜRPHILOSOPHIE, gehalten im Sommer 1901 an der Universität Leipzig von Wilhelm Ostwaid. Dritte, Lex. 8. 1905. vermehrte Auflage. geh. 12 M, geb. in Halbfranz 14 M 50 Pf. Die „Vorlesungen über Naturphilosophie" des berühmten Chemikers sind eine der interessantesten neuzeitlichen Erscheinungen. Die „Vorlesungen" stellen kein Lehrbuch oder System dar, sondern sind das Ergebnis umfassender Erfahrung bei Forschung und Unterricht, das durch die schöne Form, in der es geboten wird, eine außergewöhnliche Anziehungskraft auf den Leser ausübt. PSYCHOLOGIE DER NATURVOLKER. ENTWICKLUNGSPSVCHOLOOISCHE CHARAKTERISTIK DES NATURMENSCHEN IN INTELLEKTUELLER, AESTHETISCHER, ETHISCHER UND RELIGIÖSER BEZIEHUNG. Eine natürliche Schöpfungsgeschichte des menschl. Vorstellens, Wollens und Glaubens von Dr. Fritz Schultze, ordentlicher Professor der Philosophie an der Technischen Hochschule zu Dresden, gr. 8. 1900. geh. 10 M. WIRTSCHAFT UND RECHT nach der materialistischen Geschichtsauffassung. Eine sozialphilosophische Untersuchung von Dr. Rudolf Stammler, o. 5. Professor an der Universität Halle a. S. Vitam impendere vero. Zweite, verbesserte Auflage. M Lex. 8. 1906. geh. 15 M, geb. in Halbfranz 17 50 Pf. Die Erforschung der allgemein gültigen Gesetzmäßigkeit, unter der das Der Versoziale Leben des Menschen steht, bildet den Inhalt dieses Werkes. fasser gelangt zu dem Ergebnis, daß die allgemein gültige Richtschnur für das dem sozialen Ideal entsprechende Recht nur die Herstellung einer Gemeinschaft von Menschen sein kann, deren Wollen von bloß subjektivem Begehren frei ist, von denen vielmehr jeder die objektiven Zwecke der anderen zu den seinigen macht.
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Season and depth differences of soil moisture use for tree growth across wet and dry gradients in the Tibetan Plateau
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Season and depth differences of soil moisture use for tree growth across wet and dry gradients in the Tibetan Plateau Qian Li  Liang Jiao  (  jiaoliang@nwnu.edu.cn ) Northwest Normal University https://orcid.org/0000-0001-6066-2346 Ruhong Xue  Xichen Chen  Peng Zhang  Xuge Wang  Xin Yuan  Research Article Keywords: Tree-ring oxygen isotopes, Wet and dry gradients, Water use dynamics, Spatial and temporal variation, Tibetan Plateau Posted Date: August 23rd, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3236892/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Northwest Normal University https://orcid.org/0000-0001-6066-2346 1. Introduction Global change affects tree adaptation in a variety of ways, leading to complex changes in forest ecosystem composition, structure and function, especially in the colder northern regions (Kenneth et al. 2022; Wang et al. 2023). Global changes in tree growth are largely attributed to interactions between temperature, precipitation and radiation balance (Seddon et al. 2016; Reichstein et al. 2017). In particular, the main factor influencing the growth of trees in humid environments is temperature, while the growth of trees in arid areas is mainly limited by moisture (Amer et al. 2023). Thus, there are differences in the sensitivity of tree growth to climate change under different wet and dry conditions. Soil water is an important component of the water cycle that links the atmosphere, hydrosphere and biosphere, and is a major source of water for plants (Eslaminejad et al. 2020), which plays an important role in maintaining vegetation growth and ecosystem productivity. Soil moisture content varies with soil depth and season (Ma et al. 2020), and the water uptake mechanisms and adaptation strategies of trees to heterogeneous environments vary especially under different hydrological changes (Bertrand et al. 2014). The soil moisture was found to be the most important potential factors controlling the growth of vegetation on the Tibetan Plateau (Wang et al. 2018). The trees mainly use soil moisture during the growing season (Zeng et al. 2020; Zhang et al. 2022). However, it is important to note that the measurement and estimation of soil moisture is very complex and has great limitations for studying tree growth and water use strategies. Whereas tree rings can be used as a proxy for inversion of soil moisture changes on long time scales due to their continuous record, accurate dating, sensitivity to climate change, and wide distribution (Gao et al. 2018). For example, the oxygen isotopes in tree-ring have been used to reconstruct soil moisture in the western Himalayas over a period of nearly 100 years (Bose et al. 2016). Due to the coupled processes of water uptake and transpiration by tree roots, interannual variation in tree-ring oxygen stable isotopes (δ18OTR) can document seasonal changes in climate and the physiological-ecological response of trees to seasonal changes (Xu et al. 2020). 1. Introduction Variations in δ18OTR are mainly influenced by δ18O uptake of source water by growing trees, evaporative enrichment of leaf water, biochemical fractionation during photosynthesis, and xylem water exchange during the transport of carbohydrates to the trunk (Roden et al. 2000). This series of fractionation processes is regulated by external climate change (Brinkmann et al. 2019; Xiao et al. 2020). The δ18OTR are derived from soil water, which is ultimately derived from atmospheric water, mainly in relation to changes in precipitation, temperature and relative humidity (De et al. 2018). And changes in the atmospheric climate system are stored in soil moisture and are transferred to tree cellulose during the growing season to affect tree-ring oxygen isotopes. In addition, δ18OTR has the potential to reflect the depth of water uptake (Gessler et al. 2014). Variations in soil water δ18O at different depths are preserved in the tree rings, but the strength of the soil water isotope signal preserved in the tree rings depends on the depth of water uptake by tree roots (Huang et al. 2019, Xu et al. 2020). In monsoon-controlled areas, precipitation δ18O has a seasonal pattern and these signals are transmitted to various soil depths, causing soil water δ18O to vary in vertical gradients with seasonal strength and direction (Lindh et al. 2014). Trees convert the isotopic composition of source water to the isotopic composition of cellulose through complex non-linear physicochemical processes (Ballantyne et al. 2016). For example, it has been shown that trees in the East Asian monsoon zone significantly correlated with soil moisture during the growing season, but the seasonal response was earlier in wet areas than in dry areas, and wet areas used mainly shallow soil water while dry areas used deep soil water (Wang et al. 2021). However, other study also showed trees in the East Asian monsoon region used deep water in the dry season and shallow soil water in the non-dry season (Yang et al. 2015). Therefore, there is uncertainty in the study of water use strategies based on δ18OTR. Controlled by the Asian monsoon, the climate of the Tibetan Plateau has undergone some changes in recent years, mainly including increasing surface temperatures (Sun et al. 2022), weakening monsoons (Wong et al. 2020) and reduced potential evaporation (Fang et al. 2019) since the 1960s. Research Article Page 1/20 Abstract Soil moisture has an important influence on tree growth in climate-sensitive regions. However, the seasonality of soil moisture utilization by trees in the Tibetan Plateau, a typical climate-sensitive region, and the mechanisms of depth of uptake are still unclear. Therefore, the spatial and temporal differences in the soil moisture use dynamics of tree growth were analyzed under wet and dry gradients on the Tibetan Plateau using tree-ring δ18O (δ18OTR). The results showed that: 1) soil moisture during the growing season was the main influencing factor on tree growth under different wet and dry gradients on the Tibetan Plateau. 2) The response of δ18OTR to soil moisture had a lag in arid areas than in wet areas. 3) In wet areas, trees absorbed the soil moisture in surface, while absorbed the soil moisture in deep in arid areas. And meanwhile, trees could develop more lateral and deep root systems using soil moisture from all soil layers to cope with climatic stress. In the future, more lateral and deep root systems of trees will be better suited to survive in complex habitats on the Tibetan Plateau, and that monitoring of trees in single water use areas should be enhanced. Further, it is of great significance to study soil water use strategies of tree growth under different wet and dry gradients for predicting forest ecosystem changes in complex environments. 2.1 Study areas The six sites in the Tibetan Plateau were selected for the study such as Delingha region (DE), the Ganesh (GA) and Wache (WA) regions in the south, the Manali (MA) and Jageshwar (JA) regions in the southwest, and the Karakorun (KA) region in the west (Fig. 1). Except for the KA region which is affected by the westerly winds and the other regions are affected by the Asian summer winds. From the wettest JA region to the driest DE region, the annual precipitation decreased by 1,311 mm (Fig.S1). The six regions are divided into humid and arid areas based on annual precipitation. Specifically, the humid areas include the JA, WA and GA regions, while the arid areas include the MA, KA and DE regions. Please insert Fig. 1 about here. 1. Introduction Changes in summer wind activity affect monsoon precipitation by altering the spatial pattern of regional water vapor transport, ultimately leading to interannual variability in δ18OTR (Ge et al. 2017; Tan et al. 2018). However, current studies on the Tibetan Plateau based on δ18OTR have mainly focused on climate reconstruction (Grießinger et al. 2019; Xu et al. 2019) and physiological mechanisms (Zhang et al. 2018). Therefore, this study aims to address three main questions: (1) whether there are differences in the seasonal dynamics of tree water use under different wet Page 2/20 and dry gradients on the Tibetan Plateau, (2) whether there are differences in the ability of tree growth to use water at different soil depths under different wet and dry gradients, (3) and what are the combined adaptation mechanisms of trees based on the seasonal dynamics and soil depth of tree water use under different wet and dry gradients? and dry gradients on the Tibetan Plateau, (2) whether there are differences in the ability of tree growth to use water at different soil depths under different wet and dry gradients, (3) and what are the combined adaptation mechanisms of trees based on the seasonal dynamics and soil depth of tree water use under different wet and dry gradients? 2.2 Tree-ring isotope data The δ18OTR data in this study were downloaded from NOAA Paleoclimatology Datasets (https://www.ncei.noaa.gov/pub/data/paleo/tree- ring/isotope/asia). These δ18OTR chronologies are common to the years 1948–1998, and the specific locations, elevations, tree species, and δ18OTR chronological lengths of the sampled sites were shown in Table 1. The sampling sites were largely located in high-elevation areas, and all of the selected tree species were conifers. Tree cores were collected at breast height using increment borers. And alpha-cellulose extraction process included chemical reactions with organic reagents, sodium chlorite and sodium hydroxide to remove extractions (ash, resin, etc.), lignin and hemi- cellulose, respectively (Shi et al. 2015). Elevation gradients and age differences in the plateau had no significant effect on the climatic response of δ18OTR records (Wernicke et al. 2015; Zeng et al. 2016). Please insert Table 1 about here. Table 1 Chronological information on tree rings Sites Location Elevation Species period Rbar Date Source JA 29°38′N, 79°51′E 1870m Cedrus deodara 1621–2008 0.61–0.78 Xu et al (2018) WA 27°59′N, 90°00′E 3500m Larix griffithii 1743–2011 0.74–0.88 Sano et al (2013) GA 28°10′N, 85°11′E 3500m Abies spectabilis 1801–2000 0.56–0.78 Xu et al (2018) MA 32°13′N, 77°13′E 2700m Abies pindrow 1768–2008 0.71–0.92 Sano et al (2017) KA 35°54′N, 74°56′E 2900m Juniperus excelsa 1990–1998 0.51 Treydte et al (2006) DE 37°48′N, 97°78′E 3500m Juniperus przewalskii -4680-2011 0.40–0.76 Yang et al (2021) 2.4 Soil moisture data Soil moisture data were obtained from the Global Land Data Assimilation System (http://Idas.gsfc.nasa.gov/gldas) with a temporal resolution of 1948–2015 and a spatial resolution of 0.25°×0.25°. This dataset has a longer temporal resolution and a higher resolution for soil moisture at different depths compared to other datasets. We selected soil moisture data from 1948–1998 at three depths with 0-10cm, 10-40cm and 40- 100cm for our study. The soil moisture data we downloaded ((unit: kg/m2) had to be further converted to volumetric water content (VWC, unit: m3/m3) for data analysis by the following conversion equation: (Eq. 4) V WC = × × × kg m2 m3 1000kg 1000mm 1m 1thicknessoflayerinmm 2.3 Meteorological data Our meteorological data included the maximum, mean and minimum temperatures, actual vapor pressure, and precipitation and the Palmer drought severity index (PDSI) on monthly-scale for six regions (Fig.S1). The grid point data above were obtained from Climate Explorer (http://climexp.knmi.nl/start.cgi). Since saturation water vapor pressure deficit (VPD) and relative humidity (RH) data for the region are difficult to obtain, we calculated VPD (Murray 1966) and RH (New et al. 1999) based on the downloaded CRU TS4.04 (0.5◦×0.5◦) dataset, and the specific calculation procedure as follows: Page 3/20 Page 3/20 (Eq. 1) (Eq. 2) (Eq. 3) V PD = actualwatervapourpressure −saturatedwatervapourpressure = e0 × 10 ( ) −actualwatervaporpressure 7.5T T +237.3 RH = ( ) × 100 eesat esat = 6.108 × exp(17.27 × T/(T + 273.3) RH = ( ) × 100 eesat In the above equation, e0 = 6.11hpa, T is the monthly mean temperature (unit: ℃). In the above equation, e  = 6.11hpa, T is the monthly mean temperature (unit: ℃). 2.4 Soil moisture data 2.5 Data Analysis To investigate the elements of environmental factors that mainly affect δ18OTR, we used Pearson correlation as well as partial correlation analysis. In the meanwhile, One-way analysis of variance (ANOVA) was used to assess the variability of the δ18OTR chronology and the significance of differences was tested using Tukey's multiple comparisons. Spatial correlation analysis was performed using Matlab 2023 and mapped using ArcGIS 10.8. Please insert Fig. 4 about here. 3.1 Comparison of the spatial and temporal dynamics of δ18OTR under the wet and dry gradients Please insert Fig. 2 about here. The spatial dynamics of δ18OTR showed an overall higher value in the north and lower value in the south (Fig. 3). The lowest value of δ18OTR occurs in the southern part of the plateau (WA, 19.401‰) and the highest value is located in the northeastern part of the plateau (DE, 32.907‰). The value of δ18OTR decreased as the climate changed from arid to humid areas. Please insert Fig. 3 about here. g 3.3 Comparison of δ18OTR response to climatic and soil moisture factors under wet and dry gradients 3.3 Comparison of δ18OTR response to climatic and soil moisture factors under wet and dry gradients It is revealed that overall δ18OTR responded negatively to PDSI, precipitation and RH, and positively to temperature and VPD in different humid and arid areas (Fig. 6). However, seasonal response differences were observed in humid and arid environments, showing δ18OTR more often correlated with environmental factors in the middle of the growing season (June-August) in humid environments, and with environmental factors in the middle and late growing season (July- September) in arid areas. Further, comparing the responses of δ18OTR to soil water content at different soil depths in humid and arid areas showed variability in depth as well as time (Fig. 7). In general, the correlation was greater in wet areas with 0–10 cm and 10–40 cm in July (p < 0.01), while the arid environment δ18OTR was significantly negatively correlated with soil moisture in 10–40 cm and 40–100 cm soil layers in September (p < 0.01). The seasonal response of δ18OTR to soil moisture was lagged in the dry areas compared to the humid areas. Specifically, in humid areas, δ18OTR was significantly negatively correlated with 0–40 cm soil moisture in July and August in JA and WA (r=-0.49 to -0.41, p < 0.05), while δ18OTR in GA region was significantly negatively correlated with soil moisture at all three soil depths (r=-0.44 to -0.26, p < 0.05). However, in arid environments, δ18OTR was significantly negatively correlated with soil moisture at all three soil depths from July to September in MA (r=-0.47 to -0.31, p < 0.05), while δ18OTR in KA and DE regions were more significantly negatively correlated with soil moisture at 40–100 cm from July to September (r=-0.48 to -0.29, p < 0.05). Please insert Fig. 6 about here. 3.2 Comparison of the spatial and temporal dynamics of soil water content patterns at different depths under wet and dry gradients Soil water content at different gradients on the Tibetan Plateau showed inconsistent soil depth variation characteristics and temporal dynamics (Fig. 4). Specifically, the soil water content in the six gradient regions showed the increasing trends with increasing soil depth from 0–10 cm, 10– 40 cm to 40–100 cm (Fig. 4a). At the same time, the soil moisture content in the wet areas was significantly greater than that in the arid areas, accompanied by a greater variation in moisture content (VWCJA, WA, GA > VWCMA, KA, DE), especially in the DE area, where the moisture content of all soil layers is lower than in the other areas. In terms of time, the seasonal dynamics of soil moisture content were more pronounced in the wetter areas than in the arid areas (Fig. 4b), where the peak soil moisture content was mainly concentrated in June-September. The volumetric water content from July to September was 48.9% (JA), 49.1% (WA) and 47.8% (GA) of the year, respectively. However, the seasonal dynamics of soil moisture content in the arid areas varied more between regions. In the MA region with the double-peaked model, the volumetric water content from June to September accounts for 32.8% of the year. However, the high values of s volumetric water content in the KA region are concentrated in April-June, accounting for 32.3% of the year. But no seasonal dynamic peaks in soil moisture content were formed in the driest DE region. Please insert Fig. 4 about here. Please insert Fig. 4 about here. Page 4/20 Page 4/20 The moisture content of each soil layer in the humid areas was significantly greater than in the arid areas and decreases significantly with the decrease in regional precipitation (Fig. 5). Specifically, in the humid areas, the soil moisture in each soil layer in each regions tends to decrease and the most significant decrease was in the 40–100 cm range. In the arid areas, soil moisture showed an overall increasing trend, but the MA area shows a decreasing trend in all soil layers, which is closely related to the weakening of the monsoon in the region and the decrease in precipitation. Meanwhile, the linear fit of precipitation to soil moisture was stronger in the wetter regions than in the arid regions, with the fit becoming progressively worse from the wettest JA region to the driest DE region (Fig. S2). Please insert Fig. 5 about here. Please insert Fig.7 about here. 3.5 Comparison of spatial characteristics of δ  18  O  TR  response to soil moisture content at different soil depths under wet and dry gradients 3.5 Comparison of spatial characteristics of δ  18  O  TR  response to soil moisture content at different soil depths under wet and dry gradients 3.5 Comparison of spatial characteristics of δ  18  O  TR  response to soil moisture content at different soil depths under wet and dry gradients The spatial correlation analysis revealed that the response of δ18OTR to soil moisture at all six sites in the study area was generally negatively correlated. But the overall differences in the responses of δ18OTR to soil moisture content at 0–10 cm and 10–40 cm were not significant, and the significant difference was found at 40–100 cm (Fig. 8). And the responses of δ18OTR to soil moisture in the southern and northeastern parts of the plateau changed from negative to positive as the soil depth deepened (Fig. 8). Specifically, δ18OTR was negatively correlated with soil moisture at 0–10 cm of all sites. But the correlation showed a trend of JA > WA > GA > MA > KA > DLH. That is, the drier the climate, the lower the correlation between δ18OTR and soil moisture at 0–10 cm and 10–40 cm. However, the positive responses of δ18OTR to soil moisture at the depth of 40–100 cm increases in DE of the northeastern part and in WA of the south. Please insert Fig.8 about here. 3.4 Correlation between δ  18  O  TR  and key environmental factors during the growing season from June to September under the wet and dry gradients The VMC0-10 represents the volume moisture content of 0-10cm soil, the VMC10-40 represents the volume moisture content of 10-40cm soil, he VMC40-100 represents the volume moisture content of 40-100cm soil. Please insert Table 2 about here Please insert Table 2 about here 3.4 Correlation between δ  18  O  TR  and key environmental factors during the growing season from June to September under the wet and dry gradients 3.4 Correlation between δ  18  O  TR  and key environmental factors during the growing season from June to September under the wet and dry gradients 3.4 Correlation between δ  18  O  TR  and key environmental factors during the growing season from June to September under the wet and dry gradients The correlation between δ18OTR and soil moisture during the growing season (June-September) was found to be significantly stronger than other environmental variables through Pearson correlation analysis. Therefore, we further selected key environmental factors that strongly influenced δ18OTR in different regions during the growing season for a partial correlation analysis (Table 2). The results showed that when VPD and RH were controlled, δ18OTR was significantly negatively correlated with 0–10 cm soil moisture in the wet environment in JA (p < 0.05), while it was significantly correlated with 0–10 cm and 10–40 cm soil moisture in WA and GA (p < 0.05). When VPD and RH were controlled, δ18OTR was highly significantly negatively correlated with soil moisture at all soil depths in MA (p < 0.01), while δ18OTR was significantly negatively correlated with soil moisture at 40–100 cm in KA and DE (p < 0.05). Please insert Fig.7 about here. Page 5/20 Page 5/20 Table 2 Partial correlation analyzes between δ18OTR and environmental factors from June-September. Control variables Correlations with δ18OTR JA WA GA MA KA DE VPD VMC0 − 10 -0.396🞶🞶 -0.364🞶 -0.409🞶 -0.459🞶 -0.211 -0.145   VMC10 − 40 -0.265 -0.36🞶 -0.383🞶 -0.472🞶 -0.277 -0.286   VMC40 − 100 -0.21 -0.225 -0.241 -0.416🞶 -0.356🞶 -0.385🞶   PDSI -0.161 -0.106 -0.255 -0.264 -0.278 -0.585🞶🞶   RH 0.27 0.179 0.423🞶 0.151 -0.064 -0.163 PDSI VMC0 − 10 -0.251 -0.459🞶 -0.365🞶 -0.527🞶🞶 -0.004 0.173   VMC10 − 40 -0.213 -0.451🞶 -0.317 -0.542🞶🞶 -0.118 0.164   VMC40 − 100 -0.14 -0.156 -0.343 -0.5🞶🞶 -0.239 0.119   VPD 0.057 0.432 -0.141 -0.314 0.003 -0.021   RH 0.072 -0.396🞶 0.251 -0.028 -0.042 -0.022 RH VMC0 − 10 -0.356🞶🞶 -0.377🞶 -0.392🞶 -0.53🞶🞶 -0.213 -0.13   VMC10 − 40 -0.289 -0.38🞶 -0.365🞶 -0.542🞶🞶 -0.276 -0.274   VMC40 − 100 -0.214 -0.225 -0.28 -0.499🞶 -0.352🞶 -0.365🞶   VPD 0.296 0.259 0.367🞶 0.159 -0.042 -0.008   PDSI -0.208 -0.113 -0.242 -0.315 -0.277 -0.57🞶🞶 dicated p < 0.05 and p < 0.01, respectively. The VMC0-10 represents the volume moisture content of 0-10cm soil, the  olume moisture content of 10-40cm soil, he VMC40-100 represents the volume moisture content of 40-100cm soil. Note: * and ** indicated p < 0.05 and p < 0.01, respectively. 2 Difference strategies of season in soil moisture use dynamics for tree growth under different wet and d In climate-sensitive areas, soil moisture is critical to tree growth. A variety of factors can alter soil water uptake by trees, such as season (Rao et al. 2020), life history stage (Van et al. 2017), tree species (Schwendenmann et al. 2015), water availability and depth of plant access to water (Rao et al., 2020), and the amount of recent rainfall (Xu et al. 2011). In our study, δ18OTR was found to be significantly correlated with soil moisture during the growing season, but the seasonal response in arid and wet zones showed inconsistency, with tree-ring δ18O responding to soil moisture later in arid zones than in wet zones (Fig. 7). Specifically, in the humid areas, we found that δ18OTR significantly negatively correlated with soil moisture from June to August ((Fig. 7). Because the three humid regions are influenced by the Indian summer winds and receive more precipitation from June to August, which further affects δ18OTR by influencing soil water 18O through infiltration. The JA and GA regions showed a significant negative correlation between δ18OTR and July-September precipitation, Indian monsoon index, and monsoon circulation intensity in the region (Xu et al. 2018). And we also found a significant negative correlation between δ18OTR and soil moisture during July-August in JA and GA regions. And meanwhile, δ18OTR values of different tree species in the WA region were mainly influenced by summer precipitation since 1743 (Masaki et al. 2012). In our study, we also found that δ18OTR in the WA region was significantly negatively correlated with soil moisture in July and August. We suggest that summer winds bring abundant precipitation, and that plant roots take up water in the soil through a series of transpiration infiltration processes, finally retaining the isotopic signal of precipitation in the xylem of trees expressed through δ18OTR in humid regions. We also found the response of δ18OTR in arid regions significantly correlated with soil moisture from July to September, especially in September (Fig. 7). But the response of δ18OTR to soil moisture in arid regions was lagged compared to wet regions, which may be closely related to the extended growing season of trees due to warming and humidification of the Tibetan Plateau in recent decades (zhang et al. 2021). However, the specific response varied among the three arid regions. 4.1 The δ 18 O TR records soil water use by tree growth in different regions of the Tibetan Plateau 4.1 The δ 18 O TR records soil water use by tree growth in different regions of the Tibetan Plateau Since the 1960s, the surface temperature of the Tibetan Plateau has shown a continuous warming trend (Duan et al. 2015), and precipitation has increased in the western and eastern parts of the plateau (Bao et al. 2019; Liu et al. 2020), but weakened in the southwestern part of the plateau (Hu et al. 2021). In such a climatic change context, trees may adopt different water use strategies to maintain their growth. It has been shown that δ18OTR could reflect the seasonality and the depth of plant water uptake (Gessler et al. 2014). This means that information on the variation of soil water δ18O at different seasons and depths is stored and recorded in the tree-ring (Huang et al. 2019, Xu et al. 2020). Page 6/20 Page 6/20 The δ18OTR variations are mainly regulated by a combination of the oxygen isotope composition of the source water, and the external climate that affects fractionation processes within the tree (Roden et al. 2000). In our results, the δ18OTR was more sensitive to moisture factors than to temperature factors, and in particular δ18OTR showed the significant negative response to soil moisture during the growing season (Fig. 5). The δ18OTR is derived from soil water, but that soil water δ18O varies with soil depth (Liu et al. 2017), which is consistent with our findings. In addition, water enters the leaves from the soil through the xylem and then the lighter isotope (16O) evaporates more readily than the heavier isotope (18O), leading to an enrichment of δ18O in leaf water (Farquhar et al. 1993). Trees take up soil water directly through the root system and retain the isotopic signal in soil water in the xylem through a series of intra-tree fractionation processes. Therefore, the δ18OTR is a true reflection of soil water use by trees based on the processes of water uptake, transport and dissipation in trees. At the same time, our study found that δ18OTR values were significantly greater in arid than in humid areas (Fig. 3), due to the fact that transpiration affects leaf water enrichment by replenishing unenriched soil water to the leaves. 4.1 The δ 18 O TR records soil water use by tree growth in different regions of the Tibetan Plateau On the one hand, the δ18OTR is mainly negatively correlated with stomatal conductance, and reduced soil moisture and humidity enhance evaporative enrichment of leaf and soil water through effects on stomatal conductance and the ratio of internal leaf CO2 to atmospheric CO2 in arid environments (Mirfenderesgi et al. 2016). On the other hand, soil water evaporation is enhanced in arid environments and the source water absorbed by tree roots becomes heavier, leading to an enrichment of oxygen isotopes in leaf water (Roden et al. 2000). Sucrose produced by photosynthesis is transported through the bast to the xylem of the trunk for cellulose synthesis, and during the conversion to cellulose, oxygen isotopes in the sugar are partially exchanged with oxygen isotopes in the source water (Zhan et al. 2021), resulting in a mixed signal of both source water and transpiration-enriched oxygen isotopes in the cellulose oxygen isotope ratio of the tree-ring (Giraldo et al, 2022). These two processes result in a higher δ18OTR in arid areas than in humid areas. Similarly, a moist atmosphere reduces transpiration and thus 18O water enrichment in needles, further leading to lower δ18OTR in humid areas than in arid areas. And precipitation δ18O is inversely related to precipitation, more precipitation leads to lower precipitation δ18O values, resulting in weaker δ18O enrichment of water from water sources absorbed by trees, both of which make δ18OTR in wet areas lower than in arid areas (Yang et al. 2012). 2 Difference strategies of season in soil moisture use dynamics for tree growth under different wet and d The δ18OTR in the MA region was controlled by hydroclimatic variables such as summer monsoon precipitation, RH and PDSI (Masaki et al. 2017). Those results suggesting that monsoon precipitation has a significant effect on tree growth. Tree growth in the KA region is mainly influenced by winter snowfall, and although trees are still affected by precipitation δ18O during summer precipitation, the melting snowfall provides a more stable water source, resulting in a much lower correlation between δ18OTR and summer soil moisture in this region than in the other two regions (Kerstin et al. 2006). In the driest DE region, the δ18OTR were controlled mainly through soil moisture and precipitation variability from May to September (Yang et al. 2006), and our study provided further evidence that δ18OTR in this region was significantly negatively correlated with soil moisture in September. Page 7/20 Page 7/20 Page 7/20 In the meantime, it has been found that precipitation has a greater effect on soil moisture than temperature on the southeastern Tibetan Plateau. Soil moisture is significantly correlated with oxygen isotope growing season in the southeastern Tibetan Plateau trees (Wang et al. 2023).And the δ18OTR were found to be significantly negatively correlated with growing season precipitation in the United Kingdom (Giles et al. 2015), southern Mexico (Brienen et al. 2013), northwestern Tibetan Plateau (Zhang et al. 2021), and Ordos Plateau (Li et al. 2019), which was broadly in line with our findings that summer rainfall to supplement plant water availability during the growing season (Hahm et al. 2019). We speculated that this were two reasons for the lagging response of δ18OTR to soil moisture in arid areas relative to humid areas. On the one hand, it might be because the Indian summer winds, which firstly reached the southern region, making precipitation events occur earlier in the southern region in their northward push. Therefore, they affect the tree growth process through a series of fractionation as well as plant physiological processes, then reflecting different moisture. On the other hand, this is closely related to the warming and humidification in the western and northern parts of the plateau, where warmer temperatures and increased precipitation lead to an earlier and longer growing season for trees. 4.3 Difference strategies of depth in soil moisture use dynamics for tree growth under different wet and dry gradients on the Tibetan Plateau Precipitation on the Tibetan Plateau, where the natural environment is extremely fragile and sensitive, is highly spatially variable with annual precipitation decreasing gradually with latitude (Li et al. 2022). Therefore, trees might maintain their growth by altering their water use strategies to absorb soil moisture at different depths under different precipitation conditions. Most studies have shown that trees in arid environments tend to absorb deeper water to resist drought (Hu et al. 2021), whereas trees in wet environments tended to use shallow soil water (Holdo et al. 2018). While this was supported in our study, we have also found that trees in complex climatic environments may develop complex root systems with both lateral and deep roots, adjusting their water use strategies to maintain their growth in response to environmental changes. Our study also found that, the δ18OTR in the JA region was significantly negatively correlated with 0–10 cm soil moisture in a wet environment, while that in the WA and GA regions were significantly negatively correlated with 0–10 cm and 10–40 cm soil moisture after controlling the VPD and RH (Table 2), it suggesting that trees in humid areas tended to take up soil moisture in the surface and middle layers. Other studies also found shallow soil water (35%) and precipitation (32.5%) were the main sources of water for trees in wet areas when annual precipitation was greater than 800 mm (Zhou et al. 2011). Meanwhile, the topsoil in humid areas is the most active area for microbial activity, so that it provides nutrients to the trees (Priyadarshini et al. 2016). For example, it has been shown that trees had a relatively large range of source water and a good water use strategy in the shallow soil under humid climate conditions (Sun et al. 2019). In arid environments, the significant negative correlation between δ18OTR and 40–100 cm soil moisture in the KA and DE regions was revealed after controlling VPD and RH (Table 2), it suggesting that trees in arid environments utilize more stable deep soil moisture. And trees in DE region preferred to take up deep soil moisture through a study of cypress in the northeastern Tibetan Plateau (Grossiord et al. 2016), which validates our results. 4.3 Difference strategies of depth in soil moisture use dynamics for tree growth under different wet and dry gradients on the Tibetan Plateau In arid environments, capillary water induces trees to form deeper root systems to absorb deep soil water and groundwater to meet higher transpiration needs during the dry season, while deep root systems provide a more consistent and reliable source of water for trees (Lindh et al. 2014). But we also found the highly significant negative correlations between δ18OTR and soil moisture at all soil depths in MA, and there might be two factors. On the one hand, soil moisture δ18O fractionation occurs when precipitation infiltrates into the soil (Oerter et al. 2014), creating an isotopic gradient in the vertical profile while vigorous evaporation may mask some of the isotopic signal (Dai et al. 2015). On the other hand, it is possible that the 'bimodal' precipitation pattern in this region encourages trees to develop lateral and deep root systems, using the surface root system to obtain water from the upper soil layer when precipitation is adequate. The deep root system is used to obtain water from deep soil or groundwater when precipitation is insufficient (zhang et al. 2017). This confirms our finding that trees adjust their water use strategies in time to sustain self-sustaining growth (Zhao et al. 2018; Nolan et al. 2018). Trees under different environmental conditions have different coping strategies to climate extremes, and the resilience of trees to drought is closely related to regional soil moisture, diurnal temperature differences, and plant physiological characteristics (Fang et al. 2018). It has been reported that tree growth on the Tibetan Plateau is more vulnerable to challenges in the context of climate change (Zhang et al. 2021; Yao et al. 2023). Our results suggest that trees in arid areas are better able to cope with drought stress due to their use of more stable deep soil water, whereas trees in humid areas may be more vulnerable to drought stress in the context of climate variability due to their use of surface-middle soil water. As a result, trees in wetter areas will face greater challenges to survival in the context of a weakening southwestern monsoon with reduced precipitation and continued warming temperatures. Moreover, it is noteworthy that trees develop the water use strategy of absorbing moisture from different soil layers with the stronger lateral and deep root systems as the environment changes, which can more be beneficial to trees in coping with complex environment. 2 Difference strategies of season in soil moisture use dynamics for tree growth under different wet and d Moreover, precipitation in early in the growing season was not replenished in time, while both precipitation and temperature at the end of the growing season promoted tree growth, making the response of δ18OTR to soil moisture slightly later in the arid areas than in the humid areas (Guo et al. 2022). 5. Conclusion Page 8/20 The climate of the Tibetan Plateau has changed significantly in recent decades. It is of great significance to study the water use strategies of trees in this region for monitoring its ecological environment. Therefore, the relationship between oxygen isotope data and environmental factors in tree Page 8/20 ring on the Tibetan Plateau were investigated. We found that δ18OTR recorded spatial and temporal differences in soil moisture use dynamics by tree growth under different wet and dry gradients. Our results showed that δ18OTR responded more strongly to soil moisture than other environmental factors during the growing season under different wet and dry gradients, but the response in the arid areas had a lag compared to the humid areas. Meanwhile, trees in the humid southwestern region absorbed more shallow soil water, while trees in the arid western and northeastern regions absorbed more deep soil water. We suggest that trees use shallow soil water alone increase the risk of drought stress in a southwestern context of decreasing precipitation and increasing temperatures, whereas trees develop both lateral and deep root systems are better able to survive in complex environments. As a water tower in Asia, the Tibetan Plateau has an important influence on the climate of the Northern Hemisphere. We recommend the use of this method for larger-scale and more regional studies to research the response strategies of trees and ecological changes in the context of climate change. ring on the Tibetan Plateau were investigated. We found that δ18OTR recorded spatial and temporal differences in soil moisture use dynamics by tree growth under different wet and dry gradients. Our results showed that δ18OTR responded more strongly to soil moisture than other environmental factors during the growing season under different wet and dry gradients, but the response in the arid areas had a lag compared to the humid areas. Meanwhile, trees in the humid southwestern region absorbed more shallow soil water, while trees in the arid western and northeastern regions absorbed more deep soil water. We suggest that trees use shallow soil water alone increase the risk of drought stress in a southwestern context of decreasing precipitation and increasing temperatures, whereas trees develop both lateral and deep root systems are better able to survive in complex environments. As a water tower in Asia, the Tibetan Plateau has an important influence on the climate of the Northern Hemisphere. 5. Conclusion We recommend the use of this method for larger-scale and more regional studies to research the response strategies of trees and ecological changes in the context of climate change. Data Availability Statemen All data sources analyzed in this study are included in this paper. Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. Funding This research was supported by CAS “Light of West China” Program (2020XBZG-XBQNXZ-A), Basic Research Innovation Group Project of Gansu Province (No. 22JR5RA129), and the 2022 Major scientific Research Project Cultivation Plan of Northwest Normal University (WNU-LKZD2022-04). We also thank the anonymous referees for helpful comments on the manuscript. References 1. Amer A, Franceschi E, Hjazin A (2023). Structure and Ecosystem Services of Three Common Urban Tree Species in an Arid Climate City. Forests 14(4): 671 2. An W., Hou S, Zhang Q, Zhang W, Wu, S, Xu H, Liu Y (2017) Enhanced recent local moisture recycling on the northwestern Tibetan Plateau deduced from ice core deuterium excess records. Journal of Geophysical Research: Atmospheres 122(23): 12-541 2. An W., Hou S, Zhang Q, Zhang W, Wu, S, Xu H, Liu Y (2017) Enhanced recent local moisture recycling on the northwestern Tibetan Plateau deduced from ice core deuterium excess records. 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Zeng X, Li X, Evans MN, Wang W, An W, Xu G, Wu G (2016) Seasonal incursion of Indian Monsoon humidity and precipitation into the southeastern Qinghai–Tibetan Plateau inferred from tree ring δ18O values with intra-seasonal resolution. Earth and Planetary Science Letters 443: 9-19 85. References Environmental research 203: 111894 Page 11/20 Page 11/20 64. Su Zhou, T, Liu, Chen, Y, Shang H, Zhu L, Sha Y (2018) Linkages of the dynamics of glaciers and lakes with the climate elements over the Tibetan Plateau. Earth-Science Reviews 185: 308-324 65. Tan L, Cai Y, Cheng, Edwards LR, Lan J, Zhang H, Gao Y (2018) High resolution monsoon precipitation changes on southeastern Tibetan Plateau over the past 2300 years. Quaternary Science Reviews,195: 122-132 65. Tan L, Cai Y, Cheng, Edwards LR, Lan J, Zhang H, Gao Y (2018) High resolution monsoon precipitation changes on southeastern Tibetan Plateau over the past 2300 years. Quaternary Science Reviews,195: 122-132 66. Van T, Verheyen K, Kint V, Van E, Muys B (2017) Plasticity of tree architecture through interspecific and intraspecific competition in a young experimental plantation. Forest Ecology and Management 385: 1-9 66. Van T, Verheyen K, Kint V, Van E, Muys B (2017) Plasticity of tree architecture through interspecific and intraspecific competition in a young experimental plantation. Forest Ecology and Management 385: 1-9 67. Wang H, Tetzlaff D, Soulsby C (2018) Modelling the effects of land cover and climate change on soil water partitioning in a boreal headwater catchment. Journal of Hydrology, 558: 520-531 7. Wang H, Tetzlaff D, Soulsby C (2018) Modelling the effects of land cover and climate change on soil w catchment. Journal of Hydrology, 558: 520-531 68. Wang J, Taylor AR, D’Orangeville L (2023) Warming-induced tree growth may help offset increasing disturbance across the Canadian boreal forest. Proceedings of the National Academy of Sciences,120(2): e2212780120 69. Wan L, Liu H, Leavit S, Cresse EL, Quine TA, Shi J, Shi S (2021) Tree-ring δ18O identifies similarity in timing but differences in depth of soil water uptake by trees in mesic and arid climates. Agricultural and Forest Meteorology 308: 108569 70. Wernicke J, Grießinger J, Hochreuther P, Bräuning A (2015) Variability of summer humidity during the past 800 years on the eastern Tibetan Plateau inferred from δ 18O of tree-ring cellulose. Climate of the Past 11(2): 327-337 71. Wong J, Fong A, McVicar N, Smith S, Giambattista J, Wells D, Alexander A (2020) Comparing deep learning-based auto-segmentation of organs at risk and clinical target volumes to expert inter-observer variability in radiotherapy planning. Radiotherapy and Oncology 144: 152- 158 72. References Wu G, Liu Y, Zhang Q, Duan A, Wang T, Wan R, Liang X (2007) The influence of mechanical and thermal forcing by the Tibetan Plateau on Asian climate. Journal of Hydrometeorology 8(4): 770-789 73. Wu H, Li XY, Zhang J, Li J, Liu J, Tian L, Fu C (2019) Stable isotopes of atmospheric water vapor and precipitation in the northeast Qinghai‐ Tibetan Plateau. Hydrological Processes 33(23): 2997-3009 73. Wu H, Li XY, Zhang J, Li J, Liu J, Tian L, Fu C (2019) Stable isotopes of atmospheric water vapor and precipitation in the northeast Qinghai‐ Tibetan Plateau. Hydrological Processes 33(23): 2997-3009 74. Xiao H, Deng W, Wei G, Chen J, Zheng X, Shi T, Zeng T (2020) A pilot study on zinc isotopic compositio Geochemistry, Geophysics, Geosystems 21(11): e2020GC009430 74. Xiao H, Deng W, Wei G, Chen J, Zheng X, Shi T, Zeng T (2020) A pilot study on zinc isotopic compositions in shallow‐water coral skeletons. Geochemistry, Geophysics, Geosystems 21(11): e2020GC009430 75. Xu C, Zhao Q, An W, Wang S, Tan N, Sano M, Guo Z (2021) Tree-ring oxygen isotope across monsoon Asia: Common signal and local influence. Quaternary Science Reviews 269: 107156 76. Xu C, Zhu H, Nakatsuka T, Sano M, Li Z, Shi F, Guo Z (2019) Sampling strategy and climatic implication of tree-ring cellulose oxygen isotopes of Hippophae tibetana and Abies georgei on the southeastern Tibetan Plateau. International journal of biometeorology 63: 679-686 77. Xu G, Liu X, Sun W, Szejner P, Zeng X, Yoshimura K, Trouet (2020). Seasonal divergence between soil water availability and atmospheric moisture recorded in intra-annual tree-ring δ18O extremes. Environmental Research Letters 15(9): 094036 78. Xu G, Wu G, Liu X, Chen T, Wang B, Hudson A, Trouet V (2020) Age‐related climate response of tree‐ring δ13C and δ18O from spruce in northwestern China, with implications for relative humidity reconstructions. Journal of Geophysical Research: Biogeosciences: 125(7): e2019JG005513 78. Xu G, Wu G, Liu X, Chen T, Wang B, Hudson A, Trouet V (2020) Age‐related climate response of tree‐ring δ13C and δ18O from spruce in northwestern China, with implications for relative humidity reconstructions. Journal of Geophysical Research: Biogeosciences: 125(7): e2019JG005513 79. Xu Q, Li H, Chen J, Cheng X, Liu S, An S (2011) Water use patterns of three species in subalpine forest, Southwest China: the deuterium isotope approach. Ecohydrology 4(2): 236-244 79. References Zhan N, Wang Z, Xie Y, Shang X, Liu G, Wu Z (2021) Expression patterns and regulation of non-coding RNAs during synthesis of cellulose in Eucalyptus grandis Hill. Forests 12(11): 1565 85. Zhan N, Wang Z, Xie Y, Shang X, Liu G, Wu Z (2021) Expression patterns and regulation of non-coding RNAs during synthesis of cellulose in Eucalyptus grandis Hill. Forests 12(11): 1565 86. Zhang F, Jia W, Zhu G, Shi Y, Zhang M (2022) Using stable isotopes to investigate differences of plant water sources in subalpine habitats. Hydrological Processes 36(2): e14518 86. Zhang F, Jia W, Zhu G, Shi Y, Zhang M (2022) Using stable isotopes to investigate differences of plant water sources in subalpine habitats. Hydrological Processes 36(2): e14518 87. Zhang J, Gou X, Alexander MR, Xia J, Wang F, Zhang F, Pederson N (2021) Drought limits wood production of Juniperus przewalskii even as growing seasons lengthens in a cold and arid environment. Catena 196: 104936 Page 12/20 Page 12/20 88. Zhang J, Gou X, Manzanedo RD, Zhang F, Pederson N (2018) Cambial phenology and xylogenesis of Juniperus przewalskii over a climatic gradient is influenced by both temperature and drought. Agricultural and forest meteorology 260: 165-175 88. Zhang J, Gou X, Manzanedo RD, Zhang F, Pederson N (2018) Cambial phenology and xylogenesis of Juniperus przewalskii over a climatic gradient is influenced by both temperature and drought. Agricultural and forest meteorology 260: 165-175 89. Zhou Y, Chen S, Song W, Lu Q, Lin G (2011) Water-use strategies of two desert plants along a precipitation gradient in northwestern China. Chinese Journal of Plant Ecology 35(8): 789-80 89. Zhou Y, Chen S, Song W, Lu Q, Lin G (2011) Water-use strategies of two desert plants along a precipitation gradient in northwestern China. Chinese Journal of Plant Ecology 35(8): 789-80 Figures Figures Figure 1 Geographical locations of study area and sampling sites. The green line in the figure represents the boundary line of the Tibetan Plateau, the green dots are the tree wheel sampling points, the different shades of blue represent the different amounts of rainfall in the region, and the darker the color means more precipitation. Figure 1 Figure 1 Geographical locations of study area and sampling sites. The green line in the figure represents the boundary line of the Tibetan Plateau, the green dots are the tree wheel sampling points, the different shades of blue represent the different amounts of rainfall in the region, and the darker the color means more precipitation. Page 13/20 Page 13/20 Page 13/20 Figure 2 The time series of the δ18OTR for each site. The red solid line indicates the trend for each chronology. Figure 2 The time series of the δ18OTR for each site. The red solid line indicates the trend for each chronology. Page 14/20 Page 14/20 Page 14/20 Figure 3 Significance test for study sites. The box in the figure represents the average value of δ18OTR for each point from 1948 to 1998. Inter-sample differences in δ18OTR values were assessed using one-way ANOVA, with different letters indicating significant differences at the 0.05 confidence level. And the red dashed line represents the mean value of δ18OTR in the arid areas and the blue dashed line represents the mean value of δ18OTR in the humid areas. Figure 3 Significance test for study sites. The box in the figure represents the average value of δ18OTR for each point from 1948 to 1998. Inter-sample differences in δ18OTR values were assessed using one-way ANOVA, with different letters indicating significant differences at the 0.05 confidence level. And the red dashed line represents the mean value of δ18OTR in the arid areas and the blue dashed line represents the mean value of δ18OTR in the humid areas. Page 15/20 Figure 4 Changes of soil moisture at different soil layers and annual scales. The squares in the box line diagram represent the median volumetric water content, the black dots represent the outliers and the range of the boxes represents the 25%-75% data interval (a). The blue line in the graph represents the 40-100 cm volumetric water content, the red line represents the 10-40 cm volumetric water content and the black line represents the 0-10 cm volumetric water content (b). Figure 4 Changes of soil moisture at different soil layers and annual scales. The squares in the box line diagra content, the black dots represent the outliers and the range of the boxes represents the 25%-75% data represents the 40-100 cm volumetric water content, the red line represents the 10-40 cm volumetric w 0-10 cm volumetric water content (b). Figure 1 Figure 4 Changes of soil moisture at different soil layers and annual scales. The squares in the box line diagram represent the median volumetric water content, the black dots represent the outliers and the range of the boxes represents the 25%-75% data interval (a). The blue line in the graph represents the 40-100 cm volumetric water content, the red line represents the 10-40 cm volumetric water content and the black line represents the 0-10 cm volumetric water content (b). Page 16/20 Figure 5 Interannual variation of soil moisture. The blue line represents 40-100cm volumetric water content, the red line represents 10-40cm volumetric water content, the black line represents 0-10cm volumetric water content, and the grey dashed line represents the trend line. Figure 5 Interannual variation of soil moisture. The blue line represents 40-100cm volumetric water conte water content, the black line represents 0-10cm volumetric water content, and the grey dashed li Figure 5 Interannual variation of soil moisture. The blue line represents 40-100cm volumetric water content, the red line represents 10-40cm volumetric water content, the black line represents 0-10cm volumetric water content, and the grey dashed line represents the trend line. Page 17/20 Page 17/20 Page 17/20 Figure 6 Correlations of δ18OTR with monthly climate variables (temperature, PDSI, VPD, RH, PRCP). The “+” and “*” indicate significant correlations at the 95% and 99% level, respectively. The Tmax stands for maximum temperature, T stands for mean temperature, the Tmin stands for minimum temperature, PDSI stands for Palmer Drought Index, VPD stands for vapor pressure deficit, RH stands for relative humidity, PRCP stands for precipitation. Figure 6 Correlations of δ18OTR with monthly climate variables (temperature, PDSI, VPD, RH, PRCP). The “+” and “*” indicate significant correlations at the 95% and 99% level, respectively. The Tmax stands for maximum temperature, T stands for mean temperature, the Tmin stands for minimum temperature, PDSI stands for Palmer Drought Index, VPD stands for vapor pressure deficit, RH stands for relative humidity, PRCP stands for precipitation. Page 18/20 Figure 7 Correlations of δ18OTR with monthly soil moisture. The "+" represents p<0.05, "*" represents p<0.01, SM40-100 cm represents 40-100 cm soil moisture, SM10-40 cm represents 10-40 cm soil moisture, SM0-10 cm represents 0-10 cm soil moisture. Figure 7 Figure 8 Figure 8 The spatial correlation between the June-September VWC in each soil layer and δ18OTR. The green point in the figure represents δ18OTR sampling points and the black line is the Tibetan Plateau boundary line. Appendix.docx Figure 7 Correlations of δ18OTR with monthly soil moisture. The "+" represents p<0.05, "*" represents p<0.01, SM40-100 cm represents 40-100 cm soil moisture, SM10-40 cm represents 10-40 cm soil moisture, SM0-10 cm represents 0-10 cm soil moisture. Page 19/20 Figure 8 The spatial correlation between the June-September VWC in each soil layer and δ18OTR. The green point in the figure represents δ18OTR sampling points and the black line is the Tibetan Plateau boundary line. S l il Figure 8 The spatial correlation between the June-September VWC in each soil layer an points and the black line is the Tibetan Plateau boundary line. Figure 8 The spatial correlation between the June-September VWC in each soil layer and δ18OTR. The green point in the figure represents δ18OTR sampling points and the black line is the Tibetan Plateau boundary line. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 20/20
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How to collect non-medical data in a pediatric trial: diaries or interviews
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To cite this version: Anaïs Le Jeannic, Hassani Maoulida, Sophie Guilmin-Crépon, Corinne Alberti, Nadia Tubiana-Rufi, et al.. How to collect non-medical data in a pediatric trial: diaries or interviews. Trials, 2020, 21 (1), pp.36. ￿10.1186/s13063-019-3997-9￿. ￿inserm-02468132￿ How to collect non-medical data in a pediatric trial: diaries or interviews Anaïs Le Jeannic, Hassani Maoulida, Sophie Guilmin-Crépon, Corinne Alberti, Nadia Tubiana-Rufi, Isabelle Durand-Zaleski HAL Id: inserm-02468132 https://inserm.hal.science/inserm-02468132v1 Submitted on 5 Feb 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Le Jeannic et al. Trials (2020) 21:36 https://doi.org/10.1186/s13063-019-3997-9 Open Access Abstract Background: Non-medical data, such as the amount of time that patients and caregivers spend managing their condition, may be relevant when assessing therapeutic strategies. For chronic pediatric conditions, the time that patients and caregivers spend in seeking and providing care (which are the indirect costs in an economic evaluation) can be significantly different depending on the treatment arm. To explore methods for collecting information on the care burden for caregivers and patients, we investigated whether a patient diary provided additional information compared to retrospective investigator-led interviews and whether a diary that was completed intermittently produced more or less information than a diary completed continually. The main objective of this study was to identify which type of data collection was most effective for measuring the time spent by caregivers and for estimating indirect treatment costs over 9 months. Methods: Start-In! is a randomized controlled trial comparing the efficacy of three strategies of real-time continuous glucose monitoring for 12 months in children and adolescents with type 1 diabetes. We designed an ancillary study to assess methods of collecting information on the time spent by patients and caregivers in managing their condition (indirect costs). Data were entered retrospectively in case report forms (CRFs) by investigators during quarterly follow- up visits, which were supplemented with diaries completed prospectively by children or caregivers either continuously or intermittently. Data about absences from school and work as well as the time that caregivers spent on diabetes care were collected and the three collection methods were compared. Results: At the end of the 9-month study, 42% of the study participants failed to return their diary. For the diaries that were received, less than 10% of expected data were collected versus 82% during investigators'interviews. Based on all the information collected, we calculated that over 9 months, caregivers lost on average 3.9 days of working time (€786) and 4 days of personal time, i.e. the equivalent of €526, and spent around 15 min of time on care per day, i.e. the equivalent of €1700. Conclusions: The CRFs completed by investigators during quarterly visits cannot be replaced by a diary. Completing the diaries appeared to represent an important additional burden to children and their caregivers, and the diaries provided little additional information compared to investigators’ entries in the CRF. Trial registration: ClinicalTrials.gov, NCT00949221. Registered on 30 July 2009. How to collect non-medical data in a pediatric trial: diaries or interviews Anaïs Le Jeannic1,2,3* , Hassani Maoulida1, Sophie Guilmin-Crépon2,4,5,6,7, Corinne Alberti2,4,5,7, Nadia Tubiana-Rufi5,6 and Isabelle Durand-Zaleski1,8,9 Anaïs Le Jeannic1,2,3* , Hassani Maoulida1, Sophie Guilmin-Crépon2,4,5,6,7, Corinne Alberti2,4,5,7, Nadia Tubiana-Rufi5,6 and Isabelle Durand-Zaleski1,8,9 Background The clinical study had two phases: all subjects wore glu- cose monitors during the first 3 months, and thereafter were randomized into three groups with different strat- egies for glucose monitoring during the next 9 months. The ancillary study reported here concerns the 9-month follow-up period of the patients (Fig. 1). The Start-In! economic evaluation required informa- tion on consultations, absences from work or school due to diabetes, and the time spent by the caregiver on aver- age each day on providing diabetes care. This informa- tion was entered for all patients by the investigators in paper case report forms (CRF) at the end of each trimes- ter and represented the reference strategy. To determine the best way to collect information on care burden and time costs, we investigated whether or not a patient-completed diary would provide additional informa- tion compared to retrospective investigator-led interviews and whether a diary completed intermittently would produce more or less information than a continuous diary. We hypothesized first that a diary would enable investiga- tors to shorten their interviews, which would be helped by or even replace the diaries. We assumed that patients would tend to underreport their time costs. Our second hy- pothesis was that keeping a continuous diary would be an onerous task and could lead to fatigue and poor data collec- tion over time. However, the intermittent diary, while redu- cing the data entry burden for patients [8], has the risk that patients may fail to complete it if it was not a regular task. gy To test whether diaries would provide additional informa- tion, patients were randomized into two groups. In the con- tinuous group, children and caregivers were asked to collect data in a diary without interruption during one of the three trimesters of the study (3 months continuously). In the intermittent group, data were collected only during the last month of each of the three trimesters. Randomization was in a 3:1: ratio. In the continuous group, the diary was com- pleted daily for one of the three trimesters of the study follow-up period, allocated randomly in a 1:1:1 ratio. To simplify the work for the investigators, the randomization for the ancillary study was by center and not by patient (Figs. 2 and 3). Background In! trial (NCT00949221) that compared the efficacy and cost-effectiveness of three strategies of real-time continu- ous glucose monitoring (RT-CGM) for children with type 1 diabetes [9]. This trial was well adapted to our study ob- jective since insulin-dependent diabetes in children is a chronic condition with major time costs to children and their parents. In! trial (NCT00949221) that compared the efficacy and cost-effectiveness of three strategies of real-time continu- ous glucose monitoring (RT-CGM) for children with type 1 diabetes [9]. This trial was well adapted to our study ob- jective since insulin-dependent diabetes in children is a chronic condition with major time costs to children and their parents. g Non-medical data, such as the time spent by patients and caregivers in managing their condition, may be relevant in assessing the full burden and cost of a therapeutic strategy. The pertinence of including time costs for caregivers and patients as indirect costs in economic evaluations of thera- peutic strategies may be considered when writing the protocol and mainly depends upon the perspective of the evaluation and the type of illness being considered [1]. Productivity losses can represent a significant amount of re- sources, especially for chronic pediatric conditions because caregivers may need to seek and also provide care for chil- dren. Information on absences from work or school is not recorded in health insurance databases, so must be esti- mated, either through retrospective interviews with an in- vestigator [2] or from diaries completed by the parents or patients [3, 4]. Investigator-led interviews require additional work from the physicians, which could be reduced if the children or their caregivers completed diaries prospectively [5]. However, the use of paper or electronic diaries in clin- ical trials requires logistical management and fastidiousness from participants, especially for studies with long follow-up periods [6]. This may be more problematic for pediatric tri- als, since parental constraints may influence the acceptance of additional methods of data collection [7]. The patients in Start-In! were aged from 2 to 17 years. They had been diagnosed with type 1 diabetes for more than 1 year and were being treated by intensive insulin therapy, achieving inadequate metabolic control. The study was conducted in 11 pediatric diabetology units with expertise in pump therapy and CGM. The primary endpoint was long-term glycemic control at 12 months. Abstract Registry name: Study of Insulin Therapy Augmented by Real Time Sensor in Type 1 Children and Adolescents (START-IN!). Keywords: patient diary, data collection, time costs, investigator-led interview © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: anais.lejeannic@urc-eco.fr 1AP-HP, Groupe hospitalier Hôtel-Dieu, URC Economie de la Santé Ile de France, Paris, France 2Inserm, ECEVE UMR-S 1123, Paris, France Full list of author information is available at the end of the article * Correspondence: anais.lejeannic@urc-eco.fr 1AP-HP, Groupe hospitalier Hôtel-Dieu, URC Economie de la Santé Ile de France, Paris, France 2Inserm, ECEVE UMR-S 1123, Paris, France Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Le Jeannic et al. Trials (2020) 21:36 Le Jeannic et al. Trials (2020) 21:36 Page 2 of 7 Page 2 of 7 Background Data from the patient interviews and diaries were entered into the CRFs by investigators following the same schedule: one trimester for the continuous group and the last month of each trimester for the intermittent group. The main objective of this study was to identify which type of data collection method was most effective for obtaining information on care burden and time costs over the 9 months of a pediatric trial. The secondary objectives were to determine the patient acceptance of an intermit- tent versus a continuous diary and to check whether the amount of data collected decreased over time. Endpoint The primary endpoint was the amount of information re- corded in the diaries, compared to the amount of informa- tion given to the investigator during the quarterly follow-up visit. The Start-In! participants (patients and caregivers) were asked to record in their diary: (1) for the child, the dates of each period of absence from school or work, (2) for an adult caregiver, the dates of each period of absence from work, (3) the date of each consultation, (4) the number of caregivers present at each consultation, and (5) an estimate of the time Methods Study design Start-In! was a French multi-center trial with a random- ized controlled prospective open parallel-group design, comparing three therapeutic modalities in pediatric man- agement [9]. We designed an ancillary study to the Start- Le Jeannic et al. Trials (2020) 21:36 Le Jeannic et al. Trials Page 3 of 7 Fig. 1 Design of the Start-In! trial. The discontinuous group used intermittent RT-CGM alternating with SMBG. RT-CGM real-time continuous glucose monitoring, SMBG self-monitoring blood glucose spent on average each day by the caregivers to provide dia- betes care such, as blood glucose monitoring and insulin ad- ministration (Additional file 1). There was no minimum or maximum for the number of absences or visits that could be recorded. The same information was also collected quarterly by the investigators and entered in the CRF with the help of the diary, which was collected at the same time. entered in the diaries for each patient. If the amount of time spent by the caregiver on average each day was not completed, it was considered to be missing data rather than zero. If the other data items were not completed at all (dates of absences from school or work, and dates of consultations), we considered that the data were missing if the average time spent by the caregiver was also miss- ing. The overall amount of information recorded was compared between the intermittent and continuous groups, and with the amount of information entered by the investigators into the CRFs. Analysis y The number of absences from work or school and the number of consultations were calculated from the data Fig. 2 Groups for the Start-In! trial and the present study. Groups 1, 2, and 3 in the Start-In! trial used classic SMBG, 100% RT-CGM, and 40% RT-CGM, respectively. CG continuous group, IG intermittent group, RT-CGM real-time continuous glucose monitoring, SMBG self-monitoring blood glucose Le Jeannic et al. Trials (2020) 21:36 Page 4 of 7 Fig. 3 Timeline of data collection for each randomization group. T0 marks the end of the third month of Start-In! Blue ovals represent the continuous groups (CG1, CG2, and CG3) and orange circles represent the intermittent group (IG). CG, continuous group, CRF case report form, IG intermittent group Fig. 3 Timeline of data collection for each randomization group. T0 marks the end of the third month of Start-In! Blue ovals represent the continuous groups (CG1, CG2, and CG3) and orange circles represent the intermittent group (IG). CG, continuous group, CRF case report form, IG intermittent group Intermittent group Of the 36 patients, just under half (n = 16) returned at least one diary. Each patient should have submitted three diaries, one for each trimester, but only 41 of the 108 diaries expected were received. Of those 41 diaries, Continuous group For the indirect costs in the economic evaluation, the opportunity cost was based on the number of absences from work or school for the patients and their families. For those who were in employment, it was based on the average salary in France (€34 per hour) and for those who were unemployed, on the average salary of a house cleaner (€18.7 per hour). The average daily time spent by a caregiver administering diabetes care to a patient was valued based on the average salary of a home nurse (€28.8 per hour). All costs are in euros (€) for 2017 (1 € = 1.2US$) and were not discounted due to the short time horizon. Of the 92 patients, only 53.3% (n = 49) returned their diary. Of the 49 diaries returned, 44 were empty so data were considered to be missing. Four diaries reported that no absences had occurred and only one diary gave the date of an absence from work or school. That is, 89.8% of the diaries received had missing data. Overall, of the 92 diaries expected, 99.5% were either not re- ceived or had missing data. Results Table 1 Frequency, mean age, and ratio of boys to girls at Table 1 Frequency, mean age, and ratio of boys to girls at inclusion Centre Frequency Mean age (years) Sex ratio (boys/girls) 1 30 11.1 1.14 2 31 11.2 2.88 3 17 11.8 1.43 4 9 12.3 0.80 5 19 13.9 0.36 6 13 10.9 0.44 7 9 14.2 0.50 8 8 11.7 1.67 9 6 9.8 0.50 10 6 11.5 2.00 11 3 12.5 0.50 Overall 151 11.8 1.04 A total of 151 patients were randomized in the Start-In! trial, 52 into Group 1, 48 into Group 2, and 51 into Group 3. Their characteristics were similar at inclusion [9]. They had an average age of 12 years and there was nearly a 50:50 ratio of boys to girls, although there was large differences in age and sex between the centers, as seen in Table 1. A total of 23 patients who left the study were excluded from our ancillary analysis of the diary data, as their diaries would, obviously, be empty. Thus, diaries were expected from 128 patients for analysis, 92 in the continuous group and 36 in the intermittent group. Information on care burden was additionally col- lected on all 151 patients by the investigators during the follow-up visits. However, almost half of the 128 patients in this analysis did not return a single diary, so the infor- mation they reported to the investigators and entered in the CRF was based solely on their memory. Le Jeannic et al. Trials (2020) 21:36 Page 5 of 7 objective was to estimate the indirect medical costs in each treatment arm and identify possible differences that cannot be captured by calculating direct medical costs based upon information systematically retrieved from hospital insurance claims data. All investigators were asked to record in the CRFs data on caregiver and pa- tient burden (time), and they relied on information ob- tained from both interviews and diaries. Our study compared two prospective methods of documenting the treatment burden for caregivers and children with type 1 diabetes during a clinical trial, which included an eco- nomic evaluation. Our major finding was the magnitude of the data missing in the diaries provided to caregivers (around 99%). Results While the average cost of caregivers’ time could be estimated from investigator-led interviews to be around €3000 per patient over 9 months, the infor- mation from diaries alone did not allow a reliable estimate to be calculated. It would appear that asking families to record non-medical information in a diary during a pediatric trial of a severe chronic condition is infeasible, and we do not recommend their use. 97.6% had missing data, as only one diary was complete. Overall, 99.5% of diaries from patients were either not received or had missing data. Care burden and indirect costs Since the CRFs, which were based upon interviews and diaries, held about 80% of the indirect cost data, we calculated productivity losses and indirect costs. Over 9 months, parents lost on average 3.9 days of working time, i.e. the equivalent of €786, and 4 days of unemployed time, i.e. the equivalent of €526, and spent around 15 min of time on care per day, i.e. the equivalent of €1700. Children were absent from school for an average of 3 days over the 9 months. Many studies from different countries show a high concordance between self-reported and medical records of healthcare resource utilization in the general patient population [11, 12]. We were interested in pediatric tri- als, in which the diaries would be completed by care- givers, who may be tired from caring for a chronically ill child in addition to their jobs and may have other chil- dren to take care of. In fact, family members of someone with diabetes seems to be at higher risk of depression [13] and parents of a child with type 1 diabetes not only struggle with depression [14, 15] but also worry about hypoglycemia [16], which can lead to parental emotional Investigator-completed CRF The same information was collected retrospectively from the patients and caregivers by investigators, who entered it into the CRFs at the trimestral follow-up visits. The information was, potentially, supplemented by the data entered prospectively in the diaries. Table 2 shows the numbers of CRFs completed for each data item by the investigators for patients whose diaries were missing or empty, by randomization group (continuous or intermittent). For example, for the con- tinuous group, regarding absences from work, among the 87 patients who either returned an empty diary or no diary, this information was recorded for 73 in their CRF. For the consultations section, among the 35 patients from the intermittent group with empty or no diaries, this information was recorded for 34 in their CRF. We calculated the percentage of data missing from the CRFs in the same way we did for diaries. The continuous group had more data missing in their CRFs than the intermittent group (14% vs. 5%), which is far less than the around 99% of data missing from the diaries. In con- clusion, it appeared that for the diaries that were re- ceived, less than 10% of expected data were collected versus 82% during investigators'interviews. The amount of data entered by investigators for patients in the intermittent group was slightly higher than for the continuous group (95% vs. 86%). This may be because the participants were asked to remember information for only one month at the time of an interview instead of for three [10]. In this trial, the CRFs always contained more data than the diaries since the CRFs were based upon interviews supplemented by the diaries (if the diaries had data). In fact, diaries can be a reliable source of data to complement usual reporting methods, as shown by Dunn et al. [5], but we were not able to confirm this conclusion because of the amount of missing diary data. References 1. Haute Autorité de Santé - Choix méthodologiques pour l’évaluation économique à la HAS [Internet]. [cité 30 juill 2018]. Disponible sur: https:// www.has-sante.fr/portail/jcms/r_1499251/fr/choix-methodologiques-pour-l- evaluation-economique-a-la-has 1. Haute Autorité de Santé - Choix méthodologiques pour l’évaluation économique à la HAS [Internet]. [cité 30 juill 2018]. Disponible sur: https:// www.has-sante.fr/portail/jcms/r_1499251/fr/choix-methodologiques-pour-l- evaluation-economique-a-la-has Additional file 1. Data collected by patients in the diaries and also in the CRFs. 2. Laidi C, Prigent A, Plas A, Leboyer M, Fond G, Chevreul K, et al. Factors associated with direct health care costs in schizophrenia: Results from the FACE-SZ French dataset. European Neuropsychopharmacology. 2018;28(1):24–36. Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions ALJ wrote the manuscript. ALJ and HM carried out the analysis. IDZ supervised the methodology and statistical analysis. ALC, IDZ, SGC, CA and NTR contributed to the methodology. All authors revised the manuscript. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank Meryl Darlington for reviewing this work, as well as Neil Jouanjean and Céline Quelen for their help in managing the diary data. 3. Lin C-WC, March L, Crosbie J, Crawford R, Graves S, Naylor J, et al. Maximum recovery after knee replacement--the MARKER study rationale and protocol. BMC Musculoskelet Disord. 2009;10:69. 4. Rösch M, Leidl R, Tirpitz C, Reinshagen M, Adler G, König HH. Cost measurement based on a cost diary in patients with inflammatory bowel disease. Z Gastroenterol. 2002;40(4):217–28. Competing interests The authors declare that they have no competing interests. Received: 29 August 2019 Accepted: 13 December 2019 Received: 29 August 2019 Accepted: 13 December 2019 Received: 29 August 2019 Accepted: 13 December 2019 Discussion Some trials require non-medical data that are not readily available from patients’ charts or an insurance database. Investigators must either rely on patients’ recollection at the time of a follow-up visit or provide diaries. Our Table 2 Frequency of CRFs completed for each data item for patients not returning diaries or returning empty diaries Data item Continuous group, n (%) N = 87 Intermittent group, n (%) N = 35 Absences from school 78 (90) 34 (97) Absences from work 73 (84) 32 (91) Consultations 81 (93) 34 (97) Time spent on diabetes care 68 (78) 33 (94) Le Jeannic et al. Trials (2020) 21:36 Le Jeannic et al. Trials (2020) 21:36 Page 6 of 7 distress [17]. Difficulties are encountered with adoles- cent patients, who are becoming more independent but have troubled relationships with their parents [18] and may have transition issues, like treatment adherence [19, 20]. In those circumstances, reporting non-medical data, such as school absences, may not be a priority for the family. Funding This study was supported by Direction générale de l’offre de soin and sponsored by Assistance Publique, Hôpitaux de Paris (AP-HP). Unfortunately, the patients and their caregivers were not asked to give their opinions about the different methods of data collection, so we can only speculate about why so much data were missing. This study has other limitations. The amount of missing data prevented us from performing statistical tests or evaluating our secondary objectives of patient acceptance and fatigue. Moreover, with such little data, the quality of the infor- mation provided in the diaries could not be assessed and thus, only the quantity was explored in our trial. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-019-3997-9. y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-019-3997-9. Author details 1 1AP-HP, Groupe hospitalier Hôtel-Dieu, URC Economie de la Santé Ile de France, Paris, France. 2Inserm, ECEVE UMR-S 1123, Paris, France. 3URC Eco IdF (Paris health economics and health services research unit) and Inserm, ECEVE UMR-S 1123, Paris, France. 4AP-HP, Hôpital Universitaire Robert Debré, Unité d’Epidémiologie clinique, Paris, France. 5Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France. 6AP-HP, Hôpital Robert Debré, Service d’Endocrinologie-Diabétologie pédiatrique et Centre de référence des Maladies Endocriniennes Rares de la Croissance, Paris, France. 7CIC-EC 1426, Paris, France. 8AP-HP, Groupe hospitalier Albert Chenevier- Henri Mondor, Service de Santé Publique, Créteil, France. 9Inserm METHODS CRESS UMR 1153, Paris, France. Conclusion Trials that require non-medical data may need to rely on patients and families for information. In our study of a pediatric chronic condition, investigator-led interviews provided much more information than patient diaries. The completion of diaries appears to represent an import- ant additional burden to children and their families, who are already struggling with many competing issues. Notwithstanding, the large indirect costs associated with the loss of parental productivity underlines the import- ance of collecting these data, in one way or another. Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request. Start-in! Study Group Nadia Tubiana-Rufi, Sophie Guilmin-Crépon, Jean-Bruno Lobut, Patricia Sierra, Christine Delcroix (all CHU Robert Debré, AP-HP); Véronique Sulmont (CHU Reims, CH Annemasse); Anne-Sophie Salmon, Pierre-François Souchon (both CHU Reims); Claire Le Tallec (CHU Purpan, Toulouse); Régis Coutant, Natacha Bouhours, Sylvie Dufresne, Frederique Gatelais (all CHU Angers); Fabienne Dalla-Vale, Denis Morin (both CHU Arnaud de Villeneuve, Montpellier); Claire Stuckens, Louis Montagne, Marie Dupre, Stéphanie Coopman (all CHU Lille); Hélène Bony-Trifunovic, Karine Braun (both CHU Amiens); Hélène Crosnier, Claire Personnier (both CH Intercommunal de Poissy); François Kurtz (CH Saint-Avold); Florentia Kaguelidou, Laurence Corvez, Véronique Beruer, Eve- lyne Jacqz-Aigrain (all Centre d’Investigation Clinique CIC 1426, Inserm, Rob- ert Debré Hospital); Rolande Ducrocq (Laboratoire de Biochimie Métabolique, CHU Robert Debré, AP-HP); Adyla Yacoubi, Tania Rilcy, Cécile Hoffart- Jourdain, Akim Souag, Béatrice Andriss, Damir Mohamed, Isabelle Durand- Zaleski (all Unité de Recherche Clinique Robert Debré, Unité de Recherche Clinique Economie de la Santé and Direction de la Recherche Clinique et de l’Innovation, AP-HP). 5. Dunn J, Harris B, Iqbal G, Wood J, Mistry P, O’Beirne-Elliman J, et al. Use of patient diaries in conjunction with standard reporting methods: duplication of data or a valuable resource? Trials. 2015;16(2):P183. 6. Hendriks MRC, Al MJ, Bleijlevens MHC, van Haastregt JCM, Crebolder HFJM, van Eijk JTM, et al. Continuous versus intermittent data collection of health care utilization. Med Decis Mak. 2013;33(8):998–1008. 7. von Niederhäusern B, Saccilotto R, Schädelin S, Ziesenitz V, Benkert P, Decker M-L, et al. Validity of mobile electronic data capture in clinical studies: a pilot study in a pediatric population. BMC Med Res Methodol. 2017;17(1):163. 7. von Niederhäusern B, Saccilotto R, Schädelin S, Ziesenitz V, Benkert P, Decker M-L, et al. Validity of mobile electronic data capture in clinical studies: a pilot study in a pediatric population. BMC Med Res Methodol. 2017;17(1):163. 8. Seidl H, Meisinger C, Wende R, Holle R. Empirical analysis shows reduced cost data collection may be an efficient method in economic clinical trials. BMC Health Serv Res. 2012;12:318. 8. Seidl H, Meisinger C, Wende R, Holle R. Empirical analysis shows reduced cost data collection may be an efficient method in economic clinical trials. BMC Health Serv Res. 2012;12:318. 9. Guilmin‐Crépon S, Carel J-C, Schroedt J, Sulmont V, Salmon A-S, Tallec CL, et al. Is there an optimal strategy for real-time continuous glucose monitoring in pediatrics? A 12-month French multi-center, prospective, controlled randomized trial (Start-In!). Pediatric Diabetes [Internet]. [cité 21 9. Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval and consent to participate The AFSSAPS (National Agency for the Safety of Medicines and Health Products) approved the protocol of the START-IN! trial (2007-AO1330-53). In- formed consent was obtained from all study participants. Consent for publication Not applicable. févr 2019];0(0). Disponible sur: https://onlinelibrary.wiley.com/doi/abs/1 0.1111/pedi.12820 févr 2019];0(0). Disponible sur: https://onlinelibrary.wiley.com/doi/abs/1 0.1111/pedi.12820 févr 2019];0(0). Disponible sur: https://onlinelibrary.wiley.com/doi/abs/1 0.1111/pedi.12820 10. Heyer GL, Perkins SQ, Rose SC, Aylward SC, Lee JM. Comparing patient and parent recall of 90-day and 30-day migraine disability using elements of the PedMIDAS and an Internet headache diary. Cephalalgia. 2014;34(4):298–306. 11. Lubeck DP, Hubert HB. Self-report was a viable method for obtaining health care utilization data in community-dwelling seniors. J Clin Epidemiol. 2005; 58(3):286–90. 12. van den Brink M, van den Hout WB, Stiggelbout AM, Putter H, van de Velde CJH, Kievit J. Self-reports of health-care utilization: diary or questionnaire? Int J Technol Assess Health Care. 2005;21(3):298–304. 13. Jia Z, Li X, Yuan X, Zhang B, Liu Y, Zhao J, et al. Depression is associated with diabetes status of family members: NHANES (1999–2016). J Affect Disord. 2019;249:121–6. 14. Jaser SS, Whittemore R, Ambrosino JM, Lindemann E, Grey M. Mediators of Depressive Symptoms in Children with Type 1 Diabetes and their Mothers. J Pediatr Psychol. 2008;33(5):509–19. 15. Driscoll KA, Johnson SB, Barker D, Quittner AL, Deeb LC, Geller DE, et al. Risk Factors Associated with Depressive Symptoms in Caregivers of Children with Type 1 Diabetes or Cystic Fibrosis. J Pediatr Psychol. 2010;35(8):814–22. 16. Aalders J, Hartman E, Nefs G, Nieuwesteeg A, Hendrieckx C, Aanstoot H-J, et al. Mindfulness and fear of hypoglycaemia in parents of children with Type 1 diabetes: results from Diabetes MILES Youth – The Netherlands. Diabet Med. 2018;35(5):650–7. 17. Haugstvedt A, Wentzel-Larsen T, Graue M, Søvik O, Rokne B. Fear of hypoglycaemia in mothers and fathers of children with Type 1 diabetes is associated with poor glycaemic control and parental emotional distress: a population-based study. Diabet Med. 2010;27(1):72–8. 18. Jaser SS, Grey M. A Pilot Study of Observed Parenting and Adjustment in Adolescents with Type 1 Diabetes and their Mothers. J Pediatr Psychol. 2010;35(7):738–47. 19. Changes in Treatment Adherence and Glycemic Control During the Transition to Adolescence in Type 1 Diabetes | Diabetes Care [Internet]. [cité 22 août 2019]. Disponible sur: https://care.diabetesjournals.org/content/35/6/1219 20. Lotstein DS, Seid M, Klingensmith G, Case D, Lawrence JM, Pihoker C, et al. Transition From Pediatric to Adult Care for Youth Diagnosed With Type 1 Diabetes in Adolescence. Pediatrics. 2013;131(4):e1062–70. Start-in! Study Group Guilmin‐Crépon S, Carel J-C, Schroedt J, Sulmont V, Salmon A-S, Tallec CL, et al. Is there an optimal strategy for real-time continuous glucose monitoring in pediatrics? A 12-month French multi-center, prospective, controlled randomized trial (Start-In!). Pediatric Diabetes [Internet]. [cité 21 Page 7 of 7 Le Jeannic et al. Trials (2020) 21:36 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Effectiveness of Link Prediction for Face-to-Face Behavioral Networks
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Abstract Research on link prediction for social networks has been actively pursued. In link prediction for a given social network obtained from time-windowed observation, new link formation in the network is predicted from the topology of the obtained network. In contrast, recent advances in sensing technology have made it possible to obtain face-to-face behavioral networks, which are social networks representing face-to-face interactions among people. However, the effectiveness of link prediction techniques for face-to-face behavioral networks has not yet been explored in depth. To clarify this point, here we investigate the accuracy of conventional link prediction techniques for networks obtained from the history of face-to-face interactions among participants at an academic conference. Our findings were (1) that conventional link prediction techniques predict new link formation with a precision of 0.30–0.45 and a recall of 0.10–0.20, (2) that prolonged observation of social networks often degrades the prediction accuracy, (3) that the proposed decaying weight method leads to higher prediction accuracy than can be achieved by observing all records of communication and simply using them unmodified, and (4) that the prediction accuracy for face-to-face behavioral networks is relatively high compared to that for non-social networks, but not as high as for other types of social networks. Citation: Tsugawa S, Ohsaki H (2013) Effectiveness of Link Prediction for Face-to-Face Behavioral Networks. PLoS ONE 8(12): e81727. doi:10.1371/ journal.pone.0081727 Editor: Peter Csermely, Semmelweis University, Hungary Received August 2, 2013; Accepted October 15, 2013; Published December 10, 2013 Received August 2, 2013; Accepted October 15, 2013; Published December 10, 2013 Copyright:  2013 Tsugawa, Ohsaki. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2013 Tsugawa, Ohsaki. This is an open-access article distributed under the terms of the Creative Commons Att unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by JSPS KAKENHI Grant Number 25280030. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: s-tugawa@cs.tsukuba.ac.jp Link prediction for face-to-face behavioral networks should be useful for developing novel services and performing sociological analyses. Abstract Link prediction is promising for predicting communica- tions that are likely to occur; this can be viewed as potential communication demands. Hence, link prediction in face-to-face behavioral networks may contribute toward realizing novel services such as friendship recommendation in real-world envi- ronments, which is already common in online environments. Link prediction techniques may also be useful for analyzing the evolutionary dynamics of social networks in real-world environ- ments, an important topic in social science. Sho Tsugawa1*, Hiroyuki Ohsaki2 ation and Systems, University of Tsukuba, Ibaraki, Japan, 2 School of Science and Technology, Kwansei Gakuin University, Hyogo, Japa of Engineering, Information and Systems, University of Tsukuba, Ibaraki, Japan, 2 School of Science and Technology, Kwansei Gakuin U Effectiveness of Link Prediction for Face-to-Face Behavioral Networks Sho Tsugawa1*, Hiroyuki Ohsaki2 December 2013 | Volume 8 | Issue 12 | e81727 Link Prediction Techniques To clarify the effectiveness of link prediction techniques for face- to-face behavioral networks, here we investigate the accuracy of conventional link prediction techniques for networks obtained from the SocioPatterns dataset [20]. We extend our preliminary work [24] and investigate the prediction accuracy under various conditions. Our results support the findings in [1], and provide new findings, such as that incorporating temporal information about communication is essential for improving prediction accuracy. Moreover, we discuss the effectiveness of link prediction techniques for face-to-face behavioral networks compared with other networks through experiments with the Enron email dataset [26], which is one of the largest available email corpora. Among various link prediction techniques proposed in the literature, the common neighbor (CN) [9], Adamic/Adar (AA) [10], preferential attachment (PA) [9], Jaccard coefficient (JC) [27], and resource allocation (RA) [11] are widely used and their accuracies have been explored for several types of networks [3,12,13,28]. For each node pair (i, j), a link prediction technique gives li, j, an estimate of the likelihood of link formation between nodes i and j. In other words, a link prediction technique predicts the likelihood of future link formation as li, j. In the following paragraphs, we briefly introduce definitions of the link prediction score li, j for the following types of link prediction techniques for unweighted and weighted networks: CN [9] and weighted CN (WCN) [4,28], AA [10] and weighted AA (WAA) [4,28], PA [9] and weighted PA (WPA) [28], JC [27] and weighted JC (WJC) [1], and RA [11] and weighted RA (WRA) [4]. In what follows, C(i) denotes the set of nodes adjacent to node i, w(i, j) is a weight assigned to link (i, j), and a is a parameter for controlling the contribution of link weights to the link prediction score li, j. The main contributions of this study can be summarized as follows. N We investigated the prediction accuracy of conventional link prediction techniques for face-to-face behavioral networks by using publicly available datasets. N We showed that in order to achieve high link prediction accuracy for face-to-face behavioral networks, it is essential to incorporate temporal information and to appropriately tune the length of the training period, the period spanned by the records analyzed; good tuning results in increased link prediction accuracy. Common neighbor. Introduction Research on link prediction for social networks has been actively pursued [1–8]. In link prediction for a given social network obtained from time-window observation, new link formation in the network is predicted from the topology of the observed network. A social network is represented as a graph where individuals are represented as nodes and social ties among them are represented as links. In the literature, several link prediction techniques have been proposed [4,5,9–11]. These techniques can be used to predict new link formation by estimating the likelihood of link formation between two nodes on the basis of the observed network topology. Social ties can be defined in a number of ways, and the accuracy of link prediction techniques has been investigated for several types of social networks such as coauthorship networks [3], email networks [12], and friendship networks [13]. Link prediction techniques are expected to be utilized for several applications such as recommendation [3], anomaly detection [14], network modeling [15], missing link detection [6], evaluation of network evolution mechanisms [16], reconstruction of networks [17], and classification of partially labeled networks [18,19]. However, the effectiveness of link prediction techniques for face- to-face behavioral networks has not yet been fully explored. Our research group has been investigating the effectiveness of link prediction techniques for face-to-face behavioral networks using a publicly available dataset called the SocioPatterns dataset [20], which contains the history of face-to-face interactions among participants at an academic conference. Our preliminary results are presented in [24,25]. In addition, Scholz et al. [27] experi- mentally investigated the accuracy of conventional link prediction techniques for face-to-face behavioral networks by using their own datasets. They also investigated the effectiveness of combining face-to-face behavioral networks with other types of social networks for link prediction [2]. To understand the effectiveness of link prediction techniques when applied to real-world social networks, however, more extensive investigation is necessary. For instance, the effects of the observation period of social networks on Recent advances in sensing technology have made it possible to obtain face-to-face behavioral networks, which are social networks representing face-to-face interactions among people [20–23]. For instance, in the SocioPatterns project, social networks representing face-to-face interactions among participants at an academic conference are constructed using badge-shaped sensing devices [20]. Introduction December 2013 | Volume 8 | Issue 12 | e81727 1 PLOS ONE | www.plosone.org Link Prediction for Face-to-Face Networks Link Prediction for Face-to-Face Networks the prediction accuracy have not been discovered. The results in [1] should be verified using other datasets. li, j ~ jC(i)\C(j)j (in CN), ð1Þ Overview of Experiments We evaluate the effectiveness of link prediction techniques through experiments using the SocioPatterns dataset [20], which contains the history of face-to-face communication among 110 participants over two and a half days at an academic conference (ACM Hypertext 2009). At that conference, face-to-face commu- nication was detected and stored on radiofrequency identification (RFID) devices embedded in the conference badges worn by the participants. Badges periodically broadcasted ultra-low power radio packets that contained the participant’s ID. Each badge collected packets every 20 s, and stored a list of IDs and timestamps contained in the packets. Radio packet exchange was possible only when two people were close (1.0–1.5 [m] apart) and facing each other [20]. Face-to-face communication was detected by the IDs stored in the badges. An interval of 20 s is considered to be short enough to detect the occurrence of face-to- face communication at social gatherings [20]. li, j ~ w(i,k)azw( j,k)a log(1z X k0[C(k) w(k0,k)a) (in WAA): ð4Þ ð4Þ Preferential attachment. The preferential attachment pre- dicts new link formation from the idea that a high-degree node has a higher chance of forming new links [9]. In PA [9] and WPA [4,28], li,j is given by li, j ~ jC(i)j|jC( j)j (in PA), ð5Þ ð5Þ li, j ~ X k[C(i) w(i,k)a| X k[C( j) w( j,k)a (in WPA): ð6Þ ð6Þ For comparison purposes, we also use the Enron email dataset [36] that contains 252,759 email messages with headers and body texts exchanged between 151 employees of the Enron Corpora- tion. Jaccard coefficient. Similarly to CN, the Jaccard coefficient predicts new link formation from the number of common adjacent nodes, but the link prediction score is normalized [27]. In JC [27] and WJC [1], li, j is given by In our experiments, we divide the history of communication into a training period and a testing period, and investigate how accurately the occurrence of communications in the testing period can be predicted using the history of communications in the training period. Figure 1 shows an overview of the evaluation method. li, j ~ jC(i)\C( j)j jC(i)|C( j)j (in JC), ð7Þ ð7Þ First, as an input for link prediction techniques, an undirected graph G~(V, E), where each link weight is the number of communications, is obtained from the history of communication between times t0 and t1. The period between t0 and t1 corresponds to the training period. Link Prediction Techniques The common neighbor predicts new link formation from the idea that the existence of many common adjacent nodes between two nodes implies a high probability of new link formation between those two nodes [10]. In CN [10] and WCN [4], li, j, an estimate of the likelihood of link formation between node i and j, is given by N We showed that the proposed decaying weight method can lead to higher prediction accuracy than can be achieved by observing all records of communication and simply using them unmodified. The prediction accuracy of the decaying weight method is comparable to the accuracy achievable with an appropriately tuned training period. ð1Þ Figure 1. Overview of evaluation of link prediction techniques. New link formation is predicted from the topology of a social network obtained from communications in the training period. We investigate the accuracy of link prediction techniques by comparing the predicted links with actual new links created within the testing period. doi:10.1371/journal.pone.0081727.g001 Figure 1. Overview of evaluation of link prediction techniques. New link formation is predicted from the topology of a social network obtained from communications in the training period. We investigate the accuracy of link prediction techniques by comparing the predicted links with actual new links created within the testing period. doi:10.1371/journal.pone.0081727.g001 December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 2 Link Prediction for Face-to-Face Networks Table 1. Definitions of true positive (TP), true negative (TN), false positive (FP), and false negative (FN). ble 1. Definitions of true positive (TP), true negative (TN), false positive (FP), and false negative (FN). Number of communications is more than or equal to S Number of communications is less than S li, j is larger than or equal to T TP FP li, j is smaller than T FN TN TP, TN, FP, and FN are the numbers of node pairs (i,j) satisfying the conditions shown in this table. doi:10.1371/journal.pone.0081727.t001 Resource allocation. The resource allocation predicts new link formation from an idea similar to that in AA [11]. In RA [11] and WRA [4], li, j is given by ð2Þ li, j~ X k[C(i)\C(j) 1 jC(k)j (in RA), ð9Þ ð9Þ Adamic/Adar. The Adamic/Adar predicts new link forma- tion from the idea that many common adjacent nodes with small degree between two nodes implies a high probability of new link formation between the nodes [10]. Link Prediction Techniques Similarly to CN, AA predicts new link formation on the basis of the number of common adjacent nodes, but assign a weight to li, j based on the degrees of common adjacent nodes. In AA [10] and WAA [4], li, j is given by li, j~ w(i,k)azw( j,k)a X k0[C(k) w(k0,k)a (in WRA): ð10Þ ð10Þ li, j ~ X k[C(i)\C( j) 1 logjC(k)j (in AA), ð3Þ ð3Þ December 2013 | Volume 8 | Issue 12 | e81727 Overview of Experiments In the graph G, a link (i, j) represents the existence of a communication between individuals i and j within the training period. The weight assigned to link (i, j) is defined as the number of communications between individuals i and j within the training period. li, j~ X k[C(i)\C(j) (w(i,k)azw( j,k)a) X k[C(i) w(i,k)az X k[C(j) w( j,k)a (in WJC): ð8Þ ð8Þ PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 3 Link Prediction for Face-to-Face Networks Figure 2. The number of face-to-face communications recorded in the SocioPatterns dataset over periods of one hour. The onset of sensing face-to-face interactions is indicated by 0. doi:10.1371/journal.pone.0081727.g002 Precision evaluates the correctness, and recall evaluates the completeness of link prediction Generally there is a tradeoff Figure 2. The number of face-to-face communications recorded in the SocioPatterns dataset over periods of one hour. The onset of sensing face-to-face interactions is indicated by 0. doi:10.1371/journal.pone.0081727.g002 Figure 3. The number of email communications recorded in the Enron email dataset over periods of one month. The onset (indicated by 0) is April 1, 2000. doi:10.1371/journal.pone.0081727.g003 Figure 3. The number of email communications recorded in the Enron email dataset over periods of one month. The onset (indicated by 0) is April 1, 2000. doi:10.1371/journal.pone.0081727.g003 Figure 3. The number of email communications recorded in the Enron email dataset over periods of one month. The onset (indicated by 0) is April 1, 2000. doi:10.1371/journal.pone.0081727.g003 Figure 2. The number of face-to-face communications recorded in the SocioPatterns dataset over periods of one hour. The onset of sensing face-to-face interactions is indicated by 0. doi:10.1371/journal.pone.0081727.g002 Precision evaluates the correctness, and recall evaluates the completeness of link prediction. Generally, there is a tradeoff between precision and recall, whereby a larger threshold T increases precision and decreases recall. AUC is defined as the area under the ROC curve [32]. The ROC curve is obtained by plotting the false positive rate (FP=(FNzTN)) versus recall by changing the threshold T. AUC is 0.5 when the prediction accuracy is equal to a random prediction, and is 1.0 with perfect prediction accuracy [32]. Next, we calculate the link prediction score li, j for each node pair (i, j) that is not associated with any communication before time t1 by using one of the link prediction techniques introduced in the previous section. Overview of Experiments Since li, j for CN, AA, PA, JC, and RA are defined for an unweighted graph, we obtain li, j by simply ignoring link weights in G. As a final step, we examine how accurately we can predict which node pairs will have at least S communications between times t2 and t3 despite not having any communication before time t1. We perform this prediction by extracting node pairs whose link prediction scores li, j meet or exceed a threshold T. The period between times t2 and t3 corresponds to the testing period. It is typical in studies of this kind to evaluate the accuracy of link prediction techniques for S~1 only [4,28]. We introduce the parameter S, and evaluate the effectiveness of link prediction techniques not only for predicting new link formation but also for predicting the formation of strong ties (i.e., links with large weights). We perform experiments by changing S and a. Unless explicitly stated we use S~1 and a~1. We first investigate the accuracy of conventional link prediction techniques for face-to-face behavioral networks using the SocioPatterns dataset, and then discuss the effectiveness of link prediction techniques for face-to-face behav- ioral networks compared to other networks through experiments with the Enron email dataset. Figure 4. Degree distribution of a network obtained from the entire history of face-to-face communications in the Socio- Patterns dataset. In the network, each node represents a participant at the conference, and each link between two nodes represents the occurrence of face-to-face communication between participants during the conference. doi:10.1371/journal.pone.0081727.g004 The link prediction problem can be formulated as ranking or classification problems. Following prior work [1,6,13,29–31], we use precision and recall, which are commonly used for evaluating ranking problems, as well as area under the receiver operating characteristic (ROC) curve (AUC) [32], which is commonly used for evaluation of classification problems. Precision P and recall R are defined as in Eqs. (11) and (12) using the concepts of true positive (TP), true negative (TN), false positive (FP), and false negative (FN). TP, TN, FP, and FN, which represent the respective numbers of node pairs satisfying the corresponding conditions in Table 1. P~ TP TPzFP ð11Þ R~ TP TPzFN ð12Þ P~ TP TPzFP ð11Þ R~ TP TPzFN ð12Þ ð11Þ Figure 4. Degree distribution of a network obtained from the entire history of face-to-face communications in the Socio- Patterns dataset. Comparison of Ten Link Prediction Techniques Comparison of Ten Link Prediction Techniques To investigate the accuracy of link prediction techniques for a face-to-face behavioral network, we find the precision and recall of ten link prediction techniques while varying the threshold T. Precision-recall curves for ten link prediction techniques are shown in Fig. 6. The AUC scores of the ten link prediction techniques are shown in Fig. 7. The training period is time 0–30 [h] (t0~0, t1~30) and the testing period is time 30–60 [h] (t2~30, t3~60). Figure 6 shows that for instance, RA achieves a precision of 0.30–0.45 and a recall of 0.10–0.20 with an appropriate threshold T for a face-to-face behavioral network obtained from the SocioPatterns dataset. Unsurprisingly, this result suggests that link prediction tech- niques cannot perfectly predict future link formation, but can nevertheless achieve considerable accuracy. It is therefore expected that conventional link prediction techniques can be applicable to services such as friendship recommendation that require a moderate level of prediction accuracy. For instance, it is reported in [29] that future friendship formation in an online social networking service called LiveJournal can be predicted with a precision of 0.18 and a recall of 0.18. Since networks in LiveJournal and the SocioPatterns dataset are rather different in terms of the number of nodes and links, direct comparison of the results for those two networks is impossible. However, we expect that a precision of 0.30–0.45 and a recall of 0.10–0.20 are sufficient accuracy for a friendship recommendation service. Figure 5. Degree distribution of a network obtained from the entire history of email communication in the Enron email dataset. In the network, each node represents an employee of the Enron Corporation, and each link between two nodes represents the occurrence of email communication between the corresponding employees from April 2000 to March 2002. doi:10.1371/journal.pone.0081727.g005 Results and Discussion y p Focusing on the differences among link prediction techniques, we find that RA and PA achieve the highest accuracy. Figure 7 shows that the AUC scores of RA and PA are the highest. The DeLong test [33] shows that the AUC scores of RA and PA are significantly higher than those for other methods ( pv0:01), and that there is no significant difference between RA and PA ( pw0:1). The high accuracy of RA is expected since the existence of many common neighbors between two individuals intuitively means a high probability of face-to-face communication. The higher accuracy of RA compared to CN and AA, which are similar to RA, is due to the existence of high-degree nodes (Fig. 4). RA successfully considers such high-degree nodes, which results in high prediction accuracy. In [11], it is shown that the performance of RA is higher than that of AA for networks containing nodes with notably high degrees. The high accuracy of PA is also due to the existence of high-degree nodes. Such nodes, which represent eminent participants at the conference, tend to have many communication events, which results in the high accuracy of PA. We can also see that the accuracies of techniques for the weighted Overview of Experiments In the network, each node represents a participant at the conference, and each link between two nodes represents the occurrence of face-to-face communication between participants during the conference. doi:10.1371/journal.pone.0081727.g004 R~ TP TPzFN ð12Þ ð12Þ PLOS ONE | www.plosone.org 4 December 2013 | PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e81727 4 Link Prediction for Face-to-Face Networks Figure 5. Degree distribution of a network obtained from the entire history of email communication in the Enron email dataset. In the network, each node represents an employee of the Enron Corporation, and each link between two nodes represents the occurrence of email communication between the corresponding employees from April 2000 to March 2002. doi:10.1371/journal.pone.0081727.g005 Characteristics of Datasets Before evaluating the effectiveness of link prediction techniques, we analyze the characteristics of datasets used in this paper. Figure 2 shows the number of face-to-face communications recorded in the SocioPatterns dataset over intervals of one hour, and Fig. 3 shows the number of email communications recorded in the Enron email dataset over intervals of one month. The degree distributions of networks constructed from all communication logs in the SocioPatterns dataset and the Enron email dataset are shown in Figs. 4 and 5, respectively. Figure 2 shows that the number of face-to-face communications fluctuates in the SocioPatterns dataset, and Fig. 3 shows that there is no such tendency in email communications in the Enron email dataset. Figures 4 and 5 show that the degree distribution is vastly different in the two networks. For that reason, the effectiveness of link prediction techniques might be different for the two networks. Figure 6. Relation between precision and recall for ten link prediction techniques. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), parameter for link prediction: a~1, dataset: SocioPatterns dataset). doi:10.1371/journal.pone.0081727.g006 Figure 6. Relation between precision and recall for ten link prediction techniques. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), parameter for link prediction: a~1, dataset: SocioPatterns dataset). doi:10.1371/journal.pone.0081727.g006 December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 5 Link Prediction for Face-to-Face Networks Figure 7. Comparison of the AUC scores of the ten link prediction techniques. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), parameter for link prediction: a~1, dataset: SocioPatterns dataset). doi:10.1371/journal.pone.0081727.g007 Figure 7. Comparison of the AUC scores of the ten link prediction techniques. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), parameter for link prediction: a~1, dataset: SocioPatterns dataset). doi:10.1371/journal.pone.0081727.g007 Figure 7. Comparison of the AUC scores of the ten link prediction techniques. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), parameter for link prediction: a~1, dataset: SocioPatterns dataset). doi:10.1371/journal.pone.0081727.g007 networks (WCN, WAA, WPA, WJC, and WRA) are relatively lower than techniques for the unweighted networks (CN, AA, PA, JC, and RA). Lu¨ et al. [4] report the same phenomenon, which is analyzed in greater depth in the next section by varying the parameter of the link prediction techniques. Effects of Link Prediction Parameters To investigate the effects of link prediction technique param- eters on the prediction accuracy, we obtain the AUC scores of WCN, WAA, WPA, WJC, and WRA while varying the parameter g p q p eters on the prediction accuracy, we obtain the AUC scores of WCN, WAA, WPA, WJC, and WRA while varying the parameter Figure 8. AUC scores of five link prediction techniques obtained while varying the parameter a, which controls the contribution of link weights to the link prediction score. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), dataset: SocioPatterns dataset). a~0 is equivalent to ignoring link weights, and a~1 is equivalent to simply using the number of face- to-face communications as the link weight. doi:10.1371/journal.pone.0081727.g008 Figure 9. AUC scores of five link prediction techniques obtained while varying the threshold S. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), dataset: Socio- Patterns dataset). doi:10.1371/journal.pone.0081727.g009 PLOS ONE | www.plosone.org 6 December 2013 | Volume 8 | Issue 12 | e81727 Figure 8. AUC scores of five link prediction techniques obtained while varying the parameter a, which controls the contribution of link weights to the link prediction score. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), dataset: SocioPatterns dataset). a~0 is equivalent to ignoring link weights, and a~1 is equivalent to simply using the number of face- to-face communications as the link weight. doi:10.1371/journal.pone.0081727.g008 Figure 9. AUC scores of five link prediction techniques obtained while varying the threshold S. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), dataset: Socio- Patterns dataset). doi:10.1371/journal.pone.0081727.g009 December 2013 | Volume 8 | Issue 12 | e81727 Figure 8. AUC scores of five link prediction techniques obtained while varying the parameter a, which controls the contribution of link weights to the link prediction score. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), dataset: SocioPatterns dataset). a~0 is equivalent to ignoring link weights, and a~1 is equivalent to simply using the number of face- to-face communications as the link weight. doi:10.1371/journal.pone.0081727.g008 Figure 9. AUC scores of five link prediction techniques obtained while varying the threshold S. (training period: 0–30 (t0~0, t1~30), testing period: 30–60 (t2~30, t3~60), dataset: Socio- Patterns dataset). doi:10.1371/journal.pone.0081727.g009 December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 6 Link Prediction for Face-to-Face Networks Figure 10. AUC score of CN obtained while varying the training period length. Effects of Link Prediction Parameters (t1 and t2 are fixed to 12, 24, or 36 [h], and t0 is varied. The testing period length is 3, 5, and 10 h. Dataset: SocioPatterns dataset). doi:10.1371/journal.pone.0081727.g010 Figure 10. AUC score of CN obtained while varying the training period length. (t1 and t2 are fixed to 12, 24, or 36 [h], and t0 is varied testing period length is 3, 5, and 10 h. Dataset: SocioPatterns dataset). d i 10 1371/j l 0081727 010 Figure 10. AUC score of CN obtained while varying the training period length. (t1 and t2 are fixed to 12, 24, or 36 [h], and t0 is varied. The testing period length is 3, 5, and 10 h. Dataset: SocioPatterns dataset). doi:10.1371/journal.pone.0081727.g010 a, which controls the contribution of link weights to the link prediction score (Fig. 8). formation of strong ties, and a small S means that we predict the formation of links including weak ties (links with small weights). We show the results for only CN, AA, PA, JC, and RA in this investigation. This figure shows that appropriate link weights improve the accuracy of link prediction. However, as reported in [4], the prediction accuracy obtained when defining the link weight as the number of face-to-face communications and simply using it unmodified is even lower than that achieved when link weights are ignored altogether. All five techniques achieve the highest accuracy when a is approximately 0.3. This result suggests that the accuracy of link prediction can be improved by increasing the relative contribution of weak ties (links with small weights) to the link prediction score li, j. More detailed analysis is, however, required to determine the optimal value of a. This figure shows that the accuracy is higher when predicting only the formation of strong ties than when predicting link formation including weak ties. This suggests that link prediction techniques are more effective at predicting the formation of strong ties than weak ties. This result is considered natural since weak ties may include noisy links, which are difficult to predict. Note that this tendency is observed in different face-to-face behavioral networks [1]. However, we should note that AUC scores do not change significantly when S is six or higher. December 2013 | Volume 8 | Issue 12 | e81727 Effects of Training Period Length We next obtain the AUC score while varying the threshold S to investigate the accuracy of each technique to predict the formation of strong ties (links with large weights) (Fig. 9). In this investigation, the training period and the testing period are time 0–30 [h] and 30–60 [h], respectively. A large S means that we predict only the In this section, we examine the effectiveness of using temporal information about communication in addition to the network topology for link prediction. By utilizing temporal information, we can change the contribution of each communication event to the link prediction score, which may result in more accurate December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 7 Link Prediction for Face-to-Face Networks Figure 11. Relation between the number of face-to-face contacts used for link prediction and AUC score of CN. t1 and t2 are fixed to 12, 24, or 36 [h], and t0 varies based on the number of contacts used for link prediction. 1000, 2000, and 5000 contacts are predicted immediately following the training period, using the SocioPatterns dataset. doi:10.1371/journal.pone.0081727.g011 Figure 11 Relation between the number of face to face contacts used for link prediction and AUC score of CN t and t are f Figure 11. Relation between the number of face-to-face contacts used for link prediction and AUC score of CN. t1 and t2 are fixed to 12, 24, or 36 [h], and t0 varies based on the number of contacts used for link prediction. 1000, 2000, and 5000 contacts are predicted immediately following the training period, using the SocioPatterns dataset. doi:10.1371/journal.pone.0081727.g011 Figure 10 shows that simply increasing the training period length does not necessarily improve the prediction accuracy, and can even degrade it. Although it is intuitively expected that using more information should improve prediction accuracy, this result suggests otherwise. prediction. A simple way to utilize temporal information is to change the training period length. By changing the onset time of the training period, we can evaluate the prediction accuracy when the contribution of old communications are ignored. The effect of the length of the training period on the prediction accuracy has not been fully explored in existing work [1] or our preliminary work [24,25]. When applying link prediction techniques to face-to- face behavioral networks, it is important to know how to determine the training period. December 2013 | Volume 8 | Issue 12 | e81727 Effects of Training Period Length Figure 10 also shows that the optimal training period length that maximizes the AUC score depends on the starting time of the testing period t2. Note that the optimal training period length does not depend on the length of the testing period. We obtain the AUC scores of the link prediction techniques while varying the training period length for several starting times of the testing period t2 and lengths of the testing period. In this investigation, the testing period immediately follows the training period. Figure 10 shows the relation between the training period length and AUC score for CN. Note that the results when using other link prediction techniques are similar to the results shown in Fig. 10. Since few communications take place at times 12–24 and 36–48 [h] (see Fig. 2), we use the data for periods 0–12, 24–36, and 48–60 [h]. We next determine the training period length not by time, but by the number of face-to-face contacts used for link prediction, and perform a similar experiment to investigate the effect of training period length. Since the number of face-to-face contacts recorded in the dataset is different from hour to hour, this may explain why the optimal training period length depends on the starting time of the testing period. Figure 11 shows the relation between the number of face-to-face contacts used for link prediction and AUC score. In this investigation, the testing period is also determined by the number of face-to-face contacts to predict. PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e81727 December 2013 | Volume 8 | Issue 12 | e81727 8 Link Prediction for Face-to-Face Networks Figure 12. Comparison of the AUC scores of WCN for four types of weighting methods. t1 and t2 are fixed to 12, 24, or 36 [h]. d~10{6. Parameter for link prediction: a~0:3, Dataset: SocioPatterns dataset. doi:10.1371/journal.pone.0081727.g012 Figure 12. Comparison of the AUC scores of WCN for four types of weighting methods. t1 and t2 are fixed to 12, 24, or 36 [h]. d~10{6. Parameter for link prediction: a~0:3, Dataset: SocioPatterns dataset. doi:10.1371/journal.pone.0081727.g012 Figure 12. Comparison of the AUC scores of WCN for four types of weighting methods. t1 and t2 are fixed to 12, 24, or 36 [h]. d~10{6. Parameter for link prediction: a~0:3, Dataset: SocioPatterns dataset. doi:10.1371/journal.pone.0081727.g012 prediction is increased. December 2013 | Volume 8 | Issue 12 | e81727 Effects of Training Period Length Hence, if we know that person A met person B, we can infer that A will tend to communicate with persons near B and not with persons located far away from B. However, as time elapses, this information becomes irrelevant since persons A and B may move. Therefore, recent information is more important than old information, and the relevance of information about a particular time segment decreases with time. cases, using approximately 3,000 contacts achieves the highest accuracy. These results show that the decaying weight method achieves higher accuracy than the flat weight method with t0~0. In particular, the accuracy of the exponential decaying weight method is comparable to that of the flat weight method with the optimal training period length. The DeLong test [6] shows that there are significant differences in AUC score between the exponential decaying weight method and the flat weight method with t0~0 when t1~t2~12 and t1~t2~24 ( pv0:01). The DeLong test also shows that there are no significant differences in AUC score between the exponential decaying weight method and the flat weight method with the optimal training period length ( pw0:01). This result suggests that the decaying weight method can be useful when the optimal training period length is unknown. Th ff i f h d i i h h d i d h These observations suggest that prolonged observation of face- to-face contacts for link prediction often degrades the prediction accuracy. We cannot draw general conclusions from our results alone, but in this experiment, using approximately 3,000 contacts (30 contacts per person) is optimal in many cases. Analysis using other datasets is required in order to determine the optimal training period length in the general case. We next investigate the prediction accuracy when using a decaying weight method, which changes the contribution of each contact on the link prediction score by weighting each face-to-face contact. In the above experiments, all contacts used for link prediction are weighted equally for link prediction score calcula- tion. However, our experimental results suggest that simply using all contacts often degrades the prediction accuracy. We therefore change the contribution of each contact on the link prediction score calculation by assigning a heavy weight to new information. In this experiment, we use two types of decaying weight methods, linear and exponential. Let contacts between time t0 and time t1 be c0, c1, c2,…, cn. Effects of Training Period Length In the linear decaying weight method, for a contact ck a link weight in the graph G(V,E), which is used for link prediction score calculation, is increased by k=n, and in the exponential decaying weight method a link weight is increased by eb(k{n). In exponential decaying weight method, b is determined as e{bn~d, where d is a sufficiently small positive real number. The effectiveness of the decaying weight method is due to the spatiotemporal locality of face-to-face communication. The mobility of people is restricted by the space and their moving speed. Hence, if we know that person A met person B, we can infer that A will tend to communicate with persons near B and not with persons located far away from B. However, as time elapses, this information becomes irrelevant since persons A and B may move. Therefore, recent information is more important than old information, and the relevance of information about a particular time segment decreases with time. Effects of Training Period Length We carefully investigated the results for other starting times in the testing period, and found that prediction accuracy increases as the number of contacts used for prediction increases when the testing period includes the third day. In other These results show that in most cases, the prediction accuracy is highest when using approximately 3,000 contacts. However, when the starting time of the testing period is 36 [h], the prediction accuracy increases together with the number of contacts used for Figure 13. Relation between precision and recall of ten link prediction techniques. (training period: April 2000–March 2001, testing period: April 2001–March 2002, parameter for link prediction: a~1, dataset: Enron email dataset). doi:10.1371/journal.pone.0081727.g013 Figure 13. Relation between precision and recall of ten link prediction techniques. (training period: April 2000–March 2001, testing period: April 2001–March 2002, parameter for link prediction: a~1, dataset: Enron email dataset). doi:10.1371/journal.pone.0081727.g013 December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 9 Link Prediction for Face-to-Face Networks Figure 14. Comparison of the AUC scores of the ten link prediction techniques. (training period: April 2000–March 2001, testing period: April 2001–March 2002, parameter for link prediction: a~1, dataset: Enron email dataset). doi:10.1371/journal.pone.0081727.g014 Figure 14. Comparison of the AUC scores of the ten link prediction techniques. (training period: April 2000–March 2001, testing period: April 2001–March 2002, parameter for link prediction: a~1, dataset: Enron email dataset). doi:10.1371/journal.pone.0081727.g014 These results show that the decaying weight method achieves higher accuracy than the flat weight method with t0~0. In particular, the accuracy of the exponential decaying weight method is comparable to that of the flat weight method with the optimal training period length. The DeLong test [6] shows that there are significant differences in AUC score between the exponential decaying weight method and the flat weight method with t0~0 when t1~t2~12 and t1~t2~24 ( pv0:01). The DeLong test also shows that there are no significant differences in AUC score between the exponential decaying weight method and the flat weight method with the optimal training period length ( pw0:01). This result suggests that the decaying weight method can be useful when the optimal training period length is unknown. The effectiveness of the decaying weight method is due to the spatiotemporal locality of face-to-face communication. The mobility of people is restricted by the space and their moving speed. Comparison with the Enron Email Dataset We next compare the results for the SocioPatterns dataset and those for the Enron email dataset to investigate the characteristics of link prediction techniques for face-to-face behavioral networks. Figure 13 shows the relation between precision and recall for each link prediction technique for a network constructed from the Enron email dataset. Figure 14 shows the AUC scores of the ten link prediction techniques. In this investigation, the training period Figure 12 shows AUC scores of WCN when using flat weight method, which equally weights all contacts, with the optimal length of the training period, flat weight method with t0~0, linear decaying weight method, and exponential decaying weight method. PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e81727 10 Link Prediction for Face-to-Face Networks Figure 15. Relation between the number of email messages used for link prediction and the AUC score of CN. (t1 and t2 are fixed to the 3rd month, 6th month, or 12th month, and t0 is changed based on the number of email messages used for link prediction. Predictions are made for 1000, 2000, and 5000 messages immediately after the training period. Dataset: Enron email dataset). doi:10.1371/journal.pone.0081727.g015 Figure 15. Relation between the number of email messages used for link prediction and the AUC score of CN. (t1 and t2 are fixed to the 3rd month, 6th month, or 12th month, and t0 is changed based on the number of email messages used for link prediction. Predictions are made for 1000, 2000, and 5000 messages immediately after the training period. Dataset: Enron email dataset). doi:10.1371/journal.pone.0081727.g015 and the testing period are April 2000 to March 2001 and April 2001 to March 2002, respectively. there are significant differences in AUC score between PA and WPA and other techniques ( pw0:1). RA achieves the highest accuracy for networks obtained from the SocioPatterns dataset, but its accuracy is comparable to that of JC and AA for the network obtained from the Enron email dataset. The DeLong test shows that there are no significant differences in AUC score among AA, JC, and RA ( pv0:01). In the network obtained from the Enron email dataset, the number of high-degree nodes is smaller than that in the network obtained from the SocioPatterns dataset (Figs. 4 and 5). Comparison with the Enron Email Dataset Therefore, the link prediction scores of RA tend to be similar to those of other similar techniques (i.e., AA and JC), which determines the similarity in accuracy between these techniques. From Fig. 13, we find that, for instance, JC achieves a precision of 0.30–0.45 and a recall of 0.17–0.39 with an appropriate value of the threshold T for an email network obtained from the Enron email dataset. This result shows that conventional link prediction techniques achieve higher precision and recall for email networks than for face-to-face networks. Moreover, we should note that the number of nodes in the network obtained from the Enron email dataset is greater than that obtained from the SocioPatterns dataset. Hence, link prediction techniques have higher performance for email networks than for face-to-face networks. The topological structure of a network may affect the accuracy of link prediction techniques for the network. We next investigate the AUC score of CN for networks obtained from the Enron email dataset by changing the number of email messages used for prediction (Fig. 15). In this investigation, we compare the effectiveness of tuning the training period length for a face-to-face network and an email network. We also compare the AUC scores of each weighting method (Fig. 16) and investigate the Moreover, Fig. 14 shows that the accuracy of link prediction for the email network is significantly different among the ten link prediction techniques. The accuracy of PA and WPA for the network obtained from the Enron email dataset is significantly lower than that for other techniques. The DeLong test shows that December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 11 Link Prediction for Face-to-Face Networks Figure 16. Comparison of the AUC scores of WCN for four types of weighting methods. t1 and t2 are fixed to the 3rd, 6th, or 12th month. d~10{6. parameter for link prediction: a~0:3, Dataset: Enron email dataset. doi:10.1371/journal.pone.0081727.g016 gure 16. Comparison of the AUC scores of WCN for four types of weighting methods. t1 and t2 are fixed to the 3rd, 6th, or 12th mo Figure 16. Comparison of the AUC scores of WCN for four types of weighting methods. t1 and t2 are fixed to the 3rd, 6th, or 12th month. d~10{6. parameter for link prediction: a~0:3, Dataset: Enron email dataset. doi:10.1371/journal.pone.0081727.g016 effectiveness of incorporating temporal information with regard to link prediction. behavioral network (see Fig. Acknowledgments The authors would like to thank Dr. Makoto Imase for valuable discussions and Mr. Youhei Kobayashi for his kind support. The authors would like to thank Dr. Makoto Imase for valuable discussions and Mr. Youhei Kobayashi for his kind support. Conclusions To clarify the effectiveness of link prediction techniques for face- to-face behavioral networks, we have investigated the accuracy of conventional link prediction techniques for such networks under various conditions. We have performed several experiments utilizing the SocioPatterns dataset containing a history of face- to-face interactions among participants at an academic confer- ence. Comparison with Other Networks We lastly compare the prediction accuracy for a face-to-face behavioral network with that for other networks. Table 2 shows the structural characteristics of several types of networks and the AUC scores of CN for those networks. The AUC scores for terrorist, and protein, and food web networks are taken from Fig. 3 in [6], and that for Facebook user networks is taken from Tables 3 and 5 in [13]. One possible method that can be used to avoid the need for tuning the training period length in link prediction for face-to-face behavioral networks is using the decaying weight method. Our results showed that an exponential decaying weight method achieves comparable accuracy with the method using the optimal training period length, suggesting that the decaying weight method can be useful when the optimal length of the training period is unknown. Table 2 shows that CN achieves a higher AUC score for face-to- face networks than protein networks, but achieves similar or lower AUC scores compared to other networks. From this result, we conclude that the accuracy of link prediction techniques for face- to-face behavioral networks is higher than non-social networks, but not so high among other types of social networks. We plan to investigate the causes of this result in future research. We plan to investigate the causes of differences in accuracy between different types of social networks. It is also important to investigate the optimal training period length in other face-to-face behavioral networks in future work. Link Prediction for Face-to-Face Networks sufficient for email networks. The DeLong test shows that there is no statistically significant difference between the AUC scores of the two methods ( pw0:1). This result also suggests that, particularly for a face-to-face network, the training period should be tuned carefully to improve the prediction accuracy. As discussed in the previous subsection, in face-to-face networks, using temporal information about communications is important since face-to-face communication exhibits spatiotemporal locality. On the contrary, since email communication is not conducted in real time and email messages are exchanged over the Internet, the relevance of such locality for email networks is lower than that for face-to-face networks. results for the SocioPatterns dataset and other datasets showed that the accuracy of link prediction techniques for face-to-face behavioral networks is relatively higher than that for non-social networks, but not particularly high among other types of social networks. These results suggest that conventional link prediction techniques are applicable to services, such as friendship recom- mendation, which require a moderate level of prediction accuracy. The experiments also reveal a unique characteristic of link prediction for face-to-face behavioral networks whereby prolonged observation of social networks often degrades the prediction accuracy. For an email network, it is sufficient to use a naive method that observes all records of email messages and simply uses them unmodified. In contrast, for face-to-face behavioral net- works, it is essential to determine an appropriate training period length to ensure high prediction accuracy. In most cases in our experiments, observing approximately 30 communications per person provided high prediction accuracy. However, further analysis is necessary in order to determine the optimal training period length in the general case. References 10. Adamic LA, Adar E (2003) Friends and neighbors on the Web. Social networks 25: 211–230. 1. Scholz C, Atzmueller M, Stumme G (2012) On the predictability of human contacts: Inuence factors and the strength of stronger ties. In: Proceedings of the 4th IEEE International Conference on Social Computing (SocialCom 2012). pp. 312–321. 11. Zhou T, Lu¨ L, Zhang YC (2009) Predicting missing links via local information. The European Physical Journal B 71: 623–630. 2. Scholz C, Atzmueller M, Barrat A, Cattuto C, Stumme G (2013) New insights and methods for predicting face-to-face contacts. In: Proceedings of the 7th International AAAI Conference on Weblogs and Social Media (ICWSM’13). 12. Huang Z (2006) Link prediction based on graph topology: The predictive value of the generalized clustering coefficient. In: Proceedings of the Workshop on Link Analysis: Dynamics and Static of Large Networks (LinkKDD 2006). 3. Liben-Nowell D, Kleinberg J (2007) The link-prediction problem for social networks. Journal of the American Society for Information Science and Technology 58: 1019–1031. 13. Fire M, Tenenboim L, Lesser O, Puzis R, Rokach L, et al. (2011) Link prediction in social networks using computationally efficient topological features. In: Proceedings of the 3rd IEEE International Confernece on Social Computing (SocialCom 2011). pp. 73–80. 4. Lu¨ L, Zhou T (2010) Link prediction in weighted networks: The role of weak ties. EPL (Europhysics Letters) 89: 18001. 14. Huang Z, Zeng DD (2006) A link prediction approach to anomalous email detection. In: Proceed- ings of the 2006 IEEE International Conference on Systems, Man, and Cybernetics (SMC 2006). pp. 1131–1136. 5. Tylenda T, Angelova R, Bedathur S (2009) Towards time-aware link prediction in evolving social networks. In: Proceedings of the 3rd Workshop on Social Network Mining and Analysis (SNAKDD 2009). pp. 9:1–9:10. 15. Song HH, Cho TW, Dave V, Zhang Y, Qiu L (2009) Scalable proximity estimation and link prediction in online social networks. In: Proceedings of the 9th ACM SIGCOMM Conference on Internet Measurement Conference (IMC 2009). pp. 322–335. 6. Clauset A, Moore C, Newman MEJ (2008) Hierarchical structure and the prediction of missing links in networks. Nature 453: 98–101. 7. Kashima H, Kato T, Yamanishi Y, Sugiyama M, Tsuda K (2009) Link propagation: A fast semi- supervised learning algorithm for link prediction. In: Proceedings of the 10th SIAM International Conference on Data Mining. pp. 1099–1110. 16. Wang WQ , Zhang QM, Zhou T (2012) Evaluating network models: A likelihood analysis. Author Contributions The experiments showed that conventional link prediction techniques predict new link formation in a face-to-face behavioral network with a precision of 0.30–0.45 and a recall of 0.10–0.20 through the use of an appropriate threshold. Comparison of the Conceived and designed the experiments: ST HO. Performed the experiments: ST. Analyzed the data: ST HO. Contributed reagents/ materials/analysis tools: ST. Wrote the paper: ST HO. Conceived and designed the experiments: ST HO. Performed the experiments: ST. Analyzed the data: ST HO. Contributed reagents/ materials/analysis tools: ST. Wrote the paper: ST HO. Comparison with the Enron Email Dataset 11), and suggests that the training period must be determined carefully in order to improve the prediction accuracy, particularly for a face-to-face behavioral network. Figure 16 shows that the decaying weight method is not effective, and that the naive method (i.e., the flat weight method) is Figure 15 shows that the prediction accuracy for an email network is improved when the training period length is extended. This tendency is different from the results for a face-to-face Table 2. Structural characteristics of several types of networks and the AUC score of CN for those networks. network number of nodes average degree clustering coefficient average path length AUC face-to-face (SocioPatterns dataset) 110 38.9 0.53 2.0 0.5–0.7 email (Enron email dataset) 151 9.3 0.70 6.0 0.6–0.85 terrorists [34] – 4.9 0.36 2.6 0.5–0.8 [6] Facebook [35] 63,731 24.3 – – 0.8–0.94 [13] protein [36] – 4.8 0.06 3.7 0.5–0.58 [6] food web [37] – 3.0 0.17 3.3 0.5–0.7 [6] doi:10.1371/journal.pone.0081727.t002 PLOS ONE | www plosone org 12 December 2013 | Volume 8 | Issue 12 | e81727 Table 2. Structural characteristics of several types of networks and the AUC score of CN for those netw 12 12 Link Prediction for Face-to-Face Networks g y ( ) pp 6. Clauset A, Moore C, Newman MEJ (2008) Hierarchical structure and the prediction of missing links in networks. Nature 453: 98–101. Link Prediction for Face-to-Face Networks Link Prediction for Face-to-Face Networks 19. Gallagher B, Tong H, Eliassi-Rad T, Faloutsos C (2008) Using ghost edges for classi_cation in sparsely labeled networks. In: Proceedings of the 14th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining (KDD’08). pp. 256–264. 28. Murata T, Moriyasu S (2007) Link prediction of social networks based on weighted proximity measures. In: IEEE/WIC/ACM International Conference on Web Intelligence. pp. 85–88. g pp 29. Corlette D, Shipman III F (2010) Link prediction applied to an open large-scale online social network. In: Proceedings of the 21st ACM Conference on Hypertext and Hypermedia (HT 2010). pp. 135–140. ( ) pp 20. Isella L, Stehle´ J, Barrat A, Cattuto C, Pinton JF, et al. (2011) What’s in a crowd? Analysis of face-to-face behavioral networks. Journal of Theoretical Biology 271: 166–180. 30. Backstrom L, Leskovec J (2011) Supervised random walks: Predicting and recommending links in social networks. In: Proceedings of the 4th ACM International Conference on Web Search and Data Mining (WSDM 2011). 635–644. 21. Olguı´n DO, Waber BN, Kim T, Mohan A, Ara K, et al. (2009) Sensible organizations: Technology and methodology for automatically measuring organizational behavior. IEEE Transactions on Systems, Man, and Cybernetics, Part B: Cybernetics 39: 43–55. 31. Kashima H, Abe N (2006) A parameterized probabilistic model of network evolution for supervised link prediction. In: Proceedings of the 6th International Conference on Data Mining (ICDM 2006). pp. 340–349. 22. Eagle N, Pentland A, Lazer D (2009) Inferring friendship network structure by using mobile phone data. Proceedings of the National Academy of Sciences 106: 15274–15278. g ( ) pp 32. Hanley J, McNeil B (1982) The meaning and use of the area under a receiver operating characteristic (ROC) curvel. Radiology 143: 29–36. 23. Tsugawa S, Ohsaki H, Itoh Y, Ono N, Kagawa K, et al. (2011) Quasi-realtime social network construction with heterogeneous sensors in ambient environment. In: Proceedings of the 8th International Conference on Advances in Computer Entertainment Technology (ACE 2011). pp. 75:1–75:2. 33. DeLong ER, DeLong DM, Clarke-Pearson DL (1988) Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 44: 837–845. gy ( ) pp 24. Tsugawa S, Ohsaki H, Imase M (2012) Effectiveness of link prediction techniques for face-to-face behavioral networks. In: Proceedings of the 2012 Japan-Ireland Workshop on Intelligence and Networks (JIWIN 2012). pp. 98– 107. 34. Krebs VE (2002) Mapping networks of terrorist cells. References EPL (Europhysics Letters) 98: 28004. 17. Guimera` R, Sales-Pardo M (2009) Missing and spurious interactions and the reconstruction of complex networks. Proceedings of the National Academy of Sciences 106: 22073–22078. 8. Lu¨ L, Zhou T (2011) Link prediction in complex networks: A survey. Physica A: Statistical Mechanics and its Applications 390: 1150–1170. 8. Lu¨ L, Zhou T (2011) Link prediction in complex networks: A survey. Physica A: Statistical Mechanics and its Applications 390: 1150–1170. 18. Zhang QM, Shang MS, Lu L (2010) Similarity-based classi_cation in partially labeled networks. International Journal of Modern Physics C 21: 813–824. 9. Newman MEJ (2001) Clustering and preferential attachment in growing networks. Physical Review E 64: 025102. 9. Newman MEJ (2001) Clustering and preferential attachment in growing networks. Physical Review E 64: 025102. December 2013 | Volume 8 | Issue 12 | e81727 13 PLOS ONE | www.plosone.org Link Prediction for Face-to-Face Networks CONNECTIONS 24: 43– 52. 35. Viswanath B, Mislove A, Cha M, Gummadi KP (2009) On the evolution of user interaction in Facebook. In: Proceedings of the 2nd ACM SIGCOMMWork- shop on Social Networks (WOSN’09). pp. 37–42. 25. Kobayashi Y (2012) On the effectiveness of link prediction techniques for face- to-face behavioral networks. Bachelor thesis (in Japanese), School of Engineering and Science, Osaka University. 36. Huss M, Holme P (2007) Currency and commodity metabolites: Their identi_cation and relation to the modularity of metabolic networks. IET Systems Biology 1: 280–285. y 26. Shetty J, Adibi J (2004) The Enron email dataset database schema and brief statistical report. Technical report, Information Sciences Institute, University of Southern California. 37. Dawah H, Hawkins B, Claridge M (1995) Structure of the parasitoid communities of grass-feeding chalcid wasps. Journal of Animal Ecology 64: 708–720. 27. Salton G, McGill MJ (1986) Introduction to modern information retrieval. McGraw-Hill, Inc. PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e81727 PLOS ONE | www.plosone.org 14
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ChemicalToolBoX and its application on the study of the drug like and purchasable space
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Lucas et al. Journal of Cheminformatics 2014, 6(Suppl 1):P51 http://www.jcheminf.com/content/6/S1/P51 Open Access ChemicalToolBoX and its application on the study of the drug like and purchasable space Xavier Lucas*, Björn A Grüning, Stefan Günther From 9th German Conference on Chemoinformatics Fulda, Germany. 10-12 November 2013 computed several physicochemical descriptors to dis- cover general trends applying to each subset. The ever increasing amount of data and computational capabilities in the cheminformatics field has led to a sce- nario where efficient techniques for storage and proces- sing in an integrated, modular, and easily accessible platform are in vital demand. Here, we present Chemical- ToolBoX, a compilation of more than 30 tools integrated into a single computational chemistry and cheminfor- matics platform based on the Galaxy workflow manage- ment system [1,2]. We have recently designed a workflow within the ChemicalToolBoX to generate a library of compounds containing around 70 million unique com- mercially available small molecules, i.e. the purchasable space [3]. Subsequently, we have used filtering rules based on structural patterns and chemical alarms to dis- card problematic molecules, representing a very large portion of the drug-like and purchasable space, along with other drug discovery data sets including more than 2 million fragments (Figure 1). Furthermore, we have Published: 11 March 2014 References 1. [https://github.com/bgruening/galaxytools/tree/master/chemicaltoolbox]. 2. Grüning BA, et al:, submitted. 3. Lucas X, et al:, manuscript in preparation. doi:10.1186/1758-2946-6-S1-P51 Cite this article as: Lucas et al.: ChemicalToolBoX and its application on the study of the drug like and purchasable space. Journal of Cheminformatics 2014 6(Suppl 1):P51. Published: 11 March 2014 Published: 11 March 2014 References 1. [https://github.com/bgruening/galaxytools/tree/master/chemicaltoolbox]. 2. Grüning BA, et al:, submitted. 3. Lucas X, et al:, manuscript in preparation. doi:10.1186/1758-2946-6-S1-P51 Cite this article as: Lucas et al.: ChemicalToolBoX and its application on the study of the drug like and purchasable space. Journal of Cheminformatics 2014 6(Suppl 1):P51. References doi:10.1186/1758-2946-6-S1-P51 Cite this article as: Lucas et al.: ChemicalToolBoX and its application on the study of the drug like and purchasable space. Journal of Cheminformatics 2014 6(Suppl 1):P51. Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ * Correspondence: xavier.lucas@pharmazie.uni-freiburg.de Pharmaceutical Bioinformatics, Institute of Pharmaceutical Sciences, Albert- Ludwigs-University, Freiburg, D-79104, Germany Figure 1 © 2014 Lucas et al; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: xavier.lucas@pharmazie.uni-freiburg.de Pharmaceutical Bioinformatics, Institute of Pharmaceutical Sciences, Albert- Ludwigs-University, Freiburg, D-79104, Germany Figure 1 © 2014 Lucas et al; licensee Chemistry Cent Attribution License (http://creativecommo any medium, provided the original work i creativecommons.org/publicdomain/zero/ Figure 1 Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Publish with ChemistryCentral and every scientist can read your work free of charge Publish with ChemistryCentral and every scientist can read your work free of charge Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. W. Jeffery Hurst, The Hershey Company. * Correspondence: xavier.lucas@pharmazie.uni-freiburg.de Pharmaceutical Bioinformatics, Institute of Pharmaceutical S Ludwigs-University, Freiburg, D-79104, Germany © 2014 Lucas et al; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Preparedness and Costing on Fire Safety Installation in Commercial Buildings
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ABSTRACT Purpose: The study was carried out to analyze the awareness among the management, occupants and concerned authority of selected commercial buildings at Birtamode, jhapa and prepare the tentative cost of additional tools and equipment’s needed. Design/Methodology/Approach: This research is focused on two commercial buildings; Hanuman central and One stop mall (Birtamode Jhapa). These building are selected for research as both of the buildings are considered as largest shopping malls and they also vary in planning and construction technology. The fire safety preparedness and awareness of management, occupants were analyzed. After then preparedness and awareness of concerned authority like fire fighters, Municipality and Nepal police were analyzed. Data was collected from occupants, management and concerned authority based on the questionnaires survey, site visits with check list and key informant interview. Information regarding fire safety preparedness and awareness was presented in tabular form, charts and Key informants interview and questionnaire. f f q Findings/Result: Fire safety preparedness were lacking due to lack awareness on evacuation procedure and equipment’s operation. Management seems to be more focused on financial aspect rather than safety. Lack of proper firefighting equipment’s, equipment’s in poor condition, unmanaged electrical wires is the existing problem in the buildings. Concerned authority (Birtamode Municipality) had a fire Fighting team with a six fire men with a tank and fire fighting vehicle. But the new technologies like GPS were not introduced to firefighter. There were many cases reported where due to communication gap between the fire fighter and residents, the response was late which resulted more damage. Lack of fire hydrant near the major commercial buildings created problem for fire fighter to refill their water tank. Narrow roads inside the municipality, poor implementation of fire safety code were major problem for fire fighters. The tentative cost for the installation of fire safety tools and equipment’s Rs 5, 15,000 and Rs 9, 50,000 for one stop mall and Hanuman central respectively. Originality/Value: The study may be a guiding document for professionals, entrepreneurs and occupants for improving awareness and preparedness by obtaining cost components against fire safety. Paper Type: Research paper Keywords: Fire safety installation, Safety in commercial buildings, Preparedness and costing. 1 INTRODUCTION : Keywords: Fire safety installation, Safety in commercial buildings, Preparedness and costing. Keywords: Fire safety installation, Safety in commercial buildings, Preparedness and costing. 2 Professor, Institute of Management & Commerce, Srinivas University, Mangalore, India, OrcidID: 0000-0002-4691-8736; E-mail:psaithal@gmail.com Subject Area: Project Management. Type of the Paper: Conceptual Research. Type of Review: Peer Reviewed as per |C|O|P|E| guidance. Indexed In: OpenAIRE. DOI: https://doi.org/10.5281/zenodo.7108648 Google Scholar Citation: IJAEML International Journal of Applied Engineering and Management Letters (IJAEML) A Refereed International Journal of Srinivas University, India. Crossref DOI: https://doi.org/10.47992/IJAEML.2581.7000.0152 Received on: 04/09/2022 Published on: 24/09/2022 © With Authors. This work is licensed under a Creative Commons Attribution-Non-Commercial 4.0 International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. How to Cite this Paper: Mishra, A. K., & Aithal, P. S., (2022). Preparedness and Costing on Fire Safety Installation in Commercial Buildings. International Journal of Applied Engineering and Management Letters (IJAEML), 6(2), 182-196. DOI: https://doi.org/10.5281/zenodo.7108648 © With Authors. This work is licensed under a Creative Commons Attribution-Non-Commercial 4.0 International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. This work is licensed under a Creative Commons Attribution-Non-Commercial 4.0 International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. This work is licensed under a Creative Commons Attribution-Non-Commercial 4.0 International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. PAGE 182 A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 PUB International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION As the development works are rapidly increasing day by day fire safety should become a topic to be considered. OHS should be given first priority by concerned authority and management. Although firefighting is top priority for management in big metropolitan city like Kathmandu (capital city of Nepal) and Pokhara, newly emerged city like Damak, Biratnagar, Birtamode are seems to be unaware and negligent in this topic. Management, concerned authority and occupants are also seems to be unaware on this topic and fire has been kept on the least priority by them. Firefighting preparedness is not a priority for government compared to earthquake (UNDP, 2009) [4]. Fire wellbeing code was formed in 1994 A.D. what's more, after then it has quite recently turned into a piece of report and furthermore was never overhauled till now. Indeed, UNDP has expressed the requirement for present day and state-of-the-art fire code. Since NBC 107:1994 [5] is definitely not a specialized yet in addition an authoritative record, it should be refreshed in light of changing conditions to guarantee fire safety. So the researcher has selected two commercial building and assessed fire safety in terms of awareness and preparedness of Management, Concerned authority and Occupants and also calculated the tentative cost of installation of fire safety tools and equipment’s in the buildings. 1. INTRODUCTION : Design is the mother of construction [1]. Design of Modern Malls and complex in Urban Areas seem to consider the fire safety in some manner. But the available facility and measures are more like a formality for owners. Date expired extinguisher, lack of emergency exit, poor design, more commercial space than flowing space, owners motive of profit maximization, lack of awareness, lack of electrical drawings, poor electrical connection, low quality electrical material, unmanaged use of electrical appliances due to weather, less knowledge on fire safety, etc. are the major issues seen [2, 3]. PAGE 183 A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION The study area is one stop mall and Hanuman Central located at Birtamode, Jhapa, Nepal. It can be important document for all three parties for increasing their awareness and preparedness in case of emergency. It can be helpful for the management about the cost of additional fire safety tools and equipment’s that can be quickly installed. It can be helpful for new Designers and Developers about the fire safety practice and importance of it in case of emergency. 3. OBJECTIVES : The Objective is to assess the status of awareness and preparedness of management, concerned authority and occupants along with the cost of installation of fire safety tools and equipment’s for Selected Commercial Buildings at Birtamode, Jhapa, Nepal. 4. LITERATURE REVIEW : 4.1 Fire Safety Preparedness: Readiness involves adjusting one's conduct to decrease the effect of catastrophes on people (Drabek, 1986) [3, 8]. It is a constant pattern of arranging, making due, sorting out, preparing, preparing, working out, making, assessing, checking, and further developing exercises to guarantee powerful coordination and the improvement of capacities of concerned associations in forestalling, safeguarding against, answering, recuperating from, making assets, and moderating the impacts of catastrophic events, demonstrations of psychological oppression, and other man-made calamities [3, 9]. During the preparedness phase, emergency managers create strategies to manage and mitigate their risks, as well as take steps to acquire the required capabilities to put those plans into effect. Common preparedness measures include: Common preparedness measures include: • Communication plans that use vocabulary and methods that are simple to understand. • Emergency services should be properly maintained and trained. • Emergency services should be properly maintained and trained. • The creation and testing of emergency population warning systems, as well as emergency shelters and evacuation strategies. • Establish and maintain an emergency communication system that can assist in determining the nature of an emergency and providing instructions as necessary (Ogajo, 2013) [3, 10]. 4.2 Fire Safety Management: 2. STATEMENT OF PROBLEMS : In July 27, 2019 Fire erupted in Hanuman Central at Birtamode which incurred loss of 20 million. The building caught a flame due to short circuit in an air conditioner machine of shoe shop on ground floor of six storey building [6]. A few months back fire erupted in Sri Krishna market which incurred loss of 15 million. Fire was started from an electronic shop go down around 1:00 – 2:00 am and was taken under control only in 7 am in the morning. Fire fighters from Birtamode Municipality and Mechinagar Municipality along with Nepali Army were able to control the fire. Fig. 1: Fire at Namaste Ply indsurty at chaitubari, Birtamode -2 (RSS) [7] Fig. 1: Fire at Namaste Ply indsurty at chaitubari, Birtamode -2 (RSS) [7] In Dec 4, 2020 A gigantic fire broke out at the Namaste Handle Industry at Chaitubari, Birtamod-2 in Jhapa locale and destroyed property worth Rs 3 million. According to Representative Director of Police of Region Police Office Jhapa, Rakesh Thapa, a short out prompted the fire.As police got the data, a joint group comprising the Region Police Office Anarmani, the Territory 1 Debacle The board Organization Chandragadhi and the Furnished Police Power Headquarters Sanischare went for the rescue.Locals and security staff cooperated to drench the fire utilizing fire motors from Damak, Bhadrapur and Birtamod. In case of Nepal, Safety seems to be of less priority either in design or in operational phase. Owner doesn’t seem to be aware and prepared of unforeseen situation. Concerned authority seems weak in implementing the fire safety awareness and fire safety code. NBC Fire code has become just a document. This research mainly focuses on current fire safety condition existing commercial building and awareness and preparedness of management, occupants and concerned authority and prepare a tentative budget for installation of fire safety tools and equipment’s. PAGE 184 A. K. Mishra, et al. (2022); www.srinivaspublication.com 4.2 Fire Safety Management: In terms of fire prevention and safety, management is critical. A building's fire safety design consists of a range of measures relating to the layout, structure, and other requirements, some of which are activated in the event of a fire (Malhotra, 1993) [11]. Standard investigation, support, posting of notification and exit coordinated signs, normal fire works out, departure methodologies, and the presence of fire superintendents are all essential for fire security the executives (Ogajo, 2013) [10]. The essential objective of fire security the board is to ensure that all open fire wellbeing measures are accessible for people to use in order to aid their escape (Baker, 2003) [12]. y g Exit Requirement: q An entryway, hall, or path prompting an inside flight of stairs, an outside flight of stairs, a verandah going to the road, the top of a structure, or the road is viewed as an exit. The egress could perhaps lead to another structure in the area The exit should to another structure in the area. The exit should to another structure in the area. The exit should • Meet the minimum size criteria and enable for the evacuation of all people in a reasonable amount of time. • Be devoid of any impediments and provide no resistance to movement. devoid of any impediments and provide no resistance to movement. • Be easily seen, especially with appropriate indicators. Be consistent and do not encroach on personal space. i • Be consistent and do not encroach on personal space. Stairs: • Be consistent and do not encroach on personal space. Stairs: • At least two fire escape staircases, one internal and the other external. • When a building's plinth area reaches 500 m2, more steps must be built in proportion to the increased plinth space. • The minimum width is 1.5 meters. • The minimum width is 1.5 meters. • The distance between any point in a hallway and a building's stairway must not exceed 20 meters. Fire Escapes: The Building having 5 or more stories must provide a separate escape with of at least 75 cm width Each riser on the fire escape must be no more than 19 cm high and 20 cm width The Building having 5 or more stories must provide a separate escape with of at least 75 cm width Each riser on the fire escape must be no more than 19 cm high and 20 cm width. The Building having 5 or more stories must provide a separate escape with of at least 75 cm w • Each riser on the fire escape must be no more than 19 cm high and 20 cm width. • Each riser on the fire escape must be no more than 19 cm high and 20 cm width. • The number of risers each flight must not exceed 15. • This type of fire escape should lead users to an open area. • This type of fire escape should lead users to an open area. s type of fire escape should lead users to an open area. Exit Doors: Leave entryways should open to a way or a hallway, however not to a way or a passageway, and they should open outwards without impeding the development of individuals passing external the entryway. The greatest distance between any point in the course and such a leave entryway is 20 meters. The width and level of the leave entryway should not be under 90 cm and 180 cm, separately. 4.5 Nepal National Building Code NBC 107: 1994: p g Nepal national building code NBC 107:1994 provides fundamental requirements for fire safety in ordinary buildings (GoN, 1994). Summary of NBC 107: 1994 are listed below [2, 5]. Exit Requirement: Nepal national building code NBC 107:1994 provides fundamental requirements for fire safety in ordinary buildings (GoN, 1994). Summary of NBC 107: 1994 are listed below [2, 5]. Exit Requirement: International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION 4.4 Fire Safety Code: 4.3 Fire Safety Management Roles and Responsibilities: Fire Safety Management Roles and Responsibilities: re Safety Management Roles and Responsibilities: In a business facility, pre-planned processes for dealing with a fire emergency are required. The roles and responsible of fire safety management are listed below. Roles and Responsibilities: In a business facility, pre-planned processes for dealing with a fire emergency are required. The roles and responsible of fire safety management are listed below. Roles and Responsibilities: p Select a fire security chief to guarantee that fire wellbeing conventions are followed and that upkeep needs are met. Guarantee that staff knows about the wellbeing strategies and the tenants complete their work in a protected way. An upkeep timetable ought to be produced for all fire recognition/caution frameworks, fire dousers, hose reels, and other comparable things, and records of their review and fix ought to be safeguarded. Hold regular fire drills for representatives to guarantee that they know about the legitimate method for get out, leave areas, and courses to take in case of a fire. Make arrangements for evacuation, fire suppression, and aid to the fire department when they arrive. The strategy should be addressed with the fire department, and their agreement on the planned processes should be acquired [2, 3, 11]. PAGE 185 A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION 4.4 Fire Safety Code: A building code is a rule or guideline that establishes the minimum standards for the design and construction of structures and buildings. The goal of establishing those basic requirements is to safeguard society's health and safety [13]. In new and existing buildings, structures, and processes, the fire safety code addresses fire prevention, fire protection, life safety, and the proper storage and use of hazardous materials. They give a comprehensive approach to hazard control in all structures and sites (International Code Council, 2009). The fire safety requirements for buildings were researched using NBC 107: 1994 [2, 5]. 5.1 Study Area: y To assess the awareness and preparedness of Management, concerned authority and occupants Buildings selected were one stop mall and Hanuman Central Birtamode. To assess the awareness and preparedness of Management, concerned authority and occupants Buildings selected were one stop mall and Hanuman Central Birtamode. One Stop Mall: International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION Hanuman central is also located at Birtamode, Jhapa touching national highway (Mahendra Highway). It is also known as Hanuman Complex. It was the first Commercial complex build in Birtamode. It was opened in 2012. p It is a Rectangular shaped building with atrium in the center. Vertical circulation is aided by elevators and stairwells, whereas horizontal circulation is aided by walkways and lobby. There are 3 staircases in the building with 3 elevators. 5.2. Research Philosophy: p y The descriptive along with field based empirical research design was adopted in this research work. Figure 1 depicts the different stages applied in the research [14]. p y The descriptive along with field based empirical research design was adopted in this research work. Figure 1 depicts the different stages applied in the research [14]. Figure 1 depicts the different stages applied in the research [14]. Fig. 1: Research Onion The overall research philosophy is Pragmatism is in which only "solution" obtained from observation with reliable assessment and objective interpretation. The study outcomes in these sorts of investigations are frequently apparent and quantitative. Abduction approach for logical reasoning was followed through inductive and deductive ways. Regarding methodological choice, both qualitative as well as quantitative methods were followed. So it’s a mixed method. The research strategy was survey. As the data were collected through the different Key informant Interview, Survey and observation, this study was ex-post facto field based. The survey was done on longitudinal time horizon. The data collection methods were observation method, survey, and interview. Pragmatism Abductive Mixed (Qualitative+Quantitative) Survey Longitudinal Observation & Survey Fig. 1: Research Onion g The overall research philosophy is Pragmatism is in which only "solution" obtained from observation with reliable assessment and objective interpretation. The study outcomes in these sorts of investigations are frequently apparent and quantitative. Abduction approach for logical reasoning was followed through inductive and deductive ways. Regarding methodological choice, both qualitative as well as quantitative methods were followed. So it’s a mixed method. The research strategy was survey. As the data were collected through the different Key informant Interview, Survey and observation, this study was ex-post facto field based. The survey was done on longitudinal time horizon. The data collection methods were observation method, survey, and interview. g One Stop Mall: One stop Mall is Located at Birtamode, Jhapa. It is located at North- west side from mukti chowk (Central part of Birtamode). The building, which is a shopping complex with six stories above ground, was finished in 2016. Three floors including the ground are used for shops and top three floors are used for Cinema purpose. Building is U-shaped with a large steel structure at front connecting to building. The structure has a variety of shops selling various goods and services, as well as leisure venues such as a movie, a game center, and a pub. Underground Parking Facility is also available but the building structural components don’t have any connection with the parking below. Vertical circulation is assisted by escalators, elevators, and stairwells, while horizontal circulation is assisted by walkways and lobby. C l Hanuman Central: Hanuman Central: Hanuman Central: PAGE 186 A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION Questionnaire Survey Questionnaire Survey Q y Based on the information from the literature review questionnaire was formulated. A survey of the occupants of selected business buildings was done. Management of selected commercial buildings and concerned authority. It was conducted to understand fire safety awareness and preparedness of all three parties. Several references were takes into considerations [15, 16, 17, 18, 19, 20, and 21]. Key Informants Interview PAGE 187 A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION Key informants interview was done with Head of management of Hanuman central and One stop mall, head of fire department( Birtamode Municipality) and Engineer of Birtamode Municipality( Building Department) [15, 16, 17, 18, 19, 20, and 21]. International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION Key informants interview was done with Head of management of Hanuman central and One stop mall, head of fire department( Birtamode Municipality) and Engineer of Birtamode Municipality( Building Department) [15, 16, 17, 18, 19, 20, and 21]. 5.4 Methodology in Brief: Table 1: Summary of Methodology Objective Activities Analysis 1. To identify the level of awareness among management, occupants and concerned authority. 1. Key Informant Interview 2. Questionnaire survey 1. Qualitative Analysis 2. Quantitative Analysis 2. To calculate the tentative cost of installation of fire safety tools and Equipment’s. 1. Rate collection and calculation of cost. 1. Qualitative Analysis Table 1: Summary of Methodology 6.1 Fire Safety Preparedness of Management, Occupants and Concerned Authority: 6.1 Fire Safety Preparedness of Management, Occupants and Concerned Authority: The data from the questionnaire was used to create the graphs below, which show the inhabitants', management's, and concerned authority's level of fire safety readiness. 6.1.1 Awareness and Preparedness of Occupants on Fire Safety: 6.1.1 Awareness and Preparedness of Occupants on Fire Safety: The table 2 shows the level of fire safety readiness among the tenants of all chosen business buildings, i.e., one Stop Mall and Hanuman Central Birtamode. Table 2: Awareness on Building Feature Hanuman Central One stop Mall Awareness on building feature Yes No Can’t Say Yes No Can’t Say Building Design 10 12 18 20 19 1 Location of exit 28 11 1 27 13 0 Visibility of emergency sign 4 22 14 26 10 4 ndents were asked to state whether or not they were aware of a certain building feature. Respondents were asked to state whether or not they were aware of a certain building feature. The respondent's response is shown in Table 4.21. Ten Hanuman Central respondents were aware of the building design, 28 were aware of the position of the exit, and four respondents had emergency signs visible. 12 respondents were unfamiliar of the building layout, 11 were unaware of the position of the escape, and 22 respondents did not see an emergency sign. 18 respondents had no clue about the structure plan, 1 had no data about the position of the exit, and 14 had no clue about the crisis sign. 6.1.3 Awareness of Emergency Egress: 20 respondents of Hanuman Central knew about crisis clearing, 12 respondents knew about get together point. 18 respondents didn't know crisis clearing, 16 were uninformed about gathering point. Though, 2 respondents had no clue about crisis departure, 12 respondents had no clue about gathering point. Table 3: Awareness of Emergency Egress Hanuman Central (nos. of occupants) One stop mall (nos. of occupants) Awareness on emergency Egress Yes No Can’t Say Yes No Can’t Say Emergency evacuation 20 18 2 22 7 11 Assembly point 12 16 12 14 20 6 52.5% of respondents were found aware, 31.25% of respondents were not aware and 16.25% can’t say about emergency evacuation. 32.5% were aware, 45% were aware and 22.5% cants ay about Assembly point. It shows that tenants needed mindfulness on crisis departure. A. K. Mishra, et al. (2022); www.srinivaspublication.com PAGE 189 Table 3: Awareness of Emergency Egress Hanuman Central (nos. of occupants) One stop mall (nos. of occupants) Awareness on emergency Egress Yes No Can’t Say Yes No Can’t Say Emergency evacuation 20 18 2 22 7 11 Assembly point 12 16 12 14 20 6 52.5% of respondents were found aware, 31.25% of respondents were not aware and 16.25% can’t say about emergency evacuation. 32.5% were aware, 45% were aware and 22.5% cants ay about Assembly point. It shows that tenants needed mindfulness on crisis departure. Table 3: Awareness of Emergency Egress Hanuman Central (nos. of occupants) One stop mall (nos. of occupants) Awareness on emergency Egress Yes No Can’t Say Yes No Can’t Say Emergency evacuation 20 18 2 22 7 11 Assembly point 12 16 12 14 20 6 52.5% of respondents were found aware, 31.25% of respondents were not aware and 16.25% can’t say about emergency evacuation. 32.5% were aware, 45% were aware and 22.5% cants ay about Assembly point. It shows that tenants needed mindfulness on crisis departure. 52.5% of respondents were found aware, 31.25% of respondents were not aware and 16.25% can’t say about emergency evacuation. 32.5% were aware, 45% were aware and 22.5% cants ay about Assembly point. It shows that tenants needed mindfulness on crisis departure. 52.5% of respondents were found aware, 31.25% of respondents were not aware and 16.25% can’t say about emergency evacuation. 32.5% were aware, 45% were aware and 22.5% cants ay about Assembly point. It shows that tenants needed mindfulness on crisis departure. A. K. Mishra, et al. 6.1.2 Awareness of Building Feature: Twenty one-stop mall respondents were aware of the building design, 27 were aware of the position of the exit, and 26 respondents reported that emergency signs were visible. The building design was PAGE 188 A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION unknown to 19 respondents, the location of the exit was unknown to 13 respondents, and the emergency sign was not visible to 10 respondents. One individual had no clue about the structure format, 0 had no clue about the position of the exit, and 4 had no clue about the crisis sign. Fig. 2: Awareness of Building Feature Percentage of respondants 80 70 60 50 40 30 20 10 0 Building Plan Location of Exit Visibility of Emergency Cannot Say 23.75 1.25 22.5 International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION unknown to 19 respondents, the location of the exit was unknown to 13 respondents, and the i i ibl 10 d O i di id l h d l b h International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION unknown to 19 respondents, the location of the exit was unknown to 13 respondents, and the emergency sign was not visible to 10 respondents. One individual had no clue about the structure format, 0 had no clue about the position of the exit, and 4 had no clue about the crisis sign. wn to 19 respondents, the location of the exit was unknown to 13 respondents, and the ency sign was not visible to 10 respondents. One individual had no clue about the structure , 0 had no clue about the position of the exit, and 4 had no clue about the crisis sign. emergency sign was not visible to 10 respondents. One individual had no clue about the structure format, 0 had no clue about the position of the exit, and 4 had no clue about the crisis sign. Fig. 6.1.2 Awareness of Building Feature: 2: Awareness of Building Feature Percentage of respondants 80 70 60 50 40 30 20 10 0 Building Plan Location of Exit Visibility of Emergency Cannot Say 23.75 1.25 22.5 Fig. 2: Awareness of Building Feature 37.5% of respondents knew about building Plan, 38.75% of respondents don’t know about plan and 23.75 cannot say. 68.75% have knowledge about exit, 30% don’t know and 1.25 cannot say about it, similarly 37.5% knows about emergency sign, 40% haven’t seen and 22.5% cannot say about that. 6.1.3 Awareness of Emergency Egress: International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION Supervisory group ought to much of the time arrange normal fire drills to guarantee that inhabitants stay mindful of the proper method for get out and where they will stand by after crisis departure. The results are in similar trends to that of study conducted in Kathmandu [3], 22 respondents of LABIM Shopping center knew about crisis clearing, 14 respondents knew about get together point. 7 respondents didn't know about crisis clearing, 20 were uninformed about gathering point. Though, 11 respondents had no clue about crisis departure, 6 respondents had no clue about gathering point. A. K. Mishra, et al. (2022); www.srinivaspublication.com 6.1.3 Awareness of Emergency Egress: (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION Supervisory group ought to much of the time arrange normal fire drills to guarantee that inhabitants stay mindful of the proper method for get out and where they will stand by after crisis departure. The results are in similar trends to that of study conducted in Kathmandu [3], 22 respondents of LABIM Shopping center knew about crisis clearing, 14 respondents knew about get together point. 7 respondents didn't know about crisis clearing, 20 were uninformed about gathering point. Though, 11 respondents had no clue about crisis departure, 6 respondents had no clue about gathering point. International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION 6.1.4 Provision of Assistance: 21 respondents needn't bother with help to arrive at spot of wellbeing, 12 respondents accepted help won't be given during fire crisis to arrive at spot of security. Though, 14 respondents couldn't say assuming they required help, and 20 respondents had no clue in the event that help would be given during fire crisis. Table 4: Provision of Assistance Hanuman Central (nos. of occupants) One Stop Mall (nos. of occupants) Provision of assistance Yes No Can’t say Yes No Can’t say Need emergency assistance 15 10 15 5 21 14 Assistance provided 7 17 16 8 12 20 5 respondents of One stop Shopping center required help to arrive at spot of security, 8 respondents expected help will be given during fire crisis to arrive at spot of wellbeing. 21 respondents needn't bother with help to arrive at spot of wellbeing, 12 respondents accepted help won't be given during fire crisis to arrive at spot of security. Though, 14 respondents couldn't say assuming they required help, and 20 respondents had no clue in the event that help would be given during fire crisis. 5 respondents of One stop Shopping center required help to arrive at spot of security, 8 respondents expected help will be given during fire crisis to arrive at spot of wellbeing. 21 respondents needn't bother with help to arrive at spot of wellbeing, 12 respondents accepted help won't be given during fire crisis to arrive at spot of security. Though, 14 respondents couldn't say assuming they required help, and 20 respondents had no clue in the event that help would be given during fire crisis. 5 respondents of One stop Shopping center required help to arrive at spot of security, 8 respondents expected help will be given during fire crisis to arrive at spot of wellbeing. 21 respondents needn't bother with help to arrive at spot of wellbeing, 12 respondents accepted help won't be given during fire crisis to arrive at spot of security. Though, 14 respondents couldn't say assuming they required help, and 20 respondents had no clue in the event that help would be given during fire crisis. p p p g g Fig. 3: Awareness on Assistance 5 0 4 5 4 0 3 5 3 0 2 5 2 45 36 .2 Can't Say 36 .2 38 .7 No 18 .7 25 Yes Asistance Provided Need Emergency Assistance Percentage of Respondants A. K. 6.1.4 Provision of Assistance: 15 respondents of Hanuman Central assumed they need help to arrive at spot of security, 12 respondents expected help will be given during fire crisis to arrive at spot of wellbeing. 10 respondents needn't bother with help to arrive at spot of wellbeing, 17 respondents expected help won't be given during fire crisis to arrive at spot of security. Though, 15 respondents couldn't say assuming they required help, and 16 respondents had no clue in the event that help would be given during fire crisis. Table 4: Provision of Assistance Hanuman Central (nos. of occupants) One Stop Mall (nos. of occupants) Provision of assistance Yes No Can’t say Yes No Can’t say Need emergency assistance 15 10 15 5 21 14 Assistance provided 7 17 16 8 12 20 5 respondents of One stop Shopping center required help to arrive at spot of security, 8 respondents expected help will be given during fire crisis to arrive at spot of wellbeing. 21 respondents needn't bother with help to arrive at spot of wellbeing, 12 respondents accepted help won't be given during fire crisis to arrive at spot of security. Though, 14 respondents couldn't say assuming they required help, and 20 respondents had no clue in the event that help would be given during fire crisis. Fig. 3: Awareness on Assistance 5 0 4 5 4 0 3 5 3 0 2 5 2 45 36 .2 Can't Say 36 .2 38 .7 No 18 .7 25 Yes Asistance Provided Need Emergency Assistance Percentage of Respondants Table 4: Provision of Assistance Hanuman Central (nos. of occupants) One Stop Mall (nos. of occupants) Provision of assistance Yes No Can’t say Yes No Can’t say Need emergency assistance 15 10 15 5 21 14 Assistance provided 7 17 16 8 12 20 Table 4: Provision of Assistance Hanuman Central (nos. of occupants) One Stop Mall (nos. of occupants) Provision of assistance Yes No Can’t say Yes No Can’t say Need emergency assistance 15 10 15 5 21 14 Assistance provided 7 17 16 8 12 20 5 respondents of One stop Shopping center required help to arrive at spot of security, 8 respondents expected help will be given during fire crisis to arrive at spot of wellbeing. 6.1.5 Awareness on Fire Safety Measures: Respondents of Hanuman Central were mindful about the presence of fire douser, 10 respondents knew about the presence of water Hydrant in building. 10 respondents were uninformed about the presence of fire quencher, 12 respondents were ignorant about the presence of water hydrant and 16 respondents didn't know about fire douser, 18 respondents didn't know about water hydrant. Table 5: Awareness on Fire Safety Measures Hanuman Central (nos. of occupants) One Stop Mall (nos. of occupants) Fire safety Measures Exist Do not exist Not sure Exist Do not exist Not sure Fire extinguisher 14 10 16 15 3 22 Water hose reel/water hydrant 10 12 18 12 10 18 Table 5: Awareness on Fire Safety Measures Respondents of One stop knew about the presence of fire douser, 12 respondents knew about the presence of water hose reel in building. 3 respondents were uninformed about the presence of fire quencher, 10 respondents were ignorant about the presence of water hose reel and 22 respondents didn't know about fire douser, 18 respondents didn't know about water hose reel. International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION 25% of respondents expected help to lead them to place of wellbeing during fire crisis, 38.75% needn't bother with any help and 36.25% tenants can't say regarding that. 18.75% accepted that help would be given to during crisis, 36.25% of respondents imagine that help wouldn't be given during crisis and 45% can't say regarding that. 6.1.5 Awareness on Fire Safety Measures: A. K. Mishra, et al. (2022); www.srinivaspublication.com 6.1.4 Provision of Assistance: Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION 25% of respondents expected help to lead them to place of wellbeing during fire crisis, 38.75% needn't bother with any help and 36.25% tenants can't say regarding that. 18.75% accepted that help would be given to during crisis, 36.25% of respondents imagine that help wouldn't be given during crisis and 45% can't say regarding that. International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION 6.1.6 Ability to Operate Fire Safety Equipment: y p y q p 10 respondents of Hanuman Central think they had the option to work fire quencher, 5 respondents expected they had the option to work water hose reel/water hydrant. 22 respondents couldn't work fire douser, 25 respondents couldn't work water hose reel. While, 8 respondents were don't know whether they could work fire quencher, 10 respondents were don't know whether they could work water hose reel. A. K. Mishra, et al. (2022); www.srinivaspublication.com PAGE 191 Table 6: Ability to Operate Fire Safety Equipment Hanuman Central (nos. of occupants) One Stop Mall (nos. of occupants) Ability To Operate Fire Equipment Able to Not able to Not sure Able to Not able to Not sure Fire extinguisher 10 22 8 13 18 9 Water hose reel/water hydrant 5 25 10 2 30 8 13 respondents of One stop Shopping center accepted they had the option to work fire douser, 2 respondents expected they had the option to work water hose reel/water hydrant. 18 respondents Table 6: Ability to Operate Fire Safety Equipment Hanuman Central (nos. of occupants) One Stop Mall (nos. of occupants) Ability To Operate Fire Equipment Able to Not able to Not sure Able to Not able to Not sure Fire extinguisher 10 22 8 13 18 9 Water hose reel/water hydrant 5 25 10 2 30 8 13 respondents of One stop Shopping center accepted they had the option to work fire douser, 2 respondents expected they had the option to work water hose reel/water hydrant. 18 respondents Table 6: Ability to Operate Fire Safety Equipment 13 respondents of One stop Shopping center accepted they had the option to work fire douser, 2 respondents expected they had the option to work water hose reel/water hydrant. 18 respondents 13 respondents of One stop Shopping center accepted they had the option to work fire douser, 2 respondents expected they had the option to work water hose reel/water hydrant. 18 respondents A. K. Mishra, et al. (2022); www.srinivaspublication.com PAGE 191 International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION SRINIVAS PUBLICATION couldn't work fire douser, 30 respondents couldn't work water hose reel. While, 9 respondents were don't know whether they could work fire quencher, 8 respondents were don't know whether they could work water hose reel/water hydrant.28.75% of respondents had the option to work fire quencher, half of respondent couldn't work and 21.25% were don't know. 8.75% had the option to work water hose reel/water hydrant, 68.75 couldn't and 22.5% were don't know. Table 6 shows that of tenants needed capacity to work the firefighting hardware's. Consequently, appropriate preparation on it will be given to the inhabitants habitually so the misfortune during fire can be decreased. Equipment's with sensor system could be also an effective way as in case of theft most of shopkeeper have found to install sensor based siren which alerts the police on real time about its unauthorized opening. For this step, direct interview was conducted with management. Management seems more focused on financial part of building. Fire extinguisher was only tool for fire safety. There was smoke detector and alarm system installed in One Stop Mall. Management of both the buildings have requested for installation of fire hydrant to Municipality many times. Water hydrant installed in both the building weren’t in working condition. Fire fighter of fire department of Birtamode had many problems regarding communication, training and information about new technology. Concerned authority (Birtamode Municipality) seems weak in implementation of code and law. They are not aware about the fire safety condition of existing commercial buildings. No any seminar or awareness program has been conducted by municipality for fire safety. Even the Municipality building didn’t have proper sign for safe evacuation. Fire fighter seems always prepared but due to lack of direct communication they find hard to locate the site. Training for firefighter has been very often. Municipality seems just happy in collecting the fire tax while registration of new house. A. K. Mishra, et al. (2022); www.srinivaspublication.com 6.2.1 Cost estimate For One Stop Mall: 6.2.1 Cost estimate For One Stop Mall: Rates are collected from different website like (Daraz.com and sastodeal.com) and other rates are collected from expert and experience of researcher. p Rates are collected from different website like (Daraz.com and sastodeal.com) and other rates are collected from expert and experience of researcher. Table 7: Cost estimate For One Stop Mall S. No. Description No Unit Rate Quantity Remarks 1 Exit Signs and boards(Digital) 10 per piece 2500 25000 2 Underground water tank for water hydrant (maintenance) 1 L/s 100000 100000 for functioning of existing water tank 3 Fire extinguisher (4.5 kg) CO2 5 per piece 12000 60000 4 Fire Blanket 5 per piece 5000 25000 for each floor 5 Foam fire extinguisher 5 per piece 5000 25000 for each floor 6 Smoke detector 40 per piece 2500 100000 10 for each floor up to fourth floor 7 Smoke detector Accessories and installation charge 1 job 50000 50000 8 Fire drills 1 job 100000 100000 expert fee, management, accessories fee 9 Awareness program 1 job 25000 25000 expert fee, management, accessories fee 10 Coordination with concerned authority 1 job 5000 5000 regular visit for firefighter, interaction International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION Total 515000 Amount in words: - Five lakh fifteen thousand Only Note: - The above estimates are done on current market rate which may subject to change anytime. As mentioned it is a tentative estimate, so for exact estimate detail cost calculation should be done. Table 8: Cost estimate For Hanuman central S. No. 6.2.1 Cost estimate For One Stop Mall: Description No Unit Rate Quantity Remarks 1 Exit Signs and boards(Digital) 20 per piece 2500 50000 - 2 Overhead water tank for water hydrant 1 L/s 250000 250000 for functioning of existing water tank on top floor 3 Fire Blanket 12 per piece 5000 60000 for 4 floor , at staircase i:e, 3 staircase 4 Foam fire extinguisher 5 per piece 5000 25000 for each floor 5 Smoke detector 150 per piece 2500 375000 for each shop 6 Smoke detector Accessories and installation charge 1 job 10000 10000 - 7 Fire drills 1 job 100000 100000 expert fee, management, accessories fee 8 Awareness program 1 job 25000 25000 expert fee, management, accessories fee 9 Coordination with concerned authority 1 job 5000 5000 regular visit for authority(Fire brigade) 10 Emergency alarm system 1 job 50000 50000 Total 950000 Amount in words: - Nine lakh fifty thousand Only Amount in words: - Five lakh fifteen thousand Only Note: - The above estimates are done on current market rate which may subject to change anytime. As mentioned it is a tentative estimate, so for exact estimate detail cost calculation should be done. Rates are collected from different website like (Daraz.com and sastodeal.com) and other rates are collected from expert and experience of researcher. The cost of installation is not meaningful in comparison to that of safety assured. This once again verifies the standard theory of safety is investment with positive return, not expenditure as extra burden. Assume chance of fire is very less but the impact once caught is very high so we must assure fire safety system. For concerned authority: (1) Fire safety code NBC 107:1994 should be strictly implemented. (1) Fire safety code NBC 107:1994 should be strictly implemented. (2) Municipality should develop their own guideline according to the geography and literacy. (3) Municipality should organize site visit for firefighter to important commercial and public buildings. (4) Municipality should strictly implement the fire safety code for newly registered buildings inside its area. (5) The narrow road should be expanded as soon as possible. (6) New technology should be introduced to firefighter so that they can find exact location, should organize regular training to the fire fighters. g g g g (7) Municipality should organize regular awareness program and seminar using different media. (8) Municipality should install fire hydrant in front of important commercial and public building (8) Municipality should install fire hydrant in front of important commercial a International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVA PUBLICATION management in existing commercial buildings, no fire drills were held, and inhabitants received no awareness or training. There was huge communication gap between all three parties. Management and occupants don’t have direct communication (phone number) with fire brigade. In case of emergency people use to call Nepal police and Nepal police then call the fire fighter. The gap creates the delay for response. Also due to lack of new technology fire fighter finds hard to find the exact location. There was a problem for refilling the tank as there was no any fire hydrant installed by municipality. Fire fighters have to travel at least one kilometre to refill their tank. Due to unmanaged planning and narrow road fire fighter find hard to reach the location. There was no any coordination among the management, occupants and concerned authority. Lack of fire expert in the Birtamode area was also a major problem for management to perform fire drills for occupants. The addition of fire safety tools and equipment’s to the existing buildings seems less as compared to the loss that had occurred in the past due to fire in the buildings. Also the weakness of Municipality in implementing fire safety codes and guidelines for the large commercial buildings was a major problem seen during study. Fire extinguisher and Fire-fighter from municipality seems to be only tool to fight in case of emergency. The following points can be recommended for the Management: The following points can be recommended for the Management: (1) The calculated cost of the addition of fire safety tools and equipment’s seems less as compared to loss that might occur, so more fire safety tools and equipment’s should be added as soon as possible. (2) Fire drill and awareness program should be conducted frequently in coordination of concerned authority with the help from expert. y p p (3) The installed system (Water hydrant and water hose reels) should be made functional as soon as possible in both buildings. (3) The installed system (Water hydrant and water hose reels) should be made functional as soon as possible in both buildings. (4) Management should also focus on safety of occupants and put safety on top priority list. (5) Different signs and boards should be installed for awareness of occupants. For occupant (5) Different signs and boards should be installed for awareness of occupants. For occupants (6) Occupants should always involve in safety program organized by authority and management (6) Occupants should always involve in safety program organized by authority and management (7) Occupants also should be aware about building features, fire safety tools and equipment’s. ( ) p y y p g g y y g (7) Occupants also should be aware about building features, fire safety tools and equipmen ( ) p g y q p (8) Occupants should coordinate with the management for safe and healthy environment. Recommendation for further research work: Some issues were identified for future research: • Construction and specification of fire resistance • Problems based on human behavior. • Importance of fire safety for all the party. • Cost of fire safety before and after the incidence. 9. ACKNOWLEDGEMENT : The author is thankful to all the key informants and students of Madan Bhandari Memorial Academy Nepal 1th Batch for helping in collecting data. The author is thankful to all the key informants and students of Madan Bhandari Memorial Academy Nepal 1th Batch for helping in collecting data. REFERENCES : REFERENCES : 7. CONCLUSION : All three parties (occupants, management, and concerned authorities) lacked awareness and preparedness. The residents of commercial buildings lacked fire safety knowledge and preparedness. They were uninformed about the building's general features, such as the floor plan and egress placement. In the same way, the residents were unaware of the assembly point and emergency evacuation method. They will also require aid in getting to a safe location. They were completely unaware of the building's safety features and were unable to use them. There was no proper fire safety PAGE 193 A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION REFERENCES : A. K. Mishra, et al. (2022); www.srinivaspublication.com International Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION [1] Mishra, A. K., & Aithal, P. S., (2022). Causes and Effects of Design Change of Construction Projects. International Journal of Applied Engineering and Management Letters (IJAEML), 6(1), 87-99. DOI: https://doi.org/10.5281/zenodo.6085346. Google Scholar [2] Mishra, A. K., Shrestha A. 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B.A. School of Built Environment, University of Nairobi.http://erepository.uonbi.ac.ke/bitstream/handle/11295/58816/ Ogajo_Influence%20of%20fire%20disasters%20on%20mitigation%20and%20preparedness%2 0in%20commercial%20premises%20in%20Kenya.pdf?sequence=3 [11] Malhotra, H. L., (1993). Fire Safety in Buildings, [Online] Available at: http://www.lmc.ep.usp.br/grupos/gsi/wp- content/artigos1/Malhotra.pdf [Accesses 24 July 2017]. [12] Baker, J., (2013). The Relationship Between Fire Damage and Fire Safety Management, MPhil. Lounghborough University. https://repository.lboro.ac.uk/articles/thesis/ The_relationship_between_ fire_damage_and_fire_safety_management/9457682. [13] Tseng, W. W., Shen, T. S., Liao, S. F., Tseng, C. C. (2017). To Improve the Care Environments by Using Fire Safety Engineering for Existing Small-Scale Hospitals in Taiwan. In: Harada, K., Matsuyama, K., Himoto, K., Nakamura, Y., Wakatsuki, K. (eds) Fire Science and Technology 2015. Springer, Singapore. 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(2022); www.srinivaspublication.com PAGE 195 nternational Journal of Applied Engineering and Management Letters (IJAEML), ISSN: 2581-7000, Vol. 6, No. 2, September 2022 SRINIVAS PUBLICATION 17] Madhesshavani, (2017). [Online] Available at: http://madheshvani.com/en/details/1218/madhesh-news [Accesses 24 July 2017]. 18] Mehaffey, J. R., (1987). Flammability of building materials and fire growth, Designing for Fire Safety: The Science and its Application to Building Codes, Canada: NRC [Online] Available at: http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/fulltext/?id=a2f1e26c-8d11-4481-a1a2- 4b9afd5478e8 [Accesses 24 July 2017]. 19] OSHA a., [Online] Available at: https://www.osha.gov/OshDoc/data_General_Facts/emergency- exit- routes-factsheet.pdf [Accesses 24 July 2017]. 20] OSHA b, [Online] Available at: https://www.osha.gov/ shpguidelines/hazard-prevention.html [Accesses 24 July 2017]. 21] Yevgen Martyn et al, (2020). 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[19] OSHA a., [Online] Available at: https://www.osha.gov/OshDoc/data_General_Facts/emergency- exit- routes-factsheet.pdf [Accesses 24 July 2017]. [19] OSHA a., [Online] Available at: https://www.osha.gov/OshDoc/data_General_Facts/emergency- exit- routes-factsheet.pdf [Accesses 24 July 2017]. [19] OSHA a., [Online] Available at: https://www.osha.gov/OshDoc/data_General_Facts/emergency- exit- routes-factsheet.pdf [Accesses 24 July 2017]. [20] OSHA b, [Online] Available at: https://www.osha.gov/ shpguidelines/hazard-prevention.html [Accesses 24 July 2017]. [20] OSHA b, [Online] Available at: https://www.osha.gov/ shpguidelines/hazard-prevention.html [Accesses 24 July 2017]. [21] Yevgen Martyn et al, (2020). Software for Shelter’s Fire Safety and Comfort Levels Evaluation Data Stream Mining & Processing, Volume 1158. DOI: 10.1007/978-3-030-61656-4_31. [21] Yevgen Martyn et al, (2020). Software for Shelter’s Fire Safety and Comfort Levels Evaluation Data Stream Mining & Processing, Volume 1158. DOI: 10.1007/978-3-030-61656-4_31. PAGE 196
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Response of carbon fluxes to water relations in a savanna ecosystem in South Africa
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Clark University Clark University Clark Digital Commons Clark Digital Commons Geography Faculty Works by Department and/or School 1-1-2008 Response of carbon fluxes to water relations in a savanna Response of carbon fluxes to water relations in a savanna ecosystem in South Africa ecosystem in South Africa W. L. Kutsch Max Planck Institute for Biogeochemistry N. Hanan Colorado State University B. Scholes The Council for Scientific and Industrial Research I. McHugh Monash University W. Kubheka Colorado State University See next page for additional authors Follow this and additional works at: https://commons.clarku.edu/faculty_geography Part of the Geography Commons Repository Citation Repository Citation Kutsch, W. L.; Hanan, N.; Scholes, B.; McHugh, I.; Kubheka, W.; Eckhardt, H.; and Williams, Christopher A., "Response of carbon fluxes to water relations in a savanna ecosystem in South Africa" (2008). Geography. 921. https://commons.clarku.edu/faculty_geography/921 This Article is brought to you for free and open access by the Faculty Works by Department and/or School at Clark Digital Commons. It has been accepted for inclusion in Geography by an authorized administrator of Clark Digital Commons For more information please contact larobinson@clarku edu cstebbins@clarku edu Clark University Clark University Clark Digital Commons Clark Digital Commons Geography Faculty Works by Department and/or School 1-1-2008 Response of carbon fluxes to water relations in a savanna Response of carbon fluxes to water relations in a savanna ecosystem in South Africa ecosystem in South Africa W. L. Kutsch Max Planck Institute for Biogeochemistry N. Hanan Colorado State University B. Scholes The Council for Scientific and Industrial Research I. McHugh Monash University W. Kubheka Colorado State University See next page for additional authors Follow this and additional works at: https://commons.clarku.edu/faculty_geography Part of the Geography Commons Repository Citation Repository Citation Kutsch, W. L.; Hanan, N.; Scholes, B.; McHugh, I.; Kubheka, W.; Eckhardt, H.; and Williams, Christopher A., "Response of carbon fluxes to water relations in a savanna ecosystem in South Africa" (2008). Geography. 921. https://commons.clarku.edu/faculty_geography/921 This Article is brought to you for free and open access by the Faculty Works by Department and/or School at Clark Digital Commons. It has been accepted for inclusion in Geography by an authorized administrator of Clark Digital Commons. For more information, please contact larobinson@clarku.edu, cstebbins@clarku.edu. Clark University Clark University Clark Digital Commons Clark Digital Commons Geography 1-1-2008 Response of carbon fluxes to wa Response of carbon fluxes to wa ecosystem in South Africa ecosystem in South Africa W. L. Kutsch Max Planck Institute for Biogeochemistry N. Hanan Colorado State University B. Scholes The Council for Scientific and Industrial Research I. McHugh Monash University W. Kubheka Colorado State University See next page for additional authors Follow this and additional works at: https://common Part of the Geography Commons Repository Citation Repository Citation Kutsch, W. L.; Hanan, N.; Scholes, B.; McHugh, I.; Kub "Response of carbon fluxes to water relations in a s Geography. 921. https://commons.clarku.edu/faculty_geography/92 This Article is brought to you for free and open access by Digital Commons. It has been accepted for inclusion in Ge Commons. For more information, please contact larobins Authors Authors W. L. Kutsch, N. Hanan, B. Scholes, I. McHugh, W. Kubheka, H. Eckhardt, and Christopher A. Williams sch, N. Hanan, B. Scholes, I. McHugh, W. Kubheka, H. Eckhardt, and Christopher A. Williams This article is available at Clark Digital Commons: https://commons.clarku.edu/faculty_geography/92 This article is available at Clark Digital Commons: https://commons.clarku.edu/faculty_geography/921 1 Introduction Abstract. The principal mechanisms that connect carbon fluxes with water relations in savanna ecosystems were stud- ied by using eddy covariance method in a savanna ecosys- tem at Kruger National Park, South Africa. Since the annual drought and rewetting cycle is a major factor influencing the function of savanna ecosystems, this work focused on the close inter-connection between water relations and carbon fluxes. Data from a nine-month measuring campaign lasting from the early wet season to the late dry season were used. Savannas are defined as tropical and sub-tropical vegetation types where woody plants and grasses co-dominate. They are characterized by a seasonal change of water availability with distinct rainy and dry seasons. Decades of research have identified not only water, but also nutrients, herbivory, and fire as major drivers of savanna vegetation dynamics (Walter, 1939, 1971; Scholes and Walker, 1993; Scholes and Archer, 1997; Sankaran et al., 2004; 2005; Beerling and Osborne, 2006). Total ecosystem respiration showed highest values at the onset of the growing season, a slightly lower plateau during the main part of the growing season and a continuous de- crease during the transition towards the dry season. Despite large land surface coverage (∼20% of the global land surface and 40% of the African surface), important bio- diversity (∼8000 of 13 000 African plant species are savanna endemic, White, 1980) and substantial land use change in savannas, there have been relatively few projects focusing on the patterns and processes controlling savanna carbon and water vapour exchange (Levy, 1995; Verhoef et al., 1996; Lloyd et al., 1997; Hanan et al., 1998; Scanlon and Albert- son, 2004; Veenendaal et al., 2004; Williams and Albertson, 2004, 2005, for African savannas; Meir et al., 1996; Miranda et al., 1997; Meinzer et al., 1999; da Rocha et al., 2002; Santos et al. 2003, for South American savannas; Hutley et al., 2000, 2001, 2005; Chen et al., 2002, 2003; Beringer et al. 2003, for Australian savannas; Baldocchi et al., 2004 for temperate Mediterranean savannas in California). The regulation of canopy conductance was changed in two ways: changes due to phenology during the course of the growing season and short-term acclimation to soil water con- ditions. Clark University Clark University Clark Digital Commons Clark Digital Commons Faculty Works by Department and/or School Follow this and additional works at: https://commons.clarku.edu/faculty_geography Repository Citation Repository Citation Kutsch, W. L.; Hanan, N.; Scholes, B.; McHugh, I.; Kubheka, W.; Eckhardt, H.; and Williams, Christopher A., "Response of carbon fluxes to water relations in a savanna ecosystem in South Africa" (2008). Geography. 921. https://commons.clarku.edu/faculty_geography/921 This Article is brought to you for free and open access by the Faculty Works by Department and/or School at Clark Digital Commons. It has been accepted for inclusion in Geography by an authorized administrator of Clark Digital Commons. For more information, please contact larobinson@clarku.edu, cstebbins@clarku.edu. Biogeosciences, 5, 1797–1808, 2008 www.biogeosciences.net/5/1797/2008/ Biogeosciences © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. Response of carbon fluxes to water relations in a savanna ecosystem in South Africa W. L. Kutsch1, N. Hanan2, B. Scholes3, I. McHugh4, W. Kubheka2, H. Eckhardt5, and C. Williams6 1Max-Planck-Institute for Biogeochemistry, P.O. Box 10 01 64, 07701 Jena, Germany 2Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, USA 3Council for Scientific and Industrial Research (CSIR), Natural Resources and the Environment, P.O. Box 395, 0001 Pretoria, South Africa 4School of Geography and Environmental Science, Monash University, Clayton, Australia 5South African National Parks (SANParks), Scientific Services, Skukuza, South Africa 6Graduate School of Geography, Clark University Worcester, MA, USA 4School of Geography and Environmental Science, Monash University, Clayton, Australia 5South African National Parks (SANParks), Scientific Services, Skukuza, South Africa 6Graduate School of Geography, Clark University Worcester, MA, USA Received: 17 March 2008 – Published in Biogeosciences Discuss.: 27 May 2008 Revised: 23 September 2008 – Accepted: 6 October 2008 – Published: 23 December 2008 W. L. Kutsch et al.: Savanna water-carbon relations W. L. Kutsch et al.: Savanna water-carbon relations savannas in global carbon cycles, as well as informing poli- cies related to the role of African countries in Kyoto Protocol. African savannas (Scholes and Archer, 1997). The site was part of the SAFARI 2000 experiment to understand the in- teractions between the atmosphere and the land surface in southern Africa (Otter et al., 2002; Scholes et al., 2001; Shugart et al., 2004). The location of the tower is about 13 km WS W of Skukuza in the N’waswitshaka catchment, an ephemeral tributary of the Sabie River. The altitude of the site is 365 m a.s.l. The underlying parent material is Ar- chaean granite and gneiss. The landscape shows a typical un- dulating form with drainage lines about 3 km apart and ridge tops about 40 m above valley floors. This leads to a cate- nal pattern of soils and vegetation, with nutrient-poor Com- bretum savannas on the crests, and nutrient-enriched Acacia savannas on the lower parts of the slope.The flux tower is lo- cated at the boundary of the two plant communities. When the wind blows from the SE (105◦–196◦), a fine-leaf Acacia savanna is sampled, and when it blows from the N (270◦– 76◦), a broad-leaf Combretum savanna is sampled. Sectoral analyses of eddy covariance measurements have been con- ducted successfully in previous studies (e.g. Kutsch et al., 2005). One of the few infrastructures enabling the study carbon and water relations of African savannas is the flux tower in the Kruger National Park, installed between Skukuza and Pretoriuskop in 2000. In this study we investigate principal mechanisms that connect carbon fluxes with water relations in savanna ecosystems and differences between the two sa- vanna types. Since the annual drought and rewetting cycle is a major factor influencing the function of savanna ecosys- tems, this work is focused on two questions concerning water relations: (1) How do water availability and seasonality drive ecosystem respiration?; and (2) how does the close inter- connection between canopy conductance and photosynthesis, influence the carbon uptake by the vegetation during times of different water availability? Ecosystems respiration as measured by the eddy covari- ance technique is the sum of soil, plant, and animal respira- tion. In biomes with a pronounced rainfall seasonality and/or water limitation, the temperature response may be secondary to the response to water availability. W. L. Kutsch et al.: Savanna water-carbon relations 1 the monthly precip- itation of the growing season 2002/2003 is compared to the long-term mean. The 2002/2003 growing season was very dry. Only half of the long-term amount of rain fell (276 mm). By combining the regulation of respiration on one hand and stomatal conductance and photosynthesis on the other we provide a principle analysis of the temporal dynamics in the regulation of savanna carbon fluxes by water relations. We used data from a nine months period between January and September 2003 for this study. For annual balances of this site and improved gap-filling procedures we refer to Archibald et al. (2008). W. L. Kutsch et al.: Savanna water-carbon relations Detailed soil and vegetation analyses of the two types are given in Scholes et al. (2001). They show that the soil in the Acacia savanna contains more clay, more nutrients such a ni- trogen and phosphorous and also a higher cation exchange capacity than in the Combretum savanna. Volumetric field capacity (WCFC) is about 0.12 in the Combretum- and 0.2 in the Acacia-savanna. Volumetric water content at −1500 kPa (“wilting point”, WCWP) was 0.06 and 0.1, respectively. Rel- ative plant available water (RPAW) was calculated from the actual volumetric water content for the layers 0–10 cm and 0–30 cm by Ecosystem photosynthesis is likewise controlled by a com- plex interaction of processes that connect carbon fluxes and water relations. The control of CO2 and water vapour ex- change between ecosystems and the atmosphere by the stom- atal aperture of plant leaves is the hinge between the two cy- cles – particularly notable in arid and semi-arid biomes. However, the same stomata do not always react to the short-term (diurnal) variations of VPD in the same way. Leaf stomatal sensitivity and maximum conductance can be mod- ified within periods of several days to few weeks by phenol- ogy and by acclimation to factors such as soil water status (Halldin et al., 1984). Kutsch et al. (2001a) assessed the influence of this mid-term stomatal acclimation on ecosys- tem fluxes by calculating canopy water and carbon balances for several years under different climates. In this context it is important to consider that canopy photosynthetic capacity (FP,sat) is strongly correlated to maximum surface conduc- tance (Gs,sat; Schulze et al. (1994)). RPAW = (WCakt −WCWP) (WCFC −WCWP) · 100 (1) (1) Rainfall at the site averages 547.1 mm but with significant interannual variability. The first precipitation events of the rainy season generally occur in late September or October, with last rains in April or early May. However, much of the interannual variability in total precipitation arises with vari- ation in the dates of first and last rains. Figure 1 shows the long-term monthly means of minimum and maximum tem- perature and the mean precipitation at Skukuza (data from 1960–1999, after Scholes et al. (2001)). Precipitation is shown as cumulative curve. In order to show a complete growing season the data are shown from August to July. Most of the annual rainfall occurs during the summer (Octo- ber to April). In the lower part of Fig. 1 Introduction The most constant parameter was water use efficiency that was influenced by VPD during the day but the VPD response curve of water usage did change only slightly during the course of the growing season and decreased by about 30% during the transition from wet to dry season. The regulation of canopy conductance and photosynthetic capacity were closely related. This observation meets re- cent leaf-level findings that stomatal closure triggers down- regulation of Rubisco during drought. Our results may show the effects of these processes on the ecosystem scale. Increasing our understanding of the physical and physio- logical controls on savanna functioning using eddy covari- ance measurements is of great importance both for funda- mental ecological understanding as well as for improved management and conservation of these important ecosys- tems. Such measurements will, furthermore provide ba- sic data for refining models of ecosystem carbon dynamics and facilitate improved understanding of the role of tropical Correspondence to: W. L. Kutsch (wkutsch@bgc-jena.mpg.de) management an tems. Such m sic data for refi and facilitate im Published by Copernicus Publications on behalf of the European Geosciences Un Correspondence to: W. L. Kutsch (wkutsch@bgc-jena.mpg.de) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 1798 2.3 Supplemental measurements Additional weather measurements on the main tower in- cluded measurements of incident and reflected shortwave radiation (300–1100 nm, W m−2, CM14, Kipp and Zonen, Delft, The Netherlands), incident and reflected near-infrared (CM14 600–1100 nm, W m−2, Kipp and Zonen, Delft, The Netherlands) and incoming and emitted longwave radiation (CG2, >3.0 nm, W m−2, Kipp and Zonen, Delft, The Nether- lands). Radiation measurements were made at 20 s inter- vals and then recorded in the data-logger as 30 min aver- ages. Precipitation events were recorded with a tipping- bucket raingauge (Texas Instruments TE525, mm) totalled for each 30 min period. In addition to the meteorological and flux measurements on the main tower, we deployed supplemental microclimate stations in the adjacent Acacia and Combretum savannas to measure air temperature, air humidity, soil temperature and volumetric soil moisture. In each savanna we deployed TDR probes (Campbell Scientific CS615, cm3 water cm−3 soil) and temperature probes (Campbell Scientific 108,◦C) in pro- files with 4 or 5 measurement depths from near the soil sur- face to just above bedrock. Fig. 1. Long-term monthly means of minimum and maximum tem- perature (dotted lines) and the mean precipitation (solid line) at Skukuza (data from 1960–1999, after Scholes et al. 2001). Pre- cipitation is shown as cumulative curve. In order to show a com- plete growing season the data are printed from August to July. In the lower part the monthly precipitation of the growing season 2002/2003 (hatched bars) is compared to the long-term mean (black bars). Fig. 1. Long-term monthly means of minimum and maximum tem- perature (dotted lines) and the mean precipitation (solid line) at Skukuza (data from 1960–1999, after Scholes et al. 2001). Pre- cipitation is shown as cumulative curve. In order to show a com- plete growing season the data are printed from August to July. In the lower part the monthly precipitation of the growing season 2002/2003 (hatched bars) is compared to the long-term mean (black bars). 2.4 Data treatment Post-processing of the raw high frequency (10 Hz) data to obtain fluxes representative of thirty minute periods involved the standard spike filtering, planar rotation of velocities, lag correction for CO2 and water vapour concentrations, and cal- culation of mass fluxes using conventional equations (Mon- crieff et al., 1997; Aubinet et al., 2000). area average of approximately 1.0 (i.e. within canopy LAI ∼3.0) three or four weeks after the beginning of the rainy season and then remaining at that level until several weeks after the last rainfall. Thereafter, the trees in the Combretum savanna dropped their leaves, whereas the Acacia species re- mained leafy during the dry season. Herbaceous layer pro- ductivity and LAI is more dynamic with inter-annual vari- ability in total rainfall, with average LAI of the herbaceous layer similar to the tree layer (i.e. 1.0), but in this case spread out more or less evenly across the landscape rather than being confined within the tree canopies. Carbon dioxide flux (FCO2) was also corrected to include the canopy storage flux using the simple approach of assum- ing that the CO2 is uniformly distributed within the air col- umn between the soil surface and the inlet of the tube: F ′ CO2 = FCO2 + 1CO2 · h · 1000 η · t (2) (2) (2) where F ′ CO2 is the corrected flux (µmol CO2 m−2 s−1), FCO2 is the flux measured by the eddy covariance system (µmol CO2 m−2 s−1), 1CO2 is the difference in CO2 concentration between t0 and t−1 (ppm), h is the height of the air column (16 m), η is molar volume of an ideal gas (22.4 l mol−1), the factor 1000 converts l into m3, and t is the time step of 1800 s. 2.1 Research site 2.1 Research site The vegetation of the study site is open woodland with ap- proximately 30% tree canopy cover in both Acacia and Com- bretum savanna types (Scholes et al., 2001). Leaf area index of the trees in both savanna types is similar, increasing to an The Skukuza flux tower was established in early 2000 to study the carbon, water and energy dynamics of semi-arid Biogeosciences, 5, 1797–1808, 2008 www.biogeosciences.net/5/1797/2008/ W. L. Kutsch et al.: Savanna water-carbon relations 1799 0 5 10 15 20 25 30 35 40 Temperature (°C) 0 150 300 450 600 Cumulative precipitation (mm) 0 10 20 30 40 50 60 70 80 90 Monthly precipitation (mm) A S O N D J F M A M J J Long- term mean 2002/2003 Fig. 1. Long-term monthly means of minimum and maximum tem- perature (dotted lines) and the mean precipitation (solid line) at Skukuza (data from 1960–1999, after Scholes et al. 2001). Pre- cipitation is shown as cumulative curve. In order to show a com- plete growing season the data are printed from August to July. In the lower part the monthly precipitation of the growing season 2002/2003 (hatched bars) is compared to the long-term mean (black bars). 0 5 10 15 20 25 30 35 40 Temperature (°C) 0 150 300 450 600 Cumulative precipitation (mm) 0 10 20 30 40 50 60 70 80 90 Monthly precipitation (mm) A S O N D J F M A M J J Long- term mean 2002/2003 measure carbon dioxide (µmol mol−1) and water vapour (mmol mol−1) concentrations. The gas samples were drawn at 6 l min−1 through a 6 m teflon-coated tube to the gas ana- lyzer enclosed in a weather-proof box. The sonic anemome- ter and gas analyser measurements were recorded at 10 Hz. Cumulative precipitation ( ) 2.2 Eddy covariance system Ecosystem-level fluxes of water, heat and carbon diox- ide were made using a closed-path eddy covariance sys- tem mounted at 16 m. This consisted of a 3-dimensional sonic anemometer (Gill Wind Master Pro, Gill Instruments, Lymington, UK) to measure fluctuations in horizontal and vertical wind speeds (m s−1) and temperature (K), and a gas analyzer (LiCor 6262, LiCor, Lincoln, Nebraska, USA) to After calculation of the fluxes the eddy-covariance data were combined with and fitted against microclimate data. Nighttime carbon dioxide flux (Fn), a surrogate for ecosys- tem respiration (FR), was regressed against soil tempera- ture at 7 cm depth (TS) and soil moisture (RPAW, Eq. (1). Biogeosciences, 5, 1797–1808, 2008 www.biogeosciences.net/5/1797/2008/ 1800 W. L. Kutsch et al.: Savanna water-carbon relations Only data with a coincident friction velocity (u∗) greater than 0.25 m s−1 were used for this analysis. leaf area index, and the friction velocity (Jensen and Hum- melshøj, 1995). Finally, surface conductance was calculated as the reciprocal of the residual resistance (Eq. 9): The data set was fitted to the ecosystem respiration model described by Reichstein et al. (2002). This model pro- vides response curves for soil moisture and temperature, and changes the Q10-value of the temperature response curve with changing soil moisture as observed in this study: GS = VPD E − u u∗2 −rb  (9) (9) If VPD is given in g m−3, E in g m−2, u and u∗in m s−1, and rb in s m−1, GS is obtained in m s−1. To con- vert GS from m s−1 into the common plant physiological unit of mmol m−2 s−1, a multiplication factor has to be applied which is roughly 40 000. FR = FR,ref · f (Tsoil, RSWC) · g(RSWC) (3) f (Tsoil, RSWC) = eE0(RSWC)·( 1 Tref−T0 − 1 Tsoil−T0 ) (4) FR = FR,ref · f (Tsoil, RSWC) · g(RSWC) (3) (3) FR = FR,ref · f (Tsoil, RSWC) · g(RSWC) f (Tsoil, RSWC) = eE0(RSWC)·( 1 Tref−T0 − 1 Tsoil−T0 ) (4) f (Tsoil, RSWC) = eE0(RSWC)·( 1 Tref−T0 − 1 Tsoil−T0 ) (4) (4) In order to obtain an indication of the sensitivity of the regulation of canopy conductance to ambient humidity, GS was plotted against the actual water vapour pressure deficit of the air (VPD) for distinct periods of between two and four weeks with different levels of soil moisture. 2.2 Eddy covariance system Each data set was fitted separately to the following equation: g(RSWC) = RSWC −RSWC0 (RSWC1/2 −RSWC0 ) + (RSWC −RSWC0) (5) (5) where E0 was not fixed but a linear function of relative water content (RSWC) allowing for a RSWC effect on temperature sensitivity. RWSC used as by Reichstein et al. (2002) was derived from the RPAW for the 0–10 cm layer by: GS = GS,max c · VPD2 (10) GS = GS,max c · VPD2 (10) RWSC = RPAW 100 (6) where GS,max is a theoretical maximum conductance that occurs when VPD (mbar) is zero (set constantly to 2000 mmol m−2 s−1 in that study), and c is an empirical co- efficient that provides a measure of the sensitivity of the regulation of GS. Envelope functions covering 90% of the data were fitted to the data by quantile regression (Cade and Noon, 2003). The seasonal course of the coefficient c in Eq. (10) reveals how the canopy acclimates to changing con- ditions. This function is simpler and more empirical than the “Ball/Berry-Function” (Ball et al., 1987) that is commonly used but c is defining the shape of the response curve in the same way and reveals the same seasonal pattern as the co- efficient D0 in the Ball/Berry approach does. We chose the more empirical function to avoid deriving physiological pa- rameters top-down which is inappropriate for a physiologi- cally heterogeneous system like a savanna. (6) The modelled daytime ecosystem respiration (FR) was then used to calculate canopy assimilation (FP ) from the daytime fluxes: FP = F ′ CO2 −FR (7) (7) Canopy assimilation was then fitted against global radia- tion measured above the canopy by means of a tangens hy- perbolicus (tan h) function (von Stamm, 1994): Canopy assimilation was then fitted against global radia- tion measured above the canopy by means of a tangens hy- perbolicus (tan h) function (von Stamm, 1994): FP = FP,sat · tan h( k · I FP,sat ) (8) (8) where k is an empirical coefficient describing the initial slope of the light response curve and I is the incoming short-wave radiation (W m−2). 3 Results Surface conductance, GS, defined as the surface conduc- tance to water vapour transfer at a canopy scale, was cal- culated from the eddy flux data as described by Jensen and Hummelshøj (1995) and Herbst et al. (2002). First, the total resistance, rs, of the system was calculated as the quotient of vapour pressure deficit, VPD, of the air above the forest and water vapour flux, E. Analogously, the resistance owing to turbulent transport in the atmosphere, ra, (assumed to be equal for momentum and water vapour) was determined as the quotient of mean horizontal windspeed, u, and the square of the friction velocity, u∗2. The resistance through the vis- cous sublayers on the individual leaves, rb, was estimated as a function of the kinematic viscosity of the air, the molec- ular diffusivity of water vapour, the size of the leaves, the Biogeosciences, 5, 1797–1808, 2008 3.1 Climate and flux overview Since 2002/2003 was a dry year, periods with low rainfall during the wet season resulted in periods of low soil moisture even during the typical vegetation growth period (Fig. 2a, Ta- ble 1). The dry periods can also be seen in decreases during the annual course in the fAPAR (Fig. 2a). The net ecosystem exchange (Fig. 2b) and its components (canopy photosynthe- sis, FP , Fig. 2c, and ecosystem respiration FR, Fig. 2d) were strongly influenced by the soil moisture dynamics. There- fore, the observational data during the growing season were divided in the following sub-sets for further eco(system)- physiological analyses. Biogeosciences, 5, 1797–1808, 2008 www.biogeosciences.net/5/1797/2008/ W. L. Kutsch et al.: Savanna water-carbon relations 1801 J F M A M J J A S 1 3 2 4 5 6 7 8 Periods: 2 4 6 8 -5 0 5 10 15 20 -20 -15 -10 -5 0 5 FC (µmol m-2 s-1) FP (µmol m-2 s-1) FR (µmol m-2 s-1) 0 0.1 0.2 Soil moisture 0-10 cm (Vol %) 0.3 8 7 6 5 4 3 2 1 (a) (b) (c) (d) 0 0.1 0.2 0.3 0.4 fAPAR Fig. 2. Seasonal courses of volumetric soil water content for the layers 0–10 cm (a) in the Acacia savanna (grey line) and the Com- bretum savanna (black line) and of fAPAR of two Modis grid cells around the tower (a), of net ecosystem exchange (NEE, (b), canopy photosynthesis (FP , (c) and total ecosystem respiration (FR, (d). The periods defined in Table 1 are marked in the Fig. J F M A M J J A S 1 3 2 4 5 6 7 8 Periods: 2 4 6 8 -5 0 5 10 15 20 -20 -15 -10 -5 0 5 FC (µmol m-2 s-1) FP (µmol m-2 s-1) FR (µmol m-2 s-1) 0 0.1 0.2 Soil moisture 0-10 cm (Vol %) 0.3 8 7 6 5 4 3 2 1 (a) (b) (c) (d) 0 0.1 0.2 0.3 0.4 fAPAR Table 1. Sub-sets for data evaluation in 2003. Dataset No. 3.1 Climate and flux overview Period Characterization Mean RPAW 0–10(1) 1 25 Jan–8 Feb Wet soil, early wet season 30.6 2 10–20 Feb Dry soil, early wet season 16.1 3 23 Feb–29 Mar Wet soil, mid wet season 28.2 4 1–25 Apr Dry soil, mid wet season 11.6 5 27 Apr–11 May Wet soil, transition period 39.1 6 14 May–5 Jun Dry soil, transition period 12.7 7 14 Jun–12 Jul Wet soil, dry season 28.2 8 28 Jul–3 Sep Dry soil, dry season 9.7 (1) Averages were build on the basis of only those datasets that con- tained full information (climate and EC) after quality control and filtering. They might slightly differ from the “real” averages. Kutsch et al. Fig 3 (1) Averages were build on the basis of only those datasets that con- tained full information (climate and EC) after quality control and filtering. They might slightly differ from the “real” averages. Kutsch et al. Fig 3 (1) Averages were build on the basis of only those datasets that con- tained full information (climate and EC) after quality control and filtering. They might slightly differ from the “real” averages. Kutsch et al. Fig 3 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 FP (µmol CO2 m-2 s-1) Global radiation (W m-2) Acacia-savanna Combretum-savanna FC (µmol CO2 m-2 s-1) 0 250 500 750 1000 1250 Fig. 3. Light response curves of net ecosystem exchange (FC, up- per graph) and canopy photosynthesis (FP , lower graph) measured during the peak of the growing season (Period 3 in March 2003). The open symbols show data from the Acacia-site, the filled sym- bols from the Combretum-site. -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 FP (µmol CO2 m-2 s-1) Global radiation (W m-2) Acacia-savanna Combretum-savanna FC (µmol CO2 m-2 s-1) 0 250 500 750 1000 1250 Fig. 2. Seasonal courses of volumetric soil water content for the layers 0–10 cm (a) in the Acacia savanna (grey line) and the Com- bretum savanna (black line) and of fAPAR of two Modis grid cells around the tower (a), of net ecosystem exchange (NEE, (b), canopy photosynthesis (FP , (c) and total ecosystem respiration (FR, (d). The periods defined in Table 1 are marked in the Fig. 3.4 Seasonal characteristics of canopy conductance and photosynthesis 3.4 Seasonal characteristics of canopy conductance and photosynthesis Fig. 4. Temperature response curves of nighttime respiration for dif- ferent intervals of soil moisture. Filled cycles symbolize data from the main growing season (January–March 2003), grey cycles from the transition time (April–May 2003) and open symbols from the dry period (June–September 2003). The values of the exponential fits and the regression coefficient are related to the whole datasets comprising data from all seasons. VPD response curves of GS were plotted for each of the de- fined periods as envelope curves based on a 90% quantile regression (Fig. 6). The response curves are paired. Each pair shows data from a period with high soil water content (left) and data from a dryer period thereafter (right). In or- der to demonstrate the degree of stomatal acclimation the re- sponse curve from the left dataset is also shown in the right one as grey line. The graphs showed that stomatal sensitiv- ity changed drastically within a few days when soil moisture varied. Stomatal conductance was highest in January (6a) and March (6c). The short period of drought during Febru- ary (6b) slightly increased sensitivity to VPD, whereas dur- ing April (6d) the drought led to much-reduced canopy con- ductance over the whole range of VPD. After a large rain- fall event in May (6e), plants reverted the regulation pat- tern again but did not reveal as high conductance as in Jan- uary or March, indicating the onset of the senescence of the leaves that may be speeded up by the April drought period. Nevertheless, the following dry period during May (6f) re- duced stomatal conductance again while the senescence of the plants went on. Consequently, a rainfall event during late June did not reverse the performance any more (6g) and the during the dry season (August), canopy conductance was re- duced to a minimum (6h). W. L. Kutsch et al.: Savanna water-carbon relations Kutsch et al. Fig 5 W. L. Kutsch et al.: Savanna water-carbon relations Kutsch et al. Fig 5 y = 0.3624e0.0903x r2 = 0.433 0 2 4 6 8 10 y = 0.3437e0.0697x r2 = 0.4945 0 2 4 6 8 y = 0.1763e0.0701x r2 = 0.2301 0 2 4 6 8 Nighttime fluxes (µmol CO2 m-2 s-1) Soil temperature at – 7 cm (°C) Wet soil Dry soil Medium soil 10 20 30 40 Fig. 4. Temperature response curves of nighttime respiration for dif- ferent intervals of soil moisture. Filled cycles symbolize data from the main growing season (January–March 2003), grey cycles from the transition time (April–May 2003) and open symbols from the dry period (June–September 2003). The values of the exponential fits and the regression coefficient are related to the whole datasets comprising data from all seasons. y = 0.8734x + 0.3065 r2 = 0.6514 0 2 4 6 8 10 Modelled nighttime fluxes Measured nighttime fluxes (µmol CO2 m-2 s-1) (µmol CO2 m-2 s-1) 0 2 4 6 8 10 Fig. 5. Comparison between measured and modelled data of night- time respiration. Based on complete data set (January–September 2003). The solid line represents the regression (regression values written at the top), the 1:1 line is dashed. y = 0.3624e0.0903x r2 = 0.433 0 2 4 6 8 10 y = 0.3437e0.0697x r2 = 0.4945 0 2 4 6 8 y = 0.1763e0.0701x r2 = 0.2301 0 2 4 6 8 Nighttime fluxes (µmol CO2 m-2 s-1) Soil temperature at – 7 cm (°C) Wet soil Dry soil Medium soil 10 20 30 40 y = 0.8734x + 0.3065 r2 = 0.6514 0 2 4 6 8 10 Modelled nighttime fluxes Measured nighttime fluxes (µmol CO2 m-2 s-1) (µmol CO2 m-2 s-1) 0 2 4 6 8 10 Fig. 5. Comparison between measured and modelled data of night- time respiration. Based on complete data set (January–September 2003). The solid line represents the regression (regression values written at the top), the 1:1 line is dashed. Figure 5 shows the modelled versus measured rates. Re- sults indicate that this model approach explains much of the variation in flux rates and confirm the hypothesis about an indirect effect of water availability on ecosystem respiration via changes of temperature sensitivity (Q10) for which Re- ichstein et al. (2002) developed their model expression. 40 Soil temperature at – 7 cm (°C) Soil temperature at – 7 cm (°C) 3.2 Sectoral analysis and differences between savanna sites 3.2 Sectoral analysis and differences between savanna sites For a sectoral analysis data were sorted according to wind direction in order to examine differences between the two sa- vanna types. Data from the sector between 105◦–196◦were taken for the fine-leaf Acacia savanna, from the sector be- tween 270◦–76◦for the broad-leaf Combretum savanna. Data from other wind directions were discarded. There were no significant differences between the savanna types in terms of fluxes. As an example, Fig, 3a and b show light response curves of net ecosystem exchange (FC) and of canopy pho- tosynthesis (FP ) for the two sectors during the peak of the growing season (Period 3 as defined in Table 1, other pe- riods showed the same picture). Consequently, data were pooled again for further analysis. The following paragraphs will show a principle analysis of carbon flux responses to water relations in a savanna ecosystem without considering possible small differences between the sub-types. Fig. 3. Light response curves of net ecosystem exchange (FC, up- per graph) and canopy photosynthesis (FP , lower graph) measured during the peak of the growing season (Period 3 in March 2003). The open symbols show data from the Acacia-site, the filled sym- bols from the Combretum-site. Biogeosciences, 5, 1797–1808, 2008 www.biogeosciences.net/5/1797/2008/ 1802 W. L. Kutsch et al.: Savanna water-carbon relations 7. Conductance response curve of CO2-fluxes for the 8 periods as defined in Table 1. Black dots symbolize the canopy gross photo- synthetic flux, open circles the net ecosystem flux; data measured at saturating light conditions with incoming shortwave radiation being higher than 700 W m−2. Ecosystem net exchange (FC) and Canopy photosynthesis ( FP) (µmol CO2 m-2 s-1) Canopy conductance (mmol m-2 s-1) Canopy conductance (mmol m-2 s-1) Canopy conductance (mmol m-2 s-1) Fig. 6. VPD-response curves of stomatal conductance for the 8 periods as defined in Table 1. Data measured at saturating light conditions with incoming shortwave radiation being higher than 700 W m−2. The grey curves in the situations with dry soil (right graphs) symbolize the curves from corresponding situations with wet soils (left graphs). Fig. 7. Conductance response curve of CO2-fluxes for the 8 periods as defined in Table 1. Black dots symbolize the canopy gross photo- synthetic flux, open circles the net ecosystem flux; data measured at saturating light conditions with incoming shortwave radiation being higher than 700 W m−2. Each of these parameters showed a seasonal trend (dashed grey line) that was overlaid by mid-term responses to the re- spective drought periods, but the specific characteristics were different. Each of these parameters showed a seasonal trend (dashed grey line) that was overlaid by mid-term responses to the re- spective drought periods, but the specific characteristics were different. Light saturated canopy photosynthesis was limited strongly by canopy conductance below a threshold of 250 mmol m−2 s−1 during all of the periods under consider- ation (Fig. 7a–h). During the periods with low soil mois- ture (right figures) canopy conductance did almost never ex- ceeded this threshold and CO2 uptake by the canopy was al- most constantly reduced. In addition, the few data sets with a canopy conductance around 250 mmol m−2 s−1 suggest a de- crease of the photosynthetic capacity, which means that pho- tosynthetic capacity was strongly coupled to stomatal sensi- tivity. The seasonal trend of the coefficient c revealed a broad minimum from January to March and constantly increased during the transition period towards the dry season. C also revealed mid-term responses to the drought periods: during water stress, c was increased and showed the more conserva- tive regulation of the stomata. W. L. Kutsch et al.: Savanna water-carbon relations Data measured at saturating light conditions with incoming shortwave radiation being higher than PD (bar) 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 (b) Dry soil, early wet season (d) Dry soil, mid wet season (f) Dry soil, transition period (h) Dry soil, dry season 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 Ecosystem net exchange (FC) and Canopy photosynthesis ( FP) (µmol CO2 m-2 s-1) (a) Wet soil, early wet season (b) Dry soil, early wet season (c) Wet soil, mid wet season (d) Dry soil, mid wet season (e) Wet soil, transition period (f) Dry soil, transition period (g) Wet soil, dry season (h) Dry soil, dry season 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Canopy conductance (mmol m-2 s-1) -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 Ecosystem net exchange (FC) and Canopy photosynthesis ( FP) (µmol CO2 m-2 s-1) (a) Wet soil, early wet season (c) Wet soil, mid wet season (e) Wet soil, transition period (g) Wet soil, dry season 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Canopy conductan -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 Ecosystem net exchange (FC) and Canopy photosynthesis ( FP) (µmol CO2 m-2 s-1) (a) Wet soil, early wet season (b) Dry soil, early wet season (c) Wet soil, mid wet season (d) Dry soil, mid wet season (e) Wet soil, transition period (f) Dry soil, transition period (g) Wet soil, dry season (h) Dry soil, dry season 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Canopy conductance (mmol m-2 s-1) Fig. W. L. Kutsch et al.: Savanna water-carbon relations 1803 t al. Fig 7 VPD (bar) 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Canopy conductance (mmol m-2 s-1) (a) Wet soil, early wet season (b) Dry soil, early wet season (c) Wet soil, mid wet season (d) Dry soil, mid wet season (e) Wet soil, transition period (f) Dry soil, transition period (g) Wet soil, dry season (h) Dry soil, dry season 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 Fig. 6. VPD-response curves of stomatal conductance for the 8 periods as defined in Table 1. Data measured at saturating light conditions with incoming shortwave radiation being higher than 700 W m−2. The grey curves in the situations with dry soil (right graphs) symbolize the curves from corresponding situations with wet soils (left graphs). W. L. Kutsch et al.: Savanna water-carbon relations VP 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Canopy conductance (mmol m-2 s-1) (a) Wet soil, early wet season (c) Wet soil, mid wet season (e) Wet soil, transition period (g) Wet soil, dry season 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 VPD (bar) 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Canopy conductance (mmol m-2 s-1) (a) Wet soil, early wet season (b) Dry soil, early wet season (c) Wet soil, mid wet season (d) Dry soil, mid wet season (e) Wet soil, transition period (f) Dry soil, transition period (g) Wet soil, dry season (h) Dry soil, dry season 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 Fig. 6. VPD-response curves of stomatal conductance for the 8 periods as defined in Table 1. Data measured at saturating light conditions with incoming shortwave radiation being higher than 700 W m−2. The grey curves in the situations with dry soil (right graphs) symbolize the curves from corresponding situations with wet soils (left graphs). W. L. Kutsch et al.: Savanna water-carbon relations -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 -24 -20 -16 -12 -8 -4 0 4 Ecosystem net exchange (FC) and Canopy photosynthesis ( FP) (µmol CO2 m-2 s-1) (a) Wet soil, early wet season (b) Dry soil, early wet season (c) Wet soil, mid wet season (d) Dry soil, mid wet season (e) Wet soil, transition period (f) Dry soil, transition period (g) Wet soil, dry season (h) Dry soil, dry season 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 0 250 500 750 1000 Canopy conductance (mmol m-2 s-1) Fig. 7. Conductance response curve of CO2-fluxes for the 8 periods as defined in Table 1. Black dots symbolize the canopy gross photo- synthetic flux, open circles the net ecosystem flux; data measured at saturating light conditions with incoming shortwave radiation being higher than 700 W m−2. VPD (bar) 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 Canopy conductance (mmol m-2 s-1) (a) Wet soil, early wet season (b) Dry soil, early wet season (c) Wet soil, mid wet season (d) Dry soil, mid wet season (e) Wet soil, transition period (f) Dry soil, transition period (g) Wet soil, dry season (h) Dry soil, dry season 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 Fig. 6. VPD-response curves of stomatal conductance for the 8 periods as defined in Table 1. 3.3 Night time respiration In a first approach, the night time data sets identified for the analysis of the nocturnal fluxes after u∗filtering (1247 records) were divided into three classes of relative soil water content. Within each of these classes an exponential temper- ature response curves was generated. The analysis revealed a clear exponential increase of ecosystem respiration with increasing temperature and also a positive influence of soil moisture (Fig. 4). In addition, the data showed that the tem- perature response was modified by soil moisture: at low soil moisture the Q10-value of the temperature function was re- duced in comparison to high soil moisture. Thus, the data set was fitted to the ecosystem respiration described by Re- ichstein et al. (2002) which provides response curves for soil moisture and temperature, and changes the Q10-value of the temperature response curve with changing soil moisture as observed in this study. www.biogeosciences.net/5/1797/2008/ Biogeosciences, 5, 1797–1808, 2008 W. L. Kutsch et al.: Savanna water-carbon relations www.biogeosciences.net/5/1797/2008/ Biogeosciences, 5, 1797–1808, 2008 W. L. Kutsch et al.: Savanna water-carbon relations Relations between photosynthetic capacity (FP,sat) and canopy conductance at FP,sat (a) and between FP,sat and the co- efficient c describing stomatal response sensitivity to VPD (b) for the 8 periods under consideration. This might be a sampling issue, because the area is very patchy (Archibald and Scholes, 2007). However, the two sa- vanna types extend over at least 1 km in the respective foot- print area. We suspect that the reasons for the functional convergence of the savanna sub-types could only be found by detailed studies on leaf-level eco-physiology. Kutsch (un- published data) could show a spatial correlation between soil properties and eco-physiological strategies of the grass species. It was found that different species optimize their fluxes by balancing stomatal conductance and photosynthetic capacity between water availability (lower in the Acacia- site soils) and nutrient availability (higher in the Acacia-site soils). The resulting convergence allows to treat the data as one dataset in the following principle analysis of water- carbon interactions of fluxes in an African savanna ecosys- tem. Fig. 8. Annual courses of the coefficient c describing stomatal response sensitivity to VPD, of maximum canopy photosynthesis (FP,sat) average nighttime respiration (Rav) and R15 as derived from the model. Each point represents the center of a period de- fined in Table 1. The dashed line shows a hypothetical seasonal course without intermediate drought periods. during the drought periods, in particular in April. The ini- tially high respiration rate is typical for seasonal ecosystems facing drought or frost periods (Arneth et al., 2006; Schimel and Mikan, 2005). Respiratory patterns showed a strong dependency on tem- perature and soil moisture. In addition higher rates during the early stages of the wet season and a decrease thereafter even in periods of high soil moisture was detected. Sea- sonal changes in ecosystem respiration rates due to changes in biomass and activity were hard to detect because soil tem- peratures differed between the phenological stages. In addi- tion, changes in activity correlate with changes in tempera- ture and rainfall patterns and are “masked” by the tempera- ture and soil moisture response curves (Kutsch et al., 2001b). The relatively high Q10-value for wet conditions (∼2.5) in- dicates a hidden seasonal change in activity. W. L. Kutsch et al.: Savanna water-carbon relations The seasonal trend of FP,sat was an increase until March and a decrease thereafter – a typical performance for a plant canopy during the course of the growing season. This trend was overlaid by a reduction during drought. The seasonal trends of all important parameters are sum- marized in Fig. 8a–c. The coefficient c (Eq. 10) is an in- dicator for the sensitivity of surface conductance to VPD, FP,sat for the photosynthetic capacity. Rav is the average nighttime respiration for each period and R15 the normal- ized respiration rate at 15◦C and optimum water conditions. Night time respiration seemed to be highest at the on- set of the growing season, was stable on a slightly lower level during the main part of the growing season and de- creased thereafter. This trend was overlaid by depressions Biogeosciences, 5, 1797–1808, 2008 Biogeosciences, 5, 1797–1808, 2008 www.biogeosciences.net/5/1797/2008/ W. L. Kutsch et al.: Savanna water-carbon relations 1804 0 0 1 2 3 4 5 10 15 20 J F M A M J J A S Coefficient c 0 0.02 0.04 0.06 0.08 0.1 FP,max (µmol CO2 m-2 s-1) Rav and R15 (µmol CO2 m-2 s-1) R15 Rav Fig. 8. Annual courses of the coefficient c describing stomatal response sensitivity to VPD, of maximum canopy photosynthesis (FP,sat) average nighttime respiration (Rav) and R15 as derived from the model. Each point represents the center of a period de- fined in Table 1. The dashed line shows a hypothetical seasonal course without intermediate drought periods. 0 0 1 2 3 4 5 10 15 20 J F M A M J J A S Coefficient c 0 0.02 0.04 0.06 0.08 0.1 FP,max (µmol CO2 m-2 s-1) Rav and R15 (µmol CO2 m-2 s-1) R15 Rav 0 5 10 15 20 25 0 5 10 15 20 25 FP,max (µmol CO2 m-2 s-1) FP,max (µmol CO2 m-2 s-1) 0 5 10 15 0 0.05 0.1 Coefficient c GS at FP,max (mm s-1) Fig. 9. Relations between photosynthetic capacity (FP,sat) and canopy conductance at FP,sat (a) and between FP,sat and the co- efficient c describing stomatal response sensitivity to VPD (b) for the 8 periods under consideration. 0 5 10 15 20 25 0 5 10 15 20 25 FP,max (µmol CO2 m-2 s-1) FP,max (µmol CO2 m-2 s-1) 0 5 10 15 0 0.05 0.1 Coefficient c GS at FP,max (mm s-1) Fig. 9. W. L. Kutsch et al.: Savanna water-carbon relations This was con- firmed by comparing mean night time fluxes throughout the year: highest respiration rates were found at the onset of the wet season in January and February, followed by constant de- crease thereafter that was overlaid by a less severe influence of water shortage during the growing period (Fig. 8c). Figure 9 summarizes the close relationship between stom- atal regulation and photosynthetic capacity by the close re- lationship between canopy photosynthetic capacity and sur- face conductance (Fig. 9a) and, in addition, a strong correla- tion between coefficient c and canopy photosynthetic capac- ity (Fig. 9b) for the 8 periods under consideration. 4 Discussion The convergence of the two savanna types in terms of flux properties may be the most surprising result of this study. Sectoral analysis of flux data has been conducted success- fully in other studies, where distinct differences in fluxes be- tween sectors could be related to differences in properties such as species composition or LAI of the canopy (Kutsch et al., 2005). At this site the fluxes were identical in spite of different soil properties and plant species composition as described by Scholes et al. (2001). It is assumed that this is due to the growth respiration of the new biomass and the mineralization of easily decompos- able organic matter that has been accumulated during the dry season. Augustine and McNaughton (2005) showed re- cently that the new growing plant biomass at the onset of the Biogeosciences, 5, 1797–1808, 2008 www.biogeosciences.net/5/1797/2008/ 1805 W. L. Kutsch et al.: Savanna water-carbon relations Kutsch et al. (2001a) showed by means of model calcu- lations that stomatal acclimation allows the plant to opti- mize for high carbon gain during periods of sufficient wa- ter supply and to save water during drought. It is impor- tant to note that decreases in stomatal conductance in many plants are inter-related with decreases in photosynthetic ca- pacity (Hall and Schulze, 1980; Schulze and Hall, 1982). Whereas those authors assumed an un-coordinated response of stomatal conductance and photosynthetic capacity to soil water stress (Schulze and Hall, 1982), Flexas et al. (2006a, b) showed recently on the molecular level that stomatal closure triggers down-regulation of Rubisco either directly through its effects on chloroplast CO2 concentration or mediated by some common signalling (e.g. a response to abscisic acid). Data from this study were obtained by eddy covariance at the ecosystem scale and show the same pattern of inter-relation between stomatal conductance and photosynthetic capacity as leaf-level studies. growing season is also favoured by a large inorganic nitro- gen pool that has accumulated during the dry season, dur- ing which mineralization does not completely cease. Xu et al. (2004) found a similar pattern oak/grass savanna ecosys- tem in California. They also observed a “respiratory flush” after a rainfall event, as did Levine et al. (1996) at Preto- riuskop (30 km south of the Skukuza site) and Scholes et al. (1997) in savannas at Nylsvley, South Africa. 4 Discussion Maximum canopy-scale carbon dioxide fluxes at light sat- uration (FC,sat:−17 µmol CO2 m−2 s−1) and canopy-scale photosynthetic capacity (FP,sat:−22 µmol CO2 m−2 s−1) re- ported in this study are comparable with other eddy- covariance studies of African savanna ecosystems, particu- larly when bearing in mind that the 2002/2003 growing sea- son at Skukuza was characterised by an extremely low pre- cipitation. Scanlon and Albertson (2004) measured fluxes from four savanna ecosystems along a precipitation transect across the Kalahari sands. They report values of FP,sat be- tween −15 and −20 µmol CO2 m−2 s−1 for the “drier end” of the transect (mean annual rainfall 365 and 407 mm). A similar range is also reported in Williams and Albertson (2004) for a mixed Acacia-Combretum savanna in Botswana receiving a mean annual rainfall of 400 mm, and by Veenen- daal et al. (2004) for a Mopani-woodland in Botswana with 464 mm mean annual precipitation. It is important to note that the decrease in stomatal con- ductance goes together with the down-regulation of photo- synthetic capacity. As a consequence, Ci/Ca may remain rel- atively stable and the isotopic fractionation will not be af- fected by drought as severely as expected. Therefore, pre- dicted values of isotopic fractionation from large scale mod- eling may be wrong. The correlation between canopy photo- synthetic capacity and maximum surface conductance seems to be relevant for scaling processes from leaf to ecosystem level and even to higher scales (e.g. Lloyd et al., 2008). Scanlon and Albertson (2004), who collected only short- term data of a few days during the 2000 wet season, inter- preted differences in the light response curves of FC and the VPD response curve of water use efficiency between the sites as a long-term adaptation of the different vegetation types to the mean precipitation at each location. In our study we found a highly dynamic ecosystem response to environmen- tal factors that may also be part of this adaptation. Our approach of defining different periods according to rainfall characteristics and soil moisture provided valuable qualita- tive insights. However, it is important to stress that theories developed for individual plants or leaves cannot be scaled simply to the vegetation level because different water use strategies and life cycles may compete for the available water and in this situation a conservative strategy may simply lead to a higher water usage by the competing neighbour (Schulze et al., 2005). 4 Discussion Moreover, different plant functional types have different traits and life cycles that may influence the ecosys- tem properties much more than leaf scale acclimation. De- creases in the fAPAR from remote sensing as shown in Fig. 2a may indicate that the LAI is reduced during the dry periods. However, this observation has to be interpreted very care- fully, because the correlation between fAPAR and LAI is scat- tering (Huemmrich et al., 2005) and influenced by changing optical properties of leaves during drought stress. Therefore, further studies including detailed process analyses and multi- species canopy modelling are highly required to explain the adaptive inter-relation of canopy conductance and photosyn- thetic capacity which is only poorly represented in higher- scale models. In particular, it showed that the response of canopy con- ductance to VPD followed the soil moisture conditions very dynamically and that conductance and photosynthetic capac- ity were strongly coupled. Three findings are noteworthy: 1. The short-term regulation of canopy conductance was modified by a general seasonal trend and some mid-term acclimation to soil water conditions. This can be seen in the variation of the coefficient c in Figs. 8 and 9. 2. The most constant parameter was water use efficiency (data not shown) that was influenced by VPD during the day but the VPD response curve of water usage did change only slightly during the course of the growing season and decreased by about 30% during the transi- tion from wet to dry season. 5 Conclusion 3. The regulation of canopy conductance and photosyn- thetic capacity were closely related, suggesting a com- plex regulation pattern (Fig. 9). The eddy covariance measurements at a flux tower close to Skukuza in the Kruger National Park, South Africa, revealed the strong influence of water relations on the carbon fluxes www.biogeosciences.net/5/1797/2008/ Biogeosciences, 5, 1797–1808, 2008 W. L. Kutsch et al.: Savanna water-carbon relations 1806 in savanna ecosystems. While Merbold et al. (2008) showed the close relationship between canopy conductance and Fpsat being constant over space, this study gave useful insights into the temporal dynamics of acclimation to changing con- ditions. The mid-term ecophysiological response to climate and/or soil moisture, respectively, is an important observa- tion which is not included in any of the common models. change of forests: The euroflux methodology, Adv. Ecol. Res., 30, 113–175, 2000. Augustine, D. J. and McNaughton, S. J.: Temporal asynchrony in soil nutrient dynamics and plant production in a semiarid ecosys- tem, Ecosystems, 7, 829–840, 2005. Baldocchi, D. D., Xu, L. K., and Kiang, N.: How plant functional- type, weather, seasonal drought, and soil physical properties alter water and energy fluxes of an oak-grass savanna and an annual grassland, Agr. Forest Meteorol., 123, 13–39, 2004. Acknowledgements. Eddy covariance measurements in the Kruger Park were supported by the US National Aeronautics and Space Administration (NASA, Grant NAG5-8705 and TE/03-000-0008 to NPH), the National Science Foundation (EAR-0120630 to NPH), NOAA (Grant NA17RJ1228 to NPH), by the South African National Research Foundation (RJS) and Center for Scientific and Industrial Research (RJS). WLK was funded by the German Science Foundation (DFG, DFG Ku 1099/2-1) and the European Commission (FP6, Project “CarboAfrica”, Nr. 037132 and strongly supported by the Christian-Albrechts-University, Kiel. Construc- tion of the micrometeorological tower near Skukuza was made possible through NASA funding to Jeff Privette for participation in EOS Validation and SAFARI-2000. The scientific collaboration of Mary Scholes, Harold Annegarn, Lackson Marufu, Pierre Ngok and numerous SAFARI-2000 personnel is gratefully acknowledged. 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RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand
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RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand Laetitia Maestroni, Julien Audry, Pierre Luciano, Stephane Coulon, Vincent Géli, Yves Corda a Maestroni, Julien Audry, Pierre Luciano, Stephane Coulon, Vincent Géli, Yves Corda To cite this version: Laetitia Maestroni, Julien Audry, Pierre Luciano, Stephane Coulon, Vincent Géli, et al.. RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand. Cell Stress, 2020, 4, pp.48 - 63. ￿10.15698/cst2020.03.214￿. ￿hal-03012937￿ Research Article www.cell-stress.com HAL Id: hal-03012937 https://hal.science/hal-03012937v1 Submitted on 18 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand Laetitia Maestroni1, Julien Audry1, Pierre Luciano1, Stéphane Coulon1, Vincent Géli1,* and Yves Corda1,* 1 Aix-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille, France. Equipe Labellisée par la Ligue Nationale contre le Cancer. etitia Maestroni1, Julien Audry1, Pierre Luciano1, Stéphane Coulon1, Vincent Géli1,* and Yves C x-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille, France. Equipe Labellisée par la Ligue Nat Cancer. 1 Aix-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille, France. Equipe Labellisée par la Ligue le Cancer. * Corresponding Authors: p g Vincent Géli, Aix-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille, France. Equipe Labellisée par la Ligue Nationale contre le Cancer ; E-mail: vincent.geli@inserm.fr; Yves Corda, Aix-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille, France. Equipe Labellisée par la Ligue Nationale contre le Cancer ; E-mail: yves.corda@inserm.fr doi: 10.15698/cst2020.03.214 Received originally: 25.09.2018; in revised form: 03.01.2020, Accepted 07.01.2020, Published 17.01.2020. ABSTRACT In Saccharomyces cerevisiae, the absence of Pif1 helicase induces the instability of G4-containing CEB1 minisatellite during leading strand but not lagging strand replication. We report that RPA and Pif1 cooperate to maintain CEB1 stability when the G4 forming strand is either on the leading or lagging strand templates. At the leading strand, RPA acts in the same pathway as Pif1 to maintain CEB1 stability. Consistent with this result, RPA co- precipitates with Pif1. This association between Pif1 and RPA is affected by the rfa1-D228Y mutation that lowers the affinity of RPA in particular for G-rich single-stranded DNA. At the lagging strand, in contrast to pif1∆, the rfa1-D228Y mutation strongly increases the frequency of CEB1 rearrange- ments. We explain that Pif1 is dispensable at the lagging strand DNA by the ability of RPA by itself to prevent formation of stable G-rich secondary struc- tures during lagging strand synthesis. Remarkably, overexpression of Pif1 res- cues the instability of CEB1 at the lagging strand in the rfa1-D228Y mutant indicating that Pif1 can also act at the lagging strand. We show that the ef- fects of the rfa1-D228Y (rpa1-D223Y in fission yeast) are conserved in Schiz- osaccharomyces pombe. Finally, we report that RNase H1 interacts in a DNA- dependent manner with RPA in budding yeast, however overexpression of RNase H1 does not rescue CEB1 instability observed in pif1∆ and rfa1-D228Y mutants. Collectively these results add new insights about the general role of RPA in preventing formation of DNA secondary structures and in coordinating the action of factors aimed at resolving them. RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand Keywords: RPA, Pif1, human minisatellite CEB1, G-rich structures, G-quadruplex. Keywords: RPA, Pif1, human minisatellite CEB1, G-rich structures, G-quadruplex. Abbreviations: Co-IP – Co-immunoprecipitation, RPA – replication protein A, ssDNA – single-stranded DNA, WT – wild type. INTRODUCTION INTRODUCTION fragments by governing the sequential action of Dna2 and Fen1 [5]. During Pol δ synthesis most flaps generated on the lagging strand, by strand displacement, are normally cleaved by Fen1. However, a minor fraction escapes cleav- age [6], and the 5' ssDNA flaps on Okazaki fragments get extended by Pif1, a 5' to 3' helicase, to create substrates for RPA binding that inhibits Fen1’s cleavage [5,7-11]. RPA is next displaced by Dna2 which cleaves the long flap, gen- erating a short flap structure that undergoes cleavage via Fen1 [12-14]. Replication protein A (RPA) is the major eukaryotic single- stranded DNA (ssDNA) binding protein that consists of 70, 32, and 14 kDa subunits [1]. RPA plays a key role in coordi- nating DNA synthesis, repair, and DNA damage signalling through binding to single-stranded DNA (ssDNA) interme- diates generated during these processes [2]. RPA primarily maintains ssDNA in an unfolded state through different binding modes that are characterized by the length of the interacting ssDNA [3]. Thus, RPA binds to ssDNA with high affinity preventing the formation of DNA secondary struc- tures and annealing of homologous sequences [4]. Among its activities, RPA is involved in the maturation of Okazaki Interestingly, it has been reported in vitro, that RPA binds to and unwinds G4 structures in a 5’ to 3’ direction [15]. G4 are polymorphic and consist of four-stranded Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 48 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) mutant (rpa1-D223Y in fission yeast) also possesses a low- er affinity for ssDNA and reduced ability in removing sec- ondary structure from ssDNA [39, 40]. In this study, we investigated the role of RPA in maintaining the stability of CEB1 when the G-quadruplex-forming strand is either on the leading or lagging strand template. Our results indicate that both RPA and Pif1 cooperate at the leading strand to maintain the stability of CEB1. Consistent with this hy- pothesis, RPA co-precipitates with Pif1. In contrast to Pif1, RPA is also required to stabilize CEB1 when the G-quadruplex-forming strand is the lagging strand. How- ever, under a situation that compromises RPA binding to ssDNA, overexpression of Pif1 rescued lagging-CEB1 insta- bility, suggesting that Pif1 can unwind G4 at the lagging strand. INTRODUCTION Interestingly, Mms1 which binds to G-rich/G4 re- gions and supports the binding of Pif1, is not required to maintain the stability of CEB1. Based on these data we propose a model in which RPA facilitates Pif1 action at the leading strand DNA to unwind G4 while enriched RPA at the lagging strand DNA prevents by itself formation of sta- ble G4, explaining why Pif1 is dispensable. We extended the role of RPA in preventing non-templated DNA single- stranded structure by showing that RPA interacts with RNAse H1 in a DNA-dependent manner in S. cerevisiae, as previously reported in human cells [41]. However, we found that overexpressing RNAse H1 did not restore CEB1 stability in both pif1∆ and rfa1-D228Y mutants, suggesting that CEB1 instability is not due to R-Loop formation arising during transcription. structures formed at specific G-rich motifs within DNA, RNA, and into R loops, a RNA-DNA hybrid structure, that can eventually lead to genome instability [16-19]. The core of these structures is formed by a square arrangement of four guanines held together by Hoogsteen hydrogen bonds [20, 21]. Under specific conditions, G4 structures are rec- ognized by specific factors and their formation is controlled [22, 23]. However, it has been shown that highly stable G4 structures impede fork progression. Hence, their unwind- ing by helicases is critical [17, 24, 25]. Many helicases are able to unwind G4 structures in vitro such as the RecQ hel- icases BLM, WRN and Sgs1 and other helicases such as Pif1, FANCJ or RTEL1 [24,26-30]. G4 structures are also targeted by additional proteins that protect them [23] or support the function of an helicase at G4 [22]. In budding yeast, unwinding of G4 is mainly performed by the Pif1 helicase [26]. Indeed, a particular example is the 1.8 kb G4-forming human minisatellite CEB1, a reporter of G4 formation and processing [31, 32]. In cells lacking Pif1, CEB1 is unstable when inserted into Saccharomyces cerevisiae genome, near an early origin of replication (ARS305). Instability of CEB1, which consists in 42 motifs of 39 nucleotides ar- ranged as direct repeats, was correlated to the ability of the CEB1 motif to form G4 [31, 33, 34]. Surprisingly, in pif1∆ cells CEB1 was unstable only when the G-quadruplex- forming strand was the leading strand template [33]. RESULTS We have previously shown in fission yeast that the rpa1-D223Y mutation (rfa1-D228Y in budding yeast), that exhibits a reduced affinity for ssDNA, impaired lagging strand telomere replication and provoked accumulation of secondary structures [38]. Consistently, expression of ScPif1 rescued the phenotypes associated with the rpa1- D223Y mutation [38]. These results suggested that rpa1- D223Y cells accumulated G-rich structures at lagging strand telomeres that were resolved by the heterologous expression of ScPif1. In S. cerevisiae, RPA subunits are en- coded by RFA1, RFA2, and RFA3 genes. The rfa1-D228Y INTRODUCTION This result is apparently counterintuitive since ssDNA is mostly formed during the discontinuous synthesis of the lagging strand DNA [35] and because Pif1 has been reported to act at the lagging strand [36] and binds G4 structures located in the lagging strand [22, 37]. The rfa1-D228Y mutation affects both the leading-CEB1 and lagging-CEB1 As mentioned above, the G-rich minisatellite CEB1 can be considered as a reporter of G4 formation and processing [31, 32]. We used strains previously constructed in A. Nico- las’ laboratory in which the 1.8 kb CEB1 is inserted in both directions at 2.1 kb of ARS305 and 32.6 kb away from ARS306 allowing to primarily replicate CEB1 only from the proximal ARS305 origin (Figure 1). Depending on the orien- tation of CEB1 insertion, the G4-forming strand will be rep- licated by the leading or the lagging machinery [33]. We FIGURE 1: Map of the CEB1 insertion within chromosome III at 2.1 kb from the ARS305. In orientation I, the G-quadruplex-forming strand is the template of the leading polymerase (leading CEB1). In orientation II, the G-quadruplex-forming strand is the template of the lagging polymerase (lagging CEB1). FIGURE 1: Map of the CEB1 insertion within chromosome III at 2.1 kb from the ARS305. In orientation I, the OPEN ACCESS | www.cell-stress.com 49 Cell Stress | MARCH 2020 | Vol. 4 No. 3 FIGURE 1: Map of the CEB1 insertion within chromosome III at 2.1 kb from the ARS305. In orientation I, the G-quadruplex-forming strand is the template of the leading polymerase (leading CEB1). In orientation II, the G-quadruplex-forming strand is the template of the lagging polymerase (lagging CEB1). FIGURE 1: Map of the CEB1 insertion within chromosome III at 2.1 kb from the ARS305. In orientation I, the G-quadruplex-forming strand is the template of the leading polymerase (leading CEB1). In orientation II, the G-quadruplex-forming strand is the template of the lagging polymerase (lagging CEB1). G-quadruplex-forming strand is the template of the leading polymerase (leading CEB1). In orientation II, the G-quadruplex-forming strand is the template of the lagging polymerase (lagging CEB1). Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 49 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) FIGURE 2: RPA is required to stabilize CEB1. (A) Experimental scheme. Yeast strains of interest are mated and the resulting diploid sporulated and dissec After identification of spore-colonies of interest, the spore-colony is plated on media to obtain isolated colonies. Individual colonies are placed in liquid cult until stationary growth phase. Genomic DNAs are extracted and analysed by Southern blot. (B) RPA is required to stabilize CEB1 when the G4-forming stran replicated by the leading polymerase. The rfa1-D228Y mutation affects both the leading-CEB1 and lagging-CEB1 Genomic DNAs from yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI, and southern blotted. M branes were hybridized with the CEB1-0.6 probe. (C) Genomic DNAs from yeast cells containing the leading-CEB1Gmut-1.7 were digested by ApaI and SacII, southern blotted. The membranes were hybridized with the CEB1Gmut-1.7 probe. (D) In contrast to Pif1, RPA is required to stabilize CEB1 when the G4-form strand is replicated by the lagging polymerase. Genomic DNAs from yeast cells bearing the lagging-CEB1 were digested by ApaI and NcoI, and southern blot Membranes were hybridized with the CEB1-0.6 probe. M: ladder DNA serving as size standard (kbp). The number of colonies analysed per well, the percent of rearrangement frequencies and the total numbers of colonies are indicated in Table 1 FIGURE 2: RPA is required to stabilize CEB1. (A) Experimental scheme. Yeast strains of interest are mated and the resulting diploid sporulated and disse After identification of spore-colonies of interest, the spore-colony is plated on media to obtain isolated colonies. Individual colonies are placed in liquid cu until stationary growth phase. Genomic DNAs are extracted and analysed by Southern blot. (B) RPA is required to stabilize CEB1 when the G4-forming stra replicated by the leading polymerase. Genomic DNAs from yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI, and southern blotted. M branes were hybridized with the CEB1-0.6 probe. (C) Genomic DNAs from yeast cells containing the leading-CEB1Gmut-1.7 were digested by ApaI and SacI GURE 2: RPA is required to stabilize CEB1. (A) Experimental scheme. Yeast strains of interest are mated and the resulting diploid sporulated and dis ter identification of spore-colonies of interest, the spore-colony is plated on media to obtain isolated colonies. Individual colonies are placed in liquid til stationary growth phase. Genomic DNAs are extracted and analysed by Southern blot. (B) RPA is required to stabilize CEB1 when the G4-forming s plicated by the leading polymerase. Genomic DNAs from yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI, and southern blotted anes were hybridized with the CEB1-0.6 probe. (C) Genomic DNAs from yeast cells containing the leading-CEB1Gmut-1.7 were digested by ApaI and Sa uthern blotted. The membranes were hybridized with the CEB1Gmut-1.7 probe. (D) In contrast to Pif1, RPA is required to stabilize CEB1 when the G4- and is replicated by the lagging polymerase. The rfa1-D228Y mutation affects both the leading-CEB1 and lagging-CEB1 Genomic DNAs from yeast cells bearing the lagging-CEB1 were digested by ApaI and NcoI, and southern embranes were hybridized with the CEB1-0.6 probe. M: ladder DNA serving as size standard (kbp). The number of colonies analysed per well, the per rearrangement frequencies, and the total numbers of colonies are indicated in Table 1. FIGURE 2: RPA is required to stabilize CEB1. (A) Experimental scheme. Yeast strains of interest are mated and the resulting diploid sporulated and dissected. After identification of spore-colonies of interest, the spore-colony is plated on media to obtain isolated colonies. Individual colonies are placed in liquid culture until stationary growth phase. Genomic DNAs are extracted and analysed by Southern blot. (B) RPA is required to stabilize CEB1 when the G4-forming strand is replicated by the leading polymerase. Genomic DNAs from yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI, and southern blotted. Mem- branes were hybridized with the CEB1-0.6 probe. (C) Genomic DNAs from yeast cells containing the leading-CEB1Gmut-1.7 were digested by ApaI and SacII, and southern blotted. The membranes were hybridized with the CEB1Gmut-1.7 probe. (D) In contrast to Pif1, RPA is required to stabilize CEB1 when the G4-forming strand is replicated by the lagging polymerase. Genomic DNAs from yeast cells bearing the lagging-CEB1 were digested by ApaI and NcoI, and southern blotted. Membranes were hybridized with the CEB1-0.6 probe. M: ladder DNA serving as size standard (kbp). The number of colonies analysed per well, the percentage of rearrangement frequencies, and the total numbers of colonies are indicated in Table 1. Cell Stress | MARCH 2020 | Vol. 4 No. 3 50 OPEN ACCESS | www.cell-stress.com OPEN ACCESS | www.cell-stress.com A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) rangements of the lagging-CEB1 was clearly increased in the rfa1-D228Y mutant (31%). These results suggest that when G4-forming sequences are localized at the lagging strand, RPA prevents the instability of CEB1. They are con- sistent with the known enrichment of RPA at the lagging strand [35]. Interestingly, despite the fact that pif1∆ did not affect the stability of the lagging-CEB1, combining pif1∆ with rfa1-D228Y, slightly aggravated the CEB1 insta- bility phenotype of the single rfa1-D228Y mutant (41%). This result suggests that Pif1 can be active at the lagging- CEB1. will name leading-CEB1 or lagging-CEB1 to indicate the machinery that replicates the G4-forming strand. The rfa1-D228Y mutation affects both the leading-CEB1 and lagging-CEB1 Previous results demonstrated that the helicase activity of Pif1 was required to stabilize the leading-CEB1 but that Pif1 was dispensable for the stability of the lagging-CEB1 suggesting the existence of different mechanisms to resolve G4 when the G4-forming strand is replicated by the leading or the lagging machinery [33]. We thought to test the role of RPA in the CEB1 stability by determining the effect of the rfa1-D228Y mutant whose DNA-binding activity, in particular to G-rich ssDNA, is com- promised [38, 39]. We used an experimental scheme (Fig- ure 2A) adapted from Lopes et al. [33]. Briefly, tetrads ob- tained from the sporulation of heterozygous diploids were dissected. Spore colonies carrying the appropriated muta- tion were then resuspended in water and streaked on YPD plates in order to obtain about 200 isolated colonies. Colo- nies were inoculated in YPD liquid cultures, grown to satu- ration, and genomic DNAs were prepared from the liquid cultures. The size of CEB1 was monitored by Southern blot. We calculated the frequency of instability by monitoring CEB1 size variations (contractions and expansions). We considered that CEB1 was unstable when the intensity of the band(s) was superior to the one of the parental band. We therefore partially discriminated between early or late events of CEB1 rearrangements. Overexpression of Pif1 in rfa1-D228Y cells rescues lagging- CEB1 instability To further understand the functional interaction between RPA and Pif1 in CEB1 instability, we overexpressed Pif1 in rfa1-D228Y cells bearing leading-CEB1 or lagging-CEB1. Because strong Pif1 overexpression impairs cell viability [42], we used a low-copy centromeric plasmid in which the nuclear form of Pif1 is under the control of the GAL1 pro- moter [43]. We first noticed that Pif1 overexpression was slightly deleterious in rfa1-D228Y cells (Figure 3A). To es- timate Pif1 overexpression we measured the expression of PIF1 by reverse transcription followed by quantitative PCR. Our data indicate that PIF1 is overexpressed in a similar level in WT and rfa1-D228Y cells expressing PIF1 under the control of the GAL1 promoter (Figure 3B, left and 3C, left). Instability of both leading- and lagging-CEB1 was tested in rfa1-D228Y cells grown in galactose (SGal) allowing the overexpression of Pif1. We found that Pif1 overexpression had only a modest effect on leading-CEB1 stability in rfa1- D228Y cells while it had no effect in WT cells (Figure 3B, right). Surprisingly, Pif1 overexpression completely rescued the instability of lagging-CEB1 in rfa1-D228Y cells (Figure 3C, right). We concluded that when the G4-forming strand was replicated by the lagging machinery in cells in which the ssDNA binding activity of RPA is compromised, overex- pressed Pif1 could act at the lagging strand to unwind G4. We first analysed the stability of the leading-CEB1 (ori- entation I) in wild type (WT), pif1∆, rfa1-D228Y, and pif1∆ rfa1-D228Y cells (Figure 2B). As shown in representative gels and as previously reported [31], the leading-CEB1 was stable in the WT strain but extremely unstable in the pif1∆ mutant (53% of rearrangements) (Figure 2B, Table 1). In most cases, we obtained contractions except in one case (Figure 2B, second panel). In the rfa-1D228Y mutant, lead- ing-CEB1 was also unstable (51% of rearranged colonies). Interestingly, the double mutant pif1∆ rfa1-D228Y exhibit- ed a level of instability in a similar range of the two single mutants (43%), suggesting that RPA and Pif1 could act in similar pathways to unwind G4 and stabilize CEB1 when the G4-forming strand is replicated by the leading poly- merase (Figure 2B). The rfa1-D228Y mutation affects the interaction between Pif1 and RPA Our results indicate that RPA and Pif1 could cooperate to stabilize the leading-CEB1 and the lagging-CEB1. This prompted us to test whether Pif1 interacts with RPA. Pif1- myc was immunoprecipitated with anti-Myc antibody and tested for the presence of RPA with a polyclonal antibody directed against Rfa1 or against Rfa2 (Figure 4). Pif1-myc was efficiently co-immunoprecipitated with both Rfa1 and Rfa2 but to a lesser extend with rfa1-D228Y. To address whether the robust Co-immunoprecipitations (Co-IP) of Pif1-myc with Rfa1 and Rfa2 were dependent on the pres- ence of DNA, DNase1 was added in the lysate. As shown in Figure 4, DNA digestion affected the Co-IP suggesting that the presence of DNA is required for the robust co- precipitation of Pif1 and RPA. These results suggest that the robust association of Pif1 with RPA relies on specific structures on the DNA. We think that the interaction be- tween Pif1 and RPA is not only due to unspecific interac- tions mediated by DNA since the interaction between Pif1 We next analysed the effect of pif1∆, rfa1-D228Y, and pif1∆ rfa1-D228Y on the stability of CEB1-Gmut, a version of CEB1 mutated for its G-quadruplex-forming sequences [31, 32]. The CEB1-Gmut was inserted at the same location and in the same orientation as the leading-CEB1. As re- ported, the CEB1-Gmut was stable in pif1∆ cells [33]. In the rfa1-D228Y and pif1∆ rfa1-D228Y mutants, the % of CEB1- Gmut rearrangements dropped to 22% and 20%, respec- tively (Figure 2C). We concluded that the high leading- CEB1 instability observed in the rfa1-D228Y mutant partly relies on its ability to form G4. However, the residual CEB1- Gmut instability suggests that the rfa1-D228Y mutation affects the replication of the 42 repeated motifs of CEB1- mut irrespectively of the presence of G4. We next examined the stability of the lagging-CEB1 (orientation II) in the same mutants as above. As reported [33], deleting PIF1 had no effect on the stability of the lag- ging-CEB1 (Figure 2D). In contrast, the frequency of rear- Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 51 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) L. Maestroni et al. (2020) L. Maestroni et al. (2020) A critical role for RPA in CEB1 stability FIGURE 3: Pif1 overexpression rescues lagging-CEB1 instability in the rfa1-D228Y mutant. The rfa1-D228Y mutation affects the interaction between Pif1 and RPA (A) Colonies containing the leading- or lagging-CEB1, overexpressing Pif1 (Pif1 overexpression) or not (Empty vector) were plated on galactose medium and subsequently incubated at 30°C. (B) Left: c was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was determined using qPCR. (-): Empty vector, (+): Pif1 overexp sion. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 (Pif1 overexpression) and containing the leading-CEB1 w treated as in Figure 2. M: ladder DNA. (C) Left: cDNA was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was de mined using qPCR. (-): Empty vector, (+): Pif1 overexpression. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 ( overexpression) and containing the lagging-CEB1 were treated as in Figure 2. M: ladder DNA. Yeast strains were grown in medium containing galac (2%). The number of colonies analysed per well, the percentage of rearrangement frequencies, and the total numbers of colonies are indicated in T 1. FIGURE 3: Pif1 overexpression rescues lagging-CEB1 instability in the rfa1-D228Y mutant. (A) Colonies containing the leading- or lagging-CEB1, overexpressing Pif1 (Pif1 overexpression) or not (Empty vector) were plated on galactose medium and subsequently incubated at 30°C. (B) Left: cD was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was determined using qPCR. (-): Empty vector, (+): Pif1 overexp sion. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 (Pif1 overexpression) and containing the leading-CEB1 w treated as in Figure 2. M: ladder DNA. (C) Left: cDNA was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was de mined using qPCR. (-): Empty vector, (+): Pif1 overexpression. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 ( overexpression) and containing the lagging-CEB1 were treated as in Figure 2. M: ladder DNA. Yeast strains were grown in medium containing galact (2%) h b f l i l d ll h f f i d h l b f l i i di d i FIGURE 3: Pif1 overexpression rescues lagging-CEB1 instability in the rfa1-D228Y mutant. (A) Colonies containing the leadin FIGURE 3: Pif1 overexpression rescues lagging-CEB1 instability in the rfa1-D228Y mutant. (A) Colonies containing the leading- or lagging-CEB1, and overexpressing Pif1 (Pif1 overexpression) or not (Empty vector) were plated on galactose medium and subsequently incubated at 30°C. Mms1 is not required to maintain the leading-CEB1 and the lagging-CEB1 stability Mms1 supports Pif1 helicase binding to G4 structures [22]. We thus analysed the importance of Mms1 in CEB1 stabil- ity. We found that in mms1∆ cells, leading-CEB1 and lag- ging-CEB1 were rather stable, showing 0% (0/116) and 3% (3/116) of rearrangements, respectively (Figure 5A, Table 1). These results show that Mms1 is not required for the stability of both leading-CEB1 and lagging-CEB1. They sug- gest that CEB1 minisatellites are not targeted by Mms1. Moreover, they indicate that Mms1 does not support Pif1 function at CEB1 and, together with our previous results showing that Pif1 and RPA interact, suggest that RPA could contribute to the recruitment of Pif1 to CEB1, likely by di- rectly recruiting Pif1. RNase H1 interacts with RPA but its overexpression doesn't rescue CEB1 instability in the absence of Pif1 or reduced levels of RPA Transcription by RNA polymerase can form a three- stranded structure called R-loop [46], which can facilitate or stabilize secondary structure formation in the exposed ssDNA [16, 47, 48]. Thus, G4 structures may also result from exposure of the G4 forming strand by formation on the other strand of RNA:DNA hybrids. This may create a complex structure involving G4 DNA on one strand and a RNA:DNA hybrid on the other strand [49]. Interestingly, Pif1 has been proposed to have a patrolling role that re- moves any G4 or RNA/DNA structure [50]. Indeed, Pif1 regulates R-loop formation at specific genomic loci [51] and potentially complements RNAse H for R-loop resolu- tion [52]. On the other side, systematic analysis of protein complexes in S. cerevisiae have shown that RPA interacts with RNase H1 [53]. Finally, RPA was recently proposed to act as a sensor of R-loop in human cells and to recruit and stimulate RNase H1 to counteract R-loops [41]. To further document the potential cooperation of RPA with Pif1, we first tested whether RPA interacts with RNase H1 in a DNA- dependent manner in budding yeast. Expression of HA- Rnh1 was induced by the addition of galactose and its in- teraction with Rfa1 was probed by Co-IP. The results shown in Figure 6 indicate that Rnh1 interacts with RPA in a way that is stimulated by the presence of DNA, thereby extending the results of Gavin et al. [53] and Nguyen et al. [41]. Interestingly, we found that the rfa1-D228Y mutant had a less efficient ability to bind Rnh1 (Figure 6). These results suggest that the leading-CEB1 instability observed on the leading strand in absence of Pif1 and by reducing the association of RPA with ssDNA (rfa1-D228Y and rtt105∆ mutants) could be the consequence of the presence of R- Loops. To determine if R-loops were responsible of CEB1 instability we analysed the stability of the leading-CEB1 and lagging-CEB1 in WT, pif1∆, rfa1-D228Y, and rtt105∆ cells overexpressing RNase H1, the enzyme responsible to resolve R-loops. We found that RNase H1 overexpression did not affect CEB1 stability in WT cells, and did not rescue CEB1 stability in pif1∆, rfa1-D228Y, and rtt105∆ mutants Mms1 is not required to maintain the leading-CEB1 and the lagging-CEB1 stability The rfa1-D228Y mutation affects the interaction between Pif1 and RPA (B) Left: cDNA was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was determined using qPCR. (-): Empty vector, (+): Pif1 overexpres- sion. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 (Pif1 overexpression) and containing the leading-CEB1 were treated as in Figure 2. M: ladder DNA. (C) Left: cDNA was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was deter- mined using qPCR. (-): Empty vector, (+): Pif1 overexpression. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 (Pif1 overexpression) and containing the lagging-CEB1 were treated as in Figure 2. M: ladder DNA. Yeast strains were grown in medium containing galactose (2%). The number of colonies analysed per well, the percentage of rearrangement frequencies, and the total numbers of colonies are indicated in Table 1. FIGURE 3: Pif1 overexpression rescues lagging-CEB1 instability in the rfa1-D228Y mutant. (A) Colonies containing the leading- or lagging-CEB1, and overexpressing Pif1 (Pif1 overexpression) or not (Empty vector) were plated on galactose medium and subsequently incubated at 30°C. (B) Left: cDNA was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was determined using qPCR. (-): Empty vector, (+): Pif1 overexpres- sion. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 (Pif1 overexpression) and containing the leading-CEB1 were treated as in Figure 2. M: ladder DNA. (C) Left: cDNA was prepared from the indicated strains. The ration of PIF1 transcript to that of ACT1 was deter- mined using qPCR. (-): Empty vector, (+): Pif1 overexpression. Right, genomic DNAs from wild-type (WT) or rfa1-D228Y cells overexpressing Pif1 (Pif1 overexpression) and containing the lagging-CEB1 were treated as in Figure 2. M: ladder DNA. Yeast strains were grown in medium containing galactose (2%). The number of colonies analysed per well, the percentage of rearrangement frequencies, and the total numbers of colonies are indicated in Table 1. Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 52 L. Maestroni et al. (2020) A critical role for RPA in CEB1 stability and rfa1-228Y is lost despite the fact that the rfa1-D228Y still binds to DNA, although with lower affinity [38]. RNase H1 interacts w doesn't rescue CEB1 ins reduced levels of RPA FIGURE 4: The rfa1-D228Y muta- tion affects the interaction be- tween Pif1 and RPA. The rfa1-D228Y mutation affects the interaction between Pif1 and RPA Co- immunoprecipitation experiments were performed in triplicate. Pif1- myc is immunoprecipitated with an anti-Myc antibody (9E10). The presence of RPA in the Pif1-myc IP is monitored with an anti-Rfa1 or an anti-Rfa2 antibody. (+): cell extracts treated with benzonase. The asterisk (*) indicates a non- specific band. MW: molecular weight. FIGURE 4: The rfa1-D228Y muta- tion affects the interaction be- tween Pif1 and RPA. Co- immunoprecipitation experiments were performed in triplicate. Pif1- myc is immunoprecipitated with an anti-Myc antibody (9E10). The presence of RPA in the Pif1-myc IP is monitored with an anti-Rfa1 or an anti-Rfa2 antibody. (+): cell extracts treated with benzonase. The asterisk (*) indicates a non- specific band. MW: molecular weight. and rfa1-228Y is lost despite the fact that the rfa1-D228Y still binds to DNA, although with lower affinity [38]. RNase H1 interacts with RPA but its overexpression doesn't rescue CEB1 instability in the absence of Pif1 or reduced levels of RPA Rtt105 is required to stabilize both the leading-CEB1 and lagging-CEB1 Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates RPA loading onto ssDNA [44]. Consequently, RTT105 inactivation reduces the association of RPA with ssDNA generated during DNA transactions and affects multiple RPA functions [44, 45]. We examined the importance of Rtt105 on the stability of the leading-CEB1 and lagging-CEB1. We found that CEB1 is extremely unsta- ble in rtt105∆ cells. Remarkably, we found that the fre- quency of rearrangements reaches 100% in both leading- and lagging-CEB1 (Figure 5B, Table 1). These results reveal the importance of RTT105 in promoting the replication of G4-forming CEB1 minisatellite during leading and lagging strand synthesis and more generally, to maintain genome stability. They confirm our previous results obtained with rfa1-D228Y mutant indicating that RPA is crucial to remove G-rich structures at both leading and lagging strand. OPEN ACCESS | www.cell-stress.com OPEN ACCESS | www.cell-stress.com Cell Stress | MARCH 2020 | Vol. 4 No. 3 53 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) FIGURE 5: Importance of Mms1 and Rtt105 on CEB1 stability. (A) Mms1 is not required to stabilize CEB1. Left, genomic DNAs from mms1∆ yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI and southern blotted. Right, genomic DNAs from mms1∆ yeast cells bearing the lag- ging-CEB1 were digested by ApaI and NcoI and southern blotted. Membranes were hybridized with the CEB1-0.6 probe. (B) Rtt105 is required to stabilize both leading-CEB1 and lagging-CEB1. Left, genomic DNAs from rtt105∆ yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI and southern blotted. Right, genomic DNAs from rtt105∆ yeast cells bearing the lagging-CEB1 were digested by ApaI and NcoI and southern blotted. Membranes were hybridized with the CEB1-0.6 probe. WT: wild-type genomic DNA. The arrows show the position of stable leading-CEB1 (left) and stable lagging-CEB1 (right). The horizontal dashed lines indicate the theoretical position of stable CEB1. M: ladder DNA serving as size standard (kbp). The number of colonies analysed per well, the percentage of rearrangement frequencies, and the total numbers of colonies are indicated in Table 1. FIGURE 5: Importance of Mms1 and Rtt105 on CEB1 stability. (A) Mms1 is not required to stabilize CEB1. Left, genomic DNAs from mms1∆ yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI and southern blotted. Rtt105 is required to stabilize both the leading-CEB1 and lagging-CEB1 Right, genomic DNAs from mms1∆ yeast cells bearing the lag- ging-CEB1 were digested by ApaI and NcoI and southern blotted. Membranes were hybridized with the CEB1-0.6 probe. (B) Rtt105 is required to stabilize both leading-CEB1 and lagging-CEB1. Left, genomic DNAs from rtt105∆ yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI and southern blotted. Right, genomic DNAs from rtt105∆ yeast cells bearing the lagging-CEB1 were digested by ApaI and NcoI and southern blotted. Membranes were hybridized with the CEB1-0.6 probe. WT: wild-type genomic DNA. The arrows show the position of stable leading-CEB1 (left) and stable lagging-CEB1 (right). The horizontal dashed lines indicate the theoretical position of stable CEB1. M: ladder DNA serving as size standard (kbp). The number of colonies analysed per well, the percentage of rearrangement frequencies, and the total numbers of colonies are indicated in Table 1. FIGURE 5: Importance of Mms1 and Rtt105 on CEB1 stability. (A) Mms1 is not required to stabilize CEB1. Left, genomic DNAs from mms1∆ yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI and southern blotted. Right, genomic DNAs from mms1∆ yeast cells bearing the lag- ging-CEB1 were digested by ApaI and NcoI and southern blotted. Membranes were hybridized with the CEB1-0.6 probe. (B) Rtt105 is required to stabilize both leading-CEB1 and lagging-CEB1. Left, genomic DNAs from rtt105∆ yeast cells bearing the leading-CEB1 were digested by ApaI and XhoI and southern blotted. Right, genomic DNAs from rtt105∆ yeast cells bearing the lagging-CEB1 were digested by ApaI and NcoI and southern blotted. Membranes were hybridized with the CEB1-0.6 probe. WT: wild-type genomic DNA. The arrows show the position of stable leading-CEB1 (left) and stable lagging-CEB1 (right). The horizontal dashed lines indicate the theoretical position of stable CEB1. M: ladder DNA serving as size standard (kbp). The number of colonies analysed per well, the percentage of rearrangement frequencies, and the total numbers of colonies are indicated in Table 1. Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 54 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) FIGURE 6: Rnh1 interacts with RPA. Co-immunoprecipitation experiments were performed in triplicate. HA-Rnh1 has been overexpressed by galactose addition and immunoprecipitated with an anti-HA antibody. The presence of RPA in the HA-Rnh1 IP is monitored with an anti-Rfa1 antibody. Cell extracts treated with nuclease (Thermofisher) are indicated (+). Rtt105 is required to stabilize both the leading-CEB1 and lagging-CEB1 Yeast strains were grown in medium containing 2% glucose (-) or 2% galactose (+) when mentioned. Total proteins on the membrane were stained with Ponceau S as a loading control (Bottom). MW: mo- lecular weight. FIGURE 6: Rnh1 interacts with RPA. Co-immunoprecipitation experiments were performed in triplicate. HA-Rnh1 has been overexpressed by galactose addition and immunoprecipitated with an anti-HA antibody. The presence of RPA in the HA-Rnh1 IP is monitored with an anti-Rfa1 antibody. Cell extracts treated with nuclease (Thermofisher) are indicated (+). Yeast strains were grown in medium containing 2% glucose (-) or 2% galactose (+) when mentioned. Total proteins on the membrane were stained with Ponceau S as a loading control (Bottom). MW: mo- lecular weight. (Table 1). Taken together, our data suggest that the insta- bility observed in absence of Pif1 and reduced levels of RPA is not primarily related to R-loop formation. was calculated in both WT and rpa1-D223Y strains. The level of instability of CEB25-L1T was 14% and 20% in the WT (Figure 7B, left). In the rpa1-D223Y mutant, this level increased up to 62% and 74%, respectively (Figure 7B, right). Expectedly, the instability of CEB25-L1T-G12T that cannot form G4 was reduced to 4% for leading replication and 17% for lagging replication (Figure 7C). These results clearly show that like in budding yeast the RPA complex plays an important role in the replication and stability of G4-forming sequences. Furthermore, the fact that fission yeast lacks a functional homolog of Pif1 helicase, the Pfh1 helicase behaving more closely like the budding yeast Rrm3 helicase [56], may account for the prominent role of RPA complex in G4 unwinding in fission yeast. The rpa1-D223Y mutation affects the stability of CEB25- L1T in fission yeast Because the rpa1-D223Y mutation impaired replication of the G-rich lagging strand telomere in Schizosaccharomyces pombe, we investigated the stability of CEB in fission yeast. We took advantage of the minisatellite CEB25-L1T which contains 14 repeats of a 44 nucleotides-sequence (0.62 kb) that form a stable G4 [34, 54]. In this L1T version, the loop of the repeated sequence of CEB25 has been reduced to one thymine nucleotide. The shortening of the loop in- creases its thermal stability in correlation with the in vivo instability [54]. As a control the CEB25-L1T-G12T has been used in which the guanine at the 12th position of the re- peated sequence has been mutated into thymine, prevent- ing the formation of G4. We introduced the CEB25-L1T and CEB25-L1T-G12T in both orientations into the genome of yeast cells at the leu1 locus at chromosome 2. The leu1 locus is located in between ARS-II-1964 and ARS-II-1983 at a distance of 14 kb and 11 kb from CEB25, respectively (Figure 7A). These two ARS have a relatively low firing effi- ciency, 32% and 13%, respectively [55]. We monitored in fission yeast the instability of CEB25 in both orientations although this genomic context does not allow to clearly distinguish whether the G4-forming sequence is replicated by the leading or the lagging replication machinery. DISCUSSION We therefore propose that RPA cooperates with Pif1 to resolved G-quadruplexes during leading strand replication. Several interpretations can be invoked to explain the difference in CEB1 stability observed in pif1∆ cells during leading and lagging replication. It is possible that RPA re- cruits Pif1. We think that the difference in Pif1 require- ment at leading and lagging strands is related to RPA func- tion. At the leading strand, both Pif1 helicase and RPA are required. However, our results showing that mutated CEB1 (which are no longer able to form G4-strutures) and Pif1 overexpression both only partially rescue instability in rfa1- D228Y cells reveal higher requirement of Pif1 compared to RPA to maintain CEB1 stability at the leading strand. Inter- estingly the roles are inverted between RPA and Pif1 at the lagging strand. Because contrary to the leading strand the lagging strand contains longer stretch of ssDNA between elongating Okazaki fragments, RPA is present at a higher concentration at the lagging strand [59]. Consequently, by preventing formation/folding of G-quadruplex structures at the exposed ssDNA, RPA is likely to directly contribute to CEB1 stability independently of Pif1 at the lagging strand. Because rfa1-D228Y possesses a lower affinity for ssDNA and a reduced ability in preventing and removing second- ary structures from ssDNA, in rfa1-D228Y cells CEB1 be- comes unstable. In that situation Pif1, which is not initially required to remove G-quadruplex at the lagging strand, is now required. Our results showing that the Pif1 overex- pression almost fully rescues the stability of CEB1 in rfa1- D228Y mutant at the lagging strand suggest that CEB1-G- quadruplexes are formed in this mutant and can be pro- p In contrast to Pif1, which is not required for CEB1 sta- bility during lagging strand replication, we found that RPA has a prominence in maintaining lagging CEB1 stability. This finding points out the differential behaviour of the pif1∆ and rfa1-D228Y mutations according to the direction of replication. However, our results indicate that Pif1 over- expression drastically reduced the instability observed at the lagging strand in the rfa1-D228Y mutant whose ssDNA binding activity of RPA is compromised, in particular to G-rich regions [38]. Finally, we report that Mms1, which binds G4-structures and aids Pif1 binding to these struc- tures is not required for CEB1 stability at both leading and lagging strands. DISCUSSION In S. cerevisiae, Pif1 helicase processes G-rich secondary structures thereby preventing deleterious events that may lead to DNA breaks [33,38,57]. In vitro, Pif1 binds tightly to G4 structures and unwinds them very efficiently [26,58]. Previous studies demonstrated that Pif1 prevents the formation of G-quadruplex-dependent CEB1 internal rear- rangements during leading strand, but not lagging strand replication [33]. Here, we disclose that RPA cooperates with Pif1 to remove G-quadruplex structures at both lead- ing and lagging strand. We found that the rfa1-D228Y mu- tation increases the frequency of CEB1 rearrangements when the G-quadruplex forming strand is replicated by the leading polymerase. The level of rearrangements is similar to the level observed in pif1∆ cells. We report that mutat- ing G-quadruplex-forming sequences strongly decreases As depicted in Figure 2A, the size of CEB25-L1T was monitored by Southern blot and frequency of instability Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 55 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) TABLE 1. Rearrangement frequencies of CEB1 placed near ARS305 in both orientations in WT and mutant strains. Minisatellite Genotype Orientation I Orientation II CEB1-1.8 WT 1/154 (1%) 0/154 (0%) pif1∆ 57/108 (53%) 4/106 (4%) rfa1-D228Y 50/98 (51%) 36/118 (31%) pif1∆ rfa1-D228Y 43/101 (43%) 31/76 (41%) WT GAL::PIF1 0/44 (0%) 0/43 (0%) rfa1-D228Y GAL::PIF1 12/39 (31%) 2/43 (5%) mms1∆ 0/116 (0%) 3/116 (3%) rtt105∆ 116/116 (100%) 116/116 (100%) WT GAL::RNH1 1/58 (2%) 0/58 (0%) pif1∆ GAL::RNH1 30/58 (52%) ND rtt105∆ GAL::RNH1 58/58 (100%) 58/58 (100%) rfa1-D228Y GAL::RNH1 39/87 (45%) 29/87 (33%) CEB1-Gmut-1.7 WT 0/96 (0%) ND pif1∆ 0/48 (0%) ND rfa1-D228Y 21/94 (22%) ND pif1∆ rfa1-D228Y 13/66 (20%) ND ND – not determined. TABLE 1. Rearrangement frequencies of CEB1 placed near ARS305 in both orientations in WT and mutant strains. RPA cooperate to remove G-quadruplex structures at both leading and lagging strand (Figure 8). the instability of CEB1 in rfa1-D228Y while it totally abol- ishes the instability of CEB1 in pif1∆ cells. Contrary to pif1∆ cells, in rfa1-D228Y cells the CEB1 instability does not com- pletely rely on the G-quadruplex-forming sequence of CEB1, indicating that the instability arising in this mutant partially results from G-quadruplex-independent ssDNA-containing secondary structures. We obtained similar results with the double pif1∆ rfa1-D228Y mutant. Consistent with these results, Pif1 overexpression suppresses the instability of rfa1-D228Y mutant in the same proportion as G-mutated CEB1 motif. DISCUSSION In contrast Rtt105 that promotes RPA nu- clear import, and RPA-ssDNA complex formation at replica- tion forks is required to stabilize CEB1 inserted in both ori- entations. Collectively these results indicate that Pif1 and Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 56 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) estroni et al. (2020) A critical role for RPA i E 7: RPA is required to stabilize CEB25-L1T in fission yeast. (A) Map of the CEB25-L1T insertion within chromosome II at 14 kb and at 11 kb from the ARS-II-1983. (B) Genomic DNA from cells containing CEB25-L1T in orientation 1 and 2 was digested by Pvu d. Membranes were hybridized with CEB25 probe. (C) Genomic DNA from cells containing mutated CEB25-L1T-G12T in orientat ed by PvuII and southern blotted. Membranes were hybridized with a CEB25-L1T probe. The number of colonies analysed per w rearrangement frequencies, and the total number of colonies are indicated. Red stars mark unstable events. We calculated t ility by monitoring the size and the intensity of the CEB25. We considered that CEB25 was unstable when the intensity of the sh uperior to the one of the parental band or when the band disappears. The number of colonies analysed per well, the percentag frequencies, and the total numbers of colonies are indicated in Table 1. FIGURE 7: RPA is required to stabilize CEB25-L1T in fission yeast. (A) Map of the CEB25-L1T insertion within chromosome II at 14 kb from the ARS-II- 1964 and at 11 kb from the ARS-II-1983. (B) Genomic DNA from cells containing CEB25-L1T in orientation 1 and 2 was digested by PvuII and southern blotted. Membranes were hybridized with CEB25 probe. (C) Genomic DNA from cells containing mutated CEB25-L1T-G12T in orientation 1 and 2 was digested by PvuII and southern blotted. Membranes were hybridized with a CEB25-L1T probe. The number of colonies analysed per well, the percent- age of rearrangement frequencies, and the total number of colonies are indicated. Red stars mark unstable events. We calculated the frequency of instability by monitoring the size and the intensity of the CEB25. We considered that CEB25 was unstable when the intensity of the short or high band was superior to the one of the parental band or when the band disappears. DISCUSSION The number of colonies analysed per well, the percentage of rearrange- ment frequencies, and the total numbers of colonies are indicated in Table 1. Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 57 L. Maestroni et al. (2020) A critical role for RPA in CEB1 stab FIGURE 8: Mechanistic model for the unfolding of CEB1-G-rich structures by RPA and Pif1. (A) In WT cells, when the G-rich strand (light grey) leading strand (left part, CEB1 leading), stable G-quadruplexes structures are efficiently removed by Pif1, as proposed by Lopes and colleagues [33 the leading strand, RPA may cooperate with Pif1 either by preventing the refolding of the G4 structure or by recruiting Pif1 to the leading strand. W the G-rich strand (light grey) is replicated by the lagging polymerase (right part, CEB1 lagging) binding of RPA to the G-rich strand prevents formati stable G-rich secondary structures. In this context, Pif1 is dispensable. (B) In the rfa1-D228Y mutant, at the leading strand (left part), the reduced a ty of RPA for G-rich ssDNA either reduce the ability of RPA to prevent the refolding of the G4 structure or decrease Pif1 recruitment. The decrea RPA affects G4-unwinding and leads to the formation of CEB1 rearrangements. At the lagging strand (middle part), the decrease affinity of RPA f rich ssDNA facilitates the formation of stable G-rich structures, affects their unwinding, and generates CEB1 instability. In such a case, overexpre f Pif1 ( i ht t) ffi i tl i d G i h t t l di t CEB1 i ti A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) L. Maestroni et al. (2020) ( ) FIGURE 8: Mechanistic model for the unfolding of CEB1-G-rich structures by RPA and Pif1. (A) In WT cells, when the G-rich strand (light grey) is on leading strand (left part, CEB1 leading), stable G-quadruplexes structures are efficiently removed by Pif1, as proposed by Lopes and colleagues [33]. At the leading strand, RPA may cooperate with Pif1 either by preventing the refolding of the G4 structure or by recruiting Pif1 to the leading strand. When the G-rich strand (light grey) is replicated by the lagging polymerase (right part, CEB1 lagging) binding of RPA to the G-rich strand prevents formation of stable G-rich secondary structures. In this context, Pif1 is dispensable. DISCUSSION This may account for the even more prominent role of RPA complex in G4 unwinding in fission yeast. This assumption is also supported by the fact that the rpa1-D223Y mutation has a stronger effect at telomeres in S. pombe than its counterpart in S. cerevisiae [38,62]. Interestingly, we ob- served expansions of the CEB25-L1T even in the WT strain, especially when CEB25-L1T was preferentially replicated by the lagging machinery. This result opens new avenues to use fission yeast as a model organism to study expansions of G-rich sequences that have been associated with neuro- logical diseases. cessed by Pif1 indicating that RPA and Pif1 have redundant G-quadruplex-processing activities at the lagging strand and cooperate (Figure 8). Pif1 binds to G4 motifs with no preference for leading and lagging strand templates [57] but Pif1 is essential only for efficient replication through lagging strand G4s [60]. Consequently, the distinct behaviour of Pif1 at the leading and the lagging strands may be due to different confor- mations of the respective G-quadruplexes, affecting their folding and/or their processing due to the occupancy rate of RPA. Another possibility could be that the blocking G-quadruplex structures are better tolerated and bypassed by the lagging strand replication machinery due to its abil- ity to prime DNA synthesis downstream of G-quadruplexes. However, this seems improbable because CEB1 instability in rfa1-D228Y cells is fully suppressed by Pif1 overexpres- sion suggesting that the blocking structures are present and that the helicase activity of Pif1 is still required to re- move G4-structures and rescue CEB1 stability. RPA interacts with RNase H1 and colocalizes with it at R-loops raising the possibility that RPA could recruit and/or stimulate RNase H1 [41,53]. We observed that the interac- tion between RPA and RNase H1 is stimulated by the pres- ence of DNA and is slightly affected in rfa1-D228Y cells. Furthermore, Pif1 regulates R-loop formation and poten- tially complements RNase H for R-loop resolution [51, 52]. We found that RNase H1 overexpression did not rescue CEB1 stability in pif1∆, rfa1-D228Y, and rtt105∆ mutants. These results suggest that R-loops are likely not responsi- ble of CEB1 instability in these mutants, indicating that CEB1 instability is not due to transcription. Collectively, our results add new insights about the role of RPA as a general sensor of secondary structures and regulator of genomic stability. DISCUSSION (B) In the rfa1-D228Y mutant, at the leading strand (left part), the reduced affini- ty of RPA for G-rich ssDNA either reduce the ability of RPA to prevent the refolding of the G4 structure or decrease Pif1 recruitment. The decrease in RPA affects G4-unwinding and leads to the formation of CEB1 rearrangements. At the lagging strand (middle part), the decrease affinity of RPA for G- rich ssDNA facilitates the formation of stable G-rich structures, affects their unwinding, and generates CEB1 instability. In such a case, overexpression of Pif1 (right part) can efficiently unwind G-rich structures, leading to CEB1 size conservation. FIGURE 8: Mechanistic model for the unfolding of CEB1-G-rich structures by RPA and Pif1. (A) In WT cells, when the G-rich strand (light grey) is on leading strand (left part, CEB1 leading), stable G-quadruplexes structures are efficiently removed by Pif1, as proposed by Lopes and colleagues [33]. At the leading strand, RPA may cooperate with Pif1 either by preventing the refolding of the G4 structure or by recruiting Pif1 to the leading strand. When the G-rich strand (light grey) is replicated by the lagging polymerase (right part, CEB1 lagging) binding of RPA to the G-rich strand prevents formation of stable G-rich secondary structures. In this context, Pif1 is dispensable. (B) In the rfa1-D228Y mutant, at the leading strand (left part), the reduced affini- ty of RPA for G-rich ssDNA either reduce the ability of RPA to prevent the refolding of the G4 structure or decrease Pif1 recruitment. The decrease in RPA affects G4-unwinding and leads to the formation of CEB1 rearrangements. At the lagging strand (middle part), the decrease affinity of RPA for G- rich ssDNA facilitates the formation of stable G-rich structures, affects their unwinding, and generates CEB1 instability. In such a case, overexpression of Pif1 (right part) can efficiently unwind G-rich structures, leading to CEB1 size conservation. Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 58 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) the rpa1-D223Y mutant. The effects of the rpa1-D223Y mutant were even more pronounced in S. pombe. This may be due to the fact that fission yeast lacks a functional ortholog of the Pif1 helicase. Indeed Pfh1 behaves more closely to the budding yeast Rrm3 helicase than to Pif1 [56]. MATERIALS AND METHODS Strains and growth conditions MATERIALS AND METHODS DISCUSSION RPA acts through direct interactions with proteins acting at the level of specific secondary structures, which are hotspots of genomic instability. This mode of RPA ac- tion is conserved between yeasts and humans. How exactly Pif1 is recruited at the G-rich motifs and at G-quadruplexes that form at CEB1 during replication? One possibility is that Pif1 travels with the replication fork and facilitates replication by processing G-quadruplex struc- tures at the leading-CEB1. Indeed, Pif1 interacts with PCNA and Cac1, the large CAF-1 subunit, which preferentially assembles nucleosome onto replicating DNA [60, 61]. Thus, Pif1 may be targeted to the replication fork by its ability to interact with PCNA or the histone chaperone Cac1 and preserves genome stability by acting at G-rich motifs at the leading-CEB1. Alternatively, Pif1 could be directly recruited to G-rich motifs and G-quadruplex structures. Mms1 is a G4-DNA-binding protein that helps replication fork pro- gression at G4 and Pif1 binding to specific G4 structures [22, 37]. We show that Mms1 is not required to maintain CEB1 stability at both leading- and lagging-CEB1 revealing that G4 structures targeted by Mms1 are not deleterious for CEB1 stability. Our results are in good agreement with previous observations showing that Mms1 supports Pif1 function at G4 motifs only on the lagging strand [22, 37], whereas Pif1 is not required for CEB1 stabilisation [33]. Here we show that RPA recruits Pif1 to CEB1. We report that Pif1 associates with RPA and that this association is affected in the rfa1-D228Y mutant and by DNA digestion, suggesting that Pif1-RPA association relies on specific DNA structures. In addition we show that rtt105∆ mutation, which decreases the level of RPA associated with ssDNA (as the rfa1-D228Y mutation) [44, 45], strongly affects CEB1 stability at both leading and lagging strands. These results reveal the importance of Rtt105 in promoting the replica- tion of G4-forming CEB1 minisatellite, during leading strand and lagging strand synthesis, and confirm that the level of RPA is crucial for CEB1 stability. RPA protects and stabilizes ssDNA susceptible to secondary structure for- mation. It is possible that the ability of RPA to directly bind to Pif1 could be important to recruit Pif1 at specific G-rich sequences/structures and/or to stimulate Pif1 activity at G-rich motifs and G-quadruplex structures. OPEN ACCESS | www.cell-stress.com Strains and growth conditions LM361 LM411 diploïd CEB1-1.8 I rfa1-D228Y/RFA1 pif1::Kan/PIF1 diploïd CEB1-1.8 II rfa1-D228Y/RFA1 pif1::Kan/PIF1 This study This study LM349 diploïd CEB1-1.7 Gmut rfa1-D228Y/RFA1 pif1::Kan/PIF1 This study LM396 diploïd CEB1-1.8 I rfa1-D228Y/RFA1 + pVS45 (GAL::PIF1) This study LM398 diploïd CEB1-1.8 II rfa1-D228Y/RFA1 + pVS45 (GAL::PIF1) This study LM401 diploïd CEB1-1.8 I rfa1-D228Y/RFA1 + pSH380 (empty vector) This study LM404 diploïd CEB1-1.8 II rfa1-D228Y/RFA1 + pSH380 (empty vector) This study YVC600 diploïd CEB1-1.8 I mms1::TRP1/MMS1 This study YVC601 diploïd CEB1-1.8 II mms1::TRP1/MMS1 This study YVC602 diploïd CEB1-1.8 I rtt105::Kan/RTT105 This study YVC603 diploïd CEB1-1.8 II rtt105::Kan /RTT105 This study YVC604 diploïd CEB1-1.8 I rtt105::Kan/RTT105 GAL::RNH1::natMX/RNH1 This study YVC605 diploïd CEB1-1.8 II rtt105::Kan/RTT105 GAL::RNH1::natMX /RNH1 This study YVC606 diploïd CEB1-1.8 I pif1::Kan/PIF1 GAL::RNH1::natMX /RNH1 This study YVC607 diploïd CEB1-1.8 I rfa1-D228Y/RFA1 GAL::RNH1::natMX /RNH1 This study YVC608 diploïd CEB1-1.8 II rfa1-D228Y/RFA1 GAL::RNH1::natMX /RNH1 This study LM140 MATa rfa1-D228Y Pif1-myc::KanMX6 This study W1042-7C MATa can1-100,x SUP4-o::HIS3::pWJ317-CAN1-URA3 rfa1-D228Y Rothstein R LM301 pif1::KanMX6 rfa1-D228Y This study LM340 pif1::KanMX6 This study YBL103 his3∆1; leu2∆0; ura3∆0; met15∆0; pGal-3HA-RNH1::NAT Luke B. LM407 diploïd pGal-3HA-RNH1::natMX rfa1-D228Y/RFA1 This study S. pombe strains Genotype Origin JA961 h- leu1-32 ura4-D18 leu1+::pJK148-CEB25 orientation 1 This study JA963 h- leu1-32 ura4-D18 leu1+::pJK148-CEB25-G12T orientation 1 This study JA947 h- leu1-32 ura4-D18 leu1+::pJK148-CEB25 orientation 2 This study JA948 h- leu1-32 ura4-D18 leu1+::pJK148-CEB25-G12T orientation 2 This study SC387 h+ leu1-32 ura4-D18 ade6-M210 rad11-D223Y Ueno M. All LM strains used are derivatives of W303-1B. All YVC strains used are derivatives of W303. Spore colonies were generated from the diploid strains. All LM strains used are derivatives of W303-1B. All YVC strains used are derivatives of W303. Spore colonies were generated from the diploid strains. gested DNA was resolved in 1% agarose gel and blotted onto Hybond-XL membrane (GE Healthcare). After transfer, the membrane was cross-linked with UV and hybridized with CEB1-0.6 and CEB1 Gmut probes for CEB1-1.8 and CEB1Gmut - 1.7, respectively. 32P labelling of DNA probes was performed by random priming using Klenow fragment exonuclease (New England Biolabs), in presence of [α-32P]-CTP and hybridizations were performed in Church buffer at 55°C. Radioactive signals were detected using a BIORAD molecular imager FX. Correct and unique insertion of CEB25 at this locus generated two fragments of 3 and 14.4 kb. To check the correct size of CEB25-L1T and CEB25-L1T-G12T, a second digestion by PvuII on genomic DNA was performed on selected clones. Strains and growth conditions Strains used in this study are listed in Table 2. Yeast strains containing CEB1-1.8 I (orientation I), CEB1-1.8 II (orientation II), or CEB1Gmut-1.7 were mated with pif1∆, rfa1-D228Y, pif1∆ rfa1-D228Y, mms1∆, or rtt105∆ mutants. After sporulation and identification of the four resulting spores, the strains of interest were plated on rich medium at 30°C to obtain isolated colonies. Individual clones were grown in rich liquid culture at 30°C until stationary phase. For PIF1 overexpression experi- ments, WT cells and rfa1-D228Y cells were transformed with the centromeric plasmids pVS45 (expressing the nuclear form of Pif1 under the control of the GAL1 promoter) and pSH380 (a pRS315-derived vector control) provided by Virginia Zakian [43], then mated with leading-CEB1 (orientation I), or lagging- CEB1 (orientation II) cells. Individual clones were grown in SD- Leu (2% glucose), or SGal-Leu (2% galactose) media for repres- sion or overexpression of PIF1, when mentioned. The 0.62 kb CEB25-L1T and CEB25-L1T-G12T were cloned into pJK148 integrative plasmid in both orientations at Not1 site, from plasmid pPA84 and pPA84-G12T (A. Nicolas’s Labor- atory), respectively. The corresponding plasmids were linear- ized by Nde1 and transformed into S. pombe cells. To check the insertion of CEB25 at the leu1 locus, genomic DNA was digested by XbaI a nd southern blotted using a leu1 probe. Importantly, the results obtained in S. cerevisiae with the rfa1-D228Y mutant were confirmed in S. pombe with Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 59 L. Maestroni et al. (2020) A critical role for RPA in CEB1 stability Table2. Strains used in this study. S. cerevisiae strains Genotype Origin ORT6119-4 MATa CEB1-1.8 I-ARS305 Nicolas A. ORT6135-36 MATa CEB1-1.8 II-ARS305 Nicolas A. ORT6157-1 MATa CEB1-1.7 Gmut-ARS305 Nicolas A. Co-Immunoprecipitation experiment Yeast cells were grown at 30°C in YPD to OD600=0.8. Extracts were lysed with glass-beads in TMG-50 (10mM TrisHCl pH8.0, 1mM MnCl2, 10% (v/v) glycerol, 50 mM NaCl, 0.1 mM DTT) containing a protease inhibitor cocktail (Calbiochem), MG132 (Sigma Aldrich), and 0.5% (v/v) Tween-20. Pif1-Myc and Rnh1- HA proteins were immunoprecipitated with 9E10 monoclonal anti-Myc antibody (Santa Cruz, CA) and 12CA5 anti-HA mono- clonal antibody (Roche), respectively. The presence of Rfa1 Strains and growth conditions Southern blot hybridized by a CEB25-L1T probe (Not1-fragment from pPA84 plasmid), revealed a fragment of 1.1 kb. Fission yeast strains containing CEB25-L1T and CEB25-L1T-G12T were then mated with the rpa1-D223Y mutant. After sporulation and identification of the resulting spores, the stability of the CEB25 was controlled a second time by Southern blot after digestion of genomic DNA by PvuII. The strains of interest were plated on rich medium to obtain isolated colonies and individual clones were further grown in rich liquid culture at 32°C until stationary phase. ACKNOWLEDGMENTS We are very grateful to Alain Nicolas (Institut Curie) and Judith Lopes (Museum d’Histoire Naturelle) for the gift of REFERENCES 10. 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The di- Cell Stress | MARCH 2020 | Vol. 4 No. 3 OPEN ACCESS | www.cell-stress.com 60 A critical role for RPA in CEB1 stability L. Maestroni et al. (2020) and Rfa2 in the IP was checked with anti-Rfa1 and anti-Rfa2 antibodies (Agrisera, Sweden). CEB1-1.8 I, CEB1-1.8 II, and CEB1Gmut-1.7 strains, and for advices. We thank Brian Luke for the plasmid and the strain overexpressing RNH1. We also thank Nagham Ghaddar. V.G. is supported by the “Ligue Nationale Contre le Cancer (Equipe Labellisée)”. COPYRIGHT © 2020 Maestroni et al. This is an open-access article re- leased under the terms of the Creative Commons Attribu- tion (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged. Please cite this article as: Laetitia Maestroni, Julien Audry, Pierre Luciano, Stéphane Coulon, Vincent Géli and Yves Corda (2020). RPA and Pif1 cooperate to remove G-rich structures at both lead- ing and lagging strand. Cell Stress 4(3): 48-63. doi: 10.15698/cst2020.03.214 CONFLICT OF INTEREST h h d l h The authors declare that they have no conflict of interest. RNA isolation and analysis Total RNA was isolated from 2x108 cells using the hot phenol method [64]. Total RNA was treated with RNase-free DNase I (Qiagen) and reverse transcribed using the SuperscriptIII re- verse transcriptase (Life technologies) with random hexanu- cleotide primers (Sigma-aldrich). Quantitative PCR amplifica- tion of cDNA was carried out using the SYBR Premix Ex Taq II (Ozyme) with these cycling parameters: 1 cycle at 95°C for 30 sec, followed by 45 cycles at 95°C for 10 sec, 58°C for 15 sec, and 72°C for 20 sec, and a final extension step at 72°C for 5 min. PIF1 cDNA was quantified by using the oligonucleotides Pif1-forward (5’-CTGAAAACTCATTTGACCAG-3’) and Pif1- reverse (5’-GCAATCTTTTCTCCAAATTGC-3’). 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Apgar Score Is Related to Development of Atopic Dermatitis: Cotwin Control Study
Journal of Allergy
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Hindawi Publishing Corporation Journal of Allergy Volume 2013, Article ID 712090, 6 pages http://dx.doi.org/10.1155/2013/712090 Hindawi Publishing Corporation Journal of Allergy Volume 2013, Article ID 712090, 6 pages http://dx.doi.org/10.1155/2013/712090 Hindawi Publishing Corporation Journal of Allergy Volume 2013, Article ID 712090, 6 pages http://dx.doi.org/10.1155/2013/712090 Vibeke Naeser,1 Niklas Kahr,1 Lone Graff Stensballe,2 Kirsten Ohm Kyvik,3 Axel Skytthe,4 Vibeke Backer,1 Charlotte Giwercman Carson,5 and Simon Francis Thomsen6 1 Department of Respiratory Medicine, Bispebjerg Hospital, 2400 Copenhagen, Denmark 2 Danish Epidemiology Science Centre, Statens Serum Institut, 2300 Copenhagen, Denmark 3 Institute of Regional Health Services Research & Odense Patient Data Explorative Network, University of Southern Denmark, 5000 Odense, Denmarkh y f 4The Danish Twin Registry, University of Southern Denmark, 5000 Odense, Denmark 5t The Danish Twin Registry, University of Southern Denmark, 5000 Odense, Denm 5 Danish Pediatric Asthma Center, Gentofte Hospital, 2900 Hellerup, Denmarkt h 5 Danish Pediatric Asthma Center, Gentofte Hospital, 2900 Hellerup, Denmarkt ft 6Department of Dermato-Allergology, Gentofte Hospital, 2900 Hellerup, Denmark Correspondence should be addressed to Simon Francis Thomsen; sft@city.dk Received 27 May 2013; Revised 20 September 2013; Accepted 21 September 2013 Received 27 May 2013; Revised 20 September 2013; Accepted 21 September 2013 Academic Editor: S. L. Johnston Copyright © 2013 Vibeke Naeser et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aim. To study the impact of birth characteristics on the risk of atopic dermatitis in a twin population. Methods. In a population- based questionnaire study of 10,809 twins, 3–9 years of age, from the Danish Twin Registry, we identified 907 twin pairs discordant for parent-reported atopic dermatitis. We cross-linked with data from the Danish National Birth Registry and performed cotwin control analysis in order to test the impact of birth characteristics on the risk of atopic dermatitis. Results. Apgar score, OR (per unit) = 1.23 (1.06–1.44), 𝑃= 0.008, and female sex, OR = 1.31 (1.06–1.61), 𝑃= 0.012, were risk factors for atopic dermatitis in cotwin control analysis, whereas birth anthropometric factors were not significantly related to disease development. Risk estimates in monozygotic and dizygotic twins were not significantly different for the identified risk factors. Conclusions. In this population- based cotwin control study, high Apgar score was a risk factor for atopic dermatitis. This novel finding must be confirmed in subsequent studies. Research Article Apgar Score Is Related to Development of Atopic Dermatitis: Cotwin Control Study Vibeke Naeser,1 Niklas Kahr,1 Lone Graff Stensballe,2 Kirsten Ohm Kyvik,3 Axel Skytthe,4 Vibeke Backer,1 Charlotte Giwercman Carson,5 and Simon Francis Thomsen6 1. Introduction No statistically significant association was demonstrated between any of the birth-related exposures and the four allergic conditions studied, although some nonsignificant trends were noted for those born by Caesarean section and those exposed to fetal distress during labor.h Next, the risk factors that were unshared between the twins within the pair were examined in a 1 : 1 matched conditional logistic regression analysis (cotwin control analysis) in order to determine the effect of these risk factors on atopic dermati- tis. The factors entered into this analysis were birth weight, birth length, Apgar score, and sex. The matching was done with the affected (atopic dermatitis) twin in each pair being the case and the unaffected twin being the control. DZ same- sex and DZ opposite-sex twin pairs were considered together in the analysis. The matching of the twins in the cotwin control design allows an inherent adjustment for other factors that would otherwise confound the association between birth factors and atopic dermatitis such as genetic factors, atopic predisposition, maternal smoking during pregnancy, gestational age, mode of delivery, and current age, as well as effects of the rearing environment. Therefore the estimated risk of atopic dermatitis can be assumed to reflect more precisely the influence of birth factors, for example, Apgar score. The following predictions can be made for the cotwin control analysis (Figure 1): if the association between birth factors and atopic dermatitis is mediated via genetics, then we do not expect to find any significant risk of atopic dermatitis among discordant MZ twin pairs, because MZ twins are concordant for early rearing environmental and genetic factors. Alternatively, an increased risk of atopic dermatitis in discordant DZ twin pairs would suggest that genetic factors underlie the association, because DZ twins share the same rearing environment, but only half their genetic variants. Finally, a direct (causal) effect of birth factors, for example, Apgar score, on atopic dermatitis would be suggested by the finding that both discordant MZ and DZ twin pairs are also discordant on, for example, Apgar score, as the association can neither be reflected by environmental nor genetic factors [19]. Data were analyzed with the statistical package SPSS 18.0 (SPSS Inc., Chicago, IL). g The cotwin control study design is a matched case-control study that utilizes twin pairs discordant for an exposure or an outcome. 2. Methods The study population comprised all live-born twins in Den- mark between 1994 and 2000 included in the Danish Twin Registry. In 2003 when the twins were 3–9 years of age the twins’ parents responded to a two-page questionnaire concerning atopic diseases, diabetes, febrile and epileptic seizures, and mental health attributes. Atopic dermatitis was diagnosed by the question “has your child ever had eczema in the folds of the elbows or knees?” Zygosity was determined by information regarding the twins’ similarity to family, friends, teachers, and relations. This method determines zygosity cor- rectly in more than 95% of the cases compared with genetic marker information [18]. We cross-linked the information from the questionnaire with data from the Danish National Birth Registry where data on birth weight, birth length, maternal smoking during pregnancy, gestational age, mode of delivery (Caesarean section versus vaginal delivery), and Apgar score were obtained. 1. Introduction Particularly, because twin pairs are inherently matched on several confounding factors relating to early life and upbringing, this type of study design is ideal to tease out causative relationships in complex diseases. In the present study we use the cotwin control design to identify birth- associated risk factors for atopic dermatitis in twin pairs discordant for the disease. 1. Introduction of atopic dermatitis has increased both in developed [4, 5] and developing countries [6] in recent years, but the causes for this increase are still poorly understood.h Atopic dermatitis is a chronic relapsing skin disease with a lifetime prevalence of around 20%. The disease has an early onset with 60% being affected during the first year of life and 85% before the age of five [1]. Atopic dermatitis is characterized by recurrent episodes of itching and eczema occurring at typical sites such as the face and extensor aspects of the arms and legs in early childhood, flexures in later childhood, and head and neck as well as the hands in adult- hood. Atopic dermatitis is highly heritable and individual susceptibility to the disease is attributable particularly to mutations in the filaggrin gene [2], although other genetic variants [3] as well as environmental factors have been implicated. Previous studies have shown that the prevalence The onset of atopic dermatitis early in life implies that risk factors for the disease exert their effect already in utero or in very early childhood. However, previous studies of the association between perinatal factors and the risk of atopic dermatitis are conflicting. With regard to alcohol intake during pregnancy, results indicate an increased risk for atopic dermatitis in children of mothers who consume alcohol during pregnancy [7, 8]. Contrary to this, smoking during pregnancy has been inversely related to development of atopic dermatitis in the offspring [9, 10]. The associa- tion between exclusive breastfeeding/increased duration of breastfeeding and atopic dermatitis varies; while some studies 2 2 Journal of Allergy Journal of Allergy show a positive association [11] others show a negative [12] or no [13] association. Some studies [9, 14] but not all [15] have found that high birth weight increases the risk of atopic dermatitis, whereas other studies have found that a higher gestational age increases the risk of atopic dermatitis [16]. Mallen et al. investigated the association between six birth- related exposures (birth weight, mode of delivery, artificial commencement of labor, prematurity, neonatal intensive care unit admission, and fetal distress) and asthma, allergic rhini- tis, eczema, and hay fever in adulthood [17]. 3. Results The proportions of girls and boys in the population were 48.9% and 51.1%, respectively. The mean age was 5.9 years with no difference between girls and boys. The overall prevalence of atopic dermatitis was 14.9%, 14.0% in boys and 16.0% in girls, 𝑃= 0.003. Increasing birth length (𝑃= 0.038), nonsmoking of the mother during pregnancy (𝑃= 0.002), and monozygosity (0.012) were also significant predictors for atopic dermatitis in the whole sample when ignoring the familial relationship between the twins (Table 1). A total of 10,809 twin subjects participated in the ques- tionnaire study (participation rate 68%). Of these, complete information on atopic dermatitis was available for 5399 intact twin pairs: 850 monozygotic (MZ) twin pairs, 2,279 dizygotic (DZ) twin pairs of the same sex, 1,999 DZ twin pairs of opposite sex, and 271 twin pairs of unknown zygosity. There were 907 twin pairs discordant for atopic dermatitis, and of these 58 and 827 were monozygotic (MZ) and dizygotic (DZ) pairs, respectively. Within twin pairs discordant for atopic dermatitis, the affected twin had, on average, a higher Apgar score and was smaller at birth compared with the unaffected twin (Table 2). In multiple conditional logistic regression comparing the affected with the unaffected twin, Apgar score, OR (per unit) = 1.23 (1.06–1.44), 𝑃= 0.008, and female sex, OR = 1.31 (1.06– 1.61), 𝑃= 0.012, were significantly predisposing factors for atopic dermatitis, whereas birth anthropometric factors were not significantly related to development of atopic dermatitis. Risk estimates in discordant MZ and DZ twins were not significantly different (Table 3). 2.1. Statistical Analysis. First, the relationship between birth- associated risk factors and atopic dermatitis was examined in the whole population using unpaired 𝑡-tests and chi-square tests, ignoring the familial relationship between the twins. 2.1. Statistical Analysis. First, the relationship between birth- associated risk factors and atopic dermatitis was examined in the whole population using unpaired 𝑡-tests and chi-square tests, ignoring the familial relationship between the twins. 3 Journal of Allergy 3 Table 1: Characteristics of subjects with and without atopic dermatitis in a sample of Danish twin pairs, 3–9 years of age. Table 1: Characteristics of subjects with and without atopic dermatitis in a sample of Danish twin pairs, 3–9 years of age. 3. Results Atopic dermatitis OR (95% CI) 𝑃value Yes 𝑁= 1614 No 𝑁= 9195 Birth weight, g 2593 2571 22 (−9–52) 0.167 Birth length, cm 48.24 48.05 0.19 (0.01–0.38) 0.038 Apgar score 9.76 9.73 0.03 (−0.02–0.09) 0.172 Gestational age, days 254.9 254.6 0.32 (−0.61–1.26) 0.496 Caesarean section 14.4 15.3 0.93 (0.84–1.04) 0.220 Maternal smoking 12.9 15.5 0.80 (0.70–0.92) 0.002 Sex Boys 14.0 86.0 1.00 Girls 16.0 84.0 1.17 (1.05–1.30) 0.003 Age, years 5.92 5.86 0.06 (−0.05–0.17) 0.257 Zygosity DZ 14.4 85.6 1.00 MZ 16.8 83.2 1.20 (1.04–1.38) 0.012 Means and proportions are calculated from available data for the individual variables. OR is mean difference for birth weight, birth length, Apgar score, gestational age, and age. ristics of subjects with and without atopic dermatitis in a sample of Danish twin pairs, 3–9 years of age. Table 2: Distribution of perinatal risk factors for atopic dermatitis in 907 twin pairs, 3–9 years of age, discordant for atopic dermatitis. All twin pairs (𝑛= 907) MZ twin pairs (𝑛= 58) DZ twin pairs (𝑛= 827) Affected twins Unaffected twins Affected twins Unaffected twins Affected twins Unaffected twins Birth weight, g 2627 2649 2624 2625 2632 2650 Birth length, cm 48.37 48.41 48.60 48.17 48.40 48.44 Apgar score 9.81 9.68 9.80 9.75 9.80 9.67 Female sex 52.3 47.4 53.4 53.4 53.1 47.8 Genetic factors Environmental factors Apgar score Atopic dermatitis Genetic factors Environmental factors Apgar score Atopic dermatitis Genetic factors Environmental factors Apgar score Atopic dermatitis Larger effect size in DZ twins than in MZ twins discordant for atopic dermatitis Larger effect size in MZ twins than in DZ twins discordant for atopic dermatitis Equal effect sizes in MZ and DZ twins discordant for atopic dermatitis (3) Causal relationship (2) Environmental confounding (1) Genetic confounding Figure 1: Possible association between Apgar score and atopic dermatitis predicted by the cotwin control method. al risk factors for atopic dermatitis in 907 twin pairs, 3–9 years of age, discordant for atopic dermatitis. Table 2: Distribution of perinatal risk factors for atopic dermatitis in 907 twin pairs, 3–9 years of age, discordant for atopic dermatitis. 4. Discussion However, previous studies have found a harmful effect on atopic dermatitis of other birth-related factors linked to high Apgar score such as increased fetal growth [9], high socioeconomic status (SES) [26, 27], and nonsmoking of the mother [9, 10]. Particularly, Lundholm et al. examined the association between fetal growth and atopic diseases and found that the rate of atopic dermatitis increased with higher levels of the mother’s education and furthermore that children of mothers who did not smoke during pregnancy also had slightly higher rates of atopic dermatitis compared with children of mothers who smoked during pregnancy [9]. Furthermore, a Dutch study showed that smoking during pregnancy resulted in decreased birth weight and that women of low SES give birth to lighter babies [28]. These findings are inline with the assumption that atopic dermatitis is a disease affecting apparently robust children from high SES families. Because the affected (atopic) and the unaffected twin were matched on exposure status in our paired analysis, the observed positive association between atopic dermatitis and Apgar score is adjusted for confounding due to SES, parental smoking, and gestational age. Taken together, multiple factors are involved in the development of atopic dermatitis, particularly, genetics, fetal programming, and environmental effects, which makes We found an overall higher prevalence of atopic dermati- tis among the entire group of MZ twins than among the group of DZ twins. This finding could be due to confounding from factors in the MZ group not present in the DZ group, for example, a larger susceptibility gene pool among MZ twins or a more unfavorable intrauterine environment in MZ than in DZ twins. Also, these could be factors such as differences in the MZ and DZ families in terms of smoking and SES, but evidence in support of this theory is lacking. The large population size with identification of 907 twin pairs discordant for atopic dermatitis and the high response rate are strengths of this study, although the number of discordant MZ pairs was quite small (58 in total). We had complete ascertainment of all live-born twins in Denmark between 1993 and 2000 and cross-linked these data to data from the Danish Birth Registry combining information from two independent sources. Data on perinatal risk factors were obtained by professional midwifes’ records, which increased the validity of the data. 4. Discussion it difficult to deduce which risk factors represent direct effects and which represent mediatory effects. The equal effect sizes in MZ and DZ twins for Apgar score in cotwin control analysis suggest a direct effect of one or more of the factors attributable to the Apgar score in relation to atopic dermatitis. In this nationwide cohort of Danish twin pairs discordant for atopic dermatitis, a high Apgar score and female sex were significant determinants of atopic dermatitis, whereas birth weight and birth length were not significantly related to disease development. The prevalence of atopic dermatitis was higher in girls than in boys in the present study. Other studies contradict this, concluding that boys have a higher prevalence of atopic dermatitis and yet other studies find no significant difference between the prevalence of atopic dermatitis in boys and girls. Notably, a study from Taiwan found that the prevalence of atopic dermatitis in females is lower than in males before the age of 8 years but subsequently becomes higher [29], whereas studies from Japan and Italy show no significant difference in the prevalence of atopic dermatitis in boys and girls [30, 31]. A recent study of another (comparable) Scandinavian population from Sweden found a female preponderance of atopic dermatitis (14.5% in girls versus 9.4% in boys) [32]. p Apgar score is a clinical test performed on a newborn one and five minutes after birth. It is a composite measure of breathing effort, heart rate, muscle tone, reflexes, and skin colour and is an indicator of the newborn’s need for medical attention shortly after birth. A detrimental effect of high Apgar score on the risk of atopic dermatitis is, to our knowledge, a novel finding that needs to be confirmed in subsequent studies. Among Finnish children low Apgar score increased the risk of asthma [20, 21]. Contrary to this, Apgar score was not related to asthma [22] or hay fever [23] in Finnish twin children and did not interact with in vitro fertilization on the risk of asthma in children born after assisted reproduction [24]. Moreover, Gilman et al. found no significant association between Apgar score and maternal smoking [25]. It is somewhat counterintuitive that it is the twin with the highest Apgar score that has the highest risk of later development of atopic dermatitis. 3. Results Genetic factors Environmental factors Apgar score Atopic dermatitis Genetic factors Environmental factors Apgar score Atopic dermatitis Genetic factors Environmental factors Apgar score Atopic dermatitis Larger effect size in DZ twins than in MZ twins discordant for atopic dermatitis Larger effect size in MZ twins than in DZ twins discordant for atopic dermatitis Equal effect sizes in MZ and DZ twins discordant for atopic dermatitis (3) Causal relationship (2) Environmental confounding (1) Genetic confounding Figure 1: Possible association between Apgar score and atopic dermatitis predicted by the cotwin control method. Environmental factors Atopic dermatitis Environmental factors Equal effect sizes in MZ and DZ twins discordant for atopic dermatitis (3) Causal relationship Atopic dermatitis Figure 1: Possible association between Apgar score and atopic dermatitis predicted by the cotwin control method. Journal of Allergy 4 Table 3: Significant risk factors for atopic dermatitis in 907 twin pairs, 3–9 years of age, discordant for atopic dermatitis. All twin pairs MZ twin pairs DZ twin pairs ORMZ = ORDZ OR (95% CI) P value OR (95% CI) P value OR (95% CI) P value P value1 Birth weight 1.02 (0.81–1.28) 0.884 0.96 (0.12–7.86) 0.972 0.98 (0.77–1.24) 0.880 NS Birth length 1.00 (0.08–13.03) 1.00 8.31 (N/A) 0.854 1.62 (0.11–22.01) 0.758 NS Apgar score 1.23 (1.06–1.44) 0.008 1.19 (0.60–2.34) 0.615 1.21 (1.04–1.42) 0.015 NS Female sex 1.31 (1.06–1.61) 0.012 1.31 (1.07–1.61) 0.011 NS Only statistically significant predictors for atopic dermatitis are presented in the table (adjusted for birth weight, birth length). 1Test for heterogeneity between odds ratios for MZ and DZ twin pairs. NS: nonsignificant. ors for atopic dermatitis in 907 twin pairs, 3–9 years of age, discordant for atopic dermatitis. References [17] C. D. Mallen, S. Mottram, G. Wynne-Jones, and E. Thomas, “Birth-related exposures and asthma and allergy in adulthood: a population-based cross-sectional study of young adults in North Staffordshire,” Journal of Asthma, vol. 45, no. 4, pp. 309– 312, 2008. [1] C. A. Akdis, M. Akdis, T. Bieber et al., “Diagnosis and treatment of atopic dermatitis in children and adults: European Academy of Allergology and Clinical Immunology/American Academy of Allergy, Asthma and Immunology/PRACTALL consensus report,” Allergy, vol. 61, no. 8, pp. 969–987, 2006. [18] L. Christiansen, H. Frederiksen, K. Schousboe et al., “Age- and sex-differences in the validity of questionnaire-based zygosity in twins,” Twin Research, vol. 6, no. 4, pp. 275–278, 2003.fh [2] R. A. H. M. van den Oord and A. Sheikh, “Filaggrin gene defects and risk of developing allergic sensitisation and allergic disorders: systematic review and meta-analysis,” British Medical Journal, vol. 339, Article ID b2433, 2009. [19] D. L. Duffy, “The co-twin control study,” in Advances in Twin and Sib-Pair Analysis, T. D. Inspector, H. Snieder, and A. J. MacGregor, Eds., Greenwich Medical Media, 2000. [3] L. Paternoster, M. Standl, C. M. Chen et al., “Meta-analysis of genome-wide association studies identifies three new risk loci for atopic dermatitis,” Nature Genetics, vol. 44, pp. 187–192, 2011. [20] B. Xu, J. Pekkanen, and M.-R. J¨arvelin, “Obstetric complications and asthma in childhood,” Journal of Asthma, vol. 37, no. 7, pp. 589–594, 2000. [4] L. Stensen, S. F. Thomsen, and V. Backer, “Change in prevalence of atopic dermatitis between 1986 and 2001 among children,” Allergy and Asthma Proceedings, vol. 29, no. 4, pp. 392–396, 2008. [21] J. Mets¨al¨a, A. Kilkkinen, M. Kaila et al., “Perinatal factors and the risk of asthma in childhood—a population-based register study in Finland,” American Journal of Epidemiology, vol. 168, no. 2, pp. 170–178, 2008. [5] T. K. Ninan and G. Russell, “Respiratory symptoms and atopy in Aberdeen schoolchildren: evidence from two surveys 25 years apart,” British Medical Journal, vol. 304, no. 6831, pp. 873–875, 1992. [22] M. R¨as¨anen, J. Kaprio, T. Laitinen, T. Winter, M. Koskenvuo, and L. A. Laitinen, “Perinatal risk factors for asthma in Finnish adolescent twins,” Thorax, vol. 55, no. 1, pp. 25–31, 2000. [6] M. I. Asher, S. Montefort, B. Bj¨orkst´en et al., “Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry cross-sectional surveys,” The Lancet, vol. 368, no. Journal of Allergy Journal of Allergy 5 The fact that we studied twins may have had implications for the generalisability of the results to the population as a whole. Particularly, twins differ from singletons in a number of ways related to prenatal and early life that potentially could affect the risk of atopic dermatitis differentially in twins. These factors, which may be related to gestation, intrauterine environment, and mode of delivery, could also be expected to influence the variation in Apgar score. However, in a matched case-control study of 112 infant twins and singletons from the United States 5-minute Apgar score was not different in twins compared with singletons when matched for gestational age and mode of delivery [33], suggesting that results in relation to Apgar score can be extrapolated to the general singleton population. Still, we acknowledge that there may be unmeasured factors in our sample that could have interfered with our conclusions. [10] A. Linneberg, J. B. Simonsen, J. Petersen, L. G. Stensballe, and C. S. Benn, “Differential effects of risk factors on infant wheeze and atopic dermatitis emphasize a different etiology,” Journal of Allergy and Clinical Immunology, vol. 117, no. 1, pp. 184–189, 2006. [11] D. J. Purvis, J. M. D. Thompson, P. M. Clark et al., “Risk factors for atopic dermatitis in New Zealand children at 3.5 year of age,” British Journal of Dermatology, vol. 152, no. 4, pp. 742–749, 2005. [12] C. S. Benn, J. Wohlfahrt, P. Aaby et al., “Breastfeeding and risk of atopic dermatitis, by parental history of allergy, during the first 18 months of life,” American Journal of Epidemiology, vol. 160, no. 3, pp. 217–223, 2004. [13] J. F. Ludvigsson, M. Mostrom, J. Ludvigsson, and K. Duchen, “Exclusive breastfeeding and risk of atopic dermatitis in some 8300 infants,” Pediatric Allergy and Immunology, vol. 16, no. 3, pp. 201–208, 2005. [14] P. Leadbitter, N. Pearce, S. Cheng et al., “Relationship between fetal growth and the development of asthma and atopy in childhood,” Thorax, vol. 54, no. 10, pp. 905–910, 1999. We conclude that a high Apgar score may be linked to development of atopic dermatitis. Although this finding agrees with the theory that robust babies of high SES are at increased risk of atopic dermatitis, this has not been previ- ously documented and awaits support from future studies. [15] A. Sevelsted and H. Journal of Allergy Bisgaard, “Neonatal size in term children is associated with asthma at age 7, but not with atopic dermatitis or allergic sensitization,” Allergy, vol. 67, no. 5, pp. 670–675, 2012. [16] M. M. Moore, S. L. Rifas-Shiman, J. W. Rich-Edwards et al., “Perinatal predictors of atopic dermatitis occurring in the first six months of life,” Pediatrics, vol. 113, no. 3, pp. 468–474, 2004.h 4. Discussion Contrary to this, the diagnosis of atopic dermatitis was self-reported by the parents of the twins, which could have induced recall and reporting bias. Moreover, the question used to diagnose atopic dermatitis has not been systematically validated. Another limitation alludes to the differing age at ascertainment of the eczema diagnosis, which may have influenced the results. However, age was automatically eliminated as possible confounder in the cotwin control analysis model since it was the same for the two twins within the pair. References 9537, pp. 733–743, 2006. [23] M. R¨as¨anen, J. Kaprio, T. Laitinen, T. Winter, M. Koskenvuo, and L. A. Laitinen, “Perinatal risk factors for hay fever—a study among 2550 Finnish twin families,” Twin Research, vol. 4, no. 5, pp. 392–399, 2001. [24] B. K¨all´en, O. Finnstr¨om, K. G. Nygren, and P. Otterblad Olausson, “Asthma in Swedish children conceived by in vitro fertilisation,” Archives of Disease in Childhood, 2012. h [7] C. G. Carson, L. B. Halkjaer, S. M. Jensen, and H. Bisgaard, “Alcohol intake in pregnancy increases the child’s risk of atopic dermatitis. The COPSAC prospective birth cohort study of a high risk population,” PLoS ONE, vol. 7, Article ID e42710, 2012. [25] S. E. Gilman, H. Gardener, and S. L. Buka, “Maternal smoking during pregnancy and children’s cognitive and physical devel- opment: a causal risk factor?” American Journal of Epidemiol- ogy, vol. 168, no. 5, pp. 522–531, 2008. [8] A. Linneberg, J. Petersen, M. Grønbæk, and C. S. Benn, “Alcohol during pregnancy and atopic dermatitis in the offspring,” Clinical and Experimental Allergy, vol. 34, no. 11, pp. 1678–1683, 2004. [26] S. P. DaVeiga, “Epidemiology of atopic dermatitis: a review,” Allergy and Asthma Proceedings, vol. 33, pp. 227–234, 2012. [9] C. Lundholm, A. K. ¨Ortqvist, P. Lichtenstein, S. Cnattingius, and C. Almqvist, “Impaired fetal growth decreases the risk of childhood atopic eczema: a Swedish twin study,” Clinical and Experimental Allergy, vol. 40, no. 7, pp. 1044–1053, 2010. [27] R. Schmitz, K. Atzpodien, and M. Schlaud, “Prevalence and risk factors of atopic diseases in German children and adolescents,” Pediatric Allergy and Immunology, vol. 23, pp. 716–723, 2012. Journal of Allergy Journal of Allergy 6 [28] L. M. Silva, P. W. Jansen, E. A. P. Steegers et al., “Mother’s educa- tional level and fetal growth: the genesis of health inequalities,” International Journal of Epidemiology, vol. 39, no. 5, pp. 1250– 1261, 2010. [29] C.-Y. Hwang, Y.-J. Chen, M.-W. Lin et al., “Prevalence of atopic dermatitis, allergic rhinitis and asthma in Taiwan: a national study 2000 to 2007,” Acta Dermato-Venereologica, vol. 90, no. 6, pp. 589–594, 2010. [30] H. Saeki, H. Iizuka, Y. Mori et al., “Prevalence of atopic der- matitis in Japanese elementary schoolchildren,” British Journal of Dermatology, vol. 152, no. 1, pp. 110–114, 2005. [31] L. Naldi, F. Parazzini, and S. Gallus, “Prevalence of atopic der- matitis in Italian schoolchildren: factors affecting its variation,” Acta Dermato-Venereologica, vol. 89, pp. 122–125, 2009. References [32] N. Ballardini, I. Kull, C. S¨oderh¨all, G. Lilja, M. Wickman, and C. F. Wahlgren, “Eczema severity in preadolescent children and its relation to sex, filaggrin mutations, asthma, rhinitis, aggravating factors and topical treatment: a report from the BAMSE birth cohort,” British Journal of Dermatology, vol. 168, pp. 588–594, 2013. [33] S. A. Friedman, E. Schiff, L. Kao et al., “Do twins mature earlier than singletons? Results from a matched cohort study,” American Journal of Obstetrics and Gynecology, vol. 176, no. 6, pp. 1193–1199, 1997.
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Reverse Wedge Effect Following Intramedullary Nailing of a Basicervical Trochanteric Fracture Variant Combined with a Mechanically Compromised Greater Trochanter
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Reverse Wedge Effect Following Intramedullary Nailing of a Basicervical Trochanteric Fracture Variant Combined with a Mechanically Compromised Greater Trochanter Yu Zhang  Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital https://orcid.org/0000-0003-2274-429X Jun Hu  Jiangsu Province Hospital Xiaodong Qin  (  qinxiaodongnjmu@163.com ) Xiang Li  Jiangsu Province Hospital Guqi Hong  Jiangsu Province Hospital Research article Keywords: trochanteric fracture, intramedullary nailing, intraoperative complication Posted Date: January 20th, 2020 DOI: https://doi.org/10.21203/rs.2.14211/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Version of Record: A version of this preprint was published at BMC Musculoskeletal Disorders on March 28th, 2020. See the published version at https://doi.org/10.1186/s12891-020-03212-6. Reverse Wedge Effect Following Intramedullary Nailing of a Basicervical Trochanteric Fracture Variant Combined with a Mechanically Compromised Greater Trochanter Yu Zhang  Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital https://orcid.org/0000-0003-2274-429X Jun Hu  Jiangsu Province Hospital Xiaodong Qin  (  qinxiaodongnjmu@163.com ) Xiang Li  Jiangsu Province Hospital Guqi Hong  Jiangsu Province Hospital Research article Keywords: trochanteric fracture, intramedullary nailing, intraoperative complication Posted Date: January 20th, 2020 DOI: https://doi.org/10.21203/rs.2.14211/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Musculoskeletal Disorders on March 28th, 2020. See the published version at https://doi.org/10.1186/s12891-020-03212-6. Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Musculoskeletal Disorders on March 28th, 2020. See the published version at https://doi.org/10.1186/s12891-020-03212-6. Page 1/18 Page 1/18 Page 1/18 Abstract Background: To introduce an unreported intraoperative complication in intramedullary nailing (IN) of an anatomically reduced trochanteric fracture variant characterized by a basicervical fracture line and coronally disrupted greater trochanter (GT). Materials and Methods: A total of 414 trochanteric fractures (TF) treated with intramedullary nails from 2013 to 2017 were included in this study. After analysis of intraoperative fluoroscopy data, 33 cases, including 21 females and 12 males, with a mean age of 72.5 years (33 to 96 years) were identified for internal rotation of the cephalocervical fragment and inferior opening at the basicervical fracture line caused by nailing a satisfactorily reduced TF. The morphological features of this group of patients were analyzed on computed tomography (CT) scan. On radiograph, the magnitude of the displacement and final femoral neck-shaft angle (NSA) were measured. Results: CT analysis demonstrated that the basicervical fracture line and the posterolateral fragment (PLF) detached from the GT were the two dominant features of this cohort. They were classified according to the number of main fragments: a 3-fragmentary subgroup containing three consistent fragments (cephalocervical fragment, PLF and distal femoral shaft) and a 4-fragmentary subgroup embracing one additional fragment (lesser trochanter). The four subtypes were as follows: the 3- fragmentary S indicating a small PLF (6 cases), the 3-fragmentary M presenting a moderate PLF (3 cases), the 3-fragmentary L standing for the PLF involving whole lesser trochanter (LT) (4 cases) and the 4-fragmentary GL incorporating separated PLF and LT fragments (20 cases). Geological analysis demonstrated that the majority of the basicervical fracture lines (81.8%) just crossed the center of the piriformis fossa, while the others marginally involved the medial wall of the GT. Postoperatively, the mean width of the inferior opening at the basicervical region was 9.2±4.6 mm. The mean NSA was 135.2±7.8 degrees. The comparison between the 3- and 4-fragmentary subgroups revealed no significant differences in magnitude of displacement and NSA. Conclusion: This unreported intraoperative complication predominantly occurred in the intramedullary nailed basicervical trochanteric fracture variant combined with a PLF from the GT. The magnitude of the secondary displacement was substantial and resulted in a relative valgus reduction. This secondary displacement was caused by an impingement of the reamer with the superolateral cortex of the cephalocervical fragment and should be addressed during the operation. Level of Evidence: Therapy IV Level of Evidence: Therapy IV Introduction The increase in the aging population worldwide has resulted in a growing amount of TF among geriatric people, which presents challenges to the public health system and to orthopedic trauma surgeons [1]. Surgical intervention has been the preferred treatment for TF patients because of the benefits of effective pain control and the opportunity for early weight bearing. Compared to dynamic hip screw plates, INs are Page 2/18 gaining more popularity and could provide superior stability to unstable TF patterns that are characterized by posteromedial comminution, reverse oblique configuration, lateral wall disruption, GT disruption and basicervical variants [2, 3]. At the same time, much attention has been paid to the increasing postoperative complications associated with the IN application. Those complications include sliding screw cut-out or cut-through [4], extensive fracture collapse and reduction loss, thigh pain associated with nail tip collision, and varus malunion. However, the intraoperative complications are less noticeable. Recently, Hak reported an uncommon intraoperative complication when IN was performed to stabilize a group of TFs: a varus malreduction presented as a secondary fracture displacement caused by inserting a cephalated IN from the tip of the GT [5]. It was featured by a lateral displacement of the femoral shaft and an opening of the superior part of the primary fracture line (trochanteric region), which was later named the “wedge effect” by O’Malley [6] (Figure 1). Interestingly, we also noticed another pattern of intraoperative complications closely associated with inserting a trochanter tip starting point IN in a cohort of TFs (Figure 2). In contrast to the aforementioned “wedge effect”, the reaming/IN insertion generated internal rotation of the cephalocervical fragment and an inferiorly oriented gap at the primary fracture line (basicervical region). This pattern was named the “reverse wedge effect”. Further studies revealed that this intraoperative complication completely occurred in an uncommon TF pattern distinguished by the primary fracture line at the basicervical region (basicervical TF variant) and a detached PLF from the GT. The purpose of the present study was to investigate the incidence of the “reverse wedge effect” at a level-one tertiary trauma center, summarize its morphological features and analyze its cause and potential influence on treatment outcomes. Materials And Methods We obtained Institutional Review Board approval for this study. A total of 414 TF cases that underwent IN fixation at our hospital between January 2013 and January 2017 were included in this study. The inclusion criteria were skeletally mature, unilateral TF with complete radiographs, intraoperative fluoroscopy and preoperative CT scan which improved our understanding of the fracture characteristics and the degree of instability. The exclusion criteria were as follows: (1) patients younger than 18 years; (2) high-energy injury or multiple injuries such as car accidents or high-altitude fall injuries; and (3) patients with pathological fractures and previous histories of hip malformation or surgery of either hip. By observing intraoperative fluoroscopy, the TF cases demonstrating the “reverse wedge effect” during reaming or nail insertion were identified. We obtained Institutional Review Board approval for this study. A total fixation at our hospital between January 2013 and January 2017 were inclusion criteria were skeletally mature, unilateral TF with complete ra fluoroscopy and preoperative CT scan which improved our understand and the degree of instability. The exclusion criteria were as follows: (1) (2) high-energy injury or multiple injuries such as car accidents or high patients with pathological fractures and previous histories of hip malfo By observing intraoperative fluoroscopy, the TF cases demonstrating th reaming or nail insertion were identified. The demographic information of this cohort was extracted from patient medical records. The radiographic data were available in the Picture Archiving and Communication System (PACS), and the measurements were carried out with the built-in gauge tools. One of the senior surgeons at the hospital classified the fractures according to the AO/OTA classification on plain radiographs (H.J.). Page 3/18 Page 3/18 To eliminate the negative influences of initial displacement on the fracture morphology analysis, three- dimensional CTs (3D-CT) of both sides of the proximal femurs were reconstructed simultaneously. The fracture line at the basicervical region was designated the primary fracture line, and the coronally propagated fracture line affecting the GT was designated the secondary fracture line. The analysis of the reconstructed 3D images revealed that there were three consistent fragments: cephalocervical fragment, femoral shaft and posterolateral fragment from GT. A variable fragment (LT) was also frequently identified. Therefore, each case could be defined as either a 3-fragmentary or 4-fragmentary pattern. Then, the rationale of Shoda’s 3D-CT classification of the proximal femur was borrowed to further classify it into one of four subgroups [7]. Materials And Methods The 3-fragmentary S was the first subgroup, where “S” referred to a small PLF fragment combined with cephalocervical and shaft fragments (Figure 3A). The second subgroup was the 3-fragmentary M, in which a moderate PLF (indicated by “M”) coexisted with other two consistent fragments. Compared to the 3-fragmentary S, the secondary fracture line in the 3-fragmentary M propagated more medially and was in the vicinity of the lateral border of the intact LT (Figure 3B). In the third subgroup, one large banana-like fragment consisting of the PLF and the LT as one unit occurred in the 3-fragmentary L (Figure 3C). When the LT was also concomitantly fractured, there were four large fragments; thus, it was defined as the 4-fragmentary GL subtype (Figure 3D). On the 3D-CT, the morphological feature of the primary fracture line was specifically studied [7]. As the GT of the injured limb was routinely comminuted and the normal spatial relationship between the main fragments was altered, we utilized the mirrored image of the contralateral uninjured proximal femur as a template for the injured hip to precisely evaluate the distribution primary fracture line. After the DICOM data were entered into Materialise 3-matic (Materialise NV Inc., Leuven, Belgium), the cephalocervical fragment of the TF was maximally matched to the mirrored 3D image of the contralateral femur. On the fused images, the relationship between the primary fracture line and the GT could be analyzed (Figure 4). Thirty-one patients were stabilized by proximal femoral nail anti-rotation (PFNA, Synthes Inc., Oberderf, Switzerland) and two younger patients by a TRIGEN INTERTAN nail (Smith–Nephew, Memphis, USA). The postoperative radiography was assessed by another fellow training surgeon who did not take part in the operation (H.G.Q). The width of the secondary displacement at the inferior part of the primary fracture line and the NSA were measured on the anteroposterior (AP) view (Figure 5). All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) (ver 23.0, International Business Machines, Inc. Armonk, New York, USA). Quantitative data were expressed as median and compared among groups by Student’s t-test or Mann-Whitney U test. All P-values were two- sided and a P-value  < <lue  < 0.05 was considered to be statistically sign Surgical technique Under general anesthesia, the patient was positioned on the operation table in supination with consistent distraction of the injured limb, and the manual reduction of the fracture was performed. By persistent traction and an internal rotation maneuver, the limb length and rotational alignment were restored first. Page 4/18 After the medial cortical continuity and acceptable NSA were confirmed on AP view of the fluoroscopic image, the anterior cortex reduction was checked on lateral view. If there was negative reduction of the anterior cortex, a small elevator was percutaneously inserted to reduce it. No attempt was made to reduce an isolated LT fragment or PLF from the GT. When the reduction was satisfactory on both views, a PFNA/TRIGEN INTERTAN nail was inserted according to the manufacturer’s operation manual. After making a 5-cm incision proximal to the tip of the GT, the fasciae and muscle fibers of the gluteus medius were split. As the standard manipulation, the entry point of the nail was slightly lateral to the tip of the GT, and guide wire placement was followed by opening the femur, reaming the medullary canal and inserting a PFNA/TRIGEN INTERTAN nail. Frequently, “reverse wedge effect” occurred either during canal reaming or nail insertion. Before 2017, we resorted to enlarging the entrance of the femoral canal or overdistracting the injured limb to overcome this secondary deformity. However, the design of the reamer made the over-reaming technique extremely difficult, and the overdistraction method generally failed. Later, we chose to secure the acquired initial reduction by inserting a 3.5-mm Kirschner wire (K-wire) before reaming and PFNA/TRIGEN INTERTAN nail insertion (Figure 6E). Under image intensifier control, the K-wire was inserted along the axis of the femoral neck and closely underneath the anterior cortex. Then, the femoral canal was reamed, and the nail was inserted. A bone hook should sometimes be added to augment the maintenance of the primarily achieved reduction (Figure 6F). We found that those methods were extremely effective and dramatically shortened the operation time if the risk of the “reverse wedge effect” could be predicted preoperatively. Results A total of 33 patients met the inclusion criteria in the final analysis so that the overall incidence of the “reverse wedge effect” was 7.9%. The preoperative plain radiograph analysis revealed that all the 33 patients were basicervical TF variants while there were another 12 cases of this special fracture type did not present similar intraoperative complication. The patients included 12 males and 21 females, with a mean age of 72.5 years (33 to 96 years). The internal rotation of the cephalocervical fragment caused by reamer/IN impingement was the main underlying cause of this intraoperative complication. On the postoperative radiograph, this secondary displacement demonstrated an inferior gap at the primary fracture line and increased NSA (Figure 2B). The AO/OTA classification scheme failed to properly categorize this special fracture type properly [8]: on the plain radiograph, the coronally oriented secondary fracture line made the measurement of the lateral wall thickness unreliable and generated great confusion in differentiating AO-31 A1 GT from A2. In contrast, the classification system we devised in this study depicted each case briefly and concisely. The incidence of the four-fragment TF (4-fragmentary GL) was the highest (Table 1). In all four subgroups, the majority of the primary fracture lines transversed the center of the piriformis fossa Page 5/18 Page 5/18 Page 5/18 (83.8%) (Figure 4A). The remaining fracture lines marginally involved a small portion of the GT medial wall (Figure 4B). Therefore, all the primary fracture lines originated extracapsularly. A Mann-Whitney U test was run to determine if there were differences in magnitude of displacement and the NSA between 3-fragmentary and 4-fragmentary subgroups. Distributions of the secondary displacement and NSA for 3-fragmentary and 4-fragmentary subgroups were similar, as assessed by visual inspection. Median displacement for 3-fragmentary (8.20 mm) and 4-fragmentary (9.20 mm) was not statistically significantly different, U = 80.5, z = -1.825, p = 0.068, using an exact sampling distribution for U. While, the median NSA for 3-fragmentary (136.0 degrees) and 4-fragmentary (133.0 degrees) was not statistically significantly different, U = 116.0, z = -0.518, p = 0.624. Discussion As Cho’s study found that the incidence of the PLF in TF was as high as 88.4% [15], we observed a similar prevalence among all the 414 patients, and every “reverse wedge effect” case had a PLF. We believe that further classification and comparison would facilitate understanding and exploring the influences of those two fragments on “reverse wedge effect” formation and treatment/strategy design. Although Van Embden stated that AO/OTA classification was more comprehensive and reliable compared to the Jensen-Evans classification [16], we found that it failed to cover this special fracture pattern. According to AO/OTA classification (2018 version), AO-31A1 and A2 were designated as pertrochanteric fractures in which the main fracture line propagated through the trochanters. Saarenpaa, Watson and other authors described basicervical TF variant as fracture at the base of femoral neck that is medial to the intertrochanteric line [9, 17, 18]. So basicervical TF variant could not be classified into pertrochanteric (AO - 31A1 and A2) or intertrochanteric fracture(AO - 31A3). In contrast, in 1949, even before the clinical application of computed tomography, Evans had proposed 3- and 4-fragmentary fracture patterns in his classification scheme [19]. Considering the complexity of the fracture morphology, a three-dimensional classification system originating from Evans’ would be more pragmatic [16]. The principle from Babhulkar and Shoda’s classifications of GT fracture [2, 7] could be incorporated to further classify basicervical TF variants according to the morphology of the PLF. Although the sample size was not large enough to make statistical comparisons between the four subgroups, the measured displacements of “reverse wedge effect” and ultimate NSA were similar among them. Therefore, we could infer that the volume of the PLF minimally influenced the extent of the secondary displacement in this cohort of patients. From a mechanical view, it is the existence of the PLF contributes to the development of the “reverse wedge effect”. When the GT region is intact, the guide wire could be constrained around the tip of the GT, and the trajectory of the reamer/IN was centralized into the femoral canal. However, an incompetent GT made the guide wire and reamer float at the start site. Under an image intensifier, we observed that the reamer was prone to skew medially, especially when attempting to over-ream the superolateral corner of the cephalocervical fragment (Figure 6D). As a result, impaction between the cephalocervical fragment and the reamer/nail occurred. Discussion The purpose of the present study was to investigate the incidence, characteristics and causes of the “reverse wedge effect” during application of the IN in TF. In contrast to the previously reported “wedge effect”, which was defined as an intraoperative secondary displacement at the superior part of the pertrochanteric fracture line [6], we observed another IN-related intraoperative complication, which was demonstrated as an inferior opening at the already reduced primary fracture line. We also identified that all the cases occurred in basicervical TF variants combined with a disrupted GT. By reviewing intraoperative fluoroscopy, we noticed an impingement between the reamer/IN and the superolateral edge of the cephalocervical fragment, which caused this secondary displacement (Figure 6). Because all the 414 reviewed cases were treated by PFNA/TRIGEN INTERTAN nailing but the incidence of the “reverse wedge effect” was nearly 7.97%, we believe that anatomic factors other than implantation should play a major role. The basicervical fracture line is the most significant feature of this cohort of patients. Strictly, a basicervical fracture is defined as a 2-part fracture where the fracture line originates from the base of the femoral neck and exits above the lesser trochanter [9]. Despite the concomitant PLF or LT fracture, the primary fracture lines in this cohort of patients met this description exactly. There is little controversy about the fact that a basicervical fracture is an unstable fracture and prone to varus deformity [10]. Sliding screws were initially proposed as a treatment for these fractures [11, 12], but the recent trend favors IN fixation [9, 13]. Anatomically, the piriformis fossa is the transitional region between the hard cortex of the femoral neck and the relatively weak cancellous bone of the GT [14]. Our study demonstrated that the primary fracture lines just crossed the piriformis fossa center. This indicated that the superolateral corner of the cephalocervical fragment was primarily made from the hard cortex, which is hard enough to resist reaming and colliding with the reamer and PFNA/TRIGEN INTERTAN nail insertion. Eventually, the reamer or EFNA/TRIGEN INTERTAN nail would push the cephalocervical fragment, internally rotating and inferiorly displacing, resulting in the “reverse wedge effect”. Page 6/18 Notably, in this cohort, the PLF and LT fragment were the other two prominent anatomical features. Discussion Although mild valgus reduction is more preferred than varus reduction for allowing interfragment compression and reducing bone-implant stresses, Ciufo et al. recently showed that residual basicervical gapping was closely associated with fixation cutout [21]. Considering that the magnitude of the secondary displacement exceeded 4 mm, which was proposed by Baumgaertner as the lowest threshold for malreduction [22], we believe that this newly reported intraoperative complication is worth further investigation. There were some limitations to the present study. First, this study was a retrospective analysis. Therefore, the robustness of the analysis is undetermined. Second, a comparison between the different trochanteric fracture patterns in the incidence rate of the “reverse wedge effect” was not carried out. However, we rigorously reviewed all the intraoperative fluoroscopy findings during the study period and did not find any “reverse wedge effect” in other fracture patterns. Thus, we believe that this complication is closely correlated with the nailing basicervical TF variant combined with the PLF. Third, the treatment outcomes of this cohort of patients were not evaluated for several reasons. The primary reason is that this cohort of patients was highly heterogeneous, and some very young patients were included, which made building an age- and sex-matched control group difficult. Conclusions Our study reveals traumatic anatomic factors associated with the “reverse wedge effect” in cephalomedullary nailing in a group of trochanteric fractures. In contrast to the previously proposed “wedge effect”, this “reverse wedge effect” is characterized by an inferior opening at the primary fracture line in the trochanteric region and a mild valgus malreduction. This secondary displacement exclusively occurred during the reaming/nailing of an anatomically reduced trochanteric fracture featuring a basicervical variant type and a disrupted GT. Further analysis demonstrated that the basicervical fracture line is the major contributor to this intraoperative complication and that the impingement between the reamer/nail and the superolateral cortex tip of the cephalocervical fragment was the direct cause. Thus, the magnitude of the displacement is significant, and early postoperative weight bearing might be compromised. We advocate that this special intraoperative complication should be prevented in advance of reaming and nail insertion to optimize the final reduction quality. Discussion Since the volume of the PLF plays a relatively minor role, this fragment could be excluded during the management of the “reverse wedge effect”. In this cohort of patients, the incidence of PLF was 100% and the LT disruption was 60.6%. To investigate the role of LT disruption in this operative complication, comparisons between 3- and 4-fragmentary subgroups in the magnitude of displacement and postoperative NSA were made and revealed no significant differences. We could conclude that the appearance of LT fragment contributed minimally to the occurence and degree of the “reverse wedge effect”. When preventing the reverse wedge effect in basicervical TF variant, the lesser trochanter fragment did not have to be reduced and temporarily stabilized. It is unclear how this intraoperative complication negatively impacts the treatment outcome. The postoperative measurement demonstrated that the magnitude of the secondary displacement in the Page 7/18 “reverse wedge effect” was significant. Zhang et al. advocated the “medial positive reduction” concept to validate a stable medial cortex mismatch, but they did not quantify the threshold of the acceptable diastasis at the medial cortex [20]. It is reasonable that an obvious opening at the primary fracture line represents instability in the fracture. Thereafter, postoperative excessive collapse would occur because of failure to restore cortex interdigitation. Early postoperative weight bearing had to be postponed. The postoperative radiograph demonstrated a mild tendency of valgus reduction in the majority of patients. Although mild valgus reduction is more preferred than varus reduction for allowing interfragment compression and reducing bone-implant stresses, Ciufo et al. recently showed that residual basicervical gapping was closely associated with fixation cutout [21]. Considering that the magnitude of the secondary displacement exceeded 4 mm, which was proposed by Baumgaertner as the lowest threshold for malreduction [22], we believe that this newly reported intraoperative complication is worth further investigation. “reverse wedge effect” was significant. Zhang et al. advocated the “medial positive reduction” concept to validate a stable medial cortex mismatch, but they did not quantify the threshold of the acceptable diastasis at the medial cortex [20]. It is reasonable that an obvious opening at the primary fracture line represents instability in the fracture. Thereafter, postoperative excessive collapse would occur because of failure to restore cortex interdigitation. Early postoperative weight bearing had to be postponed. The postoperative radiograph demonstrated a mild tendency of valgus reduction in the majority of patients. Authors’ contributions QXD and ZY performed the study. HGJ and HJ participated in the operation and data collection/interpretation for the study. ZY performed the statistical analysis. QXD conceived of the study and participated in its design. LX was responsible for coordination, data collection/interpretation and proofreading the final manuscript. All authors read and approved the final manuscript. Yu Zhang will be contact to provide data and materials. Yu Zhang will be contact to provide data and materials. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. We gratefully acknowledge the support of all the doctors in our departmen No funding was obtained for this study Ethics approval and consent to participate Approved by the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) of Jiangsu Province Hospital (The First Affiliated Hospital of Nanjing Medical University) at 300 Guangzhou Road, Nanjing 210029, China. All subjects provided informed consent in written to take part in the study. Authors’ Affiliations Department of Trauma, the First Affiliated Hospital of Nanjing Medical University and Jiangsu Province Hospital Acknowledgements Acknowledgements Page 8/18 Abbreviations IN: intramedullary nailing GT: greater trochanter TF: trochanteric fractures CT: computed tomography IN: intramedullary nailing Page 9/18 AP: anteroposterior K-wire: Kirschner wire K-wire: Kirschner wire References 1. Cummings SR, Rubin SM, Black D. The future of hip fractures in the United States: numbers, costs, and potential effects of postmenopausal estrogen. Clin Orthop 1990,252:163–166 1. Cummings SR, Rubin SM, Black D. The future of hip fractures in the United States: numbers, costs, and potential effects of postmenopausal estrogen. Clin Orthop 1990,252:163–166 2. Babhulkar S. Unstable trochanteric fractures: Issues and avoiding pitfalls. Injury. 2017;48(4):803-818. 3. Sharma G, Kumar G N K, Yadav S, et al. Pertrochanteric fractures (AO/OTA 31-A1 and A2) not amenable to closed reduction: causes of irreducibility. 2014;45(12):1950-1957. 4. Kashigar A, Vincent A, Gunton MJ, et al. Predictors of failure for cephalomedullary nailing of proximal femoral fractures. Bone Joint J. 2014;96-B(8):1029-1034. 5. Hak DJ, Bilat C. Avoiding varus malreduction during cephalomedullary nailing of intertrochanteric hip fractures. Arch Orthop Trauma Surg. 2011;131(5):709-710. 6. O'Malley MJ, Kang KK, Azer E, et al. Wedge effect following intramedullary hip screw fixation of intertrochanteric proximal femur fracture. Arch Orthop Trauma Surg. 2015;135(10):1343-1347. 6. O'Malley MJ, Kang KK, Azer E, et al. Wedge effect following intramedullary hip screw fixation of intertrochanteric proximal femur fracture. Arch Orthop Trauma Surg. 2015;135(10):1343-1347. 7. Shoda E, Kitada S, Sasaki Y, et al. Proposal of new classification of femoral trochanteric fracture by three-dimensional computed tomography and relationship to usual plain X-ray classification. J Orthop Surg (Hong Kong). 2017;25(1):1-5. 8. Meinberg EG, Agel J, Roberts CS, et al. Fracture and Dislocation Classification Compendium-2018 J Orthop Trauma. 2018;32 Suppl 1:S1-S170. 9. Watson ST, Schaller TM, Tanner SL, et al. Outcomes of Low-Energy Basicervical Proximal Femoral Fractures Treated with Cephalomedullary Fixation. J Bone Joint Surg Am. 2016;98(13):1097-1102. 10. Gill JM, Johnson GR, Sher JL, et al. Biomechanical aspects of the repair of intertrochanteric fractures. J Biomed Eng. May 1989; 11 (3):235-239. 11. Baumgaertner MR, Higgins TF. Femoral neck fractures. In:Rockwood CA, Bucholz RW, Heckman JD, Green DP, eds. Rockwood and Green’s Fractures in Adults. Vol 2. 5th ed. Philadelphia, Pa:Lippincott Williams and Wilkins; 2001:1588. Page 10/18 12. Su BW, Heyworth BE, Protopsaltis TS, et al. Basicervical versus intertrochanteric fractures: an analysis of radiographic and functional outcomes. Orthopedics. 2006;29(10):919-925. 13. Hu SJ, Yu GR, Zhang SM. Surgical treatment of basicervical intertrochanteric fractures of the proximal femur with cephalomeduallary hip nails. Orthop Surg. 2013;5(2):124-129. 14. Reece GM, Poole KE, Gee AH. Imaging the femoral cortex: thickness, density and mass from clinical CT. Med Image Anal. 2012;16(5):952-65. 15. References Cho JW, Kent WT, Yoon YC, et al. Fracture morphology of AO/OTA 31-A trochanteric fractures: A 3D CT study with an emphasis on coronal fragments. Injury. 2017\;48(2):277-284. 16. Van Embden, S. Meylaerts, S. Rhemrev, et al. The Comparison of Two Classifications for Trochanteric Femur Fracture: The AO/ASIF Classification and the Jensen Classification. Injury. 2010;41(4):377-81. 17. Saarenpaa I, Partanen J, Jalovaara P. Basicervical fracture-a rare type of hip fracture. Arch Orthop Trauma Surg. 2002;122(2):69-72. 18. Okano I, Sawada T, Kushima N, et al. Treatment With Helical Blade Cephalomedullary Nail for Two- Part Basicervical Proximal Femoral Fracture in Elderly Patients: A Retrospective Observational Study. Geriatr Orthop Surg Rehabil. 2017;8(4):244–251. 19. Evans EM. The treatment of trochanteric fractures of the femur. J Bone Joint Surg Br. 1949;31 (2):190-203. 19. Evans EM. The treatment of trochanteric fractures of the femur. J Bone Joint Surg Br. 1949;31 (2):190-203. 20. Chang SM, Zhang YQ, Ma Z, et al. Fracture reduction with positive medial cortical support: a key element in stability reconstruction for the unstable pertrochanteric hip fractures. Arch Orthop Trauma Surg. 2015 Jun;135(6):811-818. 21. Ciufo DJ, Zaruta DA, Lipof JS, et al. Risk Factors Associated With Cephalomedullary Nail Cutout in the Treatment of Trochanteric Hip Fractures. J Orthop Trauma. 2017 ;31(11):583-588. 22. Baumgaertner MR, Curtin SL, Lindskog DM, et al. The value of the tip-apex distance in predicting failure of fixation of peritrochanteric fractures of the hip. J Bone Joint Surg Am 1995;77(7):1058– 1064. Table subgroups 3-fragmentary  4-fragmentary  p value Frequency 13 20 - Median Displacement (mm) 8.2 (6.90, 8.85) 9.2 (8.45, 10.95) 0.068 Median NSA (degree) 136.0 (130.5, 138.5) 133.0 (132.0, 138.8) 0.624 Table 1. The 3-fragmentary and 4-fragmentary subgroups were compared with Mann- Whitney U tests Figure s Page 11/18 Page 11/18 Figure 1 The simulated diagram depicting the “wedge effect”, where the intramedullary nail insertion causes lateralization of the femoral shaft and varus malalignment. Figure 1 The simulated diagram depicting the “wedge effect”, where the intramedullary nail insertion causes lateralization of the femoral shaft and varus malalignment. Page 12/18 Page 12/18 Figure 2 (A) The preoperative anteroposterior (AP) radiograph showing an apparently simple AO 31.A 2.2 pertrochanteric fracture. (B) Following fixation with PFNA, there was an obvious inferior gap at the basicervical region (white arrow) and moderate valgus deformity. The lucency around helix indicates a micromotion and instability of the fracture-implantation complex. Figure 2 (A) The preoperative anteroposterior (AP) radiograph showing an apparently simple AO 31.A 2.2 pertrochanteric fracture. (B) Following fixation with PFNA, there was an obvious inferior gap at the basicervical region (white arrow) and moderate valgus deformity. The lucency around helix indicates a micromotion and instability of the fracture-implantation complex. (A) The preoperative anteroposterior (AP) radiograph showing an apparently simple AO 31.A 2.2 pertrochanteric fracture. (B) Following fixation with PFNA, there was an obvious inferior gap at the basicervical region (white arrow) and moderate valgus deformity. The lucency around helix indicates a micromotion and instability of the fracture-implantation complex. Page 13/18 Page 13/18 Figure 3 Figure 3 Figure 3 Figure 3 Page 14/18 (A) The cephalocervical fragment was fused on the mirror image of the contralateral proximal femur. The primary fracture line of the injured side just crossed the piriform fossa. (B) The primary fracture line involved a small piece of the posteromedial wall of the GT. Figure 4 (A) The cephalocervical fragment was fused on the mirror image of the contralateral proximal femur. The primary fracture line of the injured side just crossed the piriform fossa. (B) The primary fracture line involved a small piece of the posteromedial wall of the GT. (A) The cephalocervical fragment was fused on the mirror image of the contralateral proximal femur. The primary fracture line of the injured side just crossed the piriform fossa. (B) The primary fracture line involved a small piece of the posteromedial wall of the GT. Page 15/18 Figure 5 Figure 5 The tangential lines to the medial cortexes of the cephalocervical and shaft fragments are drawn. The distance between the two lines represents the width of the basicervical gap. Page 16/18 e 6 B) Preoperative 3D image demonstrating a subtype 4-fragmentary GL us deformity. (C and D) The wide arrow indicates the impact between ateral cortex of the cephalocervical fragment. The resultant basicervic Figure 6 (A and B) Preoperative 3D image demonstrating a subtype 4-fragmentary GL pertrochanteric fracture with mild varus deformity. (C and D) The wide arrow indicates the impact between the reamer and the superolateral cortex of the cephalocervical fragment. The resultant basicervical gap is designated by a narrow arrow. (E) When the risk of reverse wedge effect was high, a 3.5-mm Kirschner wire (K-wire) was inserted to secure the reduction. (F) Occasionally, a single K-wire was not strong enough, and a bone hook was inserted to prevent inferior opening at the fracture line. Figure 6 (A and B) Preoperative 3D image demonstrating a subtype 4-fragmentary GL pertrochanteric fracture with mild varus deformity. (C and D) The wide arrow indicates the impact between the reamer and the superolateral cortex of the cephalocervical fragment. The resultant basicervical gap is designated by a narrow arrow. (E) When the risk of reverse wedge effect was high, a 3.5-mm Kirschner wire (K-wire) was Page 17/18 Page 17/18 Page 17/18 inserted to secure the reduction. (F) Occasionally, a single K-wire was not strong enough, and a bone hook was inserted to prevent inferior opening at the fracture line. Page 18/18 Page 18/18
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First Live-Experience Session with PET/CT Specimen Imager: A Pilot Analysis in Prostate Cancer and Neuroendocrine Tumor
Biomedicines
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Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 A pilot analysis in Prostate Cancer and Neuroendocrine Tumor. Lorenzo Muraglia1, Francesco Mattana1, Laura Lavinia Travaini1, Gennaro Musi2,3, Emilio Bertani4, Giuseppe Renne5, Eleonora Pisa5, Mahila Esmeralda Ferrari6, Uberto Fumagalli Romario4, Ottavio De Cobelli2,3, Nicola Fusco3,5, Francesco Ceci1,3* 1 Division of Nuclear Medicine, IEO European Institute of Oncology IRCCS, Milan, Italy 2 Division of Urology, IEO European Institute of Oncology IRCCS, Milan, Italy 3 Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy 4 Division of Digestive Surgery, IEO European Institute of Oncology IRCCS, Milan, Italy 5 Division of Pathology, IEO European Institute of Oncology IRCCS, Milan, Italy 6 Unit of Medical Physics, IEO European Institute of Oncology IRCCS, Milan, Italy ABSTRACT Objective: to evaluate the feasibility of the intra-operative application of a specimen PET/CT imager in a clinical setting. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 Materials and methods: this is a pilot analysis performed in three patients who received an intra- operative administration of 68Ga-PSMA-11 (n=2) and 68Ga-DOTA-TOC (n=1), respectively. Patients were administrated with PET radiopharmaceuticals to perform radio-guided surgery with a beta- probe detector during radical prostatectomy for prostate cancer (PCa) and salvage lymphadenectomy for recurrent neuroendocrine tumor (NET) of the ileum, respectively. All procedures have been performed within two ongoing clinical trials in our Institute (NCT05596851 and NCT05448157). Pathologic assessment with immunohistochemistry (PSMA-staining and SSA immunoreactivity) was considered as standard of truth. Specimen images were compared with baseline PET/CT images and histopathological analysis. Results: Patients received 1 MBq/Kg of 68Ga-PSMA-11 (PCa) or 1.2 MBq/Kg of 68Ga-DOTA-TOC (NET) prior to surgery. Specimens were collected, positioned in the dedicated specimen container, and scanned to obtain high resolution PET/CT images. In all cases a perfect match was observed between the findings detected by the specimen imager and histopathology. Overall, the PET spatial resolution was sensibly higher for the specimen images compared to the baseline whole-body PET/CT images. Furthermore, the use of the PET/CT specimen imager did not significantly interfere with any procedures, and the overall length of the surgery was not affected using the PET/CT specimen imager. Finally, the radiation exposure of the operating theater staff was lower than 40 µSv per procedure (range 26 – 40 Sv). Conclusion: the image acquisition of specimens obtained by patients who received intra-surgery injection of 68Ga-PSMA-11 and 68Ga-DOTA-TOC was feasible and reliable also in a live-experience session and has been easily adapted to surgery daily-practice. The high sensitivity, together with the evaluation of intra-lesion tumor heterogeneity, were the most relevant results since the data derived from specimen PET/CT imaging matched perfectly with the histopathological analysis. Keywords: PET/CT; PET; PET/CT specimen imager; radio-guided surgery Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444 doi:10.20944/preprints202212.0444.v2 The aim of this pilot analysis was to assess the feasibility of a PET/CT specimen imager in a live- experience session in patients who received an intraoperative injection of 68Ga-PSMA-11 and 68Ga- DOTA-TOC. The aim of this pilot analysis was to assess the feasibility of a PET/CT specimen imager in a live- experience session in patients who received an intraoperative injection of 68Ga-PSMA-11 and 68Ga- DOTA-TOC. Patient Population This pilot analysis was performed as a secondary objective of two trials currently ongoing in our Institute in Pca (NCT05596851) and NET (NCT05448157) designed to evaluate the safety and the efficacy of a positron probe detector (DROP-IN β-probe) for live intra-operative radio-guided surgery. Three patients enrolled in these trials have been considered eligible for this supplemental analysis, two affected by Pca and one affected by NET of the ileum. In all cases, patients received a baseline whole-body PET/CT to assess the burden of the disease (68Ga-PSMA-11 PET/CT and 68Ga- DOTA-TOC PET/CT respectively). The scans were performed in the Division of Nuclear Medicine of our Institute (GE Discovery MIDR) according to international procedural guidelines, 4-6 weeks prior to surgery. All patients signed informed consent form prior to enrollment and studies have been approved by local ethical committee and scientific review board. INTRODUCTION Positron Emission Tomography (PET) imaging is considered one of the leading imaging techniques to investigate oncological patients and it is considered the standard-of-care in many solid tumors and hematological malignancies (1). Recently, several technological improvements appeared in the field of PET imaging, ranging from digital detectors (e.g. silicon photomultipliers) to long axial field- of-view scanners (2). However, the attention moved also to smaller PET/computed tomography (CT) tomographs that might be implemented in the daily clinical practice of surgical oncology. Recently, a compact and mobile PET/CT specimen imager was developed to obtain full 3D high resolution images, assisting surgeons and pathologists during the surgical procedure directly in the operating theater. This specimen imager combines the picomolar sensitivity of PET (at sub-millimeter resolution) with anatomical information obtained from a high-resolution micro-CT. The first research application was in breast cancer using 18F-FDG as a PET radiopharmaceutical, where a multicenter clinical trial is currently on-going (NCT04999917). Intra-operative high-resolution PET/CT imaging has shown promising results for margin assessment in breast malignancies, where a respective sensitivity and specificity of 84% and 96% was reached for the identification of positive margins of the invasive component of invasive ductal carcinoma (3). Explorative studies in pancreatic adenocarcinoma and in head and neck cancers have also demonstrated the feasibility of intra-operatively imaging radiotracer uptake in high resolution specimen PET/CT images (4,5). Recently, novel radiopharmaceuticals emerged as game changers in prostate cancer (PCa) with prostate specific membrane antigen (PSMA) PET and in neuroendocrine tumor (NET) with somatostatin receptor (SSR) based PET. PSMA-PET showed its superiority compared to conventional imaging or other PET tracers in randomized clinical trials (6,7) or registry trials (8-10) to stage Pca or in case of disease recurrence, while SSR-agonist PET, is currently the standard-of-care to investigate gastro-entero-pancreatic NET (GEP-NET), especially in case of low-grade disease (G1-G2) (11). Furthermore, both PSMA inhibitors and SSR agonists are theranostic agents and can be labeled with isotopes for PET imaging (e.g. Gallium-68) or high-energy beta or alpha emitters (e.g. Lutetium-177 or Actinium-225) for radio-ligand therapy. Therefore, there is rising and increasing interest to explore the potential of PET/CT specimen imagers beyond 18F-FDG, applying this high-resolution imaging in patients undergoing surgery for Pca or NET. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 through a dedicated viewer interface (red square). As such the viewer does provide a 3D image of the specimen. Additionally, a 3D render is present, which can be rotated by the user. through a dedicated viewer interface (red square). As such the viewer does provide a 3D image of the specimen. Additionally, a 3D render is present, which can be rotated by the user. Specimen PET/CT imager PET/CT images were obtained using the AURA10® Specimen PET/CT imager (Xeos Medical NV, Belgium). Subsequently, optical, micro-CT and micro-PET images were acquired. Specimens were scanned in a dedicated specimen container (10 cm diameter and 4 cm height) (Figure 1). CT images were reconstructed using the Image Space Reconstruction Algorithm at 100µm voxel size. PET images were reconstructed using 20 iterations of the ordered subset expectation maximization algorithm at 400µm voxel size. The total duration between specimen insertion and visualization of the images on the device was 10 minutes. Figure 1: Overview of the PET/CT specimen imager. On the right is the device with its main constitutes: the medical display (red triangle), the specimen box container (red circle), the viewer interface (red square) and the hardware that allows a two-step acquisition (CT and PET – red star). The viewer of the PET/CT specimen imager shows the images in three different planes (coronal, sagittal and transversal) as shown in the left figure. The user can scroll through each of the planes Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 In all cases, surgical specimens were collected in the operating theater and placed in dedicated containers by the pathologist, who oriented the specimens in the same position adopted for histopathological analysis. Specimen containers were placed in the PET/CT specimen imager and one scan was performed for each specimen. PET/CT acquisitions were performed 2.5 to 4.5 hours post-injection. Such delayed acquisition times can be made due to the high detection sensitivity of the Imager. Of some specimens, several acquisitions were performed, to explore the potential of administering less activity. High-resolution fused 3D images were obtained and reviewed in a multidisciplinary setting involving nuclear medicine physicians, surgeons, and pathologists. Pathologic assessment with immunohistochemistry (PSMA-staining and SSA immunoreactivity) was considered as standard of truth. Specimen images were subsequently compared with baseline PET/CT images and histopathological analysis. Baseline whole-body PET/CT images were performed according to EANM guidelines after an intravenous injection of 2 MBq/kg of 68Ga-PSMA11 and 2,5 MBq/kg of 68Ga-DOTATOC. Baseline scans were performed within six weeks before the day of the surgery (patient-1: 4 weeks; patient- 2; 6 weeks; patient-3: 4 weeks). Description of the surgery procedure This analysis was a secondary objective of two ongoing phase II trials at our institution (NCT05596851; NCT05448157), involving the intra-operative administration of 68Ga-PSMA-11 and 68Ga-DOTATOC for radio-guided surgery with a dedicated beta-probe. Therefore, the activity used is that defined by the protocol of the trials. In this pilot study we demonstrated that high-quality images can be made with the specimen PET/CT imager, using a low activity of 68Ga-PSMA-11 and 68Ga-DOTATOC. The PCa patients received 1 MBq/kg of68Ga-PSMA-11 and the NET patient received 1.2 MBq/kg of 68Ga-DOTA-TOC. PET radiopharmaceutical administration was performed directly in the surgery theater at the beginning of the procedure. For Pca patients a robotic-assisted laparoscopic approach was adopted. Extended pelvic lymph node dissection (ePLND) was performed according to a standard template, including internal/external iliac nodes, obturatory nodes and common iliac ones. Radical prostatectomy (RP) was performed after ePLND, according to the study protocol (NCT05596851). For the ileum GEP-NET patient, open surgery was chosen as surgical approach, according to study protocol (NCT05448157). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 the areas of PSMA-uptake in the micro-PET image corresponded to prostate cancer lesions on histopathological analysis. Extra-prostatic extension, as suspected from the micro-PET images, was also confirmed. Lymph nodes that showed PSMA uptake on micro-PET were confirmed to contain metastatic tumor cells on histopathological analysis. All positive lymph nodes detected in preoperative whole-body PET/CT were resected and imaged. Patient n.2 was referred to PSMA-PET (Figure 3: A,E,I) for high-risk Pca (Gleason Score= 4+5; fusion biopsy, 21/21 positive cores bilateral, initial PSA= 8 ng/mL). PSMA-PET showed pathological and heterogeneous uptake in the whole prostate, with suspicion of extra-capsular extension, two PSMA- positive pelvic lymph nodes and one PSMA-positive extra-pelvic lymph node. According to E-PSMA reporting criteria (12) the patient was considered miT3N1M1a. The day of surgery, 90 MBq of 68Ga- PSMA-11 was administered intravenously. The prostate gland and lymph nodes were resected (Figure 2: B,F,J) and all specimens were scanned in the PET/CT specimen imager. Scans were performed sequentially, once the specimen was positioned in the container and properly oriented by the pathologist, at 206 min (prostate) and 265 min (lymph nodes) post-injection. Images (Figure 3: C,G,K) showed a high spatial resolution, and margin involvement was strongly suspected (Figure 3: C,D), in accordance with baseline PSMA-PET. PET/CT specimen images offered a high-resolution view of the distribution of PSMA inside the prostate lesion, highlighting high heterogeneity of PSMA expression in the prostate. As in the first case, areas of PSMA-uptake in the micro-PET image corresponded to prostate cancer lesions on histopathological analysis and a positive margin – as suspected from the microPET-CT images – was confirmed. Lymph nodes that showed PSMA uptake on micro-PET were confirmed to contain metastatic tumor cells on histopathological analysis. Patient n.3 underwent 68Ga-DOTA-TOC-PET (Figure 4: A) for recurrent ileum GEP-NET (G2; Ki-67= 5%). The PET scan showed the presence of a peri-pancreatic lymph node suspected of disease location. The day of surgery 70 MBq of 68Ga-DOTA-TOC was administered intravenously. The PET- positive lymph node was resected and placed in the PET/CT specimen container. The specimen was scanned in the PET/CT specimen imager (Figure 4: B,C) at 150 minutes post-injection properly showing a high-resolution view of homogeneous 68Ga-DOTA-TOC biodistribution within the lymph node. The specimen was analyzed for histopathology and immune-histochemistry assessment with SSR immune-reactivity (Figure 4: D). RESULTS Patient n.1 was referred to PSMA-PET (Figure 2: A,E) for high-risk Pca (Gleason Score= 4+4; biopsy, 6/6 positive cores bilateral, initial PSA= 10 ng/mL). PSMA-PET showed pathological and heterogeneous uptake diffusely comprising the left part of the prostate, without suspicion of extra- capsular extension. Additionally, five PSMA-positive pelvic lymph nodes were detected. According to E-PSMA reporting criteria (12) the patient was considered miT2N1M0. The day of surgery, 80 MBq of 68Ga-PSMA-11 was administered intravenously. The prostate gland and pelvic lymph nodes were resected and all specimens were scanned in the PET/CT specimen imager. Scans were performed sequentially, once the specimens were positioned in the container and properly oriented by the pathologist, at 192 min (prostate) and 273 min (lymph nodes) post-injection. High-resolution PET/CT images (Figure 2: C,G) showed higher spatial resolution compared to conventional PET images. This indicates a potential for intraoperative margin assessment, complementary to pathological analysis. Left basal margin’s involvement was strongly suspected on the prostate gland image (Figure 2: C,D). All resected lymph nodes were scanned. Histopathology analysis was performed and compared with the specimen PET/CT images (Figure 2: D,H). For the prostate gland, Patient n.1 was referred to PSMA-PET (Figure 2: A,E) for high-risk Pca (Gleason Score= 4+4; biopsy, 6/6 positive cores bilateral, initial PSA= 10 ng/mL). PSMA-PET showed pathological and heterogeneous uptake diffusely comprising the left part of the prostate, without suspicion of extra- capsular extension. Additionally, five PSMA-positive pelvic lymph nodes were detected. According to E-PSMA reporting criteria (12) the patient was considered miT2N1M0. The day of surgery, 80 MBq of 68Ga-PSMA-11 was administered intravenously. The prostate gland and pelvic lymph nodes were resected and all specimens were scanned in the PET/CT specimen imager. Scans were performed sequentially, once the specimens were positioned in the container and properly oriented by the pathologist, at 192 min (prostate) and 273 min (lymph nodes) post-injection. High-resolution PET/CT images (Figure 2: C,G) showed higher spatial resolution compared to conventional PET images. This indicates a potential for intraoperative margin assessment, complementary to pathological analysis. Left basal margin’s involvement was strongly suspected on the prostate gland image (Figure 2: C,D). All resected lymph nodes were scanned. Histopathology analysis was performed and compared with the specimen PET/CT images (Figure 2: D,H). For the prostate gland, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 A specipin was positioned in a region with high radiotracer uptake in the specimen as seen on the high-resolution PET/CT images. The positioning of this pin guided the pathologist to slice the specimen in the region of interest. This allowed an accurate comparison between the 68Ga-DOTA-TOC uptake and the histopathological SSR immune-reactivity. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 All three methodologies (baseline PET/CT, specimen PET/CT and histopathology) were fully concordant, and the lymph node was considered as a true positive finding. Table 1 and Table 2 summarize all semi-quantitative data about radiopharmaceuticals uptake, administered activity and acquisition time both in whole-body PET/CT and high-resolution PET/CT specimen imaging. Table 1 and Table 2 summarize all semi-quantitative data about radiopharmaceuticals uptake, administered activity and acquisition time both in whole-body PET/CT and high-resolution PET/CT specimen imaging. Finally, in all procedures the radiation exposure of the operating theater staff located near the surgical table was measured using an Electronic Personal Dosimeters (Thermo Scientific™ EPD Mk2™) and the absorbed dose was below 40 Sv (range 26 – 40 Sv) for each procedure. Figure 2 Patient-1 (prostate cancer): 68Ga-PSMA-11 whole-body PET/CT image showed pathological PSMA uptake (high expression, E-PSMA Visual Score=3) in the prostate and in five pelvic lymph nodes. In this figure are represented also high-resolution specimen PET/CT images and histopathological analysis in the specimens were immunohistochemical analysis (PSMA staining) was performed. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 Figure 3 Patient-2 (prostate cancer): 68Ga-PSMA-11 whole-body PET/CT images showed pathological PSMA uptake (high expression, E-PSMA Visual Score=3) in the prostate and in three pelvic lymph nodes. In this figure are represented also high-resolution specimen PET/CT images and histopathological analysis in the specimens were immunohistochemical analysis (PSMA staining) was performed. Patient-2 (prostate cancer): 68Ga-PSMA-11 whole-body PET/CT images showed pathological PSMA uptake (high expression, E-PSMA Visual Score=3) in the prostate and in three pelvic lymph nodes. In this figure are represented also high-resolution specimen PET/CT images and histopathological analysis in the specimens were immunohistochemical analysis (PSMA staining) was performed. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 Figure 4 Figure 4 Patient 3 (GEP-NET): 68Ga-DOTA-TOC whole-body PET/CT images showed pathological DOTA-TOC uptake in one single peripancreatic lymph node. High-resolution specimen PET/CT images confirmed the positivity and the presence metastasis from GEP-NET was confirmed by histopathological analysis of the specimen. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 Table 1 Semi-quantitative data measured both in pre-operative whole-body PET/CT and high-resolution specimen PET/CT images. PET/CT SUVmax Specimen Imager SUVmax Pt-1 Prostate 26.5 89.2 Pt-1 Left Obturatory Nodes 11.4 51.5 Pt-2 Prostate 36.2 57.4 Pt-2 Right Common Iliac Node 11.8 23.2 Pt-2 Left Obturatory Nodes 25.9 36.4 Pt-3 Peri-pancreatic Node 52.1 86.6 SUVmax: Maximum Standardized Uptake Value DISCUSSION DISCUSSION DISCUSSION In this pilot analysis, first we assessed the feasibility of integrating this new generation imaging approach in a live-surgery session. The collection and the scan of the specimens did not significantly interfere with any procedure, and the overall length of the surgery was not affected using the PET/CT specimen imager. Second, the radiation exposure generally measured for radio-guided surgery using gamma emitting isotopes (e.g., 99mTc or 18F) is low and this technique is extensively used in clinical practice. With 99mTc radiation doses per procedure to the surgeon’s chest is comprised between 0.2–0.8 µSv per MBq of activity present at time of surgery, while for 18F radiation exposures to the surgeon’s chest is comprised between 10 to 217 µSv for procedures. The radiation exposure for the operating theater staff measured in our pilot analysis was low as well (less than 40 Sv; range 26 – 40 Sv), confirming the feasibility of injecting positron emitting isotopes (e.g., 68Ga or 18F) directly in the surgery theater. Finally, the specimen PET images were reliable, and in full concordance with the information derived by the histopathological analysis. Moreover, they had a higher spatial resolution compared to conventional PET images. The extraction of semiquantitative parameters (e.g., SUVmax or metabolic tumor volume) was also feasible. At present, the implementation in clinical practice of this device has not been broadly explored. However, the development of PET/CT specimen imagers has a promising perspective and embraces the integration of new generation imaging with the most updated surgery techniques. This new approach might improve surgeon’s confidence in ensuring complete removal of the primary tumor. At present, the implementation in clinical practice of this device has not been broadly explored. However, the development of PET/CT specimen imagers has a promising perspective and embraces the integration of new generation imaging with the most updated surgery techniques. This new approach might improve surgeon’s confidence in ensuring complete removal of the primary tumor. Moreover, the precise correspondence of receptor-target radiopharmaceuticals uptake and immuno-histochemistry staining confirmed the precision of this imaging procedure to evaluate intra-lesion heterogeneity. Thus, the high spatial resolution of the PET specimen imager offers a deeper understanding of the bio-distribution pattern of the radiotracers inside cancer lesions. In this scenario, the use of a specimen imager might be an adjunct tool for the pathologist to better optimize resources and for a “live” assessment of specific tumor biomarkers directly during surgery. Table 2 Administered activity and acquisition time differences between standard whole body PET/CT scans and PET/CT specimen imager scans in each patient. Administered activity for baseline PET Acquisition time Baseline PET Administered activity for PET/CT Specimen Imager Acquisition time PET/CT Specimen Imager Patient-1 Pt. 1: 160 MBq 60 min 80 MBq 192-273 min Patient-2 Pt. 2: 180 MBq 60 min 90 MBq 206-275 min Patient-3 Pt. 3: 175 MBq 60 min 70 MBq 150 min Administered activity and acquisition time differences between standard whole body PET/CT scans and PET/CT specimen imager scans in each patient. Administered activity and acquisition time differences between standard whole body PET/CT scans and PET/CT specimen imager scans in each patient. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 the PET/CT specimen imager might detect micro-metastasis. Furthermore, in patient 1 extra- capsular extension was not suspected in baseline PSMA PET/CT. However, extra-capsular extension was observed with the specimen imager (and subsequently confirmed by histology), thus hinting at a potential superior sensitivity compared to the whole-body PET images used in clinical practice. Since surgical margin status is an important prognostic factor in PCa, this is a topic of particular interest. At present, even more deeply studied techniques for surgical margins assessment such as intraoperative frozen section (IFS) for the detection of positive surgical margins in PCa, did not show a significant advantage in terms of oncological outcomes and have still sub-optimal accuracy (13). In clinical practice, new generation molecular imaging has already proven its crucial role in the decision-making process of several malignancies, including PCa and NET, and the transition from pre-clinical imaging to clinical practice has been quick and clinically significant. However, new radiopharmaceuticals are emerging beyond PSMA and SSR based PET, like inhibitors of the fibroblast activating protein (FAPi PET) or ligand of the chemokine receptor 4 CXCR4 (Pentixafor PET). All these new radiopharmaceuticals will increase the number of pathways that can be explored with in vivo molecular imaging, and it will increase the understanding of tumor heterogeneity both in primary tumor and in metastatic locations. In this scenario, intra-operative PET/CT specimen imaging offers a link between molecular biology, histopathology assessment, molecular imaging and radio-guided surgery. DISCUSSION Another potential advantage might be related to the high negative predictive value (NPV) of this imaging procedure. In patient 1 and 2, histopathology confirmed the lymph node showing high PSMA expression as true positive findings. However, also PSMA negative nodes were included in the specimen container. The histopathological analysis confirmed the lack of malignant cells in these lymph nodes, thus resulting as true negative findings. The future challenge will be understanding if LIMITATIONS AND FUTURE PERSPECTIVES This is a pure explorative analysis, as this was the first experience with a PET/CT specimen imager used in prostate cancer and a neuroendocrine tumor in a live-surgery setting. Due to the limited number of patients (n=3), this analysis only provides only a first indication of the possibilities of intraoperative specimen PET/CT imaging for prostate cancer and neuroendocrine tumors. Extensive future research is needed to further investigate the clinical value of this technique. In this perspective, a deeper understanding of radiotracers’ uptake pattern inside surgical specimens through a PET/CT specimen imager could open newer research pathways that range from imaging (e.g detection of surgical margins’ involvement) to pathology (e.g. correlation with immunohistochemistry), to clinical oncology (e.g. changes in uptake patterns after treatment). Finally, nor comparative analysis with other technique (e.g. mp-MRI) or an evaluation of the impact on clinical management of the patients cannot be performed at this stage. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 CONCLUSION The use of the PET/CT specimen imager in a live-experience session was feasible and its application has been easily adapted to surgery daily-practice. In this pilot analysis, the image acquisition of specimens obtained by patients who received an intra-surgical injection of 68Ga-PSMA-11 and 68Ga-DOTA-TOC was feasible and reliable, and imaging findings matched perfectly with histopathological analysis. The high sensitivity, together with the evaluation of intra-lesion tumor heterogeneity, were the most relevant results derived from this pilot analysis. Even if far from definitive evidence, this pilot analysis showed promising results that need to be confirmed by further prospective studies in larger cohorts of oncological patients. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 February 2023 doi:10.20944/preprints202212.0444.v2 REFERENCES 1) Kapoor V, McCook BM, Torok FS. An introduction to PET-CT imaging. Radiographics. 2004 Mar- Apr;24(2):523-43. doi: 10.1148/rg.242025724. PMID: 15026598. 2) Slart RHJA, Tsoumpas C, Glaudemans AWJM, Noordzij W, Willemsen ATM, Borra RJH, Dierckx RAJO, Lammertsma AA. Long axial field of view PET scanners: a road map to implementation and new possibilities. Eur J Nucl Med Mol Imaging. 2021 Dec;48(13):4236-4245. doi: 10.1007/s00259-021- 05461-6. Epub 2021 Jun 16. PMID: 34136956; PMCID: PMC8566640. 3) Göker, M., Vergauwen, G., Van de Velde, C., Van de Vijver, K., Van de Sande, L., Keereman, V., 2021. 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https://www.degruyter.com/document/doi/10.1515/math-2022-0535/pdf
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Study on <i>r</i>-truncated degenerate Stirling numbers of the second kind
Open Mathematics
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* Corresponding author: Taekyun Kim, Department of Mathematics, Kwangwoon University, Seoul 139-701, Republic of Korea, e-mail: tkkim@kw.ac.kr Dae San Kim: Department of Mathematics, Sogang University, Seoul 121-742, Republic of Korea, e-mail: dskim@sogang.ac.kr Hyekyung Kim: Department of Mathematics Education, Daegu Catholic University, Gyeongsan 38430, Republic of Korea, e-mail: hkkim@cu.ac.kr Taekyun Kim*, Dae San Kim, and Hyekyung Kim Study on r-truncated degenerate Stirling numbers of the second kind Taekyun Kim*, Dae San Kim, and Hyekyung Kim https://doi.org/10.1515/math-2022-0535 received June 30, 2022; accepted November 24, 2022 https://doi.org/10.1515/math-2022-0535 received June 30, 2022; accepted November 24, 2022 Abstract: The degenerate Stirling numbers of the second kind and of the first kind, which are, respectively, degenerate versions of the Stirling numbers of the second kind and of the first kind, appear frequently when we study various degenerate versions of some special numbers and polynomials. The aim of this article is to consider the r-truncated degenerate Stirling numbers of the second kind, which reduce to the degenerate Stirling numbers of the second for = r 1, and to investigate their explicit expressions, some properties and related identities, in connection with several other degenerate special numbers and polynomials. Keywords: r-truncated degenerate Stirling numbers of the second kind, r-truncated degenerate Bernoulli polynomials of order alpha MSC 2020: 11B73, 11B83 Open Access. © 2022 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Open Mathematics 2022; 20: 1685–1695 is obtained by truncating the first r terms of the degenerate exponential function (see (8), (10), (35)). In the same way, the r-truncated degenerate Bernoulli polynomials of order α are defined by truncating the first r terms of the degenerate exponential function in their generating function (see (17)). In some works related to truncated degenerate special numbers and polynomials, we let the reader refer to [4,6,7,15] and refer- ences therein. The aim of this article is by using generating functions to study their explicit expressions, some proper- ties and related identities on the r-truncated degenerate Stirling numbers of the second kind, in connection with the r-truncated degenerate Bernoulli polynomials of order α, the degenerate Bernoulli numbers, the degenerate Stirling numbers of the second kind and the degenerate Bernoulli numbers of order α. The outline of this article is as follows. In Section 1, we recall the degenerate exponentials and loga- rithms. We remind the reader of the degenerate Stirling numbers of both kinds. Furthermore, we recall the degenerate Bernoulli polynomials of order α. Section 2 gives the main results of this article. We consider the r-truncated degenerate Stirling numbers of the second kind and find three explicit expressions for those numbers in Theorems 1–3. Then we introduce the r-truncated degenerate Bernoulli polynomials of order α and obtain some results in connection with the r-truncated degenerate Stirling numbers of the second kind. In Theorem 4, the degenerate Bernoulli numbers are expressed in terms of the Stirling numbers of the second kind. In Theorems 5 and 8, some identities involving the r-truncated degenerate Stirling numbers of the second kind (for r = 2 and r = 3, respectively), the degenerate Bernoulli numbers and the degenerate Stirling numbers of the second are obtained. In Theorem 6, we find an identity connecting the 2-truncated degenerate Stirling numbers of the second kind and the degenerate Bernoulli numbers. In Theorem 7, we obtain an identity relating the 2-truncated degenerate Stirling numbers of the second kind and the degen- erate Bernoulli numbers of order α. In the rest of this section, we recall the facts that are needed throughout this article. For any nonzero  ∈ λ , the degenerate exponentials are defined by he degenerate exponentials are defined by For any nonzero  ∈ λ , the degenerate exponentials are defined by ( ) ( ) ( ) ( ) ∑ = ! 1 Introduction Carlitz [1,2] initiated to study of the degenerate Bernoulli and Euler polynomials and numbers, which are degenerate versions of the Bernoulli and Euler polynomials and numbers. In recent years, studying degen- erate versions of some special numbers and polynomials has regained interests of some mathematicians and yielded quite a few interesting results (see [3–11] and references therein). It is noteworthy that studying degenerate versions is not only limited to polynomials but also extended to transcendental functions, like gamma functions (see [12,13]). It is also remarkable that the degenerate umbral calculus is introduced as a degenerate version of the classical umbral calculus (see [14]). Degenerate versions of special numbers and polynomials have been explored by various methods, including combinatorial methods, generating func- tions, umbral calculus techniques, p-adic analysis, differential equations, special functions, probability theory and analytic number theory. The Stirling number of the second ( ) S n k , 2 is the number of ways to partition a set of n objects into k nonempty subsets. The (signed) Stirling number of the first kind ( ) S n k , 1 is defined so that the number of permutations of n elements having exactly k cycles is the nonnegative integer ( ) ( ) ∣( )∣ − = −S n k S n k 1 , , n k 1 1 . The degenerate Stirling numbers of the second kind ( ) S n k , λ 2, (see (5), (8)) and of the first kind ( ) S n k , λ 1, (see (4), (7)) were introduced as degenerate versions of the Stirling numbers of the second and of the first kind, respectively. These degenerate Stirling numbers of both kinds appear frequently when we study degenerate versions of some special numbers and polynomials. Here we consider the r-truncated degenerate Stirling numbers of the second which reduce to the degenerate Stirling numbers of the second for = r 1. We note that the generating function of these numbers 1686  Taekyun Kim et al. 1686 λ α λ x n n λ α n 0 , (3) (3) When = x 0, ( ) ( ) ( ) = β β 0 n λ α n λ α , , are called the degenerate Bernoulli numbers of order α. In particular, for = α 1, ( ) ( ) ( ) = β x β x n λ n λ , , 1 are called the degenerate Bernoulli polynomials. For ≥ n 0, the degenerate Stirling numbers of the first kind are defined by When = x 0, ( ) ( ) ( ) = β β 0 n λ α n λ α , , are called the degenerate Bernoulli numbers of order α. In particular, for = α 1, ( ) ( ) ( ) = β x β x n λ n λ , , 1 are called the degenerate Bernoulli polynomials. For ≥ n 0, the degenerate Stirling numbers of the first kind are defined by When = x 0, ( ) ( ) ( ) = β β 0 n λ α n λ α , , are called the degenerate Bernoulli numbers of order α. In particular, for = α 1, ( ) ( ) ( ) = β x β x n λ n λ , , 1 are called the degenerate Bernoulli polynomials. For ≥ n 0, the degenerate Stirling numbers of the first kind are defined by ( ) ( )( ) ( [ ]) ∑ = = x S n k x , see 3 , n k n λ k λ 0 1, , (4) (4) where ( ) ( ) ( ) ( ) ( ) ( [ ]) = = − ⋯ − + ≥ x x x x x n n 1, 1 1 , 1 see 1–11, 14–22 . n 0 formula of (4), the degenerate Stirling numbers of the second kind are defined by As the inversion formula of (4), the degenerate Stirling numbers of the second kind ar ( ) ( )( ) ( ) ( [ ]) ∑ = ≥ = x S n k x n , , 0 see 3 . = + [ ] = ∞ e t x k t λt 1 see 3–7 , λ x k k λ k 0 , x λ (1) ( ) ( ) ( )( ) ( ( ) ) ( ) = = − − ⋯ − − ≥ x x x x λ x λ x n λ n 1, 2 1 , 1 . λ n λ 0, , (2) ( ) ( ) ( ) ( ) ∑ = ! = + [ ] = ∞ e t x k t λt 1 see 3–7 , λ x k k λ k 0 , x λ (1) ( ) ( )( ) ( ( ) ) ( ) ≥ x x x λ x λ x n λ n 2 1 1 (2) ( ) ( ) ( ) ( ) ∑ = ! = + [ ] = ∞ e t x k t λt 1 see 3–7 , λ x k k λ k 0 , x λ (1) (1) ( ) ( ) ( )( ) ( ( ) ) ( ) = = − − ⋯ − − ≥ x x x x λ x λ x n λ n 1, 2 1 , 1 . λ n λ 0, , (2) (2) 1, ( ) ( ) ( ) = = + e t e t λt 1 λ λ 1 λ 1 (see [8,15]). When = x 1, ( ) ( ) ( ) = = + e t e t λt 1 λ λ 1 λ 1 (see [8,15]). When = x 1, ( ) ( ) ( ) = = + e t e t λt 1 λ λ 1 λ 1 (see [8,15]). In [1,2], Carlitz considered the degenerate Bernoulli polynomials of order α given by ( ) ( ) ( ) λ λ ( [ ]) In [1,2], Carlitz considered the degenerate Bernoulli polynomials of order α given by ⎜ ⎟ ⎛ ⎝ ( ) ⎞ ⎠ ( ) ( ) ( ) ∑ − = ! = ∞ t e t e t β x t n 1 . 2 r-Truncated degenerate Stirling numbers of the second kind ! ⋯ ! + +⋯+ = ≥ S n kr k n l l l , 1 1 1 1 . λ r l l l n l r l λ l λ l λ k 2, , , , 1 2 k i k 1 2 1 2 Theorem 1. For ≥ n k , 0 with ≥ n kr, we have Theorem 1. For ≥ n k , 0 with ≥ n kr, we have Theorem 1. For ≥ n k , 0 with ≥ n kr, we have ( ) ( ) ( ) ( ) [ ] ∑ = ! ! ⋯ ! ! ⋯ ! + +⋯+ = ≥ S n kr k n l l l , 1 1 1 1 . λ r l l l n l r l λ l λ l λ k 2, , , , 1 2 k i k 1 2 1 2 From the binomial expansion, we note that ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ( ) ( ) ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ ( )( ) ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ⎛ ⎝ ⎜ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ( ) ⎞ ⎠ ⎟ ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑ ! − ! = ! − ! = ! − ! − ⋯ ! ! ⋯ ! = ! − ! ⋯ − ! ! ⋯ ! − −⋯− ! ! = − = − = − = = ∞ … = − +⋯+ = ∞ = … = − −−−⋯− k e t l t k k m e t l t k k m k m t j l l l t k k m n k m l l l n l l t n 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 . n n (9) Let  ( ) [[ ]] = ∑ ∈ = ∞ f t a t t k k k 0 . For ≥ n 0, the operator [ ] tn is defined by [ ] ( ) ( ) ( [ ]) = ≥ t f t a n , 0 see 22 . n n (9) Let  ( ) [[ ]] = ∑ ∈ = ∞ f t a t t k k k 0 . For ≥ n 0, the operator [ ] tn is defined by [ ] ( ) ( ) ( [ ]) = ≥ t f t a n , 0 see 22 . n n (9) (9) n λ k n λ k , 0 2, (5) (5) The degenerate logarithm ( )t logλ is the compositional inverse of ( ) e t λ satisfying ( ( )) ( ( )) = e t e t log log λ λ λ λ . Then we have The degenerate logarithm ( )t logλ is the compositional inverse of ( ) e t λ satisfying ( ( )) ( ( )) = e t e t log log λ λ λ λ . Then we have Study on r-truncated degenerate Stirling numbers of the second kind  1687 Study on r-truncated degenerate Stirling numbers of the second kind  1687 ( ) (( ) ) ( ) ( [ ]) ∑ + = + − = ! = ∞ − t λ t λ n t log 1 1 1 1 1 see 3 . λ λ n n n n 1 1 , λ 1 (6) (6) From (4) and (5), we note that From (4) and (5), we note that From (4) and (5), we note that ( ( )) ( ) ( ) ∑ ! + = ! ≥ = ∞ k t S n k t n k 1 log 1 , , 0 λ k n k λ n 1, (7) ( ( )) ( ) ( ) ∑ ! + = ! ≥ = ∞ k t S n k t n k 1 log 1 , , 0 λ k n k λ n 1, (7) (7) ! ! = k n n k and ( ( ) ) ( ) ( [ ]) ∑ ! − = ! = ∞ k e t S n k t n 1 1 , see 3 . λ k n k λ n 2, (8) and ( ( ) ) ( ) ( [ ]) ∑ ! − = ! = ∞ k e t S n k t n 1 1 , see 3 . λ k n k λ n 2, (8) (8) Let  ( ) [[ ]] = ∑ ∈ = ∞ f t a t t k k k 0 . For ≥ n 0, the operator [ ] tn is defined by [ ] ( ) ( ) ( [ ]) = ≥ t f t a n , 0 see 22 . 2 r-Truncated degenerate Stirling numbers of the second kind For  ∈ r , we consider the r-truncated degenerate Stirling numbers of the second kind given by ⎜ ⎟ ⎛ ⎝ ( ) ( ) ⎞ ⎠ ( ) ( ) [ ] ∑ ∑ ! − ! = ! ≥ = − = ∞ k e t l t S n kr t n k 1 1 , , 0 . λ l r l λ l k n kr λ r n 0 1 , 2, (10) (10) (10) We agree that ( ) [ ] = S n kr , 0 λ r 2, , for ≤ < n kr 0 . Note that ( ) ( ) ( ) [ ] = ≥ S n k S n k n k , , , , 0 λ λ 2, 1 2, . From (10), we note that We agree that ( ) [ ] = S n kr , 0 λ r 2, , for ≤ < n kr 0 . Note that ( ) ( ) ( ) [ ] = ≥ S n k S n k n k , , , , 0 λ λ 2, 1 2, . From (10), we note that ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ( ) ( ) ⎞ ⎠ ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ⎜ ⎜⎜ ( ) ( ) ( ) ⎞ ⎠ ⎟ ⎟⎟ ∑ ∑ ∑ ∑ ! − ! = ! ! = ! ! ⋯ ! ! ⋯ ! ! = − = ∞ = ∞ + +⋯+ = ≥ k e t l t k l t k n l l l t n 1 1 1 1 1 1 1 1 . λ l r l λ l k l r k λ l k n kr l l l n l r l λ l λ l λ k n 0 1 , , , , , 1 2 k i k 1 2 1 2 (11) (11) ⎝ ⎠ ≥ l r i Thus, by (9) and (11), we obtain the following theorem. Thus, by (9) and (11), we obtain the following theorem. Thus, by (9) and (11), we obtain the following theorem. heorem 1. For ≥ n k , 0 with ≥ n kr, we have ( ) ( ) ( ) ( ) [ ] ∑ = ! ! ⋯ ! Therefore, by comparing the coefficients on both sides of (12), we obtain the following theorem. Theorem 2. For ≥ n k , 0, we have Theorem 2. For ≥ n k , 0, we have Theorem 2. For ≥ n k , 0, we have Theorem 2. For ≥ n k , 0, we have ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ( ) ⎧ ⎨⎩ ( ) [ ] ∑ ∑ ! − ! ⋯ − ! ! ⋯ ! − −⋯− ! = ≥ ≤ < = … = − −−−⋯− k k m n k m l l l n l l S n kr if n kr if n kr 1 1 1 1 1 , , , 0, 0 . m k m l l l r l λ l λ l λ n l l l m m λ r 0 , , , 0 1 , , , 1 2 1 2, m m m λ 1 2 1 2 1 2 , When = k 1 in (10), we have ⎜ ⎟ ( ) ⎛ ⎝ ( ) ( ) ⎞ ⎠ ( ) ( )( ) ( ) ( ) [ ] [ ] [ ] ∑ ∑ ∑ ∑ ∑ ! = − ! = ! = + + ! = + ! + ! ! = ∞ = − = ∞ = ∞ + = ∞ l t e t l t S n r t n S n r r t n r t S n r r n n r t n 1 1 , , , . l r l λ l λ l r l λ l n r λ r n n λ r n r r n λ r n , 1 1 , 2, 0 2, 0 2, (13) (13) For = k 2, we have For = k 2, we have For = k 2, we have For = k 2, we have ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ⎜ ( )( ) ⎞ ⎠ ⎟ ⎛ ⎝ ( )( ) ⎞ ⎠ ⎛ ⎝ ⎜ ( ) ( ) ( ) ( ) ⎞ ⎠ ⎟ [ ] [ ] [ ] [ ] ∑ ∑ ∑ ∑∑ ! = + + ! + + ! 2 r-Truncated degenerate Stirling numbers of the second kind λ l r l λ l k m k λ k m m l r l λ l m m k j j λ j m l l l r l λ l λ l λ m l l n m k m l l l r l λ l λ l λ n l l l m m n 0 1 , 0 0 1 , 0 0 , , , , 0 1 , , , 1 2 0 0 , , , 0 1 , , , 1 2 1 m m m m m m λ 1 2 1 2 1 1 2 1 2 1 2 , (12) (12) Taekyun Kim et al. 1688  Taekyun Kim et al. 1688  Taekyun Kim et al. 1688 = + − + ! − + ! + ! ! = ∞ = ∞ = ∞ = ∞ = l t t S j r r t j r S l r r t l r t S j r r S n j r r n n j r j r t n 1 , , , , . l r l λ l r j λ r j l λ r l r n j n λ r λ r n , 2 2 0 2, 0 2, 2 0 0 2, 2, (14) ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ⎜ ( )( ) ⎞ ⎠ ⎟ ⎛ ⎝ ( )( ) ⎞ ⎠ [ ] [ ] ∑ ∑ ∑ ! = + + ! + + ! = ∞ = ∞ = ∞ l t t S j r r t j r S l r r t l r 1 , , l r l λ l r j λ r j l λ r l , 2 2 0 2, 0 2, (14) Continuing this process, we have Continuing this process, we have ⎜ ⎟ ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ⎜ ( ) ( ) ( ) ( ) ( ) ( ) ⎞ ⎠ ⎟ [ ] [ ] [ ] ∑ ∑ ∑ ! = ! + + ⋯ + + ! + ! ⋯ + ! ! = ∞ = ∞ + +⋯+ = l t t n S j r r S j r r S j r r j r j r j r t n 1 , , , . l r l λ l k kr n j j j n λ r λ r λ r k k n , 0 2, 1 2, 2 2, 1 2 k 1 2 (15) ⎜ ⎟ ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ⎜ ( ) ( ) ( ) ( ) ( ) ( ) ⎞ ⎠ ⎟ [ ] [ ] [ ] ∑ ∑ ∑ ! = ! + + ⋯ + + ! + ! ⋯ + ! ! = ∞ = ∞ + +⋯+ = l t t n S j r r S j r r S j r r j r j r j r t n 1 , , , . l r l λ l k kr n j j j n λ r λ r λ r k k n , 0 2, 1 2, 2 2, 1 2 k 1 2 (15) ⎜ ⎟ ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ⎜ ( ) ( ) ( ) ( ) ( ) ( ) ⎞ ⎠ ⎟ [ ] [ ] [ ] ∑ ∑ ∑ ! = ! + + ⋯ + + ! + ! ⋯ + ! ! = ∞ = ∞ + +⋯+ = l t t n S j r r S j r r S j r r j r j r j r t n 1 , , , . l r l λ l k kr n j j j n λ r λ r λ r k k n , 0 2, 1 2, 2 2, 1 2 k 1 2 (15) (15) On the other hand, by (10), we obtain On the other hand, by (10), we obtain On the other hand, by (10), we obtain ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ( ) ⎞ ⎠ ⎛ ⎝ ( ) ( ) ⎞ ⎠ ( ) ( )( ) ( ) ( ) [ ] [ ] [ ] ∑ ∑ ∑ ∑ ∑ ! = − ! = ! ! = ! + + ! = ! + + ! = ∞ = − = ∞ = ∞ + = ∞ t l e t l t k S n kr t n k S n kr kr t n kr t k S n kr kr n kr t 1 1 , , , . l r l l λ k λ l r l λ l k n kr λ r n n λ r n kr kr n λ r n , 1 1 , 2, 0 2, 0 2, (16) (16) Therefore, by (15) and (16), we obtain the following theorem. Therefore, by (15) and (16), we obtain the following theorem. Therefore, by (15) and (16), we obtain the following theorem. Theorem 3. For ≥ n k , 0, we have ( ) ( ) ( ) ( ) [ ] [ ] [ ] [ ] ∑ ! + = + + ⋯ + k S n kr kr S j r r S j r r S j r r , , , r λ r λ r λ r k 2, 1 2, 2 2, ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) [ ] [ ] [ ] [ ] ∑ ! + ! + = + + ⋯ + + ! + ! ⋯ + ! + +⋯+ = k n kr S n kr kr S j r r S j r r S j r r j r j r j r , , , , . λ r j j j n λ r λ r λ r k k 2, 2, 1 2, 2 2, 1 2 k 1 2 Let us consider the r-truncated degenerate Bernoulli polynomials of order α given by ( ( ) ( ) ) ( ) ( ) [ ] ∑ −∑ = ! = − ! = ∞ − t e t e t β x t n 1 . αr λ l r t l l λ α λ x n n λ r α n 0 1 , 0 , 1, l (17) (17) When = x 0, ( ) [ ] [ ] = − − β β 0 n λ r α n λ r α , 1, , 1, are called the r-truncated degenerate Bernoulli numbers of order α. Study on r-truncated degenerate Stirling numbers of the second kind  1689 ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ( ) ( ) ⎞ ⎠ ( ) ⎛ ⎝ ⎜ ( ) ( ) ⎞ ⎠ ⎟ ⎛ ⎝ ⎛ ⎝ ⎞ ⎠ ( ) ⎞ ⎠ [ ] [ ] [ ] [ ] [ ] [ ] [ ] ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑ = − ! ! = ! ! ! = ! − ! − ! ! ! = ! − ! = − = ∞ − = ∞ = ∞ − = ∞ = − − = ∞ = − − t e t t l β t l α S j αr t j β t l α S n l αr n n l l β t n α n l S n l αr β t n 1 , , , , αr λ l r l l λ α l l λ r α l j αr λ r j l l λ r α l n αr l n αr λ r l λ r α n n αr l n αr λ r l λ r α n 0 1 , 0 , 1, 2, 0 , 1, 0 2, , 1, 0 2, , 1, (18) ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ( ) ( ) ⎞ ⎠ ( ) ⎛ ⎝ ⎜ ( ) ( ) ⎞ ⎠ ⎟ ⎛ ⎝ ⎛ ⎝ ⎞ ⎠ ( ) ⎞ ⎠ [ ] [ ] [ ] [ ] [ ] [ ] [ ] ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑ = − ! ! = ! ! ! = ! − ! − ! ! ! = ! − ! = − = ∞ − = ∞ = ∞ − = ∞ = − − = ∞ = − − t e t t l β t l α S j αr t j β t l α S n l αr n n l l β t n α n l S n l αr β t n 1 , , , , αr λ l r l l λ α l l λ r α l j αr λ r j l l λ r α l n αr l n αr λ r l λ r α n n αr l n αr λ r l λ r α n 0 1 , 0 , 1, 2, 0 , 1, 0 2, , 1, 0 2, , 1, (18) where α is a positive integer. Thus, by (18), we obtain ⎛ ⎝ ⎞ ⎠ ( ) ⎧ ⎨ ⎩ ( ) [ ] [ ] ∑ − = ! ! = > = − − n l S n l αr β αr α n αr n αr , , if , 0, if . n k n k n k n k 1 2 1 1 2 1 1 2 1 {( )∣ ( ) } = … + + ⋯+ = + + ⋯+ − + = −+ −+ −+ k k k k k k k k k n k k n Λ , , , , 2 1 . n k n k n k n k 1 2 1 1 2 1 1 2 1 h {( )∣ ( ) } = … + + ⋯+ = + + ⋯+ − + = −+ −+ −+ k k k k k k k k k n k k n Λ , , , , 2 1 . n k n k n k n k 1 2 1 1 2 1 1 2 1 h We observe that ⎜ ⎟ ⎜ ⎟ ( ) ( ) ( ) ( ) ⎛ ⎝ ( ) ⎞ ⎠ ( ) ( ( ) ( ) ( ) ) ⎛ ⎝ ( ) ( ( ) ( ) ( ) )⎞ ⎠ ∑ ∑ ∑ ∑ ∑∑ + + + ⋯+ + ⋯ = − ! ! ! = + − ! … ! = + − ! … ! ! ! ! = ∞ = ∞ = ∞ = ∞ −+ −+ = ∞ = −+ t t t k k x l t k B x x x t n k B x x x t n 1 1 1 1 1 1 1 1 1 1 1 , 1 , , 1 1 1 1 , 1 , , . x λ x λ x k k λ k k k l l l λ l k k k n k n k λ λ n k n k n n k n k n k λ λ n k λ n 1 1, 2 2, 2 , 0 1 , 1 , 1 1, 2 2, 1 1 1 1 , 1 1, 2 2, 1, k 1 2 (19) ⎜ ⎟ ⎜ ⎟ ( ) ⎝ ⎠ ( ) ( ( ) ( ) ( ) ) ⎛ ⎝ ( ) ( ( ) ( ) ( ) )⎞ ⎠ ∑ ∑ ∑ ∑ ∑∑ = − ! ! ! = + − ! … ! = + − ! … ! l n αr λ r l λ r α 0 2, , 1, It is well known that the partial Bell polynomials are defined by It is well known that the partial Bell polynomials are defined by ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) ∑ ∑ ! ! = … ! = ∞ = ∞ −+ k x t l B x x x t n 1 , , , , l l l k n k n k n k n 1 , 1 2 1 where k is a nonnegative integer. Thus, we note that Thus, we note that ⎜ ⎟ ⎜ ⎟ ( ) ⎛ ⎝ ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ ⎛ ⎝( ) ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ ( ) ∑ ∑∏ … = ! ! ! ⋯ ! ! ! ⋯ − + ! = ! ! ! −+ + +⋯+ = + +⋯+ −+ = −+ −+ = −+ −+ −+ −+ B x x x n l l l x x x n k n k x j , , , 1 2 1 1 , n k n k l l l k l l n k l n n k l l n k l j n k j j k , 1 2 1 2 1 1 2 1 1 2 1 Λ 1 1 n k n k n k n k j 1 2 1 1 2 1 1 2 1 ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ∑∏ = ! ! ! = −+ n k x j 1 , j n k j j k Λ 1 1 n k j where {( )∣ ( ) } = … + + ⋯+ = + + ⋯+ − + = −+ −+ −+ k k k k k k k k k n k k n Λ , , , , 2 1 . n k n k n k n k 1 2 1 1 2 1 1 2 1 {( )∣ ( ) } = … + + ⋯+ = + + ⋯+ − + = −+ −+ −+ k k k k k k k k k n k k n Λ , , , , 2 1 . 1690 1690  Taekyun Kim et al. Then, by (19) and (20), we obtain Then, by (19) and (20), we obtain Then, by (19) and (20), we obtain ( ) ( ) ( ( ) ( ) ( ) ) ( ) ∑ … = − ! … ≥ = −+ −+ K x x k B x x x n , , 1 1 , 1 , , 1 , 1 , n λ k n k n k λ λ n k n k λ , 1 2 1 , 1 1, 2 2, 1 1, ( ) ( ) ( ( ) ( ) ( ) ) ( ) ( ) ∑ … = − ! … ≥ … = = −+ −+ K x x k B x x x n K x x , , 1 1 , 1 , , 1 , 1 , , , 1. n λ k n k n k λ λ n k n k λ λ , 1 2 1 , 1 1, 2 2, 1 1, 0, 1 2 ( ) … = K x x , , 1. λ 0, 1 2 Note that, from (19) and (20), we have Note that, from (19) and (20), we have ( ) ( ) (( ) ( ) ( ) ) ( ) ( ) ∑ ∑ … = − ! … = − ! = −+ = K k B k S n k 1, 1, 1 1 , 1 , , 1 1 , . n λ k n k n k λ λ n k λ k n k λ , 0 , 1, 2, 1, 0 2, Taking = α 1 in (17), we have Taking = α 1 in (17), we have ( ) ( ) ( ) ( ) [ ] ∑ −∑ = ! = − ! = ∞ − t e t t e t β x t n . r λ l r l l λ x n n λ r n 0 1 1 0 , 1,1 l λ , (21) ( ) ( ) ( ) ( ) [ ] ∑ −∑ = ! = − ! = ∞ − t e t t e t β x t n . = = = ∞ = ∞ −+ −+ = ∞ = −+ k k l t k B x x x t n k B x x x t n 1 1 1 1 , 1 , , 1 1 1 1 , 1 , , . k k l l k k n k n k λ λ n k n k n n k n k n k λ λ n k λ n 0 1 1 , 1 1, 2 2, 1 1 1 1 , 1 1, 2 2, 1, (19) (19) ⎜ ⎟ ⎝ ⎠ ( ) ( ( ) ( ) ( ) ) ⎛ ⎝ ( ) ( ( ) ( ) ( ) )⎞ ⎠ ∑ ∑ ∑∑ ! ! = + − ! … ! = + − ! … ! = = = ∞ = ∞ −+ −+ = ∞ = −+ k l k B x x x t n k B x x x t n 1 1 1 , 1 , , 1 1 1 1 , 1 , , . k l k k n k n k λ λ n k n k n n k n k n k λ λ n k λ n 0 1 1 , 1 1, 2 2, 1 1 1 1 , 1 1, 2 2, 1, (19) We denote (19) by We denote (19) by ( ) ( ) ∑ ∑ = … ! = ∞ ! = ∞ x t K x x t n 1 , , . n n n n n n λ n 0 1 0 , 1 2 n λ , (20) ( ) ( ) ∑ ∑ = … ! = ∞ ! = ∞ x t K x x t n 1 , , . n n n n n n λ n 0 1 0 , 1 2 n λ , (20) (20) λ x r λ l r l λ l l l λ r l r j r j λ j l l λ r l j j r λ j l l λ r l n j n j r λ n j λ r n 1 1 , 0 , 1,1 , 0 , 1,1 0 , 0 , 1,1 0 0 , , 1,1 (22) By (1) and (22), we obtain By (1) and (22), we obtain ⎜ ⎟ ( ) ⎛ ⎝ ⎞ ⎠( ) ( ) ( ) ( ) [ ] ∑ = ! + ! ≥ = + − − x n j j j r β x n 1 , 0 . n λ j n j r λ n j λ r , 0 , , 1,1 (23) ⎜ ⎟ ( ) ⎛ ⎝ ⎞ ⎠( ) ( ) ( ) ( ) [ ] ∑ = ! + ! ≥ = + − − x n j j j r β x n 1 , 0 . n λ j n j r λ n j λ r , 0 , , 1,1 (23) (23) In particular, for = n 0, [ ] ( ) = − ! β λ r r 0, 1,1 1 r λ , . By (23), we obtain In particular, for = n 0, [ ] ( ) = − ! β λ r r 0, 1,1 1 r λ , . By (23), we obtain n particular, for = n 0, [ ] ( ) = − ! β λ r r 0, 1,1 1 r λ , . By (23), we obtain ( ) ⎛ ⎝ ⎞ ⎠ ( ) ⎧ ⎨⎩ ( ) ( ) ( ) ( ) ( )( ) ( )( ( ) )⎫ ⎬⎭ [ ] [ ] = ! − − + = ! − + − + − − + + + + − − + − − β r x rλ r β r x x λ x rλ r rλ r r r rλ r λ 1 1 1 , 1 2 1 1 2 1 1 2 1 2 1 1 1 . λ r r λ λ r r λ 1, 1,1 , 2, 1,1 , 2 2 In particular, for = n 0, [ ] ( ) = − ! β λ r r 0, 1,1 1 r λ , . By (23), we obtain By (23), we obtain ( ) ⎛ ⎝ ⎞ ⎠ ( ) ⎧ ⎨⎩ ( ) ( ) ( ) ( ) ( )( ) ( )( ( ) )⎫ ⎬⎭ [ ] [ ] = ! − − + = ! − + − + − − + + + + − − + − − β r x rλ r β r x x λ x rλ r rλ r r r rλ r λ 1 1 1 , 1 2 1 1 2 1 1 2 1 2 1 1 1 . λ r r λ λ r r λ 1, 1,1 , 2, 1,1 , 2 2 ( ) ⎛ ⎝ ⎞ ⎠ [ ] = ! − − + − β r x rλ r 1 1 1 , λ r r λ 1, 1,1 , From (6), we note that ( ) ( ) ( ) ( ) ∑ ∑ + = ! = + ! = ∞ − = ∞ + t t t λ n t λ n t 1 log 1 1 1 1 1 . λ n n n n n n n n 1 1 , 0 1, λ λ 1 1 (24) (24) Replacing t by ( ) − e t 1 λ in (24), we obtain Replacing t by ( ) − e t 1 λ in (24), we obtain ( ) ⎛ ( ) ⎞ λ λ 1 1 k n k Replacing t by ( ) − e t 1 λ in (24), we obtain ( ) ( ) ( ) ( ( ) ) ⎛ ⎝ ⎜ ( ) ( )⎞ ⎠ ⎟ ∑ ∑ ∑∑ ! = = + ! − = + ! ∞ ∞ + ∞ + β t n t e t λ k k e t λ k S n k t n 1 1 1 1 1 1 1 , . n λ n k k λ k n k k λ n , 1, 1, 2, λ λ 1 1 (25) ( ) ( ) ( ) ( ( ) ) ⎛ ⎝ ⎜ ( ) ( )⎞ ⎠ ⎟ ∑ ∑ ∑∑ ! = − = + ! − = + ! r λ l r l l λ x n n λ r n 0 1 1 0 , 1,1 l λ , (21) When = r 1, ( ) ( ) [ ] = β x β x n λ n λ , 0,1 , are the degenerate Bernoulli polynomials. From (21), we note that When = r 1, ( ) ( ) [ ] = β x β x n λ n λ , 0,1 , are the degenerate Bernoulli polynomials. From (21), we note that When = r 1, ( ) ( ) [ ] = β x β x n λ n λ , 0,1 , are the degenerate Bernoulli polynomials. From (21), we note that When = r 1, ( ) ( ) [ ] = β x β x n λ n λ , 0,1 , are the degenerate Bernoulli polynomials. From (21), we note that When = r 1, ( ) ( ) [ ] = β x β x n λ n λ , 0,1 , are the degenerate Bernoulli polynomials. From (21), we note that ⎜ ⎟ ⎜ ⎟ ( ) ⎛ ⎝ ( ) ( ) ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ( ) ⎛ ⎝ ⎜ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( )⎞ ⎠ ⎟ [ ] [ ] [ ] [ ] ∑ ∑ ∑ ∑ ∑ ∑ ∑∑ = − ! ! = ! ! = + ! ! = ! + ! ! = − = ∞ − = ∞ = ∞ − = ∞ + = ∞ − = ∞ = + − − e t t e t l t β x t l t j t β x t l j r t β x t l n j j j r β x t n 1 1 1 1 1 1 . = ∞ = ∞ + = ∞ = + β t n t e t λ k k e t λ k S n k t n 1 1 1 1 1 1 1 , . n n λ n λ k k k λ k n k n k k λ n 0 , 0 1, 0 0 1, 2, λ λ 1 1 (25) ( ) ( ) ( ) ( ( ) ) ⎛ ⎝ ⎜ ( ) ( )⎞ ⎠ ⎟ ∑ ∑ ∑∑ ! = − = + ! − = + ! = ∞ = ∞ + = ∞ = + β t n t e t λ k k e t λ k S n k t n 1 1 1 1 1 1 1 , . n n λ n λ k k k λ k n k n k k λ n 0 , 0 1, 0 0 1, 2, λ λ 1 1 (25) (25) Comparing the coefficients on both sides of (25), we have Comparing the coefficients on both sides of (25), we have Comparing the coefficients on both sides of (25), we have ( ) ( ) ( ) ∑ = + ≥ = + β λ k S n k n 1 1 , , 0 . n λ k n k k λ , 0 1, 2, λ 1 (26) (26) Study on r-truncated degenerate Stirling numbers of the second kind  1691 Study on r-truncated degenerate Stirling numbers of the second kind  1691 j n λ j λ j n λ j λ j n n j k λ k j λ j n n j λ k k j λ n λ k k λ n j k k j λ j k j k λ k n λ j k k j n λ j j k n λ j k k j n λ j j k n λ j k k j n l λ l k n λ j k k j λ 0 2, 2 , 0 2, 2 , 0 0 0 1 1 1 , 1 1 1 , 1 0 2, , 1 2, (29) ⎜ ⎟ ( )⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) [ ]( ( ) ) [ ] ( ) ( ) [ ]( ( ) ) [ ] ( ) ( ( ) ) [ ] [ ] ∑ ∑ ∑ ∑ − + + = + ! − + − + ! ! = + ! − − ! − = + ! − − ! − = = = −+ = − S n j k k n k j β n k S n j k k n j k β j n k t e t t k t t e t n k t e t t k t t t e t t t t , 2 , 2 1 1 1 1 1 j n λ j λ j n λ j λ j n n j k λ k j λ j n n j λ k k j λ k 0 2, 2 , 0 2, 2 , 0 0 (29) ⎜ ⎟ ( ) [ ] ⎛ ⎝ ⎞ ⎠ ( ) ( ( ) ) ( ( ) ) ( ) ( ) [ ] ( ) ( ) ( ) [ ] ( ( ) ) ( ) ( ) ( ) ( ) ( ) ( ) [ ]( ( ) ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) [ ] ( ) ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ∑ ∑ ∑ ∑ ∑ ∑ = + ! − − ! − = − + ! ! − + + ! − + + ! − − ! − ! = − + ! ! ! + + ! − ! − ! − = − + + + ! − − ! ! − ! + − − + − ! = − + + + ! − − ! + − − + − ! = = = −+ = − = − − = − − − = − − − = − = ∞ = − S n j k k n k j β n k S n j k k n j k β j n k t e t t k t t e t n k t e t t k t t t e t n k t e t t t k t e t n k t k j e t t t k t e t n k k t t e t n k t e t j k j t n k k β n n k j k j t e t t n k k β n k j j k j t S l j j l j t n k k β n k j k j S n j j n j , 2 , 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1, 1 1 1 1 1, 1 1 . Therefore, by (29), we obtain the following theorem. Replacing t by ( ) + t log 1 λ in (3), with = = α x 1, 0, we have Replacing t by ( ) + t log 1 λ in (3), with = = α x 1, 0, we have Replacing t by ( ) + t log 1 λ in (3), with = = α x 1, 0, we have ⎜ ⎟ ( ) ( ) ( ) ( ( )) ( ) ⎛ ⎝ ( )⎞ ⎠ ∑ ∑ ∑ ∑ ∑∑ + ! = + = ! + = ! = ! = ∞ + = ∞ = ∞ = ∞ = ∞ = λ n t t t β k t β S n k t n β S n k t n 1 1 1 log 1 1 log 1 , , . n n n n λ k k λ λ k k k λ n k λ n n k n k λ λ n 0 1, 0 , 0 , 1, 0 0 , 1, λ 1 (27) (27) By comparing the coefficients on both sides of (27), we obtain By comparing the coefficients on both sides of (27), we obtain By comparing the coefficients on both sides of (27), we obtain ( ) ( ) ( ) ∑ = + ≥ = + β S n k λ n n , 1 1 , 0 . k n k λ λ n n 0 , 1, 1, λ 1 ( ) ( ) ( ) ∑ = + ≥ = + β S n k λ n n , 1 1 , 0 . k n k λ λ n n 0 , 1, 1, λ 1 (28) ( ) ( ) ( ) ∑ = + ≥ = + β S n k λ n n , 1 1 , 0 . k n k λ λ n n 0 , 1, 1, λ 1 (28) Therefore, by (26) and (28), we obtain the following theorem. Therefore, by (26) and (28), we obtain the following theorem. Therefore, by (26) and (28), we obtain the following theorem. Theorem 4. For ≥ n 0, we have Theorem 4. For ≥ n 0, we have ( ) ( ) ∑ = + = + β λ k S n k 1 1 , n λ k n k k λ , 0 1, 2, λ 1 and ( ) ( ) ∑ + = + = n λ β S n k 1 1 , . n n k n k λ λ 1, 0 , 1, λ 1 ( ) ( ) ∑ = + = + β λ k S n k 1 1 , n λ k n k k λ , 0 1, 2, λ 1 ( ) ( ) ∑ = + = + β λ k S n k 1 1 , n λ k n k k λ , 0 1, 2, λ 1 ( ) ( ) ∑ = + = + β λ k S n k 1 1 , n λ k n k k λ , 0 1, 2, λ 1 and ( ) ( ) ∑ + = + = n λ β S n k 1 1 , . n n k n k λ λ 1, 0 , 1, λ 1 From Theorem 4, we note that ⎜ ⎟ ⎜ ⎟ ( )⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) [ ]( ( ) ) [ ] ( ) ( ) [ ]( ( ) ) [ ] ( ) ( ) [ ]( ( ) ) ( ( ) ) ( ) [ ] ⎛ ⎝ ⎞ ⎠ ( ) ( ( ) ) ( ( ) ) ( ) ( ) [ ] ( ) ( ) ( ) [ ] ( ( ) ) ( ) ( ) ( ) ( ) ( ) ( ) [ ]( ( ) ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) [ ] ( ) ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) [ ] [ ] ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑ − + + = + ! − + − + ! ! = + ! − − ! − = + ! − − ! − = + ! − − ! − = + ! − − ! − = − + ! ! Therefore, by (29), we obtain the following theorem. − − ! − ! = − + ! ! ! + + ! − ! − ! − = − + + + ! − − ! ! − ! + − − + − ! = − + + + ! − − ! + − − + − ! = − − = − − − = − − − = − = ∞ = − n k t k j e t t t k t e t n k k t t e t n k t e t j k j t n k k β n n k j k j t e t t n k k β n k j j k j t S l j j l j t n k k β n k j k j S n j j n j 1 1 1 1 1 1 1 1 1 1 1 1 1 1, 1 1 1 1 1, 1 1 . n j k k j λ j k j k λ k n λ j k k j n λ j j k n λ j k k j n λ j j k n λ j k k j n l λ l k n λ j k k j λ 0 1 1 1 , 1 1 1 , 1 0 2, , 1 2, Therefore, by (29), we obtain the following theorem. Therefore, by (29), we obtain the following theorem. 1692  Taekyun Kim et al. 1692  Taekyun Kim et al. 1692  Taekyun Kim et al. Theorem 5. For ≥ n k , 0, we have Theorem 5. For ≥ n k , 0, we have Theorem 5. For ≥ n k , 0, we have ( )⎛ ⎝ ⎞ ⎠ ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) [ ] ∑ ∑ − + + = − + + + ! − − ! + − ! + − − = = − S n j k k n j k β n k k β n k j k j n j S n j j , 2 1 1 1 1, 1 . j n λ j λ n n λ j k k j λ 0 2, 2 , , 1 2, Observe that, for any formal power series ( ) = ∑= ∞ f t a t i i i 0 , we have [ ] ( ) ( ) ( )[ ] ( ) ′ = + = + + + t f t n a n t f t 1 1 . n n n 1 1 (30) (30) [ ] ( ) ( ) ( )[ ] ( ) ′ = + = + + + t f t n a n t f t 1 1 . 1692  Taekyun Kim et al. n n n 1 1 By making use of (30), we have By making use of (30), we have ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) [ ] ∑ + − − + = n k j S n j k k β 1 , 2 j n λ j λ 0 2, 2 , ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ( )[ ]( ( ) ) [ ] ( ) ( ) [ ] ⎛ ⎝ ( ( ) ) ⎞ ⎠ [ ] ( ) ( ) [ ] ( ( ) ) ( ( ) ) ( ) [ ] ( ) ( ) [ ] ( ( ) ) ( ( ) ) ( ) ( ) [ ] ( ) ( ) [ ] ( ( ) ) ( ( ) ) ( ) ( ) [ ] ( ) ( ) [ ] ( ( ) ) ( ) ( ) ( ) [ ] ( ( ) ) ( ) ( ) ( ) ( ) [ ]⎧ ⎨⎩ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ⎫ ⎬⎭ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) [ ] [ ] [ ] [ ] [ ] [ ] ∑ ∑ ∑ ∑ ∑ ∑ ∑ ∑∑ ∑∑∑ ∑ ∑∑ + − − + = + − ! − + − + − ! ! = + − ! − + − − ! − = + − ! − − ! − = + − ! − − − − ! − = + − ! − − − − − ! + − = + − ! − − − − − ! + − = + − ! − − − ! + − + − ! − − − ! + − = + − ! + − − + − ! − − + − − + − ! − − ! = + − ! + − − + − ! − − + − ! + − − − + − ! − ! 1692  Taekyun Kim et al. = = = −+ = −+ − = −+ − − − = −+ − − = − − − − − − − = ∞ = − = ∞ = = − − = − = = − − n k j S n j k k β n k S n j k k n j k β j n k n j k t e t t k t t e t n k t d dt e t t k t t e t n k t e t t e t k t t e t n k t e t t e t λt k λt t t e t n k t e t t e t λt t k λt t t e t n k t e t t t k λt n k λ t e t t t k λt t e t n k t S l k k l k λ t λ S l k k l k λ β m j t n k S l k k l k λ λ n k S l k k β λ l k n j 1 , 2 1 , 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2, 2 2 2 2, 2 2 2 1 2, 2 2 2 1 2, 2 2 2 . 1692  Taekyun Kim et al. = − − − − = ∞ = − = ∞ = = − − = − = = − − t k λt e t n k t e t t t k λt n k λ t e t t t k λt t e t n k t S l k k l k λ t λ S l k k l k λ β m j t n k S l k k l k λ λ n k S l k k β λ l k n j 1 1 1 1 1 1 1 1 1 1 1 1 1 2, 2 2 2 2, 2 2 2 1 2, 2 2 2 1 2, 2 2 2 . j λ n λ k k n λ k k λ n m k l k m λ m l m m k j k m l k j λ j l m j λ m l k n λ n l j k n l k j λ n j λ j l 0 1 2 1 2 2, 2 2, 2 , 2, 2 2, 2 , Therefore, by (31), we obtain the following theorem. Therefore, by (31), we obtain the following theorem. Theorem 6. For ≥ n k , 1, with ≥ n k, we have 1692  Taekyun Kim et al. j n λ j λ j n λ j λ j n n j k λ k j λ j n n j k λ k j λ j n n j k λ k λ λ j λ j n n j k λ k λ j λ j n n j λ k λ k j λ n λ k k n λ k k λ n m k l k m λ m l m m k j k m l k j λ j l m j λ m l k n λ n l j k n l k j λ n j λ j l 0 2, 2 , 0 2, 2 , 0 0 1 0 1 1 1 0 1 1 0 1 1 1 2 1 2 2, 2 2, 2 , 2, 2 2, 2 , ( ) ( ) ( ) [ ] ∑ = + − ! − + − + − ! ! = n k S n j k k n j k β j 1 , 2 1 j n λ j λ 0 2, 2 , (31) ( ) ( ) ( ) ( ) [ ] ( ( ) ) ( ) ( ) ( ) [ ] ( ( ) ) ( ) ( ) ( ) ( ) [ ]⎧ ⎨⎩ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ⎫ ⎬⎭ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) [ ] [ ] [ ] [ ] ∑∑ ∑∑∑ ∑ ∑∑ ! + = + − ! − − − ! + − + − ! − − − ! + − = + − ! + − − + − ! − − + − − + − ! − − ! = + − ! + − − + − ! − − + − ! + − − − + − ! − ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ⎧ ⎨⎩ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ⎫ ⎬⎭ [ ] [ ] [ ] ∑ ∑ ∑∑ + − − + = + − ! + − − + − ! − + + − − − + − ! − ! = = − = = − −+ n k j S n j k k β n k S l k k l k λ S l k k β λ l k n j 1 , 2 1 2, 2 2 2 2, 2 2 2 . j n λ j λ l k n λ n l j k n l k j λ n j λ j l 0 2, 2 , 2, 2 2, 2 , 1 Study on r-truncated degenerate Stirling numbers of the second kind  1693 Now, we observe that Now, we observe that Now, we observe that ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ( )⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) [ ]( ( ) ) [ ]⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ]( ( ) ) [ ]⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ]⎛ ⎝ ( ) ⎞ ⎠ ( ) ⎛ ⎝ ⎞ ⎠ ( ) [ ]⎛ ⎝ ( ) ⎞ ⎠ ( ) ⎛ ⎝ ⎞ ⎠ ( ) [ ] ( ) [ ] ( ) ( ) ∑ ∑ ∑ ∑ ∑ ∑ − + + = + ! − + − + ! ! = + ! − − ! − = + ! ! − − − = + ! ! − − = + ! ! − − = + ! ! − ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have − − − − = − − − − = ∞ = − = − − = − −+ n k t λ t k e t n k k t k j e t t λ t n k t k j λ β m j k t n k k t k j e t k j l λ t 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 n k t k λ n j k k j λ j j k j n m k j m j m j λ j m n j k k j λ j l k j l l j l 2 1 1 1 0 0 , 1 1 0 1 ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ( ) [ ]( ( ) ( ) ) [ ] ( ) ( ) [ ]( ( ) ( ) ) [ ]⎛ ⎝ ( ) ⎞ ⎠ [ ] [ ] ∑ ∑ ∑ ∑ + + + = + ! + + ! − ! = + ! − − − ∕ ! − = + ! − − − ∕ ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have − = − = − = + − = − n k j k S j k k β n k S j k k j k β n j n k t e t t t k t t e t n k t e t t t t k t t e t , 3 , 3 1 1 2 1 1 1 2 1 j n λ n j λ j n λ n j λ j n j k λ λ k n j λ j n j λ λ k k n j λ 0 2, 3 , 0 2, 3 , 0 2, 2 0 2, 2 (33) ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ( ) [ ]( ( ) ( ) ) [ ] ( ) ( ) [ ]( ( ) ( ) ) [ ]⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ]( ( ) ( ) ) ( ( ) ) ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ( ( ) ) ( ( ) ) [ ] [ ] ∑ ∑ ∑ ∑ ∑ + + + = + ! + + ! − ! = + ! − − − ∕ ! − = + ! − − − ∕ ! − = + ! − − − ∕ ! − = + ! − − + − ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have − − − − = − − − − = ∞ = − = − − = − −+ n k t λ t k e t n k k t k j e t t λ t n k t k j λ β m j k t n k k t k j e t k j l λ t 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 n k t k λ n j k k j λ j j k j n m k j m j m j λ j m n j k k j λ j l k j l l j l 2 1 1 1 0 0 , 1 1 0 1 (33) ⎜ ⎟ ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ( ( ) ) ( ( ) ) ∑ = + ! − − + − ! − = − − n k t k j e t t λ t k e t t 1 1 1 1 n j k k j λ j t k j λ k 0 2 2 ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ( ) [ ] ( ) ( ( ) ) ( ( ) ) ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ] ⎛ ⎝ ⎜( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ⎞ ⎠ ⎟ ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ⎛ ⎝ ⎞ ⎠ ( ) ∑ ∑ ∑ ∑ ∑ = + ! − + − ! − + + ! ! × − − + − = + ! − − − ! ! + + ! ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have = = = −+ = − = − − = − − S n j k k n k j β n k S n j k k n j k β j n k t e t t k t t e t n k k t e t t t t t e t n k k t t e t n k k k j t t e t n k k k j β n , 2 , 2 1 1 1 1 1 1 1 1 1 . j n λ j λ k j n λ j λ k j n n j k λ k j λ k j n n j λ k k j λ k n λ k j k k j n λ k j j k k j n λ k j 0 2, 2 , 0 2, 2 , 0 0 0 0 , (32) (32) Therefore, by (32), we obtain the following theorem. Therefore, by (32), we obtain the following theorem. Therefore, by (32), we obtain the following theorem. Theorem 7. For ≥ n k , 0, we have Theorem 7. For ≥ n k , 0, we have ⎜ ⎟ ⎜ ⎟ ( )⎛ ⎝ ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ ⎛ ⎝ ⎞ ⎠ ( ) [ ] ( ) ( ) ∑ ∑ − + + = + − = = − − S n j k k n k j β n k k k j β , 2 1 . j n λ j λ k j k k j n λ k j 0 2, 2 , 0 , From Theorems 4 and 5, we have From Theorems 4 and 5, we have ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ( ) [ ]( ( ) ( ) ) [ ] ( ) ( ) [ ]( ( ) ( ) ) [ ]⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ]( ( ) ( ) ) ( ( ) ) ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ( ( ) ) ( ( ) ) [ ] [ ] ∑ ∑ ∑ ∑ ∑ + + + = + ! + + ! − ! = + ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have − − − ∕ ! − = + ! − − − ∕ ! − = + ! − − − ∕ ! − = + ! − − + − ! − = − = − = + − = − = − − n k j k S j k k β n k S j k k j k β n j n k t e t t t k t t e t n k t e t t t t k t t e t n k t e t t t t t k e t n k t k j e t t λ t k e t t , 3 , 3 1 1 2 1 1 1 2 1 1 1 2 1 1 1 1 1 j n λ n j λ j n λ n j λ j n j k λ λ k n j λ j n j λ λ k k n j λ n λ λ k k λ n j k k j λ j t k j λ k 0 2, 3 , 0 2, 3 , 0 2, 2 0 2, 2 2, 2 0 2 2 (33) ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ( ) [ ] ( ) ( ( ) ) ( ( ) ) ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ] ⎛ ⎝ ⎜( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ⎞ ⎠ ⎟ ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ⎛ ⎝ ⎞ ⎠ ( ) ∑ ∑ ∑ ∑ ∑ = + ! − + − ! − + + ! ! × − − + − = + ! − − − ! ! + + ! ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have − = − = − = + − = − = − − n k j k S j k k β n k S j k k j k β n j n k t e t t t k t t e t n k t e t t t t k t t e t n k t e t t t t t k e t n k t k j e t t λ t k e t t , 3 , 3 1 1 2 1 1 1 2 1 1 1 2 1 1 1 1 1 j n λ n j λ j n λ n j λ j n j k λ λ k n j λ j n j λ λ k k n j λ n λ λ k k λ n j k k j λ j t k j λ k 0 2, 3 , 0 2, 3 , 0 2, 2 0 2, 2 2, 2 0 2 2 (33) ⎜ ⎟ ⎜ ⎟ ( ) ( ) ( ) ( ) ( ) [ ]( ( ) ( ) ) [ ] ( ) ( ) [ ]( ( ) ( ) ) [ ]⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ]( ( ) ( ) ) ( ( ) ) ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ( ( ) ) ( ( ) ) [ ] ∑ ∑ ∑ ∑ = + ! + + ! − ! = + ! − − − ∕ ! − = + ! − − − ∕ ! − = + ! − − − ∕ ! − = + ! − − + − ! Theorem 6. For ≥ n k , 1, with ≥ n k, we have − − = − = + − = − = − − n k S j k k j k β n j n k t e t t t k t t e t n k t e t t t t k t t e t n k t e t t t t t k e t n k t k j e t t λ t k e t t , 3 1 1 2 1 1 1 2 1 1 1 2 1 1 1 1 1 j λ j n λ n j λ j n j k λ λ k n j λ j n j λ λ k k n j λ n λ λ k k λ n j k k j λ j t k j λ k , 0 2, 3 , 0 2, 2 0 2, 2 2, 2 0 2 2 (33) ⎜ ⎟ ⎜ ⎟ ⎜ ⎟ ( ) [ ] ( ) ( ( ) ) ( ( ) ) ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ⎛ ⎝ ( ) ⎞ ⎠ ( ) [ ] ⎛ ⎝ ⎜( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ⎞ ⎠ ⎟ ( ) [ ] ( ) ⎛ ⎝ ⎞ ⎠ ( ( ) ) ⎛ ⎝ ⎞ ⎠ ( ) ∑ ∑ ∑ ∑ ∑ = + ! − + − ! − + + ! ! × − − + − = + ! − − − ! ! + + ! ! Theorem 8. For ≥ n k , 0, we have ⎜ ⎟ ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( )( ) ( ) [ ] ∑ ∑ ∑ ∑ + + + = − + ! − − ! ! + + ! − − ! − − + − − − − + − ! = − = − = − = n k j k S j k k β n k k j λ n j k β n k j k j k j l S n l j j λ n l j , 3 1 1 1 2 1 1, 1 1 2 1 . j n λ n j λ k j n j j n j λ j k k j l n λ l l 0 2, 3 , 0 , 1 1 2, (34) (34) Remark 9. As the counterpart of (10), we may consider the r-truncated degenerate Stirling numbers of the first kind given by Remark 9. As the counterpart of (10), we may consider the r-truncated degenerate Stirling numbers of the first kind given by ⎛ ⎝ ⎜ ( ) ( ) ⎞ ⎠ ⎟ ( ) [ ] ∑ ∑ ! + − ! = ! = − − = ∞ k t λ l t S n kr t n 1 log 1 1 , , λ l r l l l k n kr λ r n 1 1 , 1 1, λ 1 (35) (35) where r is a positive integer. These numbers will be investigated in a forthcoming article. Theorem 6. For ≥ n k , 1, with ≥ n k, we have Therefore, by (33), we obtain the following theorem. Therefore, by (33), we obtain the following theorem. Therefore, by (33), we obtain the following theorem. Theorem 8. For ≥ n k , 0, we have Theorem 8. For ≥ n k , 0, we have Theorem 6. For ≥ n k , 1, with ≥ n k, we have − − − − = − − − − = ∞ = − = − − = − −+ n k t λ t k e t n k k t k j e t t λ t n k t k j λ β m j k t n k k t k j e t k j l λ t 1 1 1 1 1 1 1 1 2 1 1 2 1 1 1 2 n k t k λ n j k k j λ j j k j n m k j m j m j λ j m n j k k j λ j l k j l l j l 2 1 1 1 0 0 , 1 1 0 1 Taekyun Kim et al. 1694  Taekyun Kim et al. ⎜ ⎟ ⎜ ⎟ ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) [ ] ( ) ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( ) ( ) ( ) ( ) ( ) ⎛ ⎝ ⎞ ⎠ ( )( ) ( ) ∑ ∑ ∑ ∑ ∑ ∑ ∑ = − + ! − − ! ! + + ! − − ! − ! − − = − + ! − − ! ! + + ! − − ! − − + − − − − + − ! = − = − = − ∞ = − −+ = − = − = n k k j λ n j k β n k t j k j S l j t l k j l λ t n k k j λ n j k β n k j k j k j l S n l j j λ n l j 1 1 1 2 1 , 1 1 2 1 1 1 2 1 1, 1 1 2 1 . k j n j j n j λ n j k k j l j λ l l k j l l l j k j n j j n j λ j k k j l n λ l l 0 , 1 1 2, 1 1 1 1 0 , 1 1 2, 1 1 Therefore, by (33), we obtain the following theorem. References [1] L. Carlitz, Degenerate Stirling, Bernoulli and Eulerian numbers, Utilitas Math. 15 (1979), 51–88. [2] L. Carlitz, A degenerate Staudt-Clausen theorem, Arch. Math. (Basel) 7 (1956), 28–33. [3] D. S. Kim and T. Kim, A note on a new type of degenerate Bernoulli numbers, Russ. J. Math. Phys. 27 (2020), no. 2, 227–235. [4] H. K. Kim, H. Baek, and D. S. Lee, A note on truncated degenerate exponential polynomials, Proc. Jangjeon Math. Soc. 24 (2021), no. 1, 63–76. 24 (2021), no. 1, 63–76. [5] T. Kim and D. S. Kim, Degenerate Whitney numbers of first and second Kind of Dowling lattices, Russ. J. Math. Phys. ( ), , [5] T. Kim and D. S. Kim, Degenerate Whitney numbers of first and second Kind of Dowling lattices, Russ. J. Math. Phys. 29 (2022), no. 3, 358–377. [6] T. Kim and D. S. Kim, Degenerate zero-truncated Poisson random variables, Russ. J. Math. Phys. 28 (2021), no. 1, 66–72. [6] T. Kim and D. S. Kim, Degenerate zero-truncated Poisson random variables, Russ. J. Math. Phys. 28 (2021), no. 1, 66–72. [7] T Kim and D S Kim Some identities on truncated polynomials associated with degenerate Bell polynomials Russ J [7] T. Kim and D. S. Kim, Some identities on truncated polynomials associated with degenerate Bell polynomials, Russ. J. Math. Phys. 28 (2021), no. 3, 342–355. [8] T. Kim, D. S. Kim, L.-C. Jang, H. Lee, and H. Kim, Representations of degenerate Hermite polynomials, Adv. A 139 (2022), 102359. [9] T. Kim, D. S. Kim, H. K. Kim, and H. Lee, Some properties on degenerate Fubini polynomials, Appl. Math. Sci. Eng. 30 (2022), no. 1, 235–248. ( ) [10] T. Kim, D. S. Kim, H. Lee, S. Park, and J. Kwon, New properties on degenerate Bell polynomials, Complexity 2021 (2021) 7648994. [11] S. K. Sharma, W. A. Khan, S. Araci, and S. S. Ahmed, New type of degenerate Daehee polynomials of the second kind, Adv. Difference Equations 2020 (2020), 428. [12] T. Kim and D. S. Kim, Degenerate Laplace transform and degenerate gamma function, Russ. J. Math. Phys. 24 (2017), no. 2, 241–248. T. Kim and D. S. Kim, Note on the degenerate gamma function, Russ. J. Math. Phys. 27 (2020), no. 3, 352–358. [14] D. S. Kim and T. Kim, Degenerate Sheffer sequences and λ-Sheffer sequences, J. Math. Anal. Appl. 493 (2021), no. 1, 124521. [15] U. Duran and M. 3 Conclusion In recent years, we have witnessed that many degenerate versions of quite a few special numbers and polynomials were investigated and some nice results were obtained by adopting various tools. In this article, we considered the r-truncated degenerate Stirling numbers of the second, which reduce to the degenerate Stirling numbers of the second for = r 1, and studied by using generating functions their explicit expressions, some properties and related identities on those numbers, in connection with several other degenerate special numbers and polynomials. As one of our future projects, we would like to continue this line of research, namely, to explore various degenerate versions of some special numbers and polynomials, and to find their applications to physics, science and engineering. Funding information: This work was supported by the Basic Science Research Program, the National Research Foundation of Korea (NRF-2021R1F1A1050151). Author contributions: All authors contributed equally to the writing of this article. All authors read and approved the final manuscript. Conflict of interest: The authors state no conflict of interest. 1695 Study on r-truncated degenerate Stirling numbers of the second kind Disclosure: An earlier version of the article has been presented as a preprint in the following link: https:// arxiv.org/abs/2206.04402. Data availability statement: No data were used to support this study. References Acikgoz, On degenerate truncated special polynomials, Mathematics 8 (2020), no. 1, 144. [16] S. Araci, A new class of Bernoulli polynomials attached to polyexponential functions and related identities, Adv. Stud. Contemp. Math. 31 (2021), no. 2, 195–204. [17] A. Bayad and J. Chikhi, Apostol-Euler polynomials and asymptotics for negative binomial reciprocals, Adv. Stud. Contemp. Math. (Kyungshang) 24 (2014), no. 1, 33–37. [18] L. Comtet, Advanced Combinatorics, D. Reidel Publishing Co., Dordrecht-Holland, 1974. [19] R. Dere and Y. Simsek, Applications of umbral algebra to some special polynomials, Adv. Stud. Contemp (Kyungshang) 22 (2012), no. 3, 433–438. ] Y. Simsek, On Boole-type combinatorial numbers and p [20] Y. Simsek, On Boole-type combinatorial numbers and polynomials, Filomat 34 (2020), no. 2, 559–565. [21] R. F. Tate and R. L. Goen, Minimum variance unbiased estimation for the truncated Poisson distribution, Ann. Math. Statist. 29 (1958), 755–765. ( ) [22] S. Roman, The umbral calculus, Pure and Applied Mathematics, Vol. 111, Academic Press, Inc. [Harcourt Brace Jovanovich Publishers], New York, 1984.
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ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME
Jurnal Pembaharuan Hukum/Jurnal pembaharuan hukum
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1 Article 1 paragraph 3 of the 1945 Constitution of the Republic of Indonesia. ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana Sultan Agung Islamic University ida.musofiana@unissula.ac.id Ida Musofiana Sultan Agung Islamic University ida.musofiana@unissula.ac.id Aji Sudarmaji Sultan Agung Islamic University aji.s@unissula.ac.id Aji Sudarmaji Sultan Agung Islamic University aji.s@unissula.ac.id Ira Alia Maerani Sultan Agung Islamic University Ira.alia@unissula.ac.id Abstract The 21st century has progressed further. As in Indonesia, currently, the development of technology is increasingly rapid. This certainly affects aspects of social life in society and its security. Today's threats are not only enemies in the military, but also non-military. Among them are the threats faced by internet users, the penetration rate shows a high cumulation, opening up opportunities for the emergence of cyber-based crimes. The development of infrastructure is in line with the development of technology. Increasingly sophisticated technology is not only used by the community for positive things. However, not a few are taking advantage of technological sophistication to reap benefits that result in losses for technology users themselves. This study aims to research, examine how legal protection for children from cybercrime. In this case, children become vulnerable to cybercrime in digital era. Why is that? Considering that the control for internet access, as well as legal products in Indonesia, cannot yet fully guarantee protection for victims of cybercrime. The method used in this research is to use the descriptive analysis approach. Assessing and analyzing legal protection for children from cybercrime based on legal theories and applicable laws and regulations. Besides, it also reveals the phenomena of the current situation in the community. urnal Pembaharuan Hukum urnal Pembaharuan Hukum JPH: Jurnal Pembaharuan Hukum Volume 7, Number 3, December 2020 A. INTRODUCTION A. INTRODUCTION The Republic of Indonesia as a state law.1 Adhere to the principles of protection law, protection of human rights for citizens country, and democratic principles, including protection of children, so it is absolute for state and government provide protection law and human rights to children as part of the nation's children who are expected to become continuation of the ideals of the struggle of the Indonesian nation. ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 201 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Precise in 2001, Indonesia stated its commitment to the Declaration of a World Worthy for Children. This is following the provisions The Convention on the Rights of the Child ratified by the government Indonesia through Presidential Decree Number 36 of 1990 and later also poured in Law Number 4 of 1979 concerning Child Welfare. Apart from that, the Law Number 23 of 2002 concerning Child Protection as amended by Law Number 35 of 2014 concerning Amendments to the Law Number 23 of 2002 concerning Protection Children and Law Number 11 of 20122 all about the Juvenile Criminal Justice System both put forward the principles general protection of children, namely non-discrimination, best interests of children, survival, and growth and respect for participation child. Entering the 21st century, any country has its challenges. The threats faced are not only in the military field but also non-military. Several threats to national security and defense are carried out by non-state actors such as insurgency, terrorism, human trafficking, cybercrime, narcotics, piracy at sea, including violations of human rights. In a conflict asymmetric warfare, carried out by non-state actors as weak parties against the government. The use of information technology, media, and communication in society, especially young students, has changed both the behavior of society and human civilization globally. The development of information and communication technology has also caused world relations to become borderless and caused significant social, economic, and cultural changes to take place so rapidly. Information technology is currently a double-edged sword because, in addition to contributing to the improvement of welfare, human progress, and civilization, it is also an effective means of acting against the law.3 g The more sophisticated the technology is not a few who use it for positive activities, also not a few who use it for negative activities. 2 Ridwan Mansur's opinion is quoted in Nevey Varida Ariani, Implementation of Law Number 11 of 2012 concerning the System Juvenile Justice to Protect Children's Interests, Media Hukum Journal, Volume 21, Nomor 1, 2014, page 108-109. 3 Ivan Fauzi Raharja, Wise to Use Social Media Among Students According to Law Number 19 of 2016 Concerning Electronic Information and Transactions, Journal Selat, Volume 6, Number 2, May 2019, page 236. 2 Ridwan Mansur's opinion is quoted in Nevey Varida Ariani, Implementation of Law Number 11 of 2012 concerning the System Juvenile Justice to Protect Children's Interests, Media Hukum Journal, Volume 21, Nomor 1, 2014, page 108-109. 3 Ivan Fauzi Raharja, Wise to Use Social Media Among Students According to Law Number 19 of 2016 Concerning Electronic Information and Transactions, Journal Selat, Volume 6, 202 ASPECTS OF LEGAL PROTECTION FOR Jurnal Pembaharuan Hukum CHILDREN FROM CYBERCRIME Volume 7 No.3 Sept-Dec 2020 Ida Musofiana Aji Sudarmaji Ira Alia Maerani 2 Ridwan Mansur's opinion is quoted in Nevey Varida Ariani, Implementation of Law Number 11 of 2012 concerning the System Juvenile Justice to Protect Children's Interests, Media Hukum Journal, Volume 21, Nomor 1, 2014, page 108-109. 3 Ivan Fauzi Raharja, Wise to Use Social Media Among Students According to Law Number 19 of 2016 Concerning Electronic Information and Transactions, Journal Selat, Volume 6, Number 2, May 2019, page 236. A. INTRODUCTION In this research technology in terms of the use of cyberspace. Along with the development of the times, the use of technology cannot be separated from people's lives. This makes technology vulnerable to crime. Children at the age categorized as children following Law Number 11 of 2012 concerning the Juvenile Criminal Justice System, namely under the age of 18 (eighteen) years. The internet is currently attached to the lives of today's children, even toddlers (babies under five years) can use gadgets to watch YouTube. According to their age, children fall into the category of groups vulnerable to cybercrime. Children can easily access the internet anywhere and anytime. Things like this open the opportunity to become victims of cybercrime more easily. It was 2001 when the crime of a pornographic website in Texas was exposed. The two perpetrators who became the perpetrators were Indonesians. In 2006 the Polda Metro Jaya arrested Peter 202 ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani W. Smith (an Australian citizen) who molested 50 (fifty) children from Indonesia, India, and Vietnam.4 W. Smith (an Australian citizen) who molested 50 (fifty) children from Indonesia, India, and Vietnam.4 The benefits of the virtual world so far provide social changes in people's lives, especially for children. Children are like a white paper, what we write down, that's where it will be drawn or formed because children have imitation qualities. Deviant behavior can occur in children. Data from the Indonesian Child Protection Commission shows that in 2016 there were 496 cases of pornography and cybercrime.5 Specifics regarding legal protection towards children, the state pays attention bypassing the Law Number 35 of 2014 which is the change of Law Number 23 of 2002 regarding Child protection. These changes are to emphasize the importance of weighting criminal sanctions and also fines for criminals against children to provide a deterrent effect, as well to encourage concrete steps for recover physically, psychologically, and also social children as victims and/or children as criminals as an anticipatory step so children as victims or not as perpetrators be the perpetrator of the same crime later day. Currently, a new legal regime has been born, known as cyberlaw or telematics law. 4 Data sourced from the Indonesian Child Protection Commission in 2011 2016. 5 Hardianto Djanggih, The conception of Legal Protection for Children as Victims of Cyber Crimes Through Penal and Non Penal Approaches, Mimbar Hukum Journal, Volume 30 Nomor 2, June 2018, page 318. 6 Ivan Fauzi, Op Cit, Journal Selat, page 237. 7 Ibid page 238 4 Data sourced from the Indonesian Child Protection Commission in 2011 – 2016. 5 Hardianto Djanggih, The conception of Legal Protection for Children as Victims of Cybe Crimes Through Penal and Non Penal Approaches, Mimbar Hukum Journal, Volume 30 Nom 2, June 2018, page 318. 6 Ivan Fauzi, Op Cit, Journal Selat, page 237. 7 Ibid., page 238 A. INTRODUCTION Cyberlaw is internationally used for legal terms related to the use of information and communication technology. Likewise, telematics law is the embodiment of the convergence of telecommunications law, media law, and informatics law. Other terms that are also used are the law of information technology, virtual world law, and cyberspace law. These terms were born considering activities carried out through computer systems networks and communication systems both locally and globally (internet) by utilizing computer system-based information technology which is an electronic system can be seen virtuallay.6 The emergence of this virtual version of social networks affects the relationship between people, generally teenagers. The community site above was created to fulfill the desire of individuals to communicate without any time and space restrictions. Not infrequently social networks often have the potential to influence a person's thinking patterns and shape individual personalities. A large number of social media users among junior and senior high school students at this time did not escape its spread to all corners of the country. However, these social media users have very little information about the existence of Law Number 19 of 2016 concerning Electronic Information and Transactions, which is a legal product that limits the freedom of social media users to be wiser in its use, especially among teenagers.7 As we know, teenagers are the largest community in Indonesian society who use social media regularly. The initial reason they are very ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 203 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani active in using social media is to seek attention, ask for opinions, and foster an image, but over time they become dependent. Although social media has a positive impact on adolescents, when it is difficult for them to escape from activities related to social media, it will have a less positive impact. Several studies show that due to the excessive use of social media, adolescents are found to experience incongruence in their self-concept.8 Indonesian Internet User Services Association revealed the results of its released survey 2016 on children of internet users. 8 Pamela Felita and Friends, Use of Social Media and Self Concept in Teens, Psikologi MANASA Journal, Volume 5 Nomor 1, March 2016, page 31 9 Ministry of Communication and Information of the Republic of Indonesia, Indonesian Internet User Penetration and Behavior, https://aptika.kominfo. go.id/index.php/berita/274-rilis-hasil-survei-penetrasi-dan-perilaku-pengguna-internet- indonesia-tahun-2018, acceded on 22 September 2020, 09.55 o’clock 10 Setiono, Understanding of Legal Research Methodology Postgraduate Law studies of Sebelas Maret University, 2010, page 20 A. INTRODUCTION The result of a survey showed 76.4% were not safe on the internet for the child.9 This is because of the internet or information technology is an instrument potential criminal instrument, and the second indicates the need for immediate Fixing the legal sector in the field of child protection as victims of cybercrime in Indonesia. The researcher in this case formulates several problem formulations including How is legal protection for children from cybercrime? Then, Providing Legal Protection for Child Victims from Cybercrime. 204 ASPECTS OF LEGAL PROTECTION FOR Jurnal Pembaharuan Hukum CHILDREN FROM CYBERCRIME Volume 7 No.3 Sept-Dec 2020 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 8 Pamela Felita and Friends, Use of Social Media and Self Concept in Teens, Psikologi MANASA Journal, Volume 5 Nomor 1, March 2016, page 31 9 Ministry of Communication and Information of the Republic of Indonesia, Indonesian Internet User Penetration and Behavior, https://aptika.kominfo. go.id/index.php/berita/274-rilis-hasil-survei-penetrasi-dan-perilaku-pengguna-internet- indonesia-tahun-2018, acceded on 22 September 2020, 09.55 o’clock 10 Setiono, Understanding of Legal Research Methodology Postgraduate Law studies of Sebelas Maret University, 2010, page 20 11 Andi Hamzah, Indonesian Criminal and Criminal System from Retribution to Reform, Printed for 2, Pardnya Paramita, Jakarta, page 16. 12 Faizin Sulitio, etc, Alternative Model of Crime of Cyber Pornography Crime, Arena Hukum Journal, Volume 9, number 3, 2016, page 359. 13 Ibid., C. RESULT AND DISCUSSION RESULT AND DISCUSSION 1. The Concept of Child Protection as Victims of Cyber Crime p y According to Phillips in his book A First Book English Law, as quoted by Andi Hamzah, which is seen as a goal the current punishment is a variation from deterrent forms, good addressed to violations of the law itself as well as those who have potential become criminals, protection to society from evil deeds, improvement or reform to criminals. The latter is the most modern and popular today, not only for purposes improve imprisonment conditions but also looking for other alternatives that are not criminal in fostering lawbreakers.11 Frequently asked questions criminal law experts in various seminars are the question of how to criminal law should or should at this time and future. This question is related to the field of criminal law politics (penal policy), namely how better criminal law should be it is created, structured, and used to organize or controlling human behavior in Public. especially to cope with crimes committed by members society as well as by the authorities.12 Crime is a deviant behavior that will always exist and cling to every form of society. Crime is the oldest social problem therefore must be overcome. Judging from the consequences of crime can disturb or destroy and hinder the achievement of national goals and also prevent optimal use of national sources Sudarto stated that the use of means penal in tackling crime cannot be released from its connection with legal policy criminal or political criminal law (criminal law policy). In this connection, then Soedarto argued that implementing criminal law politics meant making an assessment and elections to achieve statutory results criminal at best, in a sense meet the requirements of justice and efficiency. Efforts to find alternatives penalties for deprivation of liberty in the short term were also supported by the Union Nations. In the 2nd UN Congress regarding "The Prevention of Crime and Treatment of Offenders ”in 1960 London recommended that the practice is thorough removal short term imprisonment is not maybe. only realistic solutions can be achieved by reducing the number of uses.13 That gradual reduction by increasing substitute forms or alternative to conditional punishment, supervision/probation, fines, work outside the agency. and actions others that do not contain deprivation independence. B. RESEARCH METHODS Legal research is closely related to the concept of law used that according to Soetondyo Wigyosoebroto of the five legal concepts, these legal concepts are as follows: 1. Law is the principle of truth and justice that applies naturally and applies universally; 2. Law is positive norms in the statutory system; 3. Law is what is decided by the judge (in concreto) and is systemized as a judge-made law; 4. Laws are institutionalized social behavior patterns that exist as empirical social variables; and 5. Law is a manifestation of symbolic meanings of social behavior as seen in their interactions.10 5. Law is a manifestation of symbolic meanings of social behavior as seen in their interactions.10 This study uses a descriptive-analytical literature method. Analytical descriptive research can be done without using a basic supposition or proposition, but it has begun with an outline of the thoughts and problems to be studied. The literature method is one of the data collection methods used in social research methodology to trace event record data ”. The literature used in this research is sourced from journals, books, and other online literature contains the concept of legal protection for child victims of social media crimes in general as data in this paper. 204 ASPECTS OF LEGAL PROTECTION FOR Jurnal Pembaharuan Hukum CHILDREN FROM CYBERCRIME Volume 7 No.3 Sept-Dec 2020 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 8 Pamela Felita and Friends, Use of Social Media and Self Concept in Teens, Psikologi MANASA Journal, Volume 5 Nomor 1, March 2016, page 31 9 Ministry of Communication and Information of the Republic of Indonesia, Indonesian Internet User Penetration and Behavior, https://aptika.kominfo. go.id/index.php/berita/274-rilis-hasil-survei-penetrasi-dan-perilaku-pengguna-internet- indonesia-tahun-2018, acceded on 22 September 2020, 09.55 o’clock 10 Setiono, Understanding of Legal Research Methodology Postgraduate Law studies of Sebelas Maret University, 2010, page 20 Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 204 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani C. RESULT AND DISCUSSION Sudarto had a very good reason relation to cybercrime phenomenon this one of which resulted in the child being victim, so to overcome it must with the formulation and use of legislation precisely so that the purpose of that law can be achieved. In Indonesia, child ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 205 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani protection from harmful effects of cyber exploitation can be found in various laws, namely: protection from harmful effects of cyber exploitation can be found in various laws, namely: a. Article 38 paragraph (3) of the Law Number 32 of 2002 concerning The broadcast stated that “Fill broadcast must provide protection and empowerment to the public specifically, namely children and adolescents.14 b. Article 15 Law Number 44 2008 about the Pornography states that “Everyone obliged to protect children from pornography influence and prevent children's access to information pornography.15 c. Article 43 of Law Number 33 of 2009 concerning Film emphasized that "Business actors movies are prohibited from doing dubbing import movie sound into language Indonesia, except for imported films educational interests and/or research.16 d. Article 27 of Law Number 19 of 2016 concerning Change Law Number 11 of 2008 concerning Information & Transactions Electronic confirms prohibition of distribution and/or transmits and/or creates Electronic Information accessible and/or Electronic Documents has a charge that breaks decency and gambling.17 d. Article 27 of Law Number 19 of 2016 concerning Change Law Number 11 of 2008 concerning Information & Transactions Electronic confirms prohibition of distribution and/or transmits and/or creates Electronic Information accessible and/or Electronic Documents has a charge that breaks decency and gambling.17 g y g g e. Article 59 of Law Number 35 of 2014 concerning Change Law Number 23 of 2002 on Child Protection states that "the Government and other state institutions are obliged and responsible for provide special protection to children in emergencies, children in conflict with the law, children from minority groups and isolated exploited children economic and/or sexual, child who is trafficked, the child who is fall victim to abuse narcotics, alcohol, psychotropic drugs, and other addictive substances (drugs), children victims of kidnapping, sales and trafficking, victim's child.18 2. 14 Law Number 32 of 2002 concerning The broadcast. 14 Law Number 32 of 2002 concerning The broadcast. 15 Law Number 44 of 2008 about the Pornography. 16 Law Number 33 of 2009 concerning Film emphasized. 17 Law Number 19 of 2016 concerning Change Law Number 11 of 2008 concerning Information & Transactions Electronic. 18 Law Number 23 of 2002 on Child Protection. 19 Indah Setyowati, Ida Musofiana, Juridical Studies Against Diversion In Criminal Justice System, Jurnal Pembaharuan Hukum, Volume 6 Number 2, 2019, page 241. g 15 Law Number 44 of 2008 about the Pornography. 206 ASPECTS OF LEGAL PROTECTION FOR Jurnal Pembaharuan Hukum CHILDREN FROM CYBERCRIME Volume 7 No.3 Sept-Dec 202 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 14 Law Number 32 of 2002 concerning The broadcast. 15 Law Number 44 of 2008 about the Pornography. 16 Law Number 33 of 2009 concerning Film emphasized. 17 Law Number 19 of 2016 concerning Change Law Number 11 of 2008 concerning Information & Transactions Electronic. 18 Law Number 23 of 2002 on Child Protection. 19 Indah Setyowati, Ida Musofiana, Juridical Studies Against Diversion In Criminal Justice System, Jurnal Pembaharuan Hukum, Volume 6 Number 2, 2019, page 241. C. RESULT AND DISCUSSION Legal Protection for Child Victims from Cybercrime Indonesia itself as a country that has ratified the Convention on the Rights of the Child through Presidential Decree Number 36 of 1990 dated August 25, 1990, has further integrated children's rights into national law, namely into Law Number 23 of 2002 concerning Child Protection19 which has been amended by-law number 35 of 2014. Frequently asked questions criminal law experts in various seminars are the question of how to criminal law should or should at this time and future. Law Number 19 of 2016 Although this law is not specific regulates pornography, but regulations on that matter are also regulated in this law. One of them is regulated in Article 27 paragraph Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 206 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani (1), namely: "Every people knowingly and without rights distribute and/or transmit and/or make accessible Electronic Information and/or Electronic Documents has a charge that violates decency. "The meaning of decency regulated in the article includes related to pornography. The internet further supports the child sex tourism industry by providing patrons of the detours a means by which to easily connect with other like-minded steps throughput the world.20 Constitution in protecting children from cyber exploitation, according to the author has not been able to reach there is a cybercrime development resulting in children as victims. Author's reason is that there are still forms of cybercrimes that need to be criminalized and necessary also, formulate the appropriate form of punishment for cybercriminals who resulted in children as a victim. Protection efforts for victims of a criminal act is an effort to recover losses that have been obtained by the victim. This will be more interpreted if victims are directly involved in the process of settlement of criminal cases. Enforcement law is a development effort which is continuous aiming realizing national life and a safe, orderly, and peaceful state dynamic environment in the world independent.21 The purpose of providing compensation is not others to develop justice and the welfare of the victim as community members by benchmarks in practice, victims are given rights and obligation to be developed as people, children are also people. 20 Ahmad Taufan Damanik, The Efforts of ASEAN-ACWC in Addressing the Trend of Sexual Crimes Against Children Online”, Makalah, Conference on Sexual Crime against Children Online: Law Enforcement and Regional Cooperation, Jakarta, 29 – 30 October 2012. 21 Thantawi, et al., Protection of Victims of Crime Cyber Crime in the Indonesian Criminal Law System, Journal Ilmu Hukum, Vol. .2, No. 1, 2014, page. 39. 22 Hardianto Djanggih, The conception of Legal Protection for Children as Victims of Cyber Crimes Through Penal and Non Penal Approaches, Mimbar Hukum Journal, Volume 30 Number 2, June 2018, page 323. y p g 22 Hardianto Djanggih, The conception of Legal Protection for Children as Victims of Cyber Crimes Through Penal and Non Penal Approaches, Mimbar Hukum Journal, Volume 30 Number 2, June 2018, page 323. Jurnal Pembaharuan Hukum ASPECTS OF LEGAL PROTECTION FOR Volume 7 No.3 Sept-Dec 2020 CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 207 20 Ahmad Taufan Damanik, The Efforts of ASEAN-ACWC in Addressing the Trend of Sexual Crimes Against Children Online”, Makalah, Conference on Sexual Crime against Children Online: Law Enforcement and Regional Cooperation, Jakarta, 29 – 30 October 2012. 21 Thantawi, et al., Protection of Victims of Crime Cyber Crime in the Indonesian Criminal Law System, Journal Ilmu Hukum, Vol. .2, No. 1, 2014, page. 39. 22 Hardianto Djanggih, The conception of Legal Protection for Children as Victims of Cyber Crimes Through Penal and Non Penal Approaches, Mimbar Hukum Journal, Volume 30 Number 2, June 2018, page 323. C. RESULT AND DISCUSSION Therefore, required strict, simple regulations, and easy to understand so it can avoid discrimination in the application by law enforcement and intimidation from certain parties which exacerbates it the condition of the victim in its suffering prolonged. g p g In the development of laws and regulations in Indonesia, penalties for compensation disadvantages are not only the domain of civil law but has also entered into criminal law. Development this happens because it is increasing world community attention to access victims of criminal acts in the justice system criminal.22 The penalty for damages is a crime that obliges someone who has acted detrimentally to others to pay some money or goods to the injured person. The existence of the Criminal Code which is Dutch heritage that was formed in 19th century did not recognize the punishment of change loss. This is because the paradigm at the time of its formation more to retribution (retributive). However, Article 14c of the Criminal ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 207 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Code has provisions conditional criminal stating that judge in imposing conditional punishment against the perpetrator of a criminal act other than determining general conditions can also impose special conditions in the form of replacement losses that must be met before the term the experiment ended. Apart from the Criminal Code, it is deep Another positive criminal law provision about indemnity is also known. Corruption Criminal Act in Article 34 regulates additional penalties in the form of payment of replacement money in the amount as much as possible as wealth objects obtained from corruption. In this case, the compensation is given to the state because the state is the victim (collective victim). In the context of pornographycyber then this compensation sanction can also be in the form of collective victims represented by the state and their use for mental rehabilitation victims of pornography. Also, compensation can be given to people who directly fall victim to pornography cyber. Based on the results of interviews with Police investigators, a special criminal investigation Unit 1 in the section of cybercrime, explained that during the pandemic era that had started in Indonesia last March 2020. 23 Nanung, Reksrimsus Investigator At The Central Java Police, Cybercrime Unit 1, 26 October 2020 C. RESULT AND DISCUSSION The results of the police report in Unit 1, only one case of a child victim of cybercrime in the Central Java Regional Police.23 in the implementation of legal protection for victims by the articles violated by the perpetrator. since the perpetrator is a child, then diversion is done. Sanctions for damages as referred to has stated above is the sanction intended to protect victims of action criminal. Apart from that, there are still some other benefits that provide added value on compensation sanctions if included in the new KUHP. 208 ASPECTS OF LEGAL PROTECTION FOR Jurnal Pembaharuan Hukum CHILDREN FROM CYBERCRIME Volume 7 No.3 Sept-Dec 2020 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 23 Nanung, Reksrimsus Investigator At The Central Java Police, Cybercrime Unit 1, 26 October 2020 Book: Ahmad Taufan Damanik, 2012, The Efforts of ASEAN-ACWC in Addressing the Trend of Sexual Crimes Against Children Online, Paper Conference on Sexual Crime against Children Online: Law Enforcement and Regional Cooperation, Jakarta; Ahmad Taufan Damanik, 2012, The Efforts of ASEAN-ACWC in Addressing the Trend of Sexual Crimes Against Children Online, Paper Conference on Sexual Crime against Children Online: Law Enforcement and Regional Cooperation, Jakarta; D. CONCLUSION D. CONCLUSION The conclusion in this article is that it is preferable to use social media following applicable regulations and subject to the Law of the Republic of Indonesia Number 19 of 2016 concerning Amendments to Law of the Republic of Indonesia Number 11 of 2008 concerning Electronic Information and Transactions. As well as the need for an active role from the community and parents in supervising children in using social media so that they are not entangled with criminal sanctions and/or compensation. 208 ASPECTS OF LEGAL PROTECTION FOR Jurnal Pembaharuan Hukum CHILDREN FROM CYBERCRIME Volume 7 No.3 Sept-Dec 2020 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 23 Nanung, Reksrimsus Investigator At The Central Java Police, Cybercrime Unit 1, 26 October 2020 Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Journal: Hardianto Djanggih, The conception of Legal Protection for Children as Victims of Cyber Crimes Through Penal and Non Penal Approaches, Mimbar Hukum Journal, Volume 30 No. 2, June 2018; Hardianto Djanggih, The conception of Legal Protection for Children as Victims of Cyber Crimes Through Penal and Non Penal Approaches, Mimbar Hukum Journal, Volume 30 No. 2, June 2018; Ivan Fauzi Raharja, Wise to Use Social Media Among Students According to Law Number 19 of 2016 Concerning Electronic Information and Transactions, Journal Selat, Volume 6, Number 2, May 2019; Nevey Varida Ariani, Implementation of Law Number 11 of 2012 concerning the System Juvenile Justice to Protect Children's Interests, Media Hukum Journal, Volume 21, Nomor 1, 2014; Pamela Felita and Friends, Use of Social Media and Self Concept in Teens, Psikologi MANASA Journal, Volume 5 Nomor 1, March 2016; Rochmawati, Weny, Internet Utilization of Behavior (Descriptive Study of Using the Internet for Entertainment and Academic Interests among Children in the City of Surabaya), Journal Libri-Net, Vol. 1, No. 1, 2012; S., Kandi Aryani, Youth Acceptance of Pornography Discourse on Sex Sites in Online Media, Journal Penelitian Dinamika Sosial, Vol. 7, 2008; Setiono, Understanding of Legal Research Methodology Postgraduate Law studies of Sebelas Maret University, 2010; Sulistio, Faizin, et al., Alternative Model of Crime of Cyber Pornography Crime, Journal Arena Hukum, Vol. 9, No. 3, 2016; Jurnal Pembaharuan Hukum ASPECTS OF LEGAL PROTECTION FOR Volume 7 No.3 Sept-Dec 2020 CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 209 Sulistio, Faizin, et al., Alternative Model of Crime of Cyber Pornography Crime, Journal Arena Hukum, Vol. 9, No. 3, 2016; Thantawi, et al., Protection of Victims of Crime Cyber Crime in the Indonesian Criminal Law System, Journal Ilmu Hukum, Vol. .2, No. 1, 2014; Ulinnuha, Masyari, Protecting Children from Internet Negative Content: A Study of Children's Web Browsing, Jurnal Sawwa, Vol. 8, No. 3, 2013; Legislation: KUHP; Law Number 32 of 2002 concerning The broadcast; Law Number 44 of 2008 about the Pornography; Law Number 33 of 2009 concerning Film emphasized; Thantawi, et al., Protection of Victims of Crime Cyber Crime in the Indonesian Criminal Law System, Journal Ilmu Hukum, Vol. .2, No. 1, 2014; Ulinnuha, Masyari, Protecting Children from Internet Negative Content: A Study of Children's Web Browsing, Jurnal Sawwa, Vol. 8, No. Journal: 3, 2013; Jurnal Pembaharuan Hukum ASPECTS OF LEGAL PROTECTION FOR Volume 7 No.3 Sept-Dec 2020 CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 209 Legislation: KUHP; Law Number 32 of 2002 concerning The broadcast; Law Number 44 of 2008 about the Pornography; Law Number 33 of 2009 concerning Film emphasized; Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 Legislation: ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani 209 Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 209 Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Law Number 35 of 2014 Change Law Number 23 of 2002 on Child Protection; Law number 19 of 2016 change Law 11 of 2008 Concerning Electronic Information and Transactions; Website: Ministry of Communication and Information of the Republic of Indonesia, Indonesian Internet User Penetration and Behavior, https://aptika.kominfo.go.id/index.php/berita/274-rilis-hasil-survei- penetrasi-dan-perilaku-pengguna-internet-indonesia-tahun-2018; The Indonesian Child Protection Commission in 2011 – 2016. www.kpai.go.id; Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Law Number 35 of 2014 Change Law Number 23 of 2002 on Child Protection; Law number 19 of 2016 change Law 11 of 2008 Concerning Electronic Information and Transactions; Website: Ministry of Communication and Information of the Republic of Indonesia, Indonesian Internet User Penetration and Behavior, https://aptika.kominfo.go.id/index.php/berita/274-rilis-hasil-survei- penetrasi-dan-perilaku-pengguna-internet-indonesia-tahun-2018; The Indonesian Child Protection Commission in 2011 – 2016. www.kpai.go.id; Law Number 35 of 2014 Change Law Number 23 of 2002 on Child Protection; Law number 19 of 2016 change Law 11 of 2008 Concerning Electronic Information and Transactions; Law Number 35 of 2014 Change Law Number 23 of 2002 on Child Protection; Law number 19 of 2016 change Law 11 of 2008 Concerning Electronic Information and Transactions; Website: Ministry of Communication and Information of the Republic of Indonesia, Indonesian Internet User Penetration and Behavior, https://aptika.kominfo.go.id/index.php/berita/274-rilis-hasil-survei- penetrasi-dan-perilaku-pengguna-internet-indonesia-tahun-2018; The Indonesian Child Protection Commission in 2011 – 2016. www.kpai.go.id; 210 ASPECTS OF LEGAL PROTECTION FOR CHILDREN FROM CYBERCRIME Ida Musofiana, Aji Sudarmaji, Ira Alia Maerani Jurnal Pembaharuan Hukum Volume 7 No.3 Sept-Dec 2020 210
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https://www.cb.cityu.edu.hk/ef/doc/GRU/WPS/GRU%232019-030%20Benjamin.pdf
English
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Predicting mid-life capital formation with pre-school delay of gratification and life-course measures of self-regulation
Journal of economic behavior & organization
2,020
cc-by
16,267
a b s t r a c t How well do pre-school delay of gratification and life-course measures of self-regulation predict mid-life capital formation? We surveyed 113 participants of the 1967–1973 Bing pre-school studies on delay of gratification when they were in their late 40’s. They re- ported 11 mid-life capital formation outcomes, including net worth, permanent income, absence of high-interest debt, forward-looking behaviors, and educational attainment. To address multiple hypothesis testing and our small sample, we pre-registered an analysis plan of well–powered tests. As predicted, a newly constructed and pre-registered mea- sure derived from preschool delay of gratification does not predict the 11 capital formation variables (i.e., the sign-adjusted average correlation was 0.02). A pre-registered composite self-regulation index, combining preschool delay of gratification with survey measures of self-regulation collected at ages 17, 27, and 37, does predict 10 of the 11 capital formation variables in the expected direction, with an average correlation of 0.19. The inclusion of the preschool delay of gratification measure in this composite index does not affect the index’s predictive power. We tested several hypothesized reasons that preschool delay of gratification does not have predictive power for our mid-life capital formation variables. © 2019 The Authors. Published by Elsevier B.V. y This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) his is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) ✩ Authors are listed in alphabetical order. Sadly, our extraordinary friend and coauthor, Walter Mischel, who led the Bing study starting with the first experiments in 1967, passed away during the editorial process. The Bing Longitudinal Project was supported by a number of grants from NIMH and NSF to Walter Mischel and Yuichi Shoda, of which the most recent are National Institutes of Health Grant MH39349 and National Science Foundation Grant BCS-0624305 . Research reported in this publication was also supported by the National Institute on Aging of the National Institutes of Health under Award Numbers P30AG034532 and P01AG005842 and the Pershing Square Fund for Research on the Foundations of Human Behavior. The content is the sole responsibility of the authors and does not necessarily represent the official views of any of these grant agencies. For her initial work preparing the economics survey, the authors thank Dawn DeGere. Journal of Economic Behavior and Organization journal homepage: www.elsevier.com/locate/jebo Predicting mid-life capital formation with pre-school delay of gratification and life-course measures of self-regulation ✩ Daniel J. Benjamin a , b , David Laibson b , c , Walter Mischel d , Philip K. Peake e , Yuichi Shoda f , Alexandra Steiny Wellsjo g , ∗, Nicole L. Wilson h a Center for Economic and Social Research and Department of Economics, University of Southern California, Los Angeles, CA 900 United States b National Bureau of Economic Research, Cambridge, MA 02138, United States c Department of Economics, Harvard University, Cambridge, MA 02138, United States d Department of Psychology, Columbia University, New York, NY 10027, United States e Department of Psychology, Smith College, Northampton, MA 01063, United States f Department of Psychology, University of Washington, Seattle, WA 98195, United States g Department of Economics, University of California, Berkeley, CA 94720, United States h Department of Management, Lundquist College of Business, University of Oregon, Eugene, OR 97403, United States a r t i c l e i n f o Article history: Received 9 May 2018 Revised 28 May 2019 Accepted 17 August 2019 JEL Classifications: D910 D140 I310 I210 I120 Keywords: Self-regulation Delay of gratification Mid-life capital formation Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation Dan Benjamin, University of Southern California/NBER David Laibson, Harvard University/NBER Walter Mischel, Columbia University Philip K. Peake, Smith College Yuichi Shoda, University of Washington Alexandra Steiny Wellsjo, University of California, Berkeley Nicole L. Wilson, University of Oregon © 2019 by Benjamin, Laibson, Mischel, Peake, Shoda, Wellsjo & Wilson. All rights reserved. Short sections of text, not to exceed two paragraphs, may be quoted without explicit permission provided that full credit, including © notice, is given to the source. © 2019 by Benjamin, Laibson, Mischel, Peake, Shoda, Wellsjo & Wilson. All rights reserved. Short sections of text, not to exceed two paragraphs, may be quoted without explicit permission provided that full credit, including © notice, is given to the source. ARTICLE IN PRESS JID: JEBO Journal of Economic Behavior and Organization xxx (xxxx) xxx Journal of Economic Behavior and Organization xxx (xxxx) xxx E-mail address: alexsteiny@berkeley.edu (A.S. Wellsjo). 1. Introduction The capacity to self-regulate matters for a wide array of life outcomes. A critical component of self-regulation is the ability to delay gratification. Working at Stanford’s Bing Nursery School beginning in the late 1960’s, Mischel and colleagues conducted numerous experimental variations of the now classic “self-imposed delay of gratification” paradigm to examine the cognitive and contextual mechanisms that affect preschoolers’ ability to wait to obtain a more desired outcome ( Mischel et al., 1989; Mischel, 2014 ). Follow-up investigations beginning in the 1980’s reported that preschool waiting time predicted better self-regulation as teenagers, especially among children who participated in certain experimental variations of the preschool delay task ( Shoda et al., 1990 ). These “diagnostic” experimental conditions were challenging in that they com- bined the physical presence of the tempting outcomes with requiring children to rely on their own spontaneously generated strategies for coping with the frustration of waiting ( Shoda et al., 1990 ). Longitudinal research with the Bing cohort has evolved into a multi-disciplinary exploration. Research on the Bing cohort now spans roughly 50 years, making it the longest study of the delay of gratification paradigm. 1 Outcomes that have been studied include coping, social, and academic competence ( Mischel et al., 1988; Mischel et al., 1989; Shoda et al., 1990 ), SAT scores ( Shoda et al., 1990 ), substance use ( Ayduk et al., 20 0 0 ), borderline personality features ( Ayduk et al., 2008 ), BMI ( Schlam et al., 2013 ), executive functioning, and neural activation patterns ( Berman et al., 2013; Casey et al., 2011 ). The predictive power of the delay of gratification task has also been documented in other samples with childhood and adolescent outcomes such as BMI ( Connell and Francis, 2014; Francis and Susman, 2009; Seeyave et al., 2009 ), ADHD symptoms ( Campbell and von Stauffenberg, 2009 ), academic competence ( Duckworth et al., 2013 ; Watts et al., 2018 ), and social competence ( Yang and Wang, 2007 ). As one would expect, partial regression coefficients between preschool delay and subsequent life outcomes are lower when early childhood, delay-related variables such as family background and early cognitive development are added to the model as predictors ( Watts et al., 2018 ). 1. Introduction In previous research in the Dunedin cohort, other survey and observer-reported measures of early life self-control have been found to be predictive of indices of adult outcomes, including socioeconomic status, financial planfulness, and income ( Moffitt et al., 2011 ). The network of documented longitudinal relations of preschool delay of gratification are extensive. Nonetheless, both media and academic accounts of the research commonly exaggerate the scope of the actual findings ( Watts et al., 2018 ). In these accounts, preschool delay is cast as prognostic of almost all adult life milestones. In light of this common miscasting, it is important that ongoing research empirically examine whether and how self-control actually relates to later life outcomes. The current research explores one such connection: the relation to mid-life capital formation. Economists conceptualize capital formation to include any costly activity that accumulates a resource (i.e., “capital”) that generates future benefits. This definition implies that capital formation includes investments in human capital (e.g., working hard at school or on the job to obtain knowledge/experience that will pay off later), investments in social capital (e.g., developing a network of professional contacts that will increase long-run professional success), and investments in financial capital (e.g., saving now so that the household can spend more later). In the current paper, we revisit 113 individuals from the original Bing cohort, roughly 45 years after they participated in the original experiments. Within this sample, we examine associations between measures of self-regulation based on multiple assessments during the first four decades of life (including preschool delay) and a comprehensive array of mid-life measures of capital formation. In addition, we also study preschool waiting time on its own as a predictor of mid-life capital formation. To our knowledge, ours is the first attempt to look at these relationships because no cohort for which delay of gratification was measured in childhood has become old enough to survey in mid-life until now, with the exception of the Dunedin cohort, which is roughly ten years younger than the Bing cohort which we study. In the conclusion of this paper, we compare our results to the small set of partially overlapping results from the Dunedin study and find a remarkable degree of concordance despite the starkly different socioeconomic contexts of the two studies (and the somewhat younger age of the Dunedin study participants). The rest of the paper proceeds as follows. D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 2 1. Introduction In Section 2 we discuss our sample. In Section 3 we describe our key mea- sures and our pre-registered analysis plan. In Section 4 we state our pre-registered hypotheses and report our main results. In Section 5 we present additional results. In Section 6 we conclude and discuss the generalizability of our findings. The Online Appendix (OA) contains further details about sample recruitment, survey design, and analyses, as well as a number of additional and robustness analyses that we conducted. a b s t r a c t For helpful comments, the authors thank Constança Esteves-Sorenson and Charlie Sprenger; participants at the Behavioral Economics Annual Meeting, the Drawing Links between Happiness, Health, and Self-regulation conference, the LABEL-IEPR Conference on Children’s Decision Making, the NBER Summer Institute, and the RAND Summer Institute; and seminar participants at the University of California Berkeley, the University of California San Diego, and the University of Southern California. ∗Corresponding author. E-mail address: alexsteiny@berkeley.edu (A.S. Wellsjo). https://doi.org/10.1016/j.jebo.2019.08.016 0167-2681/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) https://doi.org/10.1016/j.jebo.2019.08.016 0167-2681/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) https://doi.org/10.1016/j.jebo.2019.08.016 0167-2681/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license. ( http://creativecommons.org/licenses/by/4.0/ ) Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 JID: JEBO D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 ARTICLE IN PRESS ARTICLE IN PRESS JID: JEBO D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 3 [m3Gsc; November 16, 2019;10:34 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx [ D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx Table 1 Means, medians, and standard deviations of survey measures (prior to rank-normalizations). Survey Sample ( N = 113) Full Sample ( N = 543) Mean Median SD Mean Median SD p -value Preschool measures Male 37% 48% 0.01 Age at wait task (months) 51.6 52.0 5.6 52.1 52 5.7 0.36 Raw wait time (seconds) 509.7 556.0 369.1 470.1 440 367.7 0.20 Deviation from predicted preschool wait time (ln seconds) 0.08 0.51 2.37 0.00 0.27 2.30 0.66 Demographics at economics survey Age 46.0 46.0 2.0 Married 79% Has children (among those with information) 89% Missing information on children 20% White 94% Asian 4% Capital formation measures Net worth ($ millions) 1.8 0.9 3.4 Permanent income ($ thousands) 131 78 147 Wealth-income ratio 13.5 10.1 14.6 High interest-rate debt ($ thousands) 1.2 0.0 2.6 Credit card misuse – – – Delay choice (% later choices) 85% 89% 14% Savings rate 11.0% 10.0% 9.3% Financial health – – – Educational attainment (years) 19.1 19.0 2.1 Forward-looking behaviors – – – Social Status 7.6 8.0 1.4 Notes : Entries of “-” are for measures with no natural scale. p -value is from an OLS regression of each variable on a constant and an indicator variable for being in the survey sample. See OA Section II for more detailed information on sample size for each outcome. Table 1 Means, medians, and standard deviations of survey measures (prior to rank-normalizations). During 2012–2013, we contacted the 156 Bing cohort participants for whom we had current contact information (28% of the 550 total), and we invited them to participate in an online survey. 2 The survey is reproduced in the OA. 113 participants completed it (72% of those we contacted). Table 1 shows the demographics of our survey sample, as well as summary statistics for the measures we discuss in Section 3 . ARTICLE IN PRESS Survey respondents averaged 46 years of age at the time they took the survey, 37% are male, and they are on average white, married with children, well educated, and wealthy. Table 1 also compares our survey sample with the full Bing sample on several variables we have for the full sample: sex, age during the delay of gratification task, and wait time. While our survey sample has fewer males (37% versus 48%), it does not differ substantially from the full Bing cohort on the other variables. 3.1. Capital formation measures 3.1. Capital formation measures From the survey responses, we constructed the following 11 capital formation measures, which we use as dependent variables in our study: • Net worth is the sum of assets minus the sum of debts. • Permanent income is defined as total household income (the average of reported income in the calendar year preceding the survey and the calendar year when the respondent was age 35) divided by the reported number of adults living in the household. Income at age 35 is inflation-adjusted to dollars in the year before the survey. • Permanent income is defined as total household income (the average of reported income in the calendar year preceding the survey and the calendar year when the respondent was age 35) divided by the reported number of adults living in the household. Income at age 35 is inflation-adjusted to dollars in the year before the survey. W l h i i i h di id d b i • Wealth-income ratio is net worth divided by permanent income. • High interest-rate debt (reverse scaled) is the annual amount of interest above 6% paid on high-interest rate debt. For each debt category, respondents selected an interest rate bucket for each type of debt. We assumed the interest rate is the midpoint of the selected category. For each high interest-rate debt category we calculate the amount of interest above 6% as the dollar amount of debt multiplied by the interest rate above 6%. • Credit card misuse (reverse scaled) is an index with four components: high interest-rate debt, amount of carried credit card debt (i.e., not paid off each month), a binary measure for having been denied a credit card in the last year, and number of late payments on a credit card bill in the last year. • Credit card misuse (reverse scaled) is an index with four components: high interest-rate debt, amount of carried credit card debt (i.e., not paid off each month), a binary measure for having been denied a credit card in the last year, and number of late payments on a credit card bill in the last year. 2 The survey was approved by the institutional review boards at Harvard University and the University of Washington. All participants provided informed consent. Many of the questions used to form the economic outcome variables were taken from the Cognitive Economics Project ( http://ebp-projects.isr. umich.edu/CogEcon/ ). 2. The Bing cohort and our survey sample The original Bing experiments were conducted during 1967–1973 (first described in Mischel and Ebbesen, 1970 ). They included a total of 550 students from Stanford’s Bing Nursery School, aged about 4 years old (ranging from 2 to 6). Many of the participants are children of Stanford faculty and staff. 1 Other long-running longitudinal studies with measures of self-regulation include the Dunedin Multidisciplinary Health and Development Study and the NICHD Study of Early Child Care and Youth Development (SECCYD). The Dunedin cohort was first assessed at age 3 in the mid-1970’s and most recently assessed at age 38 (with an ongoing assessment at age 45). While the Dunedin study did include parent and teacher survey measures and observer reports of self-regulation in preschool, it did not include the Mischel delay of gratification task. The NICHD-SECCYD began collecting data in the early 1990’s when children were aged 0-3 and collected the final wave of data in the mid-20 0 0’s when the children were 15 years old. The NICHD-SECCYD contains a version of the Mischel delay of gratification task as well as parent and teacher survey measures of self-regulation from birth through early adolescence. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx • Delay choice is the percent of questions for which the respondent selected the “money later” instead of the “money sooner” option in 40 tradeoff questions (20 questions with a choice between money today and thirty days from today and 20 questions with a choice between money thirty and sixty days from today). • Savings rate is the self-reported percentage of income saved over the past few years, including retirement accounts and all other savings. • Savings rate is the self-reported percentage of income saved over the past few years, including retirement accounts and all other savings. • Financial health is an index comprised of three components: self-ratings about having enough money to meet needs, difficulty in paying monthly bills, and current financial situation. • Financial health is an index comprised of three components: self-ratings about having enough money to meet needs, difficulty in paying monthly bills, and current financial situation. y p y g y • Educational attainment (years of education) is calculated from reported degrees received. • Educational attainment (years of education) is calculated from reported degrees received. • Forward-looking behaviors is an index comprised of 5 equal-weighted components: measures of diet, exercise habits, and BMI; smoking and alcohol behavior; preventative health and dental care; procrastination; and consideration of future financial consequences. • Forward-looking behaviors is an index comprised of 5 equal-weighted components: measures of diet, exercise habits, and BMI; smoking and alcohol behavior; preventative health and dental care; procrastination; and consideration of future financial consequences. • Social status is self-reported placement in society on a 10-rung ladder. The ladder is described as representing where people stand in the United States, with the top of the ladder being those who have the most money, the most education, and the most respected job. • Social status is self-reported placement in society on a 10-rung ladder. The ladder is described as representing where people stand in the United States, with the top of the ladder being those who have the most money, the most education, and the most respected job. Summary statistics for these measures are in Table 1 . D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx We applied a rank-order inverse normal transformation to all final variables – including both the capital formation vari- ables described above, as well as the self-regulation variables described below – because our statistical procedures have better small-sample properties when the variables are normally distributed (for discussion of this transformation, see, e.g., Bishara and Hittner, 2012 ). In general, the raw data for our capital formation and self-regulation variables do not appear to be normally distributed (see OA Sections I and II for distributions of the raw and transformed variables). The rank- normalization approach has the advantage that we can apply it uniformly, rather than making different parametric assump- tions for each variable (e.g., that wealth is log-normally distributed). To implement the transformation, we first calculated the rank order of the subject within the sample and then used the inverse normal cdf to fit the ranks to a standard nor- mal distribution. The transformation preserves the rank ordering of the original variables but alters their scaling and may therefore affect the correlations with other variables. However, our transformation ensures that variables are approximately normal by construction and minimizes the effects of outliers. Because the transformation relies on rank order, the variables in our analyses represent a normalized measure of relative standing. 3 In prior work, wait times were adjusted to account for experimental condition by taking the difference from the mean wait time in a condition (equivalent to a fixed-effects specification). In the current work, we instead treat the experimental conditions as random effects (as described below) because mean wait time in a condition is estimated imprecisely when the per-condition sample size is small. Essentially, the random effects model predicts the effect of an experimental condition as a weighted average of the observed mean wait time in the condition (conditional on age and sex) with the mean wait time across experimental conditions. The larger the sample size for an experimental condition, the larger the weight assigned to the observed mean wait time in that condition. In addition to the treatment of experimental conditions, RND also differs from wait time used in prior work because it accounts for the child’s age and sex, it adjusts for the fact that observed wait times are capped at 15 minutes, and we measure wait time in log-seconds rather than seconds. Finally, we note that our new rank-normalized measure may or may not be an improvement on the old absolute-deviation-from-the-mean measure. See OA for more discussion of the methodological differences. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 ARTICLE IN PRESS JID: JEBO D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 3.2. Self-regulation measures We study two measures of self-regulation. The first measure, rank-normalized delay (RND) , is derived from children’s waiting times observed while participating in experimental variants of the self-imposed delay of gratification paradigm when the children were in preschool. The second measure, rank-normalized self-regulatory index (RNSRI) , consists of four equal-weighted components, each measuring self-regulation at a different age. One of the components of RNSRI is RND. We will describe RND followed by RNSRI. RND is a new measure derived from preschool waiting that takes into account the fact that there were diverse treatment conditions in the wait-task experiments differing in a number of ways that affected how long children on average waited in each condition (including both “diagnostic” and “non-diagnostic” conditions, as we discuss below). 3 In addition, RND also addressed the fact that wait times are censored (the criterion for successfully waiting was typically set to 15 min). Specifically, we constructed the RND measure in three steps: (a) we generated an expected log wait time for each treatment condition using a tobit random-effects model, controlling for the child’s sex and age at the time of the experiment; (b) we calculated the difference between each child’s actual log wait time and this expected log wait time; (c) we rank-normalized these differences. We now explain steps (a) and (b) in more detail. For step (a), we used wait-time data from nearly all the Bing participants: a total of 543 children for whom data on wait time, age at which they did the wait-time task, and sex is available. The sample size per experimental condition is 3 to 165 participants. Our survey data, which is from a subset of 113 of these participants across 20 of the 21 experimental conditions, contains 1 to 34 participants per condition. For children who participated in multiple studies at the Bing School, we use only the wait time from their first delay of gratification study (because the psychological meaning of the waiting experiences in subsequent studies may be different from their first time). p q y ) A histogram of the 543 raw wait times is shown in Fig. 1 A. The distribution has many observations at just a few seconds, as well as a long right tail (censored at 900 s). For our model of expected wait times, we therefore adopted a log-normal Please cite this article as: D.J. Benjamin, D. Laibson and W. 3.1. Capital formation measures Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 3.2. Self-regulation measures Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 5 ARTICLE IN PRESS JID: JEBO [m3Gsc; November 16, 2019;10:34 ] [m3Gsc; November 16, 2019;10:34 ] JID: JEBO JID: JEBO D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 5 [m3Gsc; November 16, 2019;10:34 ] D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx istribution of wait times across Bing cohort participants and experimental conditions stribution of wait times from the 543 children who participated in the original Bing experiments and for whom data on wait time, ag the wait-time task, and sex is available. Figure 1A displays the distribution of individual wait times. Figure 1B displays the distributio es across the 21 experimental conditions. ution. Fig. 1 B shows a histogram of mean raw wait times in the 21 experimental conditions. This distribution 5 5 Fig. 1. Distribution of wait times across Bing cohort participants and experimental conditions Fig. 1. Distribution of wait times across Bing cohort participants and experimental conditions Notes : Distribution of wait times from the 543 children who participated in the original Bing experiments and for whom data on wait time, age at which they did the wait-time task, and sex is available. Figure 1A displays the distribution of individual wait times. Figure 1B displays the distribution of mean wait times across the 21 experimental conditions. Fig. 1. Distribution of wait times across Bing cohort participants and experimental conditions Notes : Distribution of wait times from the 543 children who participated in the original Bing experiments and for whom data on wait time, age at which they did the wait-time task, and sex is available. Figure 1A displays the distribution of individual wait times. Figure 1B displays the distribution of mean wait times across the 21 experimental conditions. Notes : Distribution of wait times from the 543 children who participated in the original Bing experiments and for w they did the wait-time task, and sex is available. Figure 1A displays the distribution of individual wait times. Figur wait times across the 21 experimental conditions. distribution. Fig. ARTICLE IN PRESS We calculated an empirical-Bayes estimate of the expected ln(wait time) for each participant as the expected value of the posterior distribution of the experimental condition’s random effect, treating the model’s parameter estimates as the true parameter values. Table 2 shows the parameter estimates. The age coefficient of 0.08 (SE = 0.02) implies that, on average, every additional month of age when participating in the wait-time task is associated with waiting roughly 8% longer. This is consistent with prior work in the Bing and other samples on the development of delay of gratification strategies ( Mischel and Mischel, 1983 ) and directionally consistent with prior results on the correlation between age and delay (e.g., Carlson et al. (2018) find a marginally significant positive correlation between wait time and age in the Bing and two other samples). The male coefficient of −0.67 (SE = 0.23) implies that, controlling for age, males on average wait less than 50% as long as females—a remarkably large difference between the sexes. Previous analyses of the Bing cohort have found mixed results with respect to gender. Mischel and Underwood (1974) do find that males on average wait less than females when the data is aggregated across experimental arms. However, other analyses of subsets of the Bing cohort have not found statistically different waiting times between boys and girls ( Mischel and Moore, 1973, 1980; Mischel et al., 1988; Ayduk et al., 20 0 0 ), perhaps due to the smaller sample sizes in these analyses. A recent exception is Carlson et al. (2018) , who find that girls waited significantly longer than boys in the diagnostic subsample of Bing participants. In a different sample of 135 preschoolers in the 1980’s, Carlson et al. (2018) find a similar, though statistically insignificant, sex difference. In a third sample of 540 preschoolers in the 20 0 0’s, they find no sex difference. Turning to step (b), for each participant we calculate ln(subject’s actual wait time) – expected ln(wait time). If the par- ticipant’s wait time was at the boundary of 900 s, we used the estimated model to calculate the expected value of the uncensored wait time (conditional on the participant waiting at least 900 s, as well as on the participant’s age and sex) and used this expected value in place of the participant’s actual wait time. ARTICLE IN PRESS D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx [m3Gsc; November 16, 2019;10:34 ] D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx [ D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx [ D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 6 Table 2 Parameter estimates from tobit random effects model of log preschool wait time, con- trolling for age, sex, and experimental condition. Wait time in log-seconds β1 : Age at delay of gratification task (months) 0.081 ∗∗∗ (0.020) β2 : Male −0.673 ∗∗∗ (0.227) α: Constant 2.289 ∗∗ (1.071) σε : SD of Individual-Specific Error 2.469 ∗∗∗ (0.101) ση : SD of Random Effect (Experimental Condition) 0.786 ∗∗∗ (0.218) N 543 Notes: Parameter estimates from a tobit random effects regression of preschool wait time (measured in log-seconds) on age at delay of gratification task, sex, and a con- stant. ση is the estimated standard deviation of the experimental condition effect, and σε is the estimated standard deviation of the individual-specific error. Standard errors are in parentheses. ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01. Table 2 Parameter estimates from tobit random effects model of log preschool wait time, con- trolling for age, sex, and experimental condition. Notes: Parameter estimates from a tobit random effects regression of preschool wait time (measured in log-seconds) on age at delay of gratification task, sex, and a con- stant. ση is the estimated standard deviation of the experimental condition effect, and σε is the estimated standard deviation of the individual-specific error. Standard errors are in parentheses. ∗p < 0.1, ∗∗p < 0.05, ∗∗∗p < 0.01. month older β1 , the effect of being male β2 , the variance of the experimental-condition random effects σ 2 η , and the variance of the individual-specific error σ 2 ε . month older β1 , the effect of being male β2 , the variance of the experimental-condition random effects σ 2 η , and the variance of the individual-specific error σ 2 ε . p ε We estimated the tobit random-effects model by maximum likelihood (using the GLLAMM procedure in Stata version 14.0). Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 4 As we report in Section 5 , contrary to what we expected, the predictive power of the self-regulation measures does not increase as the age of mea- surement gets closer to the age at which we measure the capital formation variables. 3.2. Self-regulation measures 1 B shows a histogram of mean raw wait times in the 21 experimental conditions. This distribution appears to have a long right tail, so we also assumed that the condition means are log-normally distributed. We estimated the following tobit model of expected wait time: y ∗ gi = α + β1 × ag e gi + β2 × mal e gi + ηg + ε i , where i indexes individuals; g indexes experimental conditions; y ∗ gi is the underlying, uncensored, log wait time; ag e gi is age in months at time of the delay task ; mal e gi is a binary indicator of sex; ηg is the group random effect ∼N( 0 , σ 2 η ) , and ε gi is an individual-specific error ∼N( 0 , σ 2 ε ) . We modeled the effect of experimental condition as a random effect to deal with the very small sample sizes in some conditions. We used a tobit model to account for the maximum wait time of 900 s (or 15 min) imposed in the original experiments. The model has five parameters: a constant term α, the effect of being one Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 3.3. Statistical approach With the data we had, we faced two main statistical challenges. First, our sample size is small: 113 survey respondents. Given the small magnitude of associations we anticipated, we expected to have low statistical power for detecting any particular association between a self-regulation measure and a capital formation measure. Moreover, due to low power, any association we identified as statistically significant would be likely to have an exaggerated effect-size estimate (e.g., Gelman and Carlin, 2014 ), a bias sometimes called the “statistical winner’s curse” (e.g., Ioannidis, 2008 ). Second, because the self-regulation and capital formation variables we examine could yield more than 600 different hypothesis tests, we faced a potential multiple-testing problem and its associated “data-mining” problems. Consequently, there would be a high rate of false positives at a p -value threshold of 0.05. Moreover, if we adopted a family-wise significance threshold such as a Bonferroni correction, we would exacerbate the problem of low power. To address the potential multiple-testing problem, we pre-registered and time-stamped on Open Science Framework ( https://osf.io/u39hg/ ) our coding of the variables and our planned series of analyses before calculating any correlations between independent variables and dependent variables. 5 Moreover, we distinguished between four types of analyses: (i) tests of primary hypotheses, (ii) tests of secondary hypotheses, (iii) ex-post analyses, and (iv) robustness checks. Our pre- registration plan specified our tests of primary and secondary hypotheses and our robustness checks, and we committed to identifying any ex-post analyses as such in the paper. We further committed to treat tests of secondary hypotheses as exploratory. We pre-registered only two primary hypotheses, which we describe in Section 4 . We report some of our secondary and ex-post analyses in Section 5 , and we report all of them in the OA Section V. Our planned series of analyses of our primary hypotheses were designed to address the challenge of our small sample size. We planned four analyses. First, with each of our capital formation measures as a dependent variable, we run an OLS regression on each measure of self-regulation, controlling for sex. (Regressions with the savings rate as the outcome also control for permanent income.) While we report the regression results because they are simple and serve as inputs to our other three tests, we do not use a p -value threshold to judge statistical significance due to the multiple-testing problem. D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 7 “general cognitive ability” (with the items selected based on their face validity for these concepts). We then factor-analyzed the remaining 25 items within the Smith sample using principal axis factoring and oblique rotation. We labeled the four oblique factors resulting from this analysis “attention,” “coping,” “goal pursuit,” and “concern for others”; see OA Section I-C for a list of the items included in each of the six subscales. For each participant at each age, we created rank-normalized subscale scores, giving equal weight to each item in the subscale. We then calculated a rank-normalized CCQ score for each respondent at each age, giving equal weight to each of the 6 subscales and then rank-normalizing the resulting variable. We computed RNSRI by taking an equal-weighted mean of RND and rank-normalized CCQ score at ages 17, 27, and 37, and then rank-normalizing the resulting variable. 5 In a slight abuse of terminology, we will often refer to “correlations” between our independent and dependent variables. As described below, what we will actually report are coefficients from OLS regressions of our dependent variables on measures of self-regulation, controlling for sex and other variables. Because both the independent and dependent variables are normalized, the regression coefficients can be interpreted as partial correlation coefficients. 6 For example, for an average correlation of 0.15 between our dependent variables and an independent variable, our simulations yielded on average 3.9 of 11 dependent variables associated with a p -value less than 0.05, 3.4 associated at a false discovery rate threshold of 0.1, and in 97% of simulations, we Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 ARTICLE IN PRESS We then factor-analyzed the remaining 25 items within the Smith sample using principal axis factoring and oblique rotation. We labeled the four oblique factors resulting from this analysis “attention,” “coping,” “goal pursuit,” and “concern for others”; see OA Section I-C for a list of the items included in each of the six subscales. For each participant at each age, we created rank-normalized subscale scores, giving equal weight to each item in the subscale. We then calculated a rank-normalized CCQ score for each respondent at each age, giving equal weight to each of the 6 subscales and then rank-normalizing the resulting variable. We computed RNSRI by taking an equal-weighted mean of RND and rank-normalized CCQ score at ages 17, 27, and 37, D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 7 ARTICLE IN PRESS JID: JEBO [m3Gsc; November 16, 2019;10:34 ] JID: JEBO JID: JEBO 5 In a slight abuse of terminology, we will often refer to “correlations” between our independent and dependent variables. As described below, what we will actually report are coefficients from OLS regressions of our dependent variables on measures of self-regulation, controlling for sex and other variables. Because both the independent and dependent variables are normalized, the regression coefficients can be interpreted as partial correlation coefficients. 6 For example, for an average correlation of 0.15 between our dependent variables and an independent variable, our simulations yielded on average 3.9 of 11 dependent variables associated with a p -value less than 0.05, 3.4 associated at a false discovery rate threshold of 0.1, and in 97% of simulations, we Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- ARTICLE IN PRESS We calculated this expected value using simulation, using 10,0 0 0 draws from the model under the assumption that the parameter estimates in Table 2 are the true parameter values. Our second measure, RNSRI, has four components: RND and survey measures of self-regulation measured at ages 17, 27, and 37. The latter three components are derived from the California Child Q-Set (CCQ), a set of personality descrip- tors ( Block and Block, 1980 ). Versions of the CCQ were completed by participants’ parents with respect to their children’s personality around age 17 and by the participants themselves around ages 27 and 37. 4 For each of the three ages, we constructed a measure of overall self-regulation from subsets of CCQ items, with items and weights determined by the following procedure. First, a preliminary set of 37 CCQ items relating to self-control was selected based on ratings by three of us (Peake, Shoda, and Wilson). These items were administered to an independent sample of 191 Smith College students. We conducted a principal components analysis, which found that all but one of these items loaded positively on the first unrotated principal component. In our Bing survey sample, using ratings obtained at ages 17, 27, and 37, we then conducted a confirmatory principal component analysis on the remaining 36 items. We eliminated five items from the analysis due to failure to demonstrate consistent positive loadings on the first unrotated principal component. From among the remaining 31 items, we formed two subscales, each consisting of 3 items, for “delay of gratification” and 4 As we report in Section 5 , contrary to what we expected, the predictive power of the self-regulation measures does not increase as the age of mea- surement gets closer to the age at which we measure the capital formation variables. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 7 ARTICLE IN PRESS JID: JEBO [m3Gsc; November 16, 2019;10:34 ] “general cognitive ability” (with the items selected based on their face validity for these concepts). In this Section, we describe the two primary hypotheses that represent the core of our study Hypothesis 1. We hypothesize that RNSRI will have a positive, but relatively modest, correlation with the measures of mid- life capital formation. Although we predicted that self-regulation plays a significant role in development, given that many other factors (peers, career choice, marital outcomes, family wealth, inheritance, chance, etc.) influence capital formation, we predicted that the correlation between self-regulation and capital formation is modest. Specifically, our pre-registered prior distribution for the correlation of RNSRI with each measure of mid-life capital formation had a mean of 0.15 (and standard deviation of 0.2). These predicted correlations are consistent with past results for these types of outcomes (e.g., Moffitt et al., 2011; Richard et al., 2003 ). Tests of Hypothesis 1 are reported in the top panel of Table 3 . Of the 11 capital formation measures, 10 are associated with the predicted sign. Six variables are significant at our FDR threshold of 0.1 (and the same six have p -values < 0.05): net worth, credit card misuse, financial health, forward-looking behaviors, educational attainment, and permanent income. In an F -test, the (joint) null that all 11 capital formation measures have no association is rejected (empirical Wald test p -value = 0.0 0 05). OA Section V-A and V-D contains details of all these analyses, Bayesian posterior means and standard deviations for the correlation with each outcome, as well as pre-registered robustness analyses. The average observed correlation across all 11 variables, ¯r = 0.19 (SE = 0.04), is close to our pre-registered prior mean of 0.15. The average of our Bayesian posterior means is a correlation of 0.18. Hypothesis 2. On its own, RND (measured around age four) will have only a very small correlation with the measures of mid-life capital formation. As specified in our pre-registration, we predicted very small correlations between our measure of preschool waiting and measures of mid-life capital formation for three reasons (which we discuss further in Section 5 ). First, most of the respondents to the capital formation survey were tested in experimental conditions that have previously been designated as “non-diagnostic” (e.g., the experimenter suggested a specific self-regulation strategy to the child). Theoretical considerations and earlier findings ( Shoda et al., 1990 ) led us to anticipate that variation within non-diagnostic conditions would generate less predictive power than variation within diagnostic conditions. D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 8 Note that we originally hypothesized that the relationship between preschool waiting and mid-life capital formation would be stronger in the “diagnostic” conditions of the original Bing Study (for example, see Shoda et al., 1990 ; and p. 5 of our pre-registered analysis plan). Nevertheless, in order to maximize sample size, for tests of our primary hypotheses, we pooled respondents who participated in diagnostic and non-diagnostic experimental conditions, as we did in some of the prior work ( Mischel et al., 1988; Ayduk et al., 20 0 0 , 20 08; Schlam et al., 2013 ). Our pre-registered priors and corresponding power calculations take into account this pooling across conditions. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 reject the (joint) null that all 11 variables each have no effect with an empirical Wald test p -value less than 0.05. Instead, for an average correlation of 0.05, our simulations yielded on average 0.9 of 11 dependent variables associated with a p -value less than 0.05, 0.3 associated at a false discovery rate threshold of 0.1, and in 18% of simulations, we reject the (joint) null that all 11 variables each have no effect with an empirical Wald test p -value less than 0.05. 8 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx ARTICLE IN PRESS JID: JEBO [m3Gsc; November 16, 2019;10:34 ] 8 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx ARTICLE IN PRESS JID: JEBO [m3Gsc; November 16, 2019;10:34 ] 3.3. Statistical approach Second, to identify “significance” of associations between a self-regulation measure and a specific capital formation mea- sure, we use a false discovery rate (FDR) threshold of 0.1. Compared with a family-wise error rate threshold, an FDR thresh- old has greater statistical power, albeit with a different interpretation: rather than controlling the error rate of any particular association, an FDR threshold of 0.1 means that among the set of “significant” associations, at most 10% are false positives. We compute the FDR of each association using the Benjamini and Hochberg (1995) algorithm, applied separately to the OLS p -values for each of the two self-regulation measures. Third, for each self-regulation measure, we test for the joint significance of associations with our 11 capital formation measures. We use a Wald test with a p -value threshold of 0.05. Because the standard Wald test p -value is biased downward in small samples, we calculate an empirical p -value by comparing the observed Wald test statistic to a distribution of Wald test statistics under the null, which we generate by permuting our data. Specifically, we randomly permute the values of the dependent variable within the sample 10 0 0 times, and we re-calculate the Wald test statistic each time. We calculate our empirical p -value as equal to the percentile where our observed Wald test statistic falls in the distribution of test statistics from the permuted samples. Finally, to obtain an estimate of the magnitude of the association between each self-regulation measure and each cap- ital formation measure, we report our Bayesian posterior mean and standard deviation for each coefficient. For each self- regulation measure, we pre-registered our prior about the mean and standard deviation of the OLS coefficients for the 11 capital formation measures. We calculate the posterior mean and standard deviation for each coefficient assuming normal distributions for both the prior and the observed OLS coefficient. Due to Bayesian “shrinkage” toward the prior, the Bayesian posterior means do not suffer from the “statistical winner’s curse” bias ( Ioannidis, 2008 ) and can thus serve as our “best guess” effect-size estimates. g Before pre-registering our analysis, we used Monte Carlo (i.e., simulation) methods to confirm that given our priors, our planned FDR and Wald tests were well-powered. 6 In Section 4 , we compare our pre-specified priors to what we found. In this Section, we describe the two primary hypotheses that represent the core of our study Second, as noted for Hypothesis 1, self-control is only one of the many factors that influence mid-life capital formation. Third, the assessment of RND is separated in time from the capital formation variables by over 40 years. Research and theory suggest that while many people are stable in self-control over the life-course, many exhibit change, either towards increasing or decreasing self-control ( Berman et al., 2013; Casey et al., 2011 ). In contrast, the other components of RNSRI average a separation of around 20 years from the capital formation variables and represent an average of many indicators of self-regulation. The predictive power of an index consisting of many items typically exceeds the predictive power of each of its components. Since RND is only one component of the 86- item RNSRI, we expected it to have less predictive power. Specifically, our pre-specified prior distribution for the correlation of RND with each measure of mid-life capital formation had a mean of 0.05. Tests of Hypothesis 2 are reported in the bottom panel of Table 3 . Of the 11 capital formation measures, 6 are positively correlated with RND, but none are significantly associated at our FDR threshold of 0.1 (and all have p -values > 0.05). In an F -test, the joint null of no effect for all 11 capital formation variables is not rejected (empirical Wald test p -value = 0.45). None of our pre-registered robustness analyses, including alternative measures of childhood wait time, substantively affects the conclusions from the bottom panel of Table 3 (see OA Section V-D). The average observed correlation across all 11 variables, ¯r = 0.02 (SE = 0.05), is close to our pre-registered prior mean of 0.05. The average of our Bayesian posterior means is a correlation of 0.03. ation m e contro ation m e contro 5.1. Mechanisms As explained above, three mechanisms motivated our pre-registered hypotheses that the index of self-regulatory mea- sures would be moderately predictive, while the preschool delay of gratification task would be very weakly predictive. These mechanisms flow from standard statistical properties and our understanding of the psychological processes underlying the cumulative findings of past empirical studies: 1. The index of self-regulatory measures is comprised of 86 responses per participant, whereas the preschool delay of gratification task is a single behavioral task. An index of similar measures tends to have a higher signal-to-noise ratio than its components. 1. The index of self-regulatory measures is comprised of 86 responses per participant, whereas the preschool delay of gratification task is a single behavioral task. An index of similar measures tends to have a higher signal-to-noise ratio than its components. p 2. The preschool delay of gratification task is measured using a diagnostic variant of the task for 34 of our 113 participants; the remaining 79 participants experienced a non-diagnostic variant of the pre-school delay of gratification task. Pooling across diagnostic and non-diagnostic conditions weakens the correlation with outcome variables. 2. The preschool delay of gratification task is measured using a diagnostic variant of the task for 34 of our 113 participants; the remaining 79 participants experienced a non-diagnostic variant of the pre-school delay of gratification task. Pooling across diagnostic and non-diagnostic conditions weakens the correlation with outcome variables. g g 3. The index of self-regulatory measures is comprised of questions that are measured throughout the life course up to age 37 (specifically, ages 4, 17, 27, and 37), whereas the preschool delay of gratification task is measured at age 4. Self- regulation measured closer in time to the observed outcomes will be more strongly related to them. 3. The index of self-regulatory measures is comprised of questions that are measured throughout the life course up to age 37 (specifically, ages 4, 17, 27, and 37), whereas the preschool delay of gratification task is measured at age 4. Self- regulation measured closer in time to the observed outcomes will be more strongly related to them. We now discuss (pre-registered) secondary and (non-pre-registered) ex-post analyses that shed some light on these mechanisms. We find support for the first, suggestive evidence in favor of the second, and no support for the third. Table 4 Table 4 Mean of coefficients from regressions of the 11 capital formation measures on RNSRI, RND, and individual CCQ items. Measure(s) of Self-Regulation Type of Analysis Mean of Coefficients Across Measures of Capital Formation RNSRI (includes RND and Age 17, 27, and 37 CCQ indices) Pre-registered primary 0.19 (0.04) RND Pre-registered primary 0.02 (0.05) RND (diagnostic condition) Pre-registered secondary 0.07 (0.09) RND (non-diagnostic condition) Pre-registered secondary −0.01 (0.06) All CCQ items (avg over 85 items) Ex-post 0.07 (0.03) Age 17 CCQ items (avg over 23 items) Ex-post 0.08 (0.05) Age 27 CCQ items (avg over 31 items) Ex-post 0.07 (0.02) Age 37 CCQ items (avg over 31 items) Ex-post 0.08 (0.02) Notes : All regressions control for sex, and regressions with savings rate as the dependent variable also include control for permanent income (coefficients on control variables not reported). For the regressions on CCQ items, the sign of each coefficient is adjusted to be positive before the coefficients are averaged. Bootstrapped standard errors are in parentheses. e 4 n of coefficients from regressions of the 11 capital formation measures on RNSRI, RND, and individual CCQ items. from regressions of the 11 capital formation measures on RNSRI, RND, and individual CCQ items. Notes : All regressions control for sex, and regressions with savings rate as the dependent variable also include control for permanent income (coefficients on control variables not reported). For the regressions on CCQ items, the sign of each coefficient is adjusted to be positive before the coefficients are averaged. Bootstrapped standard errors are in parentheses. 7 Some of the outcome variables, such as the total years of education and income at age 35, precede the age 37 self-regulation measurement. In ex-post analyses, we found that this does not drive our main results; excluding educational attainment, permanent income, and wealth-to-income ratio from the primary outcomes, the average correlation between RNSRI and the remaining outcomes is 0.18. This is consistent with our finding that self-regulation measured at age 37 is not a better predictor of our primary outcomes than measurements at age 17 and 27. Focusing on the three potentially problematic outcomes, while educational attainment does appear to be most strongly predicted by self-regulation at age 37, permanent income and wealth-to-income ratio are not. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 ARTICLE IN PRESS 0 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx ID: JEBO [m3Gsc; November 16, 2019;10 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx [ D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx [ 10 mation measures, controlling for sex. (7) (8) (9) Savings rate Financial health Education years −0.01 0.24 ∗∗ 0.24 ∗∗∗ (0.09) (0.09) (0.09) 103 110 110 −0.06 −0.07 0.13 (0.09) (0.09) (0.09) 106 113 113 de control for permanent income (coefficients n ation s ∗∗∗ 9) 9) ents n 5.1. Mechanisms To test the first mechanism, we evaluate the predictive power of each of the 85 components of the CCQ index compared to the predictive power of the RNSRI index taken as a whole. In this ex-post analysis, we regressed each of the capital formation measures on each of the 85 items in the RNCCQ indices (23 at age 17, 31 at age 27, and 31 at age 37) in separate regressions (i.e., 85 CCQ items x 11 outcomes = 935 regressions). In Table 4 , we report the average coefficient across all 935 regressions of a capital formation measure on a rank-normalized CCQ item. This average correlation is 0.07 (SE = 0.03), which is smaller than 0.19 (SE = 0.04), the average correlation reported above between the RNSRI index and each of the 11 capital formation variables. In a related ex-post analysis, we found stronger correlations between self-regulation measures and the capital formation measures when we use an index of capital formation variables rather than examining them separately. We constructed the capital formation index by averaging the 11 capital formation measures for the 102 subjects with no missing outcome variables and then rank-normalizing this average. The correlation between RNSRI and the capital formation index is 0.43 (SE = 0.09), and 0.13 (SE = 0.10) between RND and the capital formation index; see OA. 7 The second mechanism is a potential explanation for the weak relationships we find with RND. The preschool delay of gratification task was run in many different ways and only some of those trials were theorized to be diagnostic ( Shoda et al., 1990 ). For example, in some trials the four-year-old participants were asked to think about the rewards in a particular way that was hypothesized to facilitate delay (e.g., think of marshmallows as clouds). In other trials, the participants were not given any suggested cognitive strategies. These two methods are referenced in the literature as suggested ideation and spon- taneous ideation . In the original Bing studies, this ideation manipulation was crossed with a reward-exposure manipulation: in some cases the rewards were exposed (elevating appetitive responses and thereby making delay relatively more difficult), and in other cases the rewards were covered or removed. Experimental settings with spontaneous ideation and exposed Please cite this article as: D.J. Benjamin, D. Laibson and W. 5.1. Mechanisms Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 ARTICLE IN PRESS [m3Gsc; November 16, 2019;10:34 JID: JEBO JID: JEBO D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 11 rewards were theorized to be the most likely to be “diagnostic” of self-regulatory competencies. Indeed, it was only these settings that produced the significant correlation between preschool wait time and subsequent adolescent outcomes, includ- ing parental CCQ item ratings of adolescents employed in the current research and SAT scores ( Shoda et al., 1990 ). rewards were theorized to be the most likely to be “diagnostic” of self-regulatory competencies. Indeed, it was only these settings that produced the significant correlation between preschool wait time and subsequent adolescent outcomes, includ- ing parental CCQ item ratings of adolescents employed in the current research and SAT scores ( Shoda et al., 1990 ). Specifically, in our current data, only 34 participants were assessed in the diagnostic version of the preschool delay task. We pre-registered an analysis to examine the predictive power of RND separately for participants in the diagnostic and non-diagnostic version of the preschool delay task, but we classified it as a secondary analysis because we anticipated large standard errors due to the smaller sample sizes. Based on past results, we hypothesized that RND would have more pre- dictive power among the diagnostic participants. As shown in Table 4 , in the diagnostic condition the average correlation between RND and the 11 human capital measures is 0.07 (SE = 0.09), which, incidentally, is equal to the average correlation between the individual components of the CCQ index and the 11 human capital measures. In contrast, the average correla- tion between preschool wait time in the non -diagnostic conditions and the 11 human capital measures is −0.01 (SE = 0.06). Hence, our evidence provides suggestive support for the earlier finding that the diagnostic conditions are the relevant ones for assessing individual differences in self-regulatory competence—albeit with two caveats, both related to the large stan- dard errors: (i) the coefficients in both conditions are statistically indistinguishable from zero, and (ii) the diagnostic and non-diagnostic correlations are not statistically distinguishable from each other. 5.1. Mechanisms If we take the point estimates at face value, they suggest that once we level the playing field, by comparing single questions from the self-regulatory index to the diag- nostic variant of the preschool delay task, the predictive power of preschool waiting time is identical to the typical reported CCQ item assessing self-regulation later in the life-course. However, re-analysis of prior evidence raises questions about the consistency of the differences between diagnostic and non-diagnostic conditions. In an ex-post analysis suggested in the editorial process, we re-analyzed the correlation between BMI and preschool wait time in Schlam et al. (2013) separately for the diagnostic and non-diagnostic subsamples. Of the 164 subjects in the BMI study, 37 were in a diagnostic condition (25 of whom are also in the current survey sample). Contrary to our findings, the relationship between preschool delay (measured as the deviation from the condition mean) and BMI is weaker in the diagnostic group ( r = −0.03, SE = 0.14) than in the non-diagnostic group ( r = −0.24, SE = 0.08), though the difference is not statistically distinguishable. The correlations between BMI and delay deviation are similar if we minimize the effect of outliers by winsorizing the data. See OA Section V-C-8 for more detail. It is also worth noting that two studies using data from the NICHD-SECCYD find the expected negative relationship between wait time in the diagnostic version of the preschool task and adolescent BMI ( Seeyave et al. (2009) and Francis and Susman (2009) ). In light of all these results, evidence for the importance of the diagnostic methodology is mixed. To test the third mechanism, in an ex-post analysis we examined the relationship between the date of measurement and predictive power. Surprisingly to us, we found no relationship between the age at which the self-regulation questions from the CCQ were administered and the predictive power of those questions for mid-life capital formation. Specifically, the mean correlation with the capital formation measures for the questions administered at age 17 is 0.08 (SE = 0.05), the mean correlation from the questions administered at age 27 is 0.07 (SE = 0.02), and the mean correlation for the questions administered at age 37 is 0.08 (SE = 0.02) ( Table 4 ). 8 8 Similarly, there is limited evidence for an age trend in the predictive power of the indices of CCQ questions. In ex-post analysis, we found that the average correlations between the mid-life capital formation measures and RNCCQ at ages 17, 27, and 37 are 0.15 (SE = 0.09), 0.17 (SE = 0.06), and 0.19 (SE = 0.05), respectively (see OA). 5.2. Excluding RND from the self-regulation measure 5.2. Excluding RND from the self-regulation measure 5.2. Excluding RND from the self-regulation measure We formulated our primary hypotheses in terms of RND and RNSRI, where the former self-regulation measure is a com- ponent of the latter. Among our pre-registered secondary analyses, we also examined an RNCCQ index, which is otherwise the same as RNSRI (i.e., it is composed of the CCQ indices from ages 17, 27, and 37) but excludes RND. This RNCCQ index has correlation r = 0.16 (SE = 0.09) with RND in the survey sample. 9 Considering the predictive power of the RNCCQ index on its own, we find that its mean correlation with the capital formation measures is 0.20 (SE = 0.04) whether we control for RND (pre-registered secondary analysis) or not (ex-post analysis); see OA Sections V-B-2 and V-C-14 for more details. Thus, the inclusion of RND in the RNSRI index does not change its predictive power for the capital formation measures. 5.3. Money now vs. money later as a predictor average correlations between the mid-life capital formation measures and RNCCQ at ages 17, 27, and 37 are 0.15 (SE = 0.09), 0.17 (SE = 0.06), and 0.19 (SE = 0.05), respectively (see OA). 9 The overall correlation between RND and RNCCQ is calculated by combining RNCCQ data across ages 17, 27, and 37. The correlations for each respective age bucket are r = 0.06 (SE = 0.10), r = 0.23 (SE = 0.09), and r = 0.15 (SE = 0.10). Readers interested in other subcomponents of RNCCQ (including the factor- analytic subscales) can find the associated correlations with RND in the OA. 10 For a review and methodological critique of money earlier vs. later questions, see Cohen et al (forthcoming) . For example, in principle choices in the money earlier vs. later paradigm should reveal market interest rates, transactions costs, and trustworthiness of the experimenter, and should not measure true time preferences (because money is temporally fungible). Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 8 Similarly, there is limited evidence for an age trend in the predictive power of the indices of CCQ questions. In ex-post analysis, we found tha average correlations between the mid-life capital formation measures and RNCCQ at ages 17, 27, and 37 are 0.15 (SE = 0.09), 0.17 (SE = 0.06), and (SE = 0.05), respectively (see OA). 9 The overall correlation between RND and RNCCQ is calculated by combining RNCCQ data across ages 17, 27, and 37. The correlations for each respe age bucket are r = 0.06 (SE = 0.10), r = 0.23 (SE = 0.09), and r = 0.15 (SE = 0.10). Readers interested in other subcomponents of RNCCQ (including the fa analytic subscales) can find the associated correlations with RND in the OA. 10 For a review and methodological critique of money earlier vs. later questions, see Cohen et al (forthcoming) . For example, in principle choices i money earlier vs. later paradigm should reveal market interest rates, transactions costs, and trustworthiness of the experimenter, and should not me true time preferences (because money is temporally fungible). Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with p 9 The overall correlation between RND and RNCCQ is calculated by combining RNCCQ data across ages 17, 27, and 37. The correlations for each respective age bucket are r = 0.06 (SE = 0.10), r = 0.23 (SE = 0.09), and r = 0.15 (SE = 0.10). Readers interested in other subcomponents of RNCCQ (including the factor- analytic subscales) can find the associated correlations with RND in the OA. 6. Concluding remarks We have reported pre-registered analysis of the latest survey wave of the Bing pre-school study on delay of gratification. Respondents were in their late 40’s at the time of this latest wave and were asked to report 11 mid-life capital formation outcomes (e.g., net worth and permanent income). Our analysis plan both described our methods and predicted what we would find. As predicted, a newly constructed measure derived from preschool delay of gratification does not predict the 11 capital formation variables (i.e., the sign-adjusted average correlation was 0.02). By contrast a composite self-regulation index, combining preschool delay of gratification with survey measures of self-regulation collected at ages 17, 27, and 37, does predict 10 of the 11 capital formation variables in the expected direction, with an average correlation of 0.19. The inclu- sion of the preschool delay of gratification measure in this composite index does not affect the index’s predictive power for two reasons. Most importantly, the index of self-regulatory measures is comprised of 86 responses per participant, whereas the preschool delay of gratification task is a single behavioral task. In addition, the preschool delay of gratification task is measured using a diagnostic variant of the task for 34 of our 113 participants; the remaining 79 participants experienced a non-diagnostic variant of the pre-school delay of gratification task. The data we have analyzed is unique because the Bing cohort is the only sample where preschool delay of gratification has been studied long enough to examine relationships with mid-life outcomes. While the tests of our primary hypotheses were well powered, we caution that our sample is relatively small and not representative of the overall population—e.g., 97% of our sample has a four-year college degree (the exceptions are one participant who has a two-year college degree and two who have some college but no degree)—limiting the generalizability of the results. However, we can compare our results to the small set of overlapping analyses that have been conducted using the Dunedin cohort, which began collecting data in 1972–1973 and has childhood self-regulation measures but no preschool delay of gratification measure. 11 The cohort is from a small town in New Zealand with much lower levels of educational attainment ( Moffitt et al., 2011 ). Specifically, 29% of the Dunedin sample is college educated ( Belsky et al., 2016 ). 11 Specifically, the measure of self-regulation used in Moffitt et al. (2011) is an index of examiner ratings of lack of control at ages 3 and 5; parent and teacher ratings of impulse aggression and hyperactivity at ages 5, 7, 9, and 11; parent and teacher ratings of lack of persistence and impulsivity at ages 9 and 11; and self-reported symptoms of hyperactivity, inattention, and impulsivity at age 11. This measure does not include performance on the delay of gratification task that we use to construct our RND measure. 5.4. Other secondary, robustness, and ex-post analyses 5.4. Other secondary, robustness, and ex-post analyses In OA, we report the results from all pre-registered secondary and robustness analyses as well as all ex-post analyses we conducted. In our secondary analyses, we examine additional outcome variables, secondary sets of independent variables (e.g., breaking out the RNSRI into its components), and comparisons of diagnostic and non-diagnostic conditions. In our ex-post analyses, we report correlations between our key variables, results using individual questions from the RNCCQ as independent variables, an analysis of the characteristics of the Bing sample over time, an in-depth analysis of BMI as an outcome variable, results using delay choice as an independent variable, robustness of the primary analyses to controlling for age, results using an index of capital formation measures as the dependent variable, and an analysis of the effects of rank-normalization. We also report results from two ex-post analyses resulting from the editorial process: analyses of RNCCQ as an independent variable on its own, and a comparison of the BMI correlations in the diagnostic and non-diagnostic subsamples using BMI data collected in previous surveys. In our robustness analyses, we test the robustness of our primary analyses to measuring wait time in seconds rather than log-seconds, winsorizing RNSRI and RND, estimating a non-linear effect of RND, using alternative imputation methods for the RNCCQ indices, using the delay deviation measure from prior work, and dropping survey respondents for whom we had to make assumptions in order to assign values to any of their capital formation measures. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 12 paradigm ( Mischel and Ebbesen, 1970 ; see Peake, 2017 for discussion). Nonetheless, monetary delay choice measures (e.g., money earlier vs. later) have been popular among economists and psychologists as a tool for assessing time preferences and hence, as a putative index of self-control and impulsive behavior (e.g. Thaler, 1981 ; see Stevens, 2017 for examples). We con- sider (monetary) delay choice to be a dependent variable in our primary analysis, but in an ex-post analysis, we examined it as a predictor of the remaining capital formation variables (as in Meier and Sprenger (2010), Meier and Sprenger (2013) , and Falk et al. (2018) ). Delay choice is a rank-normalized variable measuring the percent of times a respondent selected the later response in a series of incentivized money earlier vs. later questions. The questions included 20 of the form “Which would you prefer? (A) getting $X today (B) getting $Y thirty days from today” and 20 of the form “Which would you prefer? (A) getting $X thirty days from today (B) getting $Y sixty days from today,” with values ranging from $10 to $29. y y y ( ) g g y y y g g We regressed the remaining 10 capital formation measures on delay choice, controlling for sex. Although all 10 correla- tions have the predicted sign, none is significant at the 5% level. The average correlation with the capital formation measures is 0.11 (SE = 0.06). See OA Section V-C-9 for more detailed information. 12 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx ARTICLE IN PRESS JID: JEBO [m3Gsc; November 16, 2019;10:34 ] D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx ARTICLE IN PRESS [m3Gsc; November 16, 2019;10:34 ] JID: JEBO 5.3. Money now vs. money later as a predictor One of our capital formation variables, delay choice, is based on responses to money earlier vs. later questions. 10 Early research on self-control in children relied heavily on delay choice data ( Mischel, 1958, 1961a, 1961b; Mischel and Gilli- gan, 1964; Bandura and Mischel, 1965; Mischel and Staub, 1965; Mischel and Grusec, 1967 ). Recognition of the conceptual limitations of delay choice measures was a primary motivation for the development of the preschool delay of gratification 8 Similarly, there is limited evidence for an age trend in the predictive power of the indices of CCQ questions. In ex-post analysis, we found that the average correlations between the mid-life capital formation measures and RNCCQ at ages 17, 27, and 37 are 0.15 (SE = 0.09), 0.17 (SE = 0.06), and 0.19 (SE = 0.05), respectively (see OA). ( ) p y ( ) 9 The overall correlation between RND and RNCCQ is calculated by combining RNCCQ data across ages 17, 27, and 37. The correlations for each respective age bucket are r = 0.06 (SE = 0.10), r = 0.23 (SE = 0.09), and r = 0.15 (SE = 0.10). Readers interested in other subcomponents of RNCCQ (including the factor- analytic subscales) can find the associated correlations with RND in the OA. 10 For a review and methodological critique of money earlier vs. later questions, see Cohen et al (forthcoming) . For example, in principle choices in the money earlier vs. later paradigm should reveal market interest rates, transactions costs, and trustworthiness of the experimenter, and should not measure true time preferences (because money is temporally fungible). 10 For a review and methodological critique of money earlier vs. later questions, see Cohen et al (forthcoming) . For example, in principle choices in the money earlier vs. later paradigm should reveal market interest rates, transactions costs, and trustworthiness of the experimenter, and should not measure true time preferences (because money is temporally fungible). Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 The authors declare no conflicts of interest. The authors declare no conflicts of interest. Code availability Code for data analysis is available in the Online Appendix. D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 13 to the self-regulation measures in the Bing sample. For example, Moffitt et al. (2011) found that a 1 SD increase in child- hood self-control as measured in the Dunedin study predicts a 0.24 SD increase in income at age 32. In the Bing sample, a 1 SD increase in RNSRI predicts a 0.32 SD increase in rank-normalized permanent income. Similarly, a 1 SD increase in the Dunedin self-control measure predicts a 0.12 SD decrease in credit card problems, while a 1 SD increase in the Bing RNSRI predicts a 0.18 SD decrease in rank-normalized credit card misuse. A 1 SD increase in the Dunedin self-control mea- sure predicts a 0.14 SD decrease in money management difficulties, while a 1 SD increase in the Bing RNSRI predicts a 0.24 SD increase in financial health. 12 Despite these intriguing similarities across the two samples, the issue of generalizability remains an important question to be addressed in future research as mid-life data becomes available in more childhood longitudinal cohorts. to the self-regulation measures in the Bing sample. For example, Moffitt et al. (2011) found that a 1 SD increase in child- hood self-control as measured in the Dunedin study predicts a 0.24 SD increase in income at age 32. In the Bing sample, a 1 SD increase in RNSRI predicts a 0.32 SD increase in rank-normalized permanent income. Similarly, a 1 SD increase in the Dunedin self-control measure predicts a 0.12 SD decrease in credit card problems, while a 1 SD increase in the Bing RNSRI predicts a 0.18 SD decrease in rank-normalized credit card misuse. A 1 SD increase in the Dunedin self-control mea- sure predicts a 0.14 SD decrease in money management difficulties, while a 1 SD increase in the Bing RNSRI predicts a 0.24 SD increase in financial health. 12 Despite these intriguing similarities across the two samples, the issue of generalizability remains an important question to be addressed in future research as mid-life data becomes available in more childhood longitudinal cohorts. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jebo.2019.08.016 . Data availability Data collection for the Bing Longitudinal Study now spans nearly 50 years. Due to a host of historical issues connected to the consenting procedures in this work and promises made to participants to protect both their privacy and the confiden- tiality of their responses, it has been a long-standing policy and IRB requirement that we not share the data connected with this work with outside parties or agencies. We believe it is imperative that we honor the promises made to our participants who have generously provided input to the project over the years. References b ll ff b l d h b l d f p g pp y ( ) Block, J., Block, J.H., 1980. The California Child Q-set. Consulting Psychologists Press, Palo Alto, CA. g y g g, C. , 2009. Delay and inhibition as early predictors of ADHD symptoms in third grade. J. Abnorm. Child Psychol. 37, 1–15 . ort effects in children’s delay of gratification. Dev. Psychol. 54, 1395–1407 . Campbell, S.B. , von Stauffenberg, C. , 2009. Delay and inhibition as early predictors of ADHD symptoms in third grade. J. 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Econ. 2 (1), 193–210 M i S S C 2013 Di ti fi i l lit ti f d ti i ti i fi i l d ti Meier, S. , Sprenger, C. , 2013. Discounting financial literacy: time preferences and participation in financial education programs. J. Econ. Behav. Organ. 95, 159–174 . Mischel, W. , 1958. Preference for delayed reinforcement: An experimental study of a cultural observation. J. Abnorm. Soc Mi h l W 2014 Th M h ll T M i S lf C l Li l B d C N Y k Mischel, W. , 1958. Preference for delayed reinforcement: An experimental study of a cultural observation. J. Abnorm. Soc. Psych. 56 (1), 57–61 . Mischel, W. , 2014. The Marshmallow Test: Mastering Self-Control. Little, Brown and Company, New York . Mischel, W. , 2014. The Marshmallow Test: Mastering Self-Control. Little, Brown and Company, New York . Mi h l W 1961 D l f tifi ti d f hi t d i i th lt J Ab S P h 62 (3) 543 552 Mischel, W. , 2014. The Marshmallow Test: Mastering Self-Control. Little, Brown and Company, New York . Mischel, W. , 1961a. Delay of gratification, need for achievement, and acquiescence in another culture. J. Abnorm. Soc. Ps Mischel, W. , 1961b. Preference for delayed reinforcement and social responsibility. J. Abnorm. Soc. Psych. 62 (1), 1–7 . Mischel, W. , Ebbesen, E.B. , 1970. Attention in delay of gratification. J. Pers. Soc. Psychol. 16, 329–337 . Mischel, W. , 1961b. Preference for delayed reinforcement and social responsibility. J. Abnorm. Soc. Psych. Mischel, W. , Ebbesen, E.B. , 1970. 6. Concluding remarks Despite the stark socioeconomic differences, the self-regulation measures used in the Dunedin study have similar predictive power 11 Specifically, the measure of self-regulation used in Moffitt et al. (2011) is an index of examiner ratings of lack of control at ages 3 and 5; parent and teacher ratings of impulse aggression and hyperactivity at ages 5, 7, 9, and 11; parent and teacher ratings of lack of persistence and impulsivity at ages 9 and 11; and self-reported symptoms of hyperactivity, inattention, and impulsivity at age 11. This measure does not include performance on the delay of gratification task that we use to construct our RND measure. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 13 ARTICLE IN PRESS ID: JEBO [m3Gsc; November 16, 2019;10:34 ] JID: JEBO JID: JEBO ARTICLE IN PRESS M ffi T E l 2011 A di f hildh d lf l di h l h l h d bli f I P di dient of childhood self-control predicts health, wealth, and public safety. In: Proceedings of the National Academy of Scienc merica, 108, pp. 2693–2698 . Moffitt, T.E. , et al. , 2011. A gradient of childhood self-control predicts health, wealth, and public safety. In: Proceedings of the of the United States of America, 108, pp. 2693–2698 . Peake, P.K. , 2017. Delay of gratification: Explorations of how and why children wait and its linkages to outcomes over the life course. In: Stevens, J. (Ed.), Impulsivity. Springer International Publishing, pp. 7–60 . Ri h d F D B d J C S k Z J 2003 O h d d f i l h l i i l d ib d R G P h l 7 331 363 p y p g g, pp Richard, F.D. , Bond Jr, C. , Stokes-Zoota, J. , 2003. One hundred years of social psychology quantitatively described. Rev. Gener. Psychol. 7, 331–363 . Schlam, T.R. , Wilson, N.L. , Shoda, Y. , Mischel, W. , Ayduk, Ö, 2013. Preschoolers’ delay of gratification predicts their body mass 30 years later. J. Pediatr. 162, 90–93 . Richard, F.D. , Bond Jr, C. , Stokes-Zoota, J. , 2003. One hundred years of social psychology quantitatively described. Rev. Gener. Psychol. 7, 331–363 . Schlam, T.R. , Wilson, N.L. , Shoda, Y. , Mischel, W. , Ayduk, Ö, 2013. Preschoolers’ delay of gratification predicts their body mass 30 years later. J. Pediatr. 162, 90–93 . Schlam, T.R. , Wilson, N.L. , Shoda, Y. , Mischel, W. , Ayduk, Ö, 2013. Preschoolers’ delay of gratification predicts their body mass 30 years later. J. Pediatr. 162, 90–93 . Seeyave, D.M. , Coleman, S. , Appugliese, D. , Corwyn, R.F. , Bradley, R.H. , Davidson, N.S. , Kaciroti, N. , Lumend, J.C. , 2009. Ability to delay gratification at age 4 years and risk of overweight at age 11 years. Arch. Pediatr. Adolesc. Med. 163, 303–308 . h d i h l k di i d l i i d lf l i f h l d l f ifi i id if i Seeyave, D.M. , Coleman, S. , Appugliese, D. , Corwyn, R.F. , Bradley, R.H. , Davidson, N.S. , Kaciroti, N. , Lumend, J.C. , 2009. ARTICLE IN PRESS D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx D.J. Benjamin, D. Laibson and W. Mischel et al. / Journal of Economic Behavior and Organization xxx (xxxx) xxx 14 Mischel, W. , Gilligan, C. , 1964. Delay of gratification, motivation for the prohibited gratification, and responses to temptation. J. Abnorm. Child Psychol. 69 (4), 411–417 . Mischel, W. , Gilligan, C. , 1964. Delay of gratification, motivation for the prohibited gratification, and responses to temptation. J. Abnorm. Child Psychol. 69 (4), 411–417 . Mischel, W. , Gilligan, C. , 1964. Delay of gratification, motivation for the prohibited gratification, and responses to tempt (4), 411–417 . ( ) Mischel, W. , Grusec, J. , 1967. Waiting for rewards and punishments: effects of time and probability on choice. J. Pers. Soc. Psychol. 5 (1), 24–31 . Mischel, H.N. , Mischel, W. , 1983. The development of children’s knowledge of self-control strategies. Child Dev. 54, 603–619 . ( ) Mischel, W. , Grusec, J. , 1967. Waiting for rewards and punishments: effects of time and probability on choice. J. Pers. Soc. Psychol. 5 (1), 24–31 . Mischel, H.N. , Mischel, W. , 1983. The development of children’s knowledge of self-control strategies. Child Dev. 54, 603–619 . Mischel, W. , Moore, B. , 1973. Effects of attention to symbolically presented rewards on self-control. J. Pers. Soc. Psychol. 28 ( Mischel W Moore B 1980 The role of ideation in voluntary delay for symbolically presented rewards Cognit Ther Res 4 Mischel, W. , Staub, E. , 1965. Effects of expectancy on working and waiting for larger reward. J. Pers. Soc. Psychol. 2 (5), 625–633 . Mischel, W. , Shoda, Y. , Peake, P.K. , 1988. The nature of adolescent competencies predicted by preschool delay of gratification. J. Pers. Soc. Psychol. 54, 687–696 . Mischel, W. , Shoda, Y. , Rodriguez, M.I. , 1989. Delay of gratification in children. Science 244, 933–938 . g y g Mischel, W. , Underwood, B. , 1974. Instrumental ideation in delay of gratification. Child Dev. 45 (4), 1083–1088 . ffi l d f h ldh d lf l d h l h l h d bl f d f h l d f Mischel, W. , Underwood, B. , 1974. Instrumental ideation in delay of gratification. Child Dev. 45 (4), 1083–1088 . References Attention in delay of gratification. J. Pers. Soc. Psychol. 16, 329–337 . Mischel, W. , 1961b. Preference for delayed reinforcement and social responsibility. J. Abnorm. Soc. Psych. 62 Mischel, W. , Ebbesen, E.B. , 1970. Attention in delay of gratification. J. Pers. Soc. Psychol. 16, 329–337 . 12 Other outcomes studied in Moffitt et al. (2011) at age 32 include measures of physical health, depression, substance dependence, socioeconomic status, single-parent child rearing, financial planfulness, and criminal conviction. 12 Other outcomes studied in Moffitt et al. (2011) at age 32 include measures of physical health, depression, substance dependence, socioeconomic status, single-parent child rearing, financial planfulness, and criminal conviction. Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 ARTICLE IN PRESS Ability to delay gratification at age 4 years and risk of overweight at age 11 years. Arch. Pediatr. Adolesc. Med. 163, 303–308 . Shoda, Y. , Mischel, W. , Peake, P.K. , 1990. Predicting adolescent cognitive and self-regulatory competencies from preschool delay of gratification: identifying di i di i D P h l 26 978 986 Shoda, Y. , Mischel, W. , Peake, P.K. , 1990. Predicting adolescent cognitive and self-regulatory competencies from preschool de diagnostic conditions. Dev. Psychol. 26, 978–986 . Stevens, J.R. (Ed.), 2017. Impulsivity: How Time and Risk Influence Decision Making, vol. 64. Springer International Publishing . Thaler, R. , 1981. Some empirical evidence on dynamic inconsistency. Econ. Lett. 8 (3), 201–207 . W tt T W D G J Q H 2018 R i iti th h ll t t t l li ti i ti ti li k b t l d l f tifi ti Stevens, J.R. (Ed.), 2017. Impulsivity: How Time and Risk Influence Decision Making, vol. 64. Springer International Publishing . Thaler, R. , 1981. Some empirical evidence on dynamic inconsistency. Econ. Lett. 8 (3), 201–207 . Watts, T.W. , Duncan, G.J. , Quan, H. , 2018. Revisiting the marshmallow test: a conceptual replication investigating links between early delay of gratification and later outcomes Psychol Sci 29 1159 1177 Thaler, R. , 1981. Some empirical evidence on dynamic inconsistency. Econ. Lett. 8 (3), 201 207 . Watts, T.W. , Duncan, G.J. , Quan, H. , 2018. Revisiting the marshmallow test: a conceptual replication investigating links between early delay of gratification and later outcomes. Psychol. Sci. 29, 1159–1177 . p y y ( ) Watts, T.W. , Duncan, G.J. , Quan, H. , 2018. Revisiting the marshmallow test: a conceptual replication investigating links between early delay of gratification and later outcomes. Psychol. Sci. 29, 1159–1177 . and later outcomes. Psychol. Sci. 29, 1159–1177 Yang, L. , Wang, J. , 2007. A follow-up study of self-imposed delay of gratification at age r as a predictor of children’s schoo age 9. Acta Psycholog. Sin. 39, 668–678 . Please cite this article as: D.J. Benjamin, D. Laibson and W. Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016 Please cite this article as: D.J. Benjamin, D. Laibson and W. y ng, J. , 2007. A follow-up study of self-imposed delay of gratification at age r as a predictor of children’s school-based socia ta Psycholog. Sin. 39, 668–678 . ARTICLE IN PRESS Mischel et al., Predicting mid-life capital formation with pre- school delay of gratification and life-course measures of self-regulation ✰ , Journal of Economic Behavior and Organization, https://doi.org/10.1016/j.jebo.2019.08.016
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IDEALISMO À ITALIANA: FILOSOFIA NA ITÁLIA E INFLUÊNCIA GERMÂNICA NOS SÉCULOS XIX E XX: O CASO DA INFLUÊNCIA E RECEPÇÃO DE FEUERBACH, KIERKEGAARD E NIETZSCHE NAS FILOSOFIAS DE BENEDETTO CROCE E GIOVANNI GENTILE
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IDEALISMO À ITALIANA: FILOSOFIA NA ITÁLIA E INFLUÊNCIA GERMÂNICA NOS SÉCULOS XIX E XX: O CASO DA INFLUÊNCIA E RECEPÇÃO DE FEUERBACH, KIERKEGAARD E NIETZSCHE NAS FILOSOFIAS DE BENEDETTO CROCE E GIOVANNI GENTILE Márcio Gimenes Universidade de Brasília Resumo: O objetivo do presente trabalho é investigar o panorama da filosofia italiana dos séculos XIX e XX e a respectiva influência germânica por ela sofrida, avaliando ainda a articulação do idealismo italiano e a crítica ao cristianismo produzida na Itália nesse período. Para tanto, dividiremos nosso artigo em duas partes principais: na primeira, investigaremos o idealismo italiano e a crítica à religião na Itália entre os séculos XIX e XX, dando especial ênfase a dois dos seus principais autores: Benedetto Croce e Giovanni Gentile. Já na segunda parte, investigaremos a influência de três pensadores do idealismo germânico na Itália desse período, a saber, Feuerbach, Kierkegaard e Nietzsche, bem com a sua recepção em contexto italiano e sua importância para o idealismo local. Palavras-chave: Filosofia italiana; filosofia contemporânea; idealismo; cristianismo. Abstract: The objective of this study is to investigate the panorama of Italian philosophy of the nineteenth and twentieth centuries and its Germanic influence for her suffering, still evaluating the joint Italian idealism and criticism of Christianity produced in Italy during that period. In this way, we will divide our article into two main parts: first, we will investigate Italian idealism and criticism of religion in Italy between the nineteenth and twentieth centuries, with special emphasis on two of its main authors: Benedetto Croce and Giovanni Gentile. Already in the second part, we will investigate the influence of three thinkers of Germanic idealism in Italy of that period, namely Feuerbach, Kierkegaard and Nietzsche, as well as their reception in the Italian context and their importance for local idealism. Keywords: Italian Philosophy; Contemporary Philosophy; Idealism; Christianity. Introdução A despeito de o Brasil possuir uma imensa afinidade cultural com a Itália, não apenas por partilhar a herança linguística latina, mas também por haver recebido, especialmente a partir do final do século XIX e início do século XX, inúmeros imigrantes italianos, a filosofia italiana, talvez excluindo- se aqui autores clássicos como Maquiavel, Galileu, Vico e alguns outros, ainda é, infelizmente, pouco conhecida e estudada no Brasil e, segundo avaliamos, o mesmo ocorre em Portugal e em em outros países de língua portuguesa. Obteve maior êxito entre os brasileiros – e cremos que entre os falantes de português em geral – o estudo dos autores franceses, alemães e ingleses, o que © Dissertatio [45] 184-2082017 Márcio Gimenes avaliamos como importante, mas não excludente. Afinal, a própria filosofia italiana dos séculos XIX e XX não é feita, a rigor, como filosofia italiana pura, isto é, como um produto ligado a um modo de pensar ou a uma posição geográfica, tal como aponta Roberto Esposito em sua obra O pensamento vivo – a origem e a atualidade da filosofia italiana (ESPOSITO, 2013). A filosofia, por definição, teria imensa dificuldade em ser tomada como pertencente a esse ou aquele país, uma vez que ela é, por princípio, universal. Desse modo, dissertar sobre filosofia italiana, francesa, alemã, inglesa, brasileira, latino-americana, europeia ou africana sempre comportaria, em si mesmo, um problema e uma impossibilidade. Contudo, pensamos que aqui é que se insere efetivamente o interesse do nosso trabalho, isto é, em saber como ocorre aquilo que se convencionou denominar como filosofia italiana dos séculos XIX e XX. Para tanto, daremos especial destaque ao idealismo e à crítica da religião na Itália e a analisaremos a partir do pensamento de autores como Benedetto Croce, Giovanni Gentile, Antonio Gramsci, Maurizio Viroli, Carlo Augusto Viano, Eugenio Garin e Franco Lombardi, dentre outros. Assim almejamos compreender, ao menos em linhas gerais, um pouco do panorama da filosofia italiana do período e do contexto religioso italiano da época. Julgamos tal passo, inclusive, importante para uma adequada compreensão da maneira como os italianos interpretaram a crítica religiosa do cristianismo operada pelos autores germânicos do século XIX como Feuerbach e Nietzsche (que eram alemães) e bem como Kierkegaard (que era dinamarquês), uma vez que tais autores foram centrais para o idealismo italiano e influenciaram diretamente na sua crítica ao cristianismo e na articulação intelectual do seu tipo de idealismo. Introdução Assim, o objetivo desse trabalho consiste na avaliação do problema religioso italiano no século XIX e na sua problematização, ou seja, como, também a partir dele, se pode compreender o idealismo italiano desse século e do século seguinte. Com efeito, nosso intuito é avaliar o hegelianismo e o pós- hegelianismo na Itália, para tanto nos debruçaremos especialmente sobre algumas reflexões delimitadas dos autores já citados. Avaliamos que aqui, com as delimitações adequadas, conseguiremos apresentar um quadro significativo de diferentes matizes do hegelianismo na cultura italiana dos séculos XIX e XX. Feito tal percurso, apresentaremos nossa conclusão crítica que, segundo avaliamos, pode apresentar importantes desafios para uma compreensão mais ampla da história da filosofia e para uma valorização do pensamento italiano. 185 Dissertatio [45] 184-2082017 1. O contexto do idealismo italiano e da crítica da religião na Itália entre os séculos XIX e XX: com especial atenção para Benedetto Croce e Giovanni Gentile 1. O contexto do idealismo italiano e da crítica da religião na Itália entre os séculos XIX e XX: com especial atenção para Benedetto Croce e Giovanni Gentile 1. O contexto do idealismo italiano e da crítica da religião na Itália entre os séculos XIX e XX: com especial atenção para Benedetto Croce e Giovanni Gentile Carlos Augusto Viano, em sua obra La filosofia italiana del Novecento (VIANO, 2006), apresenta, a partir do debate do idealismo italiano, notadamente a partir da polêmica das teses de Benedetto Croce e Giovanni Gentile, um inspirador retrato da filosofia italiana no início do século XX. Tal retrato é feito em forma panorâmica e, segundo avaliamos, apesar dos seus limites, pode ser importante para uma primeira compreensão de aspectos significativos da filosofia italiana do século XX. Sua apresentação, de uma forma geral, nos fornece a interpretação crociana de Hegel, Marx e também um pouco de aspectos da posição liberal do pensador napolitano. De igual modo, avalia e apresenta os principais movimentos de renovação da cultura italiana do período dando, especial ênfase, às revistas e outras produções. O autor igualmente investiga a polêmica intelectual e pessoal entre Croce e Gentile, o fascismo, a religião ensinada para a massa na escola pública, bem como a tese do Estado laico como um ideal tanto do fascismo quanto da escola pública. Afirmo, portanto, a minha profissão de fé agrade ou não agrade a quem está a me ouvir: eu sou cristão. Sou cristão porque creio na religião do espírito, mas quero rapidamente acrescentar a fim de evitar equívocos: eu sou católico. E não de hoje, fique isso bem claro. Católico, a rigor, sou desde junho de 1875, a saber, Introdução Por isso, não fortuitamente, o tema da laicidade, tão caro à filosofia moderna, foi um pilar central da reforma que Gentile operou enquanto ministro da instrução pública e acabou, na verdade, por se constituir no seu calcanhar de Aquiles nas suas polêmicas contra a Igreja. Desde o primeiro momento, Gentile, como ministro e como educador, teve uma difícil relação com o pensamento católico. A tese por ele defendida do Estado laico e da religião como educação básica para as massas incultas nunca pareceu muito aceitável aos olhos das instituições religiosas. Sua posição apontava para a Igreja como uma espécie de iniciadora dos espíritos na cultura, mas, ao mesmo tempo, afirmava, ainda indiretamente, que ela, de fato, não fazia ciência. Como era de se esperar, notadamente num contexto como o italiano, os católicos reagem e não aceitam nem as teses de Croce (da religião da liberdade), nem a posição de Gentile, nem o humanismo modernista. Contudo, também recusam o antigo tomismo. Assim é que se delinea a tumultuada relação da Igreja com o fascismo. A crise da Igreja Católica em perder o seu lugar de posto cultural, especialmente na Itália tem uma forte repercussão. É verdade que Gentile se proclamava católico e advogava a tese de uma religião estatal e racional, tal como se pode notar na sua declaração La mia religione: 186 Márcio Gimenes desde quando estou no mundo. E sou, por isso, desolado de não poder-vos anunciar também eu, uma crise, uma tempestade da alma, uma súbita conversão, um golpe fulminante. Estou, prosaicamente, desde o dia do meu nascimento, percorrendo a estrada de Damasco (GENTILE, 1965, p.406). desde quando estou no mundo. E sou, por isso, desolado de não poder-vos anunciar também eu, uma crise, uma tempestade da alma, uma súbita conversão, um golpe fulminante. Estou, prosaicamente, desde o dia do meu nascimento, percorrendo a estrada de Damasco (GENTILE, 1965, p.406). Contudo, tal posição não atenua os fatos de que, aos olhos de muitos católicos – e da estrutura eclesiástica – o máximo de sua religião alcançava um mero tipo de religião da razão ao estilo hegeliano. O mesmo ocorre com Croce ao advogar a tese da religião da liberdade. g g Na filosofia italiana do século XX, a politica sempre foi uma tônica e um ponto central. As discussões acaloradas entre liberais e socialistas foram marcantes em todo o período. Introdução Croce era, inclusive, sobrinho de Bertrando e de Silvio Spaventa, introdutores do pensamento hegeliano na Itália. Gentile foi um grande admirador e aluno de Bertrando. Ambos partem, portanto, do idealismo filosófico hegeliano. Aliás, como bem salienta Garin, o hegelianismo italiano deve ser compreendido num “quadrado” que inclui Spaventa, De Sanctis, Croce e Gentile. Com efeito, ambos renovam a vida cultural italiana, ampliam os debates, produzem inúmeras revistas, livros e retiram a Itália do seu gueto intelectual, ampliando seu escopo de discussão com autores alemães e também ingleses e franceses. p g O mesmo Garin (GARIN, 1966, p.1225-1243) aponta que os irmãos Spaventa e a chamada escola de Napóles foram extremamente importantes para a inserção do pensamento hegeliano na Itália, salientando ainda que Firenze e Palermo foram dois importantes centros da filosofia italiana no período. Os antecedentes de tal pensamento podem ser percebidos em dois pensadores: Domenico Mazzoni e Giambattista Passerini. Ambos foram sacerdotes católicos e os primeiros italianos a estudarem, ainda que de modo mais rudimentar, o pensamento de Hegel. Por isso, na concepção de Garin, o primeiro hegeliano com estudo um pouco mais aprofundado foi, na verdade, Augusto Vera. Tal autor escreveu, em Paris, no ano de 1855, uma Introduction à la philosophie de Hegel. Sua obra foi basicamente construída dentro do ambiente acadêmico francês e foi a primeira a apresentar uma certa robustez filosófica. O mesmo Garin (GARIN, 1966, p.1225-1243) aponta que os irmãos Spaventa e a chamada escola de Napóles foram extremamente importantes para a inserção do pensamento hegeliano na Itália, salientando ainda que Firenze e Palermo foram dois importantes centros da filosofia italiana no período. Os antecedentes de tal pensamento podem ser percebidos em dois pensadores: Domenico Mazzoni e Giambattista Passerini. Ambos foram sacerdotes católicos e os primeiros italianos a estudarem, ainda que de modo mais rudimentar, o pensamento de Hegel. Por isso, na concepção de Garin, o primeiro hegeliano com estudo um pouco mais aprofundado foi, na verdade, Augusto Vera. Tal autor escreveu, em Paris, no ano de 1855, uma Introduction à la philosophie de Hegel. Sua obra foi basicamente construída dentro do ambiente acadêmico francês e foi a primeira a apresentar uma certa robustez filosófica. Contudo, para Spaventa, Vera seria um hegeliano mais hegeliano do que o próprio Hegel. Introdução Nesse contexto também se pode compreender o tema da renovação moral nos debates entre Croce e Gentile. Muito se ouve falar, no contexto posterior a Segunda Guerra, da tese fascista de Gentile, da sua atuação como ministro, como político, de sua enorme produção intelectual e do desfecho trágico de sua vida e do seu assasinato em 1944 pelas forças opositoras ao regime. Contudo, avaliamos que, primeiro, cabe entender que a sua posição e a sua atuação política tem uma estreita relação com a sua posição filosófica. Em outras palavras, sua interpretação, como filósofo, do estado ético e moral hegeliano, isto é, o seu hegelianismo é quem efetivamente o coloca no centro dos acontecimentos políticos do século XX. Assim, a imagem do filosófo que é legada à posteridade, ao menos nos primeiros anos da sua morte, isto é, aquela imagem do filósofo aliado ao fascismo é verdadeira, mas não faz total justiça ao pensador, visto que, sem entrarmos no âmbito do julgamento moral ou político, Gentile foi um intelectual de fôlego, com uma imensa produção intelectual e um dos primeiros ministros a construir um plano de instrução pública para toda a Itália, além de levar adiante o empreendimento da Enciclopédia italiana, que era um desdobramento, a notar pelo seu próprio nome, da sua posição idealista em filosofia, tal como é igualmente perceptível nesse pequeno trecho da declaração La mia religione: “A religião cresce, se espande, se consolida e vive, dentro da filosofia, que elabora incessantemente o conteúdo imediato da religião e o introduz na vida da história” (GENTILE, 1965, p.424). Em outras palavras, a religião para Gentile está inserida dentro do todo do sistema hegeliano. g De igual modo, o filósofo italiano talvez mais conhecido no contexto após a Segunda Guerra é Benedetto Croce. O filósofo napolitano, antigo amigo de Gentile, com quem rompe após discordâncias políticas, é uma figura de destaque por sua forte oposição ao fascismo e por sua clara posição contrária aos intelectuais que o defenderam. Tal posição lhe confere destaque inclusive no exterior, tendo também o mérito de levar a filosofia italiana além dos seus confins. Entretanto, tal como muito bem relembra Garin, citando 187 Dissertatio [45] 184-2082017 Gramsci: “Croce não pode ser compreendido sem Gentile” (GRAMSCI apud GARIN, 1997, p.172, n.2). Assim, Gentile e Croce possuem alguns traços comuns: ambos são herdeiros da tradição hegeliana. Introdução Desse modo, ao analisar criticamente sua obra, aponta para a falta de um filosofar italiano e também percebe nas suas reflexões uma falta da compreensão de Hegel dentro de um contexto de maior liberdade e secularização. O tema da falta de liberdade para a reflexão filosófica é um aspecto central a ser percebido aqui. Para Spaventa, o fato de a Itália ser a sede do poder católico já a impederia, por natureza, a tal reflexão. Desse modo, sua posição aponta, com extrema argúcia, para uma direção que pode ser explorada conceitualmente: a filosofia italiana clássica foi, no seu entender, emigrada para autores não italianos como Espinosa, Leibniz e Kant. Esses seriam italianos sem serem italianos por nascimento. Em outras palavras, trariam em sua filosofia todo o tema clássico da filosofia italiana da liberdade de reflexão. Desse modo, Espinosa teria um equivalente com Giordano Bruno e Kant com Vico. Tal tese, que já aparece em Rodolfo Mondolfo (MONDOLFO, 1924, p.2ss), ressoa mesmo em autores como Gentile e Croce. O primeiro, por exemplo, escreve um importante trabalho sobre a filosofia de Giordano Bruno, o que não parece desprovido de significado 188 Márcio Gimenes (GENTILE, 1920). O segundo desenvolve, na obra Storia d´Europa nel secolo decimonono, o importante conceito de religião da liberdade. Com efeito, o hegelianismo italiano possui um arco que começa por Vera, os irmãos Spaventa e alcança autores como Gentile e Croce, incluindo ainda, nesse horizonte conceitual, Gramsci. Garin destaca ainda aqui a importância de autores como Antônio Tari, que, juntamente, com Spaventa e Vera teria formado a “santíssima trindade hegeliana na Itália”, incluindo ainda os nomes de Labriola e Francesco de Sanctis. Não se poderia fazer justiça à filosofia italiana do século XX sem mencionarmos o surgimento da Crítica em 1903, o famoso periódico dirigido por Croce. Nessa época, o pensador napolitano afirma-se como um autor mais maduro. Começam a se delinear aqui os seus interesses mais claros que, em geral, acompanham aqueles que abandonaram a religião, ou, ao menos, não dão mais a ela uma importância central. Em seu lugar, surgem a arte, a moral, o direito, a metodologia histórica. Introdução Em outras palavras, Croce configura-se aqui como um hegeliano típico, como aponta Garin: “Negada a filosofia da história como reencarnação da teologia, a filosofia já se fazia para Croce, compreensão da aparência da história humana ou, se se quiser, do ritmo profundo da humanidade no seu processo” (GARIN, 1997, p.184). Junto com Garin podemos observar, inclusive, diversos Croces e Gentiles, autores com diversas fases. Por influência de Labriola, ambos se tornam leitores de Hegel e também de Marx. Croce passa de tal fase para a Estética, realizando, mais ou menos, um sistema ideal no decorrer de sua obra. No fundo, sua filosofia fica na fronteira e cumpre o drama da consciência italiana: “[...] o antigo drama da consciência italiana próxima do interesse mundano, da alegria da vida, do amor das formas e dos arroubos místicos de uma outra cidade” (GARIN, 1997, p.266). Assim, avaliamos que notadamente para compreendermos melhor o tema da religião da liberdade em Croce, cumpre fazer uma avaliação daquilo que Galasso (GALASSO, 2002) denomina com a sua fase histórica. q ( ) Um dos marcos do pensamento italiano do século XX foi a publicação, em 1932, da já citada Storia d´Europa nel secolo decimono de Benedetto Croce. Ali o filósofo napolitano desenvolve um tema que já existe em outros autores que o antecedem, a saber, o tema da religião da liberdade. Contudo, sua abordagem é diferenciada. Na sua concepção, a era posterior a Napoleão possui, em todo o mundo, um anseio pela liberdade. Essa é a nova palavra de ordem. Segundo Croce, tal aspiração é oriunda de nossa herança greco- romana, tendo um acréscimo, depois da Revolução Francesa do século XVIII, dos temas da igualdade e da liberdade. Na verdade, a liberdade pode ser dita de muitos modos e possui, sem dúvida alguma, muitas facetas: liberdade civil, liberdade política, liberdade do indivíduo singular, liberdade dos indivíduos 189 Dissertatio [45] 184-2082017 coletivos. Desse modo, Croce promove nessa obra uma espécie de inventário da liberdade, discorrendo sobre ela nos gregos, nos romanos, nos filósofos, na Renascença, na Reforma Protestante, na Revolução Francesa e etc. Sua tese é, no fundo, de matriz hegeliana, a saber, a história deve ser vista como a história da liberdade. A partir dessa visão apresenta-se também a sua perspectiva acerca da moral. E, ao abordar o tema da moral, surgem outras indagações: o que é a liberdade? Para que ela serve? A quem ela pertence? Introdução Um dos erros mais grosseiros do catolicismo foi a defesa da monarquia absolutista e a manutenção de uma ideia limitada sobre o poder do 190 Márcio Gimenes governante que, aliás, ainda se pode perceber também em Lutero. A tese crociana é que, na verdade, o pensamento moderno se caracteriza por uma passagem do naturalismo e do racionalismo para uma progressiva caminhada em direção à dialética e ao idealismo. Por isso, segundo o pensador de Nápoles, o liberalismo tem forte relação com o espiritualismo e o comunismo com o materialismo mais estrito. Tal concepção forneceria ao liberalismo um pouco mais de sofisticação intelectual. Nesse contexto, pode-se entender também um importante fator constituinte dos séculos XIX e XX, isto é, a presença do romantismo que, segundo Croce, pode ser divido em dois tipos: o primeiro seria teórico e especulativo; o segundo seria prático e, por isso, mais sentimental e moral. O fato concreto é que no romantismo encontra-se uma reação ao academicismo e nele se tem, por exemplo, a geração da Estética e de suas preocupações filosóficas. Por sua vez, o romantismo de matriz religiosa e mais moral, além de ser protestante é profundamente prático. Tal romantismo sonha com um mundo capaz de conciliar a natureza e o espírito, ao contrário da estética, que privilegiará a fantasia e o sonho e também um espírito de cunho mais especulativo, que, de algum modo, sempre percorrerá uma trilha atrás de uma síntese. A partir de tal diversidade de espécies de romantismo temos, no entender de Croce, até mesmo um neopaganismo, visto que ele vai em busca, inclusive, das suas origens primitivas germânicas. Entretanto, também existe nesse romantismo um lado ético e político, que talvez possa ser melhor explorado. Seu erro básico foi, na verdade, dar uma forte ênfase no mundo da vida prática, sem compreender a importância da teoria e da especulação. Assim, a religião da liberdade crociana ultrapassaria o romantismo mas, ao mesmo tempo, se nutriria de suas principais ideais e as reelaboraria, desenvolvendo uma guerra do espírito, uma dialética diferenciada, tal como se poderá notar na resistência e na oposição ao predomínio absolutista promovido pelo liberalismo. A tese central de Croce é que ocorre uma vitória do ideal liberal em todo o mundo. O absolutismo vai pouco a pouco perdendo seu terreno. Introdução Croce assiste, no seu tempo, os diversos nacionalismos se apropriarem do conceito de liberdade e tentarem circunscrevê-lo, aos seus domínios territoriais, concretizando-o a partir do solo onde se vive. Entretanto, a liberdade é também uma ruptura com vínculos tradicionais, podendo ainda ser vista como liberdade econômica. O pensador napolitano vê, na figura do intelectual do século XX, a afirmação da liberdade por excelência. No seu entender, o intelectual seria um tipo de novo missionário em uma nova cruzada, a saber, a cruzada da liberdade. Desse modo, o pensador avalia que a liberdade é uma religião. Ela possui uma ética própria e aponta para um tipo de religião do futuro. Figuras como Sócrates e Jesus e eventos como o paganismo, o cristianismo, o agostinianismo, o calvinismo mostrariam claramente tal tese. A era da liberdade seria, no fundo, a era do espírito preconizada por Joaquim de Fiore no século II. O problema para Croce é que tal religião da liberdade não existe na Igreja Católica. No seu entender, o catolicismo é a negação da ideia liberal. Seu grande intuito é a pregação de uma religião ultramundana, ainda que, em alguns momentos, defenda ou propicie o aparecimento de defensores da religião da liberdade: “a história, que é a história da liberdade, se mostra mais forte do que a sua doutrina e seu programa, e o derrota e o força a se contradizer no campo dos fatos” (CROCE, 1965, p.23). Nesse mesmo escopo é que se pode compreender os desafios do Renascimento e da Reforma Protestante diante de uma Igreja Católica corroída. No entender do pensador, a Contra-Reforma salvou o corpo político da Igreja, mas não foi capaz de salvar a sua alma. Assim, seu domínio ficou restrito ao mundano e perdeu a sua vocação religiosa. Em decorrência de tal situação, a Igreja Católica assiste a fuga dos seus pensadores, bem como dos seus cientistas, restando nela basicamente o construto da Escolástica. Em outras palavras, ela não alcança a modernidade: não chega a autores como Lutero, Calvino, Rousseau e Voltaire. Tal instituição se configura como inimiga do liberalismo. Contudo, Croce é muito claro ao afirmar não um anti-cristianismo, mas um anti-clericalismo: “[...] a aversão era não ao catolicismo, mas ao ‘clericalismo’, ao ‘negro clericalismo’” (CROCE, 1965, p.27). Introdução Os ideais liberais de desenvolvimento, progresso, educação, civilidade, ganham corpo e se ampliam em escala mundial, ainda que isso ocorra de modo diverso, a depender do contexto. Para além do domínio político e econômico, afirma-se aqui uma tese bastante instigante: os séculos XIX e XX revelam uma vida intelectual bastante ativa. Aqui se pode claramente perceber a ênfase cultural dos ideais liberais e a reação dos ideais católicos. A curiosa posição de Croce é que na Itália (e mesmo na França) a renovação do pensamento se dá por meio dos jovens pensadores alemães: Kant, Hegel e autores pós-hegelianos. No seu entender, acontece na Igreja Católica uma queda cultural e, diante disso, o que resta a ela é o exercício do 191 Dissertatio [45] 184-2082017 poder político: “[...] a Igreja Católica não podia seguir os seus adversários na alta esfera em que se moviam e, mais e mais, se reduzia a potência prevalentemente política” (CROCE, 1965, p.84). Assim, os padres são cada vez mais vistos na companhia dos reis e dos seus ministros. Por isso, Croce percebe em tudo isso uma mistura dos interesses da sacristia e dos ideais policiais. O fato é que, no seu entender, o liberalismo consegue colher nessa época, inclusive na Igreja, os seus melhores pensadores e por isso, não despropositadamente, muitos dos pensadores dos séculos XIX e XX são oriundos da estrutura eclesiástica. A Revolução Francesa foi, sem dúvida alguma, um ponto central para a posição liberal desde o século XVIII. Croce aponta que seus ecos e repercussão podem ser percebidos na Itália em autores como Giuseppe Mazzini. Afirmam-se aqui dois curiosos tipos de catolicismo italianos: o primeiro é o tipo do católico liberal; o segundo o do anti-clerical. Ambos acontecem num período marcado por debates intensos no campo social e entre as teses de socialistas, comunistas e liberais. Percebe-se aqui, segundo Croce, a importância decisiva dos pensadores pós-hegelianos pois esses, nutridos também por autores franceses e pelo vento da Revolução Francesa promovem, em seu campo, uma reviravolta no idealismo alemão. Tal configuração torna-se decisiva para a nova compreensão do idealismo não apenas na Alemanha, mas no restante do mundo. Além disso, Croce destaca a contribuição de autores como Tocqueville, autor da célebre análise da Democracia na América, que, no seu entender, afirma-se como um profeta dos novos tempos do século XIX e da sua passagem para o século XX. Ainda naqueles primeiros meses um jovem filósofo de inspiração hegeliana, Silvio Spaventa, tematizava pensar o impensável e dizia que o infinito abstrato da Igreja e aquele vivo na nacionalidade e no Estado, o infinito da religião e o infinito da sociedade, por obra e fato de “um homem tido por infalível”, eram reconhecidos na sua unidade, na unidade de Deus, “que reina no intelecto e no coração como no próprio céu se nutre da sua luz” (CROCE, 1965, p.151). Introdução Os ideais liberais tem diante de si, especialmente na Itália, um inimigo decisivo: o papado. Como Croce bem aponta, seguindo a trilha de Silvio Spaventa, seu tio, tal guerra existe desde os dias de Maquiavel e persiste até o século XX: Ainda naqueles primeiros meses um jovem filósofo de inspiração hegeliana, Silvio Spaventa, tematizava pensar o impensável e dizia que o infinito abstrato da Igreja e aquele vivo na nacionalidade e no Estado, o infinito da religião e o infinito da sociedade, por obra e fato de “um homem tido por infalível”, eram reconhecidos na sua unidade, na unidade de Deus, “que reina no intelecto e no coração como no próprio céu se nutre da sua luz” (CROCE, 1965, p.151). Assim, a Igreja parece não conviver bem com os ideais liberais. Quando ela se encontra fragilizada, clama por maior liberdade. Contudo, quando ela é forte e poderosa, tolhe a liberdade, mostrando efetivamente o seu intuito. Tal será sempre a tônica da difícil relação entre o liberalismo e a Igreja, que piora substancialmente quando há um problema efetivo a se responder, a saber: o que fazer com o poder temporal do papa? Ou melhor, o que fazer 192 Márcio Gimenes quando tal poder, por força do avanço dos ideais liberais, acaba. Como justificá-lo? A resposta à pergunta não é simples, mas Croce ensaia uma possível: [...] porque a Igreja, sociedade perfeita, inclui com o espiritual o temporal, e a seu tempo estendia bastante largo o seu poder, e investia e coroava os príncipes da terra e os excomungava e os depunha, e se ora se via reduzida a dominar somente um pedaço da Itália, nem por isso tinha renunciado a um direito ao qual não lhe era dado renunciar sem contradizer a própria doutrina e natureza (CROCE, 1965, p.199). O fim do poder temporal eclesiástico não deixa de ser o fim de um dado tipo de teocratismo e por isso repercute de forma positiva na Itália e em toda a Europa. Tal posição ajuda, inclusive, na redescoberta de valores cristãos mais sinceros e honestos deixando mais claro, por exemplo, um debate entre as teses da Igreja triunfante (aliada ao poder) e a Igreja militante (a Igreja dos mártires, dos que sofrem). Por isso, de forma muito curiosa, mas não sem propósito, Croce nas palavras finais do seu livro usa, uma vez mais, o termo “religião da liberdade” (CROCE, 1965, p.316). Introdução Em outras palavras, completa o seu inventário da liberdade. Segundo Galasso, o período compreendido após 1870 é uma época fundamental da história europeia. Por isso, não fortuitamente, duas das obras crocianas abordam claramente tal período, a saber, Storia d´Europa nel secolo decimono e Storia d´Italia dal 1871 al 1915. Aqui, no horizonte posterior à guerra franco-prussiana afirmam-se a discussão de temas como o nacionalismo, o socialismo, o republicanismo, os valores liberais, o positivismo, bem como o hegelianismo e a sua herança. Assim, o próprio título da obra de Galasso (Croce e lo spirito del suo tempo) parece claramente demonstrar as intenções do autor. Por isso, no seu entender, Croce seria, na verdade, um autor do diagnóstico da crise do final do século XIX e daquilo que se sucederia no século XX. q q Com efeito, a obra do autor napolitano, ao seu modo, não deixa de espelhar um debate entre o romantismo e a ciência, notadamente o debate entre o positivismo e a ciência. Assim, há também aqui uma proximidade entre os temas crocianos e os temas desenvolvidos por Comte e por Hegel. Por isso, há também na obra do pensador, apesar dele nunca ter sido um positivista, fortes traços do laicismo e do anticlericalismo típicos do positivismo. Curiosamente, tal positivismo mostra, uma vez mais, que a cultura italiana é ora europeia e ora parece distante dos temas filosóficos da Europa, a depender do contexto e da perspectiva avaliada. No entender de Galasso, o positivismo foi, na Itália, muito mais um clima cultural do que uma filosofia: “O positivismo foi, com efeito, um clima cultural e moral, antes e mais do que uma filosofia” (GALASSO, 2002, p.93). 193 Dissertatio [45] 184-2082017 Do mesmo modo que o positivismo possui uma recepção particular na Itália, o mesmo ocorre com o marxismo. Aqui, seguindo a esteira dos introdutores de Hegel na Itália como Spaventa e Labriola, Croce e Gentile, por exemplo, aproximam-se dos temas da filosofia marxista. Aqui compreendemos o que se poderá chamar como idealismo italiano. Nesse contexto, tanto Croce, como também Gentile, se inserem como filósofos interessados nos temas da filosofia hegeliana e como críticos do positivismo. Assim, o Croce desse período é um autor hegeliano, estudioso da filosofia do espírito, alguém que busca recuperar os temas da filosofia da história e, nesse sentido, um crítico do positivismo. Introdução Aliás, não há distinção entre eles. Por isso, sua pista de liberdade pode nos livrar do autoritarismo tanto da religião como dos totalitários políticos de esquerda ou de direita e, nesse sentido, ele é inspirador mesmo para seus opositores como Gramsci (1949a, 1949b). Além disso, possui o mérito de inserir a filosofia italiana em um debate filosófico internacional na medida em que suas teses foram discutidas por importantes autores como Löwith e Cassirer, por exemplo. Entretanto, como bem parece salientar Viano, o idealismo crociano (e gentiliano) sempre conviveram também numa certa dubiedade: ao mesmo tempo em que pareciam integrar a filosofia italiana ao mundo, pareciam também fechá-la ao mundo, não sendo tal fechamento unicamente culpa do fascismo: Do ponto de vista filosófico-cultural em geral o predomínio da filosofia crociana, juntamente com o domínio daquela gentiliana, criou um clima de prevalência idealística: esse tinha garantido à Itália a uniformidade cultural, mas a manteve alheia aos movimentos que da Primeira Guerra Mundial até metade do século tinham caracterizado a cultura dos outros grandes países europeus e dos Estados Unidos. A segunda parte do problema de fronte ao qual se encontrava a filosofia italiana depois da guerra se configurava assim como a abolição do fechamento cultural italiano, um fechamento que o fascismo tinha reforçado e institucionalizado com a própria política cultural, mas que já antes o neoidealismo havia praticado (VIANO, 1985, p.49). Assim, abre-se aqui também um importante espaço para que se possa pensar a filosofia italiana do século XX com Croce (e Gentile), mas também contra ambos, o que sempre parece mais rico e significativo. Tal exercício é efetivado, segundo uma perspectiva gramsciana, por Piccioni (PICCIONI, 1983) que, na sua obra Ideologia e filosofia del neoidealismo italiano, aponta claramente que tanto Croce como Gentile são autores pós-positivistas e, ao mesmo tempo, autores conservadores e anti-marxistas. Como vimos, para Gentile – mas também para Croce – a religião é um dado tipo de moral que é, inclusive, necessária ao povo. Assim, o que se afirma aqui é uma clássica tese conservadora. O idealismo desenvolvido por ambos, apesar de todos os méritos, não deixa de flertar, segundo Piccioni, com um elitismo filosófico já presente na filosofia hegeliana e, a rigor, presente no próprio platonismo, que foi incorporado pela posição idealista hegeliana. Introdução Para Galasso, Croce era “idealista porque se inseria no sulco da tradição do pensamento que de Kant o tinha levado até Hegel” (GALASSO, 2002, p.153). Contudo, o Croce leitor de Hegel, mesmo da Lógica e do Sistema, nunca deixou de ter em seu horizonte o tema hegeliano por excelência: a racionalidade dentro da história. A conciliação entre a história e a filosofia. Aqui, no entender de Galasso, talvez exista uma divergência entre Croce e Gentile. É certo que ambos foram leitores de Hegel, de Marx e deram importância ao tema da história. Entretanto, para Galasso, Gentile não teria conseguido se desapegar do tema da Lógica. Assim, mais do que afirmar que Croce e Gentile romperam em função do fascismo, Galasso parece construir um argumento teórico para a primeira separação entre ambos os pensadores, o que seria mais significativo filosoficamente. Com muita propriedade, Galasso percebe na obra de Croce um momento historiográfico. Inserem-se nesse horizonte, muitas de suas obras, mas há um especial destaque para seu trabalho sobre filosofia da história denominado Teoria e storia della storiografia. Há aqui a afirmação do pensador da crise da consciência europeia, o que aproxima Croce da sua tradição e de autores como Burckhardt, Weber, Horkheimer. Desse modo, o contexto cultural italiano dos anos do fascismo e do pós-fascismo apontam para a importância de autores como Croce, Gentile e dos pensadores católicos. Aqui notamos uma questão que mereceria uma investigação mais aprofundada: por que o idealismo de Croce e de Gentile sempre se reporta aos temas da religião e da religiosidade? No entender de Galasso, tal coisa ocorre pelo fato da religião ser, de algum modo, a base do idealismo italiano, como igualmente suspeitava Viroli na sua obra Come se Dio ci fosse – Religione e libertà nella storia d´Italia (VIROLI, 2009). Assim, “de resto, fora do idealismo e do reflorescente catolicismo filosófico, o panorama filosófico italiano não oferecia muito” (GALASSO, 2002, p.322). Com efeito, nesse clima, percebemos a afirmação do liberalismo crociano, que fica mais evidente na obra Storia d´Europa nel secolo decimono, tal como constatamos. Aqui Croce, em seu momento historiográfico, afirma o tema do liberalismo e da liberdade em oposição ao catolicismo e ao 194 Márcio Gimenes fechamento político italiano dos anos do fascismo e de acordos entre o Estado e a Igreja. Devemos notar que o ciclo histórico crociano não é maior e nem menor que sua obra filosófica. Introdução Tal tipo de posição serve tanto para dar autoridade ao filósofo como aquele que pensa melhor, e por isso governa e lidera, como também pode fornecer ao mesmo filósofo as bases para sua crítica. Por isso, segundo Piccioni, Croce como filósofo idealista, está na base tanto do pré-fascismo como do anti-fascismo, sendo um conservador de raiz. Assim, seu apreço pelo tema da filosofia da história é um apreço 195 Dissertatio [45] 184-2082017 hegeliano por excelência, um ideal de progresso e superação estão contidos no seu filosofar: hegeliano por excelência, um ideal de progresso e superação estão contidos no seu filosofar: A defesa dos valores de cultura preservados daqueles empíricos – na realidade do processo histórico real portador de novos agentes da história – vem a constituir a essência do pensamento teórico e político crociano sob um plano de fechado conservadorismo e a garantir a continuidade mesma do pré-fascismo ao anti-fascismo (PICCIONI, 1983, p.60). Paolo Rossi chama a atenção para o fato de que os intelectuais italianos, formados nos anos do fascismo, foram, ainda que depois viessem a se opor a ele, inegavelmente formados dentro do seu ambiente cultural. Por isso, segundo ele, há uma reação, de certo modo doentia, ao tentar renegá-lo: Muitos italianos – certo a maioria – foram muito hábeis na arte de fazer desaparecer o fascismo da sua história pessoal. Intelectuais de várias proveniências e natureza não se comportaram de modo muito diferente dos seus compatriotas e tem ao longo aplicado essa mesma técnica à história nacional (ROSSI, 2012, p.13). Por isso, como já percebeu como argúcia Gramsci, Croce e Gentile, por exemplo, não podem ser vistos separadamente, visto que fazem parte do mesmo escopo cultural. Assim, Rossi faz uma curiosa citação, a partir de um exemplo particular, do quanto tais filósofos eram vistos conjuntamente tanto antes, como até mesmo depois do fascismo: Meu pai, um estimado e assaz culto professor de Liceu, tinha uma religião laica de trabalho intelectual e da função da escola clássica, se inspirava principalmente em Croce nos seus trabalhos de crítica dantesca, mas admirava também Gentile e lia e apostilava os seus escritos. Era muito longe (por temperamento e por formação cultural) de cada forma de intolerância e fanatismo, mas julgo que compartilhava plenamente a imagem gentiliana da história contemporânea da Itália e o diagnóstico gentiliano sobre o significado e a função história do fascismo (ROSSI, 2012, p.26). Introdução Uma vez apresentado esse muito breve panorama da filosofia italiana dos séculos XIX e XX através de dois dos seus principais expoentes, avaliamos que cabe agora investigar um dos aspectos daquilo que podemos chamar de idealismo germânico à italiana, a saber, a recepção das obras de Feuerbach, Kierkegaard e Nietzsche em solo italiano. 2. O idealismo germânico à italiana: a recepção de Feuerbach, Kierkegaard e Nietzsche na Itália O filósofo Feuerbach possui uma curiosa recepção ao seu trabalho: ora sua obra é vista e interpretada entre os hegelianos, ora entre os marxistas e ora se afirma nos debates teológicos e religiosos. Perone (PERONE, 1972) aponta que, para além de tais interpretações, parece importante ressaltar que 196 Márcio Gimenes seu legado, de fato, alcança um patamar mais elevado na medida em que tais posições são superadas e sua crítica começa a ser feita a partir de uma interpretação científica e historiográfica. Como um exemplo de tal avanço na pesquisa feuerbachiana notamos, em contexto italiano, a obra do pensador italiano Franco Lombardi, que ocorre na sequência de outras interpretações que haviam explorado a relação de Feuerbach com o idealismo: O L. Feuerbach, La Nuova Italia, Firenze 1935, de F. Lombardi, de certo modo, aproveita-se do precedente, visto que, movendo-se a partir do idealismo, intenta a sua própria superação através de Feuerbach, e está mais interessado na contribuição filosófica de Feuerbach do que na sua crítica religiosa. Por outro lado, isso se afirma sob o fundo da contemporânea situação cultural italiana, dominada pelo neoidealismo; nela o interesse por Feuerbach assume imediatamente uma veia polêmica e um significado teorético (PERONE, 1972, p.183). O L. Feuerbach, La Nuova Italia, Firenze 1935, de F. Lombardi, de certo modo, aproveita-se do precedente, visto que, movendo-se a partir do idealismo, intenta a sua própria superação através de Feuerbach, e está mais interessado na contribuição filosófica de Feuerbach do que na sua crítica religiosa. Por outro lado, isso se afirma sob o fundo da contemporânea situação cultural italiana, dominada pelo neoidealismo; nela o interesse por Feuerbach assume imediatamente uma veia polêmica e um significado teorético (PERONE, 1972, p.183). Segundo Perone, destacam-se ainda, na primeira metade do século XX, na crítica italiana de Feuerbach, alguns importantes trabalhos: dois de Pareyson (PAREYSON, 1950;1971) e as clássicas interpretações de Mondolfo (MONDOLFO, 1923; 1924), além, evidentemente, da já citada obra pioneira de Franco Lombardi. Na segunda metade do século XX temos como destaque dois importantes trabalhos de Claudio Cesa (CESA, 1963; 2003), igualmente conhecido por suas célebres traduções de obras do pensador alemão. Especialmente nessa última obra, de modo mais elaborado, começa a se desenhar um estudo de Feuerbach e da importância da temática da consciência como um ponto importante na reflexão filosófica do autor. 2. O idealismo germânico à italiana: a recepção de Feuerbach, Kierkegaard e Nietzsche na Itália Entre os anos sessenta e setenta do século XX, destacam-se ainda as obras de Rambaldi (RAMBALDI, 1970; 1966a; 1966b), Perone (PERONE, 1972), Severino (SEVERINO, 1972), Alessi (ALESSI, 1975), bem como o importante artigo de Caracciolo (CARACCIOLO, 1978). Nos anos setenta, e já adentrando nos anos oitenta, ganham destaque na recepção italiana de Feuerbach os estudos sobre os textos inéditos e póstumos do pensador, os chamados Nachlaβ. Assim, afirmam-se as importantes obras de Casini (CASINI, 1979; 1974) e Tomasoni (TOMASONI, 1982), estudioso dos inéditos do pensador alemão e tradutor de algumas obras importantes de Feuerbach, que também publicará, em 2011, a mais completa biográfica filosófica do pensador alemão disponível em língua italiana até então (TOMASONI, 2011). Já a recepção kierkegaardiana na Itália, padece daquilo que Cornélio Fabro, grande especialista e maior tradutor italiano da obra do autor dinamarquês, denominou como “o provincianismo laico-clerical da cultura italiana” (PIZZUTI, 1995, p.226). Há, contudo, no seu entender, uma honrosa exceção: “Se excetua a monografia de F. Lombardi, Kierkegaard (FIRENZE, 1936, republicada integralmente em 1967)” (PIZZUTI, 1995, p.226). Tal 197 Dissertatio [45] 184-2082017 trabalho de Lombardi pode ser melhor compreendido dentro do contexto intelectual que antecedia à Segunda Guerra Mundial e está especialmente localizado em torno dos debates culturais promovidos em Florença, tal como bem acentua Garin e, nesse sentido, trata-se de um Kierkegaard bastante específico e com a grande virtude de possuir uma abrangência intelectual: “Como não recordar, apenas para um exemplo, que Kierkegaard, a quem hoje cabe o amargo destino de servir de tema a tantas dissertações e aulas universitárias, era apresentado aos italianos propriamente no ‘Leonardo’1 e traduzido na papiniana ‘Cultura dell´anima’” (GARIN, 1997, p.22). Na esteira dessa interpretação de Lombardi, podemos ainda elencar outros trabalhos de autores italianos: Abbagnano (La struttura dell´esistenza), Paci (Principi di una filosofia dell´essere), Carlini (Il mito del realismo), Battaglia (Il problema morale nell´esistenzialismo), Stefanini (Esistenzialismo ateo ed esistenzialismo teistico), Cantoni (La coscienza inquieta), tal como acentua Pizzuti (PIZZUTTI, 1995, p.226-228) e Basso (2009, p.82-85). Entretanto, a despeito dos avanços e méritos de todos esses trabalhos, eles parecem ainda estar englobados naquilo que se convencionou denominar como a Kierkegaard Renassaince alemã. Desse modo, Pizzuti avalia que a interpretação de Kierkegaard tem um efetivo avanço em solo italiano com a obra e com as traduções de Cornélio Fabro, já na metade do século XX. 1 O Leonardo era uma revista literária. Em 1906, na sua edição de Abril-Junho, ela publicou um ensaio de Hoffding, célebre comentarista dinamarquês de Kierkegaard. Em 1907, publicou uma tradução de In Vino Veritas feita por Knud Ferlov. Se pode afirmar, em síntese, que Fabro na Itália realizou o mesmo programa que Thulstrup promoveu na Dinamarca, a diferença do estudioso dinamarquês, que ignorou quase totalmente o contributo dos estudiosos católicos, Fabro conduzido pela mesma lógica da sua linha hermenêutica sublinhou, com uma insistência que por vezes parecia (mas não era) uma afirmação confessional, a objetiva convergência de alguns pontos kierkegaardianos com a posição da 2. O idealismo germânico à italiana: a recepção de Feuerbach, Kierkegaard e Nietzsche na Itália Fabro, que era religioso e um importante conhecedor de Santo Tomás e da tradição escolástica, faz uma espécie de ligação de Kierkegaard com o mundo católico, tal coisa também se pode notar, por exemplo, na Espanha, na recepção de Unamuno. Contudo, ainda que Fabro tenha tal posição, ele não compreende, em momento algum do seu trabalho, Kierkegaard como uma espécie de católico. Para Pizzutti seu trabalho, a despeito de algumas limitações, é um avanço, por exemplo, em relação a um outro importante estudioso dinamarquês de Kierkegaard do mesmo período, Niels Thulstrup, que não coloca nenhuma possibilidade de diálogo entre o autor dinamarquês e o mundo católico, circunscrevendo-o aos domínios protestantes e nórdicos: Se pode afirmar, em síntese, que Fabro na Itália realizou o mesmo programa que Thulstrup promoveu na Dinamarca, a diferença do estudioso dinamarquês, que ignorou quase totalmente o contributo dos estudiosos católicos, Fabro conduzido pela mesma lógica da sua linha hermenêutica sublinhou, com uma insistência que por vezes parecia (mas não era) uma afirmação confessional, a objetiva convergência de alguns pontos kierkegaardianos com a posição da 198 Márcio Gimenes doutrina católica, e absolutamente tomista, não sem, todavia, abafar as razões precisas pela qual Kierkegaard não pode ser tomado nem por católico e nem por cripto-católico (PIZZUTI, 1995, p. 228). doutrina católica, e absolutamente tomista, não sem, todavia, abafar as razões precisas pela qual Kierkegaard não pode ser tomado nem por católico e nem por cripto-católico (PIZZUTI, 1995, p. 228). Karl Löwith, pensador que teve curta passagem pela Itália no período que antece à Segunda Guerra Mundial, é figura importante para compreendermos a recepção de Feuerbach, Kierkegaard e Nietzsche em solo italiano. O autor do clássico De Hegel a Nietzsche possui uma relação acadêmica com três pensadores italianos bastante importantes da época: Giovanni Gentile, Benedetto Croce e Franco Lombardi. O primeiro deles, autor com quem o pensador colaborou nas suas publicações, era, na sua concepção, um homem bastante culto e gentil, embora tivesse ligações com o fascismo. Löwith narra que, numa dada ocasião social, espantou-se com o modo como ele, a fim de defender a política externa italiana colocou “na mesma panela fascista, Maquiavel, Mazzini e Mussolini” (LÖWITH, 1988, p.117), o segundo é merecedor do seu mais alto elogio. 2. O idealismo germânico à italiana: a recepção de Feuerbach, Kierkegaard e Nietzsche na Itália Na curta experiência de um passeio em sua companhia em Nápoles, Löwith encontrou-se com Benedetto Croce, a quem tomava por um dos poucos espíritos livres em atividade numa Europa sitiada pelo nazi-fascismo, tal como se pode perceber na elogiosa menção: Eu tive uma vez a sorte de encontrar-me com Croce na sua casa junto com alguns de seus amigos. Ele depois passeou conosco, pelas ruas de Nápoles, até a meia-noite e mesmo se nós mais jovens nem sempre pudessemos concordar com sua avaliação das mutações que ocorriam em nossa época, uma coisa é certa: na Europa ele é um dos poucos espíritos que permaneceu livre e em posse de um saber e de uma cultura que fazia envergonhar todos os mais jovens (LÖWITH, 1988, p.118). Já o terceiro pensador italiano da sua relação o acompanha até o momento do seu embarque para o exílio no Japão, como podemos atestar por esse emotivo trecho da sua autobiografia: Em Nápoles passamos ainda um belo entardecer junto a Franco Lombardi, que no dia seguinte – era 11 de outubro – nos acompanhou ao navio japonês Suwa Maru. A separação da Itália foi para mim mais dolorosa porque era a separação de uma terra que tinha negado até mesmo o direito de hospitalidade a um estrangeiro (LÖWITH, 1988, p.146-147). Tratava-se de Franco Lombardi, autor das obras já citadas sobre Kierkegaard e Feuerbach. Na visão de Bobbio, suas teses sobre tais os dois autores germânicos colocam ambos os pensadores numa chave de leitura secularizada que estabelecerá um novo patamar na interpretação italiana: Com uma singular intuição antecipadora, Franco Lombardi escreve em 1935 um livro sobre Feuerbach, em 1936 um outro sobre Kierkegaard: dois filósofos que permaneceram à margem da filosofia acadêmica, reencontrarão à liberação sobre uma via mestra, um do renascimento marxista, o outro do existencialismo (BOBBIO, 1986, p.149). 199 Dissertatio [45] 184-2082017 Lombardi escreve também uma importante história da filosofia italiana compreendida nos últimos cem anos, isto é, entre 1848 e 1948. Em La filosofia italiana negli ultimi cento anni (s/data), mesmo com a forma um pouco panorâmica, pode-se perceber a importante contribuição do pensador italiano em seu inventário. É notória sua tentativa de compreender o pensamento italiano dentro de um contexto europeu e, por isso, são inúmeras as citações aos eventos e temas europeus por excelência. 2. O idealismo germânico à italiana: a recepção de Feuerbach, Kierkegaard e Nietzsche na Itália Vico, por exemplo, filósofo moderno italiano, será sempre a fonte de inspiração, coisa igualmente perceptivel na obra de Croce e Gentile. Assim, não é fortuita que, em 1848, mesmo período em que Marx e Engels redigiam o seu Manifesto do Partido Comunista, o autor situe, como ponto importante do contexto filosófico italiano, o trabalho dos jovens hegelianos de Nápoles: De Sanctis, Bertrando Spaventa, Salvatore Tomassi e Antonio Labriola. Contudo, Lombardi, que é ele próprio um leitor atento de Kierkegaard e Feuerbach, acredita, como bom pós-hegeliano, que é preciso superar a filosofia hegeliana sistemática: “a libertação da filosofia ‘do sistema’ ou a posição de uma filosofia do homem. Esta constituia, doravante, o programa que seria posto para o pensamento filosófico italiano” (LOMBARDI, s/data, p.33). Assim, para o pensador, todos os pensadores antisistemáticos são, de algum modo, irmãos de Kierkegaard. Tal como ele assim defende ao analisar a filosofia de Labriola: Não diferentemente de Kierkegaard (que de todos foi irmão na sua reação contra a filosofia “sistemática”, outra que contra a filosofia “do sistema”, isto é, contra a ideologia hegeliana), esses pensadores podiam dizer de si mesmos: “Vi um pássaro do qual se diz que prenuncia a chuva – e um tal pássaro sou eu. Se em uma geração começam a reunir-se as nuvens prenunciadoras de um temporal... como eu” (LOMBARDI, s/data, p.50-51). Se Vico foi inspirador dos filósofos italianos modernos, Lombardi sublinha que Galileu será, por sua vez, o inspirador renascentista dos autores do positivismo italiano, chegando tal influência até mesmo a autores do idealismo italiano como Croce e Gentile. Por isso, de forma muito espirituosa, ele compara Croce, o filósofo campeão da liberdade em contexto italiano com a figura do bispo Mynster como “testemunha da verdade” nos tempos de Kierkegaard (LOMBARDI, s/data, p.108, nota 1). Não menos espirituosa será a comparação feita entre Croce e os devotos napolitanos de São Gennaro: “o culto pátrio se repartiu igualmente em Nápoles entre ‘os parentes’ de São Gennaro e os parentes de Don Benedetto” (LOMBARDI, s/data, p.119). 2. O idealismo germânico à italiana: a recepção de Feuerbach, Kierkegaard e Nietzsche na Itália Kierkegaard, autor a quem Lombardi dedicou uma boa parte dos seus estudos, é o inspirador de uma contudente crítica de sua parte ao catolicismo italiano e ao modo como seus compatriotas o vivem enquanto identidade nacional: “Aqui se poderia perguntar em qual modo se pode falar de 200 Márcio Gimenes ‘convertidos ao catolicismo’ em um país como a Itália: não somos talvez nós todos se podemos perguntar tal como Kierkegaard nascidos na Itália? E não é a Itália, por assim dizer, não é talvez a Itália – um reino católico apostólico cristão? E assim não seremos todos cristãos?” (LOMBARDI, s/data, p.142). O autor dinamarquês, tomado ainda como pai do existencialismo produz em solo italiano, alguns bons comentadores e estudiosos no entender de Lombardi. Pareyson, por exemplo, seria um deles. Contudo, a despeito de reconhecer a importância do trabalho do estudioso de Kierkegaard que foi Cornélio Fabro, Lombardi é contundente em sua crítica a ele, o que o coloca numa posição direta de crítica aos kierkegaardianos e faz sua interpretação do pensador dinamarquês mais próxima dos pós-hegelianos: “Padre Cornélio Fabro que dos primeiríssimos estudos honestos e severos sobre a psicologia passou a ocupar-se ativamente do existencialismo kierkegaardiano, com um tentativa de relacioná-lo com o existencialismo kierkegaardiano, que na verdade repugna a isso, sob o velho tronco da doutrinas tomistas” (LOMBARDI, s/data, p.180). Nesse mesmo sentido é que podemos compreender a continuidade daquilo que se convecionará denominar posteriormente de existencialismo italiano. Aqui, segundo Viano, teremos destaque para os nomes de Luigi Pareyson, Bobbio, Abbagnano. O foco de tais autores se dará preferencialmente nos temas da condição humana, da finitude e, com menor ênfase no tema do irracional. Como se pode notar, são todos temas marcantes do existencialismo pós-guerra. Feita tal visão panorâmica da recepção italiana de Feuerbach e Kierkegaard, falta ainda a compreensão da obra nietzschiana na Itália. Segundo Penzo (PENZO, 1990, p.199-204), a recepção da obra nietzschiana na Itália possui, no seu primeiro momento, uma configuração literária, tal como ocorre também na Alemanha. O autor é visto como o pensador anárquico, aquele que destruiu Deus, a moral, os valores tradicionais. Assim, tal imagem oscila entre um negativo e um positivo, funcionando como um mito. A democracia se reduz então a uma luta de egoísmos vaidosos, que se desenvolvem sob o abaixamento sistemático da superioridade legítima e adquirida. É o triunfo do burguês, do filisteu, da trufa, do asno presunçoso, do pedante que faz o sábio, do idiota que se crê igual ao homem de engenho, de todas as mediocridades e todas as baixezas (D´ANNUNZIO, 1994, p.14). pedante que faz o sábio, do idiota que se crê igual ao homem de engenho, de todas as mediocridades e todas as baixezas (D´ANNUNZIO, 1994, p.14). Para o intérprete italiano de Nietzsche, a desigualdade existira na natureza humana e não reconhecê-la seria ilógico, contrário àquilo que é natural. Nesse sentido, ele justificaria um direito do mais forte, uma vez que isso é obtido pela natureza e assim justificaria, inclusive, a colonização africana (e de outras partes do mundo) pelas nações europeias. O problema mais grave para D´Annunzio residiria no fato da decadência europeia, já bem percebida por Nietzsche em sua obra: “Segundo a doutrina de Frederico Nietzsche uma entre as razões da geral decadência está nisso: que a Europa inteira recebeu a sua definitiva marca da noção de bem e de mal tomada no sentido moral dos escravos” (D´ANNUNZIO, 1994, p.14). Segundo Michelini, a interpretação apresentada por D´Annunzio é, na verdade, não apenas a primeira aparição do autor em contexto italiano, mas também prova ser a primeira interpretação feita mais num sentido cultural, isto é, ainda se trata de um Nietzsche muito geral e menos elaborado de modo acadêmico ou investigativo, tal como se poderá notar nos anos posteriores: É sobretudo o Nietzsche de D´Annunzio, isto é, o Nietzsche filtrado pelo esteticismo decadente e provincial e morbidamente sensual do poeta italiano, aquele destinado a ter maior sucesso e divulgação, próprio porque era mais fácil, menos profundo e consciente – ao contrário do original – do tormento que atravessa a inteira civilização ocidental (MICHELINI, 1978, p.18-19). É sobretudo o Nietzsche de D´Annunzio, isto é, o Nietzsche filtrado pelo esteticismo decadente e provincial e morbidamente sensual do poeta italiano, aquele destinado a ter maior sucesso e divulgação, próprio porque era mais fácil, menos profundo e consciente – ao contrário do original – do tormento que atravessa a inteira civilização ocidental (MICHELINI, 1978, p.18-19). Em 1898, surge em contexto italiano uma tradução, feita por Edmondo Weisel, de Para além de bem e mal. Note-se, conforme aponta Michelini, que tal coisa é bastante prematura, uma vez que até mesmo na Alemanha, pátria do filósofo, algumas obras ainda não haviam aparecido na integra. Aqui pode haver um aspecto singular na relação do filósofo com a Itália, também para compreensão da sua recepção entre os italianos. Nietzsche, como é conhecido, teve diversas estadias na Itália, começando-as em 1876. 2. O idealismo germânico à italiana: a recepção de Feuerbach, Kierkegaard e Nietzsche na Itália Desse modo, é significativo que a primeira configuração nietzschiana na Itália se afirme na interpretação de Gabriele D´Annunzio: “De resto, é significativo que a fortuna de Nietzsche na Itália seja ligada ao nome de D´Annunzio, que lê Nietzsche entre 1892-1894 em um senso estético-heróico” (PENZO, 1990, p.199). A interpretação feita por D´Annunzio é ainda importante pela apresentação de um Nietzsche crítico da democracia, tal como se pode perceber num texto seu de 25 de Setembro de 1892 e publicado com o instigante título de A besta eletiva: 201 Dissertatio [45] 184-2082017 pedante que faz o sábio, do idiota que se crê igual ao homem de engenho, de todas as mediocridades e todas as baixezas (D´ANNUNZIO, 1994, p.14). Como aponta Michelini: “Na Itália, Nietzsche havia encontrado a atmosfera ideal para escrever a sua obra filosófica. A longa série de estadias no nosso país foi iniciada treze anos antes, em 1876, e terminava, tragicamente, na cidade italiana que tinha mais amado, Torino” (MICHELINI, 1978, p.29). Também Buddensieg, comentador alemão da obra de Nietzsche, corrobora com a tese da importância das estadias italianas na obra de Nietzsche. Ele, na sua obra A Itália de Nietzsche, analisa de modo minucioso os nove locais por onde o filósofo esteve: Sorrento e Napoli, Genova, Veneza, Roma, Florença, Nice (hoje França), Torino. Tal como também aponta Michelini, que afirmava que Nietzsche sempre procurou viver na Itália como uma pessoa comum, sem fazer contatos acadêmicos ou de trabalho, Buddensieg aponta para o quanto o 202 Márcio Gimenes pensador sentia-se à vontade em território italiano: “Contudo, na Itália, ele vive e escreve e se sente mais como um cidadão das localidades onde habita” (BUDDENSIEG, 2006, p.19). Por isso, com extrema propriedade, D´Iorio, estudioso contemporâneo da obra nietzschiana, aponta uma curiosa ligação afetiva de Nietzsche com o sul da Itália, notadamente por ocasião da sua passagem por Sorrento: “Da varanda de seu quarto diante de Sorrento, ele avista a ilha de Ischia, ilha vulcânica, lugar real e imaginário que servirá ao filósofo de modelo para as ‘ilhas bem-aventuradas’, as ilhas dos discípulos de Zaratustra, as ilhas bem-aventuradas são as do futuro, da esperança, da juventude” (D´IORIO, 2014, p.14). Depois dessa primeira interpretação de cunho literário livre e também de um Nietzsche mais politizado ao estilo fascista, afirma-se, notadamente na primeira metade do século XX, uma interpretação com um viés existencial, notadamente presente nas obras de Banfi, Paci e alguns outros, tal como aponta Penzo (PENZO, 1990, p.199-204). Um importante apontamento sobre Nietzsche é feito, ainda em 1904, numa resenha feita pelo filósofo italiano Giovanni Papini a um livro de Francesco Orestano intitulado As ideias fundamentais de F. Nietzsche no seu progressivo desenvolvimento. Para ele, que era um crítico de Nietzsche, era preciso que se fosse além da concepção rasa de um “filósofo de manicômio” para um real compreensão de Nietzsche: A desventura maior de Nietzsche como filósofo foi, depois a loucura, a sua fortuna. Por alguns anos ele tornou-se o pensador da moda, o oráculo dos literatos, o fornecedor de frases de efeitos para cenários estéticos. pedante que faz o sábio, do idiota que se crê igual ao homem de engenho, de todas as mediocridades e todas as baixezas (D´ANNUNZIO, 1994, p.14). Houve uma grande quantidade de gente que acreditava entender o filósofo porque amava o artista, e essa admiração, dizemos assim ‘literária’, o tornavam suspeitos aos filósofos puros. Nem um e nem outro estavam totalmente errados (PAPINI, 1904, p.63). Contudo, conforme Michelini também destaca uma importante interpretação de Nietzsche em contexto italiano será aquela fornecida por Benedetto Croce, apresentada em 1907 nas páginas de A Crítica. O pensador napolitano, mesmo não sendo um comentador de estrito senso do autor alemão, e ainda que apresentando pontuais discordâncias com o seu pensamento, aponta, pela primeira vez, para um significado filosófico da obra nietzschiana, fato fundamental para uma mudança de rumos: Mas já Croce tinha providenciado, por ocasião da tradução italiana do ‘Nascimento da tragédia’, por ele patrocinada, a enquadrar na justa medida o valor do modo nietzschiano de fazer filologia. Naquele artigo, publicado na ‘Critica’, Croce coloca, como veremos, em relevo a fecundidade daquele método no estudo dos acontecimentos antigos [...] (MICHELINI, 1978, p.19). Cabe ressaltar que o momento da primeira chegada de Nietzsche na Itália, ainda nos círculos de D´Annunzio, é marcada por forte influência do 203 Dissertatio [45] 184-2082017 positivismo, o que pode ter forçado o filósofo a ser tomado pelos círculos literários ou estéticos. Fato que, conforme podemos ver em Croce, é recuperado de um modo mais preciso. De igual modo, tal como aponta Michelini, talvez esse seja um contexto adequado para compreendermos o tema nietzschiano do Super-homem na exagerada interpretação de D´Annunzio: “D´Annunzio encontrou na moral do Super-homem o meio mais adequado e próximo da sua personalidade mórbida e sensual para entrar no ciclo da literatura europeia” (MICHELINI, 1978, p.221). Uma interpretação, que se afirma na segunda metade do século XX, aproxima Nietzsche de Heidegger e pode ser percebida notadamente na intepretação de Gianni Vattimo em La fine della modernita (VATTIMO, 2014). Nesse mesmo registro, percebe-se a relação entre Nietzsche e Freud, bem como entre Nietzsche e Marx, sendo seu articulador o já citado Karl Löwith. Depois da Segunda Guerra Mundial, afirma-se o trabalho interpretativo e de tradução de G. Colli e M. Montinari, como bem aponta Marton: Ponto de inflexão maior, porém, constitui a edição crítica das obras completas do filósofo. E isto porque seus efeitos não se limitam à Itália, mas se fazem sentir nos estudos nietzschianos de maneira geral. pedante que faz o sábio, do idiota que se crê igual ao homem de engenho, de todas as mediocridades e todas as baixezas (D´ANNUNZIO, 1994, p.14). Não há dúvida de que os esforços de Giorgio Colli e Mazzino Montinari serão cruciais para trazer a pesquisa internacional sobre a filosofia de Nietzsche ao ponto em que se encontra hoje (2007, p.49). Por isso, de modo não fortuito, Garin, estudioso da história da filosofia italiana, avalia que Nietzsche ainda é um desafio para a filosofia do século XX: E assim avança o anúncio de Nietzsche, que próprio colocava aquele início de século no abismo de uma loucura que parece mais que uma crise patológica, a lúcida consequência de uma posição coerente: Deus está morto, todos os deuses estão mortos, e também o homem está morto, e a natureza com suas leis legítimas é o mundo conhecido (1997, p.31). Tal tese não parece equivocada e, na realidade, retoma o problema da morte de todos os deuses e de toda a metafísica, fato talvez já ocorrido em Kant. Contudo, o problema é retomado por outro viés interpretativo e, por isso, adquire significado filosófico e relevância ainda nos dias atuais. Considerações finais A dúvida que surge, logo após o final da Segunda Guerra, é, num dado grau, a mesma tanto na Alemanha quanto na Itália: um regime filosófico pode resultar no nazismo ou no fascismo? Nesse sentido, a crítica alemã operada por Adorno e Horkheimer em A dialética do esclarecimento teria estreita relação com a crítica italiana feita a posteriori, ou seja, Giovanni Gentile seria, no 204 Márcio Gimenes contexto italiano, o equivalente daquilo que teria sido Heidegger? Em ambos, pode-se perceber que a tomada de posição política é uma consequência do seu pensamento filosófico. O mesmo também ocorre na posição política de Croce, liberal oposto ao regime e na posição politica socialista de Franco Lombardi. Em outras palavras, a oposição ao regime possui uma raiz muito próxima do mesmo idealismo que ajudou na sustentação do regime. Assim, aquilo que chamamos como filosofia italiana entre os séculos XIX e XX é melhor percebida, talvez, com uma investigação da influência alemã. Um dado curioso é que talvez passe desapercebido é que Croce e Gentile, como bem pontua Verucci (2006) foram condenados ao mesmo tempo pela Igreja Católica, isto é, em 1932. Ambos foram tomados como autores que pouco nacionalistas e que acentuavam demais a chamada filosofia estrangeira isto é, a filosofia idealista alemã. Desse modo, “a filosofia de Croce nascia da ‘fonte teutônica’, isto é, hegeliana, e gerou uma filosofia similar e talvez também, se fosse possível, pior que a sua, isto é, o atualismo” (VERUCCI, 2006. p.145). Desse modo, se os italianos possuem uma dada recepção de Hegel, eles também possuirão uma interpretação muito peculiar dos filósofos da vida, isto é, dos pós-hegelianos que se lançam contra o mestre Hegel. Aqui podemos, incluir autores como Nietzsche, Schopenhauer, Feuerbach, Kierkegaard e inúmeros outros. Para Viano, as críticas de Croce como antiacadêmico podem ser compreendidas dentro desse espírito. D´Annunzio, precursor de uma dada interpretação nietzschiana na Itália, estaria na mesma linha, com um pouco menos de ênfase. Assim, Viano ressalta aqui um isolamento da filosofia italiana, que se perdia internamente num debate entre idealistas e positivistas (VIANO, 2006), crítica igualmente realizada por Garin (GARIN, 1997). Passadas as guerras e querelas mais marcantes, talvez seja essa a época de uma observação menos contaminada e mais ampla do que foi – e do segue sendo – a filosofia italiana e de qual seria a sua relavância no contexto atual dos estudos filosóficos. Considerações finais Curiosamente, talvez Adorno e Horkheimer, de algum modo, espelham um pouco da crítica do idealismo italiano e da sua crítica ao cristianismo: “A velhinha italiana que, em sua piedosa simplicidade, consagra uma vela a San Gennaro em favor do neto que partiu para a guerra, pode estar mais próxima da verdade do que os papas e bispos que, imunes à idolatria, abençoam as armas contra as quais San Gennaro é impotente. Mas, para a simplicidade, a própria religião torna-se um sucedâneo da religião” (ADORNO e HORKHEIMER, 1985, p.167). Logo, talvez existam mais pontes entre a Itália e a Alemanha do que se possa imaginar num primeiro exame. Referências Referências 205 Dissertatio [45] 184-2082017 ADORNO, T. W. e HORKHEIMER, M. Dialética do Esclarecimento. Rio de Janeiro: Jorge Zahar Editor, 1985. ALESSI, A. L´ateismo di Feuerbach. Roma: LAS, 1975. BASSO, I. “Italy: From a Literary curiosity to a Philosophical Comprehension”. In: Kierkegaard´s International Reception, v. 8, Tome II, Southern, Central and Eastern Europe, edited by Jon Stewart, Farnham, Ashgate, 2009 BOBBIO, N. Profilo ideológico del novecento italiano. Torino: Einaudi, 1986. CARACCIOLO, A. “Dio e spazio religioso in Feuerbach”. In: AA.VV., Dialettica e Religione, vol. I, Benucci, Perugia, 1978, p. 203-269. BUDDENSIEG, T. L´Italia di Nietzsche. Milano: Libri Scheiwiller, 2006. CASINI, L. Feuerbach postumo. Il panteismo della Lezioni di Erlangen. Firenze: Sansoni, 1979. ______. Storia e umanesimo in Feuerbach. Bologna: Il Mulino, 1974. ______. Cesa, C. Il giovane Feuerbach. Bari: Laterza, 1963. ______. Introduzione a Feuerbach. Roma: Laterza, 2003. CROCE, B. Storia d´Europa nel secolo decimono. Roma: Laterza, 1965. ______. Storia d´Italia dal 1871 al 1915. Roma: Laterza, 1977. ______. Teoria e storia della storiografia. Milano: Adelphi, 2001. D´ANNUNZIO, G. Su Nietzsche. Catania: De Martinis & C. Editori, 1994. D´IORIO. Nietzsche na Itália – a viagem que mudou os rumos da filosofia. Rio de Janeiro: Zahar, 2014. ESPOSITO, R. Pensamento vivo – origem e atualidade da filosofia italiana. Belo Horizonte: Editora UFMG, 2013. GALASSO, G. Croce e lo spirito del suo tempo. Roma: Laterza, 2002. GARIN, E. Cronache di filosofia italiana 1900-1960 v.1. Roma: Laterza, 1997. ______. Storia della filosofia italiana – volume terzo. Torino: Einaudi, 1966. GENTILE, G. Giordano Bruno e il pensiero del Rinascimento. Firenze: Vallecchi Editore, 1920. ______. La religione. Firenze: Sansoni, 1965. GRAMSCI, A. Gli intellettuali e l´organizzazione della cultura. Torino: Einaudi, 1949a. 206 Márcio Gimenes ______. Il materialismo storico e la filosofia di Benedetto Croce. Torino: Einaudi, 1949b. LOMBARDI, F. Feuerbach. Firenze: La Nuova Italia, 1935. ______. La filosofia italiana negli ultimi cento anni. Asti: Casa Editrice Arethusa, s/data. ______. Kierkegaard. Firenze: La Nuova Italia, 1936. ____. Kierkegaard. Firenze: La Nuova Italia, 1936. LOSURDO, Domenico. Dai Fratelli Spaventa a Gramsci – per una storia politico- sociale dela fortuna di Hegel in Italia. Napoli: La Città del Sole, 1987. LÖWITH, K. La mia vita in Germania: prima e dopo il 1933. Milano: Il Saggiatore, 1988. MARTON, S. (org.). Nietzsche pensador mediterrâneo: a recepção italiana. São Paulo: Discurso, 2007. MICHELINI, G. Nietzsche nell´Italia di D´Annunzio. Palermo: S. F. Flaccovio Editore, 1978. MONDOLFO, R. La Filosofia política in Italia nel secolo XIX (conferenze sulla Storia d´Italia del secolo XIX svolte nell´anno accademico 1922-1923. Padova: La Litotipo Editrice Universitaria, 1924. ______. Sulle orme di Marx. Bologna: Cappelli, 1923-1924. PAPINI, G. “Francesco Orestano – le idee fondamentali di Fed. Niezsche nel loro svolgimento”. In: La Critica. Rivista di Letteratura, Storia e Filosofia diretta da Benedetto Croce, 2, 1904, p. 63-66. PAREYSON, L. “Due possibilità: Kierkegaard e Feuerbach”. In: Esistenza e persona, Taylor, Torino, 1950. ______. Studi sull´esistenzialismo. Firenze: Sansoni, 1971. PENZO, G. Invito al pensiero di Nietzsche. Milano: Mursia, 1990. PERONE, U. Teologia ed esperienza religiosa in Feuerbach. Milano: Mursia, 1972. PICCIONI, L. Ideologia e filosofia del neoidealismo italiano. Urbino: Università degli Studi di Urbino, 1983. PIZZUTI, G.M. Invito al pensiero di Kierkegaard. Milano: Mursia, 1995. RAMBALDI, E. Feuerbach 1842 ‒ Necessità di un cambiamento. Firenze: Sansoni, 1970. ______. La critica antispeculativa di L. Feuerbach. Firenze: La Nuova Italia, 1966a. 207 Dissertatio [45] 184-2082017 ______. Le origini della sinistra hegeliana. Firenze: La Nuova Italia, 1966b. ROSSI, P. Un breve viaggio e altre storie – le guerre , gli uomini, la memoria. Milano: Raffaello Cortina Editore, 2012. ______. Le origini della sinistra hegeliana. Firenze: La Nuova Italia, 1966b. ROSSI, P. Un breve viaggio e altre storie – le guerre , gli uomini, la memoria. Milano: Raffaello Cortina Editore, 2012. SEVERINO, G. Origine e figure del processo teogonico in Feuerbach. Milano: Mursia, 1972. TOMASONI, F. Feuerbach e la dialettica dell´essere. Firenze: La Nuova Italia, 1982. ______. Ludwig Feuerbach – Biografia intellettuale. Brescia: Morcelliana, 2011. VATTIMO, G. La fine della modernità. Milano: Garzanti, 2014. VERUCCI, G. Idealisti all´Indice – Croce, Gentile e la condanna del´Sant´Uffizio. Roma: Laterza, 2006. VIANO, C. . La filosofia italiana del Novecento. Bologna: Il Mulino, 2006. VIROLI, M. Come se Dio ci fosse – Religione e libertà nella storia d´Italia. Torino: Einaudi, 2009. E-mail: marciogimenes@unb.br E-mail: marciogimenes@unb.br Recebido: 31/05/2016 Aprovado: 04/04/2017 208
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BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG | Digol, Debate in Russia on Conflict in Georgia NATO-Mitglieder bzw. ihrer Gesellschaften vor politischem Zwang zum Kernbereich des Artikels 5 gehöre. Daher führe der Kampf gegen eine terroristische Bedrohung die Allianz – wenn auch unter veränderten Vorzeichen – wieder an ihren Beginn zurück. Eine solche strategische Besinnung sei nicht nur unter sicherheits­politischen Aspekten dringend geboten, sondern ge­ währleiste auch die notwendige Legi­timation der NATO in den Mitgliedstaaten, die durch die Vielzahl von Auslands­einsätzen in den vergangenen Jahren nicht (mehr) hergestellt werden konnte. Unter dem Über­begriff »Transatlantic Homeland Defense« plädieren diese Beobachter daher für eine multidimensio­ nale Strategie der Allianz, die Elemente diplomatischer, finan­ zieller, wirtschaftlicher, geheimdienstlicher und polizeilicher Zusammenarbeit umfassen soll. Konkret soll die Zivilverteidi­ gung in die Allianzdoktrin mit dem Ziel auf­genommen wer­ den, das Territorium der NATO vor terroristischen Angriffen zu schützen.25 Ob diese Vorschläge jedoch geeignet sind, die skizzierten strukturellen Beschränkungen zu überwinden, darf bezweifelt werden. 25 Vgl. Amas, Neyla u.a., Transatlantic Homeland Defense, Center for Techno­ logy and National Security Policy – National Defense University (Mai 2006), Special Report (<www.ndu.edu/inss/press/CTNSP-INSS/spl-rpt.pdf>); sowie Hamilton, Daniel S., NATO Summit I: In Area, or in Trouble, in: International Herald Tribune, 27.11.2006. Am stärksten wird diese Position von dem ehema­ ligen spanischen Ministerpräsidenten Aznar vertreten. Vgl. Aznar, Jose-Maria, NATO – An Alliance for Freedom, in: The RUSI Journal, 151 (2006) 4, S. 38-40. Right or wrong: Debate in Russia on Conflict in Georgia Diana Digol* Abstract: The article presents a comprehensive overview of opinions in Russia on the August 2008 military conflict with Georgia. The positions presented include not only those of the political elite and political opinion-makers but also those of the cultural and social elite and of representatives of various ethnic groups involved in the conflict. The overview concludes by presenting results of public opinion polls. The period covered is between the commencement of hostilities on 8 August 2008 and the end of that year. The article is limited to Russia and does not cover the debates of neighbouring countries. Keyworlds: Russia, Georgia, conflict, opinion Russland, Georgien, Konflikt, Meinung 1. In���������� troduction F rom the start of the conflict between Georgia and Russia the leading Western news agencies dedicated several re­ ports daily to this conflict. The desire to understand the events was overwhelming. Experts on Russia and the Caucasus were in high demand by local and national news agencies. Ini­ tial assessments rapidly found their way into major European and American news channels and newspapers. The majority of the media blamed Russia for the developments and were look­ ing for details. What was happening in Russia? How were these events shown, interpreted, received and discussed within Russia, by Russians, Georgians, Ossetians and others? From the outset of the con­ flict, an increasingly intense and comprehensive debate took place on TV, radio and in print media. The mass media provid­ ed a platform to numerous persons of various political inclina­ tions as well as apolitical individuals, intellectuals, academics and artist. This article presents a comprehensive overview of the opinions in Russia on the August 2008 military conflict between Georgia and Russia. The premise is that analysing opinions of a variety of people would help to create a picture of these events from a Russian point of view. Telling this story, I speak with the voices of the people allowing myself to use summaries of their argu­ 112 | S+F (27 . Jg.) 2/2009 ments. I conclude the overview by presenting the results of public opinion polls. The opinions of the two highest ranking leaders of Russia, Dmitry Medvedev and Vladimir Putin are presented to deline­ ate the official position of the political elite. The opinion of the official opposition (parties, other than the ruling one, rep­ resented in the parliament) is presented through the voices of Gennady Zyuganov, leader of the Communist Party of the Rus­ sian Federation (KPRF), Vladimir Zhirinovsky, the leader of the Liberal Democratic Party of Russia (LDPR) and Sergei Mironov, a leader of the party “A Just Russia”. The position of other par­ ties (unofficial opposition) is presented through the voices of Mikhail Kasyanov, leader of People’s Democratic Union (PDU), Sergei Mitrokhin, a leader of the “Yabloko” party and Garry Kasparov, a leader of “United Civic Front”. The views of Natalia Narochnitskaya, from the Institute for Democracy and Cooperation, Stanislav Belkovskii, Director of the Institute for National Strategy and Alexandr Konovalov, President of the Institute of Strategic Evaluation are presented to delineate the position of Russian political scientists and ana­ lysts. The views of Russian intellectuals are presented by the journalist Vladimir Pozner and the writer Boris Strugatskii. In * Dr. Diana Digol is researcher at the Center for OSCE Research (CORE) at the Institute for Peace Research and Security Policy (IFSH), Germany.  In the current State Duma (October 2008), only these three parties, in addi­ tion to the governing party “United Russia”, are represented. https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Digol, Debate in Russia on Conflict in Georgia addition, the reactions of a group of intellectuals from St. Pe­ tersburg and of the Imperial House of Russia are presented. The Georgian elite is represented by the TV star Tina Kandelaki, the journalist Otar Kushanashvili, the singer Vakhtang Kikabidze, Nikolai Tsiskaridze, a principal dancer of the Bolshoi Ballet, and a number of others. The position of the Ossetian elite is represented by opera director Valery Gergiev and the actors Bulat Budaev and Soslan Fidarov. To conclude, the opinion of the general public is summarised, based on results of the two prominent opinion polls in Russia: the public opinion research centre VCIOM and the non-governmental organization “Le­ vada Analytical Centre” (“Levada-Center”). This article is different from other articles on similar topics in presenting not only the position of the political elite and political opinion-makers, but also that of members of the cul­ tural and social elite as well as representatives of various ethnic groups involved in the conflict. These views are then contrasted with results of the public opinion polls. The article is limited to the period between the 8 of August 2008 and the end of December 2008, when research for the article was finalised. It is confined to Russia and does not include debates of neighbour­ ing countries. 2. Political elite 2.1 Official position The official position of Russia has been framed and expressed by Medvedev and Putin in political addresses, speeches and interviews (Medvedev, 2008a, 2008b, 2008c, 2008d, 2008e, 2008f, 2008g, 2008h), (Putin, 2008a, 2008b, 2008c, 2008d). It has been broadcasted on national TV and published in na­ tional newspapers. Hence, the official position is that Georgia attacked not only the small territory of South Ossetia, but also the lightly armed peacekeepers, causing deaths mainly among Russian peacekeepers. This fact served as additional evidence for the official position that Georgia planned in advance and prepared an attack on South Ossetia and was, potentially, plann­ ing a similar one on Abkhazia. As a response, Russia was forced to intervene with the goal of preventing a humanitarian cri­ sis and a new genocide. It was argued that this decision was deeply rooted in the long term historical ties existing between Russia and the people of South Ossetia and the geographical proximity of Russia to South Ossetia, making Russia the only possible state able to intervene in a timely fashion. Thus, Russia had no choice but to intervene promptly to protect the civil­ ian population; in this respect a parallel has been drawn to the massacres in Srebrenica in July 1995, when Dutch peacekeepers preferred to retreat rather than intervene, which literally led to the deadly consequences (Putin, 2008c). It has been further argued that Russia was against any new form of confrontation and wished to avoid any new edition of the “Cold War” and global isolation of Russia. Russia expected to  Arguably, Georgian peacekeepers were informed about the imminent attacks and left their positions beforehand (“Peace enforcement: war chronicle,” 2008). | BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG find understanding in the West (Medvedev, 2008h), (Putin, 2008b). As far as the economic consequences are concerned, Russia is aware that the West can try to use leverage against Russia in the economic sphere, in particular with respect to its attempts to become a WTO member. However, it was stressed again and again that when a decision had to be taken to intervene and protect the people of South Ossetia economic considerations were of secondary importance (Medvedev, 2008g), (Putin, 2008a). Regarding the recognition of independence of South Osse­ tia and Abkhazia and related accusations of Russia applying double standards in the case of South Ossetia versus the case of Chechnya, Medvedev and Putin, drew a parallel between South Ossetia and Kosovo rather than between South Ossetia and Chechnya. The argument is that as much as Kosovo was a casus sui generis, South Ossetia and Abkhazia were also such cases (Medvedev, 2008d), (Putin, 2008c). Moreover this recog­ nition was the implementation of the UN principle of the right of self-determination of nations. The Georgian attack is believed by Russian officials to have been staged by Americans and with Americans; their physical pres­ ence in the midst of military action has been suspected and is being investigated. In the words of Medvedev, the Americans must be feeling sad that their “virtual project ’Free Georgia’ had failed” (Medvedev, 2008h). A possible link between the elec­ toral campaign in the USA and the need to support one of the presidential candidates was also mentioned (Putin, 2008c). 2.2 Official opposition The position of the official opposition is presented through the party positions of the only three opposition parties in the cur­ rent State Duma: the Communist Party of the Russian Federa­ tion (KPRF), the Liberal Democratic Party of Russia (LDPR) and “A Just Russia” Party. The position of the Communist Party of the Russian Federation (KPRF) was in line with the official position. Its leader Gennady Zyuganov argued that Europe should have been grateful to Rus­ sia for stopping a big Caucasian war. There was no doubt, he continued, that Georgia initiated military actions by assaulting and killing the Russian peacekeepers (Zyuganov, 2008b). It was Russia’s military duty to get involved in the conflict (Zyuga­ nov, 2008a). The KPRF organised a protest meeting in front of the Georgian Embassy, waving banners with slogans such as: “Freedom to South Ossetia”, “Saakashvili – to the Hague”, etc. (Vladov, 2008). Moreover, the KRPF authored an appeal to the Russian government asking them to stop the colonial regime of Saakashvili and to immediately recognise the independ­ ence of South Ossetia and Abkhazia (Zyuganov, 2008a, 2008b, 2008c).  One might recall, however, that in the case of Serbia-Kosovo, Russia was sup­ porting Serbia’s position arguing for the precedence of territorial integrity over the right of self determination. The change of the position is explained in the interview of Putin to the German channel ARD (Putin, 2008c). https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. S+F (27. Jg.) 2/2009 | 113 BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG | Digol, Debate in Russia on Conflict in Georgia The position of the Liberal Democratic Party of Russia (LDPR) was also in line with the official position, but was more radical and far-reaching in its interpretations and suggestions (Zhiri­ novsky, 2008b). Thus, the party’s leader, Vladimir Zhirinovsky, argued that a pre-emptive attack of Russia would have been a much better scenario (Zhirinovsky, 2008c). He stated that in such a case, Tskhinvali and the lives of thousands of Ossetians would have been saved. The recognition of South Ossetia was justified: never again would Ossetians be living together with Georgians (Zhirinovsky, 2008a). The party “A Just Russia” was equally united in its opinion and supportive of official Russia’s position. In the opinion of its leader, Sergei Mironov, Russia had no other choice but to inter­ vene since Georgia was systematically physically eliminating the local Ossetian and Abkhazian population. He described the actions of Saakashvili as “a strategic mistake”, further adding that “He showed the world his intentions towards his people. He is killing them” (Mironov, 2008a). Mironov believes that with his actions “Saakashvili has signed a death sentence for himself” politically (Mironov, 2008b). The party views the worsening of the relations between Russia and the EU as tem­ porary and foresees no serious economic consequences for Rus­ sia (Ponomarev, 2008). 2.3 Non-official opposition By contrast to the official positions, the former prime minister of Russia, and currently a leader of the opposition party, The People’s Democratic Union (PDU), Mikhail Kasyanov said that Russia had continuously and incessantly provoked Georgia. It was regrettable that Georgia attacked militarily; however, Rus­ sia should have stayed within its peacekeepers’ mandate. In­ stead it launched a full-scale war. He thoroughly criticised the actions of Russia’s government and argued that these actions were unprofessional and disproportionate. By these actions, he continued, Russia turned itself from a peacekeeper and media­ tor in the region into a party to the conflict, which might lead to international isolation. He was one of the first to suggest that, as a consequence, Russia might be deprived of hosting the Olympic Games in 2014 (Stack, 2008). The “Yabloko” party expressed concerns about the situation in South Ossetia. Its leader, Sergey Mitrokhin, expressed his wish that Russia should not turn from a “saviour of lives in South Ossetia” into an aggressor and should stop the war as soon as possible (Mitrokhin, 2008b). At the same time, he appealed to the NATO countries to take the issue of Georgia’s membership in NATO off its agenda (Mitrokhin, 2008a). The Russian chess grandmaster, former World Chess Champi­ on and nowadays writer and political activist as well as leader of the political movement “United Civic Front” (UCF), Garry Kasparov, partially echoed the official position. In particular, he did not doubt that it was Saakhasvili who had provoked the war (Kasparov, 2008c). However, he maintained that the postwar developments were against the interests of Russia and its citizens, that they contradict the international treaties Russia has signed and ratified and conflict with the norms of inter­ national cooperation (Kasparov, 2008a). Moreover, he blamed 114 | S+F (27 . Jg.) 2/2009 Russia for acting against the agreed-upon Sarkozy-Medvedev plan for the peaceful regulation of the conflict in South Ossetia and Abkhazia (Kasparov, 2008a). As a consequence, he envi­ sioned a growing instability in the Caucasus and the prolifera­ tion of separatist movements, the complete political isolation of Russia, the acceleration of negative tendencies in the Russian economy and the weakening of its international position. By recognising South Ossetia and Abkhazia, Russia has prepared a long-term “headache” for itself (Kasparov, 2008b). In short, he viewed this conflict and Russia’s response as a military political adventure with catastrophic political consequences. 3. Social and cultural elite A variety of divergent opinions of Russia’s intellectuals, includ­ ing those of ethnic Georgians and Ossetians are presented be­ low. 3.1 Political analysts Natalia Narochnitskaya, head of the Paris office of the Institute for Democracy and Cooperation, a Russian non-profit founda­ tion, defined the operation in South Ossetia as Russia’s victory. For the first time in the last 15 years Russia acted as suggested by its “national pride, historical obligations and own interests” (Ovchinnikov, 2008). Russia demonstrated that the opinion of its own people was more important than the opinion of the West. According to her, the whole Western rhetoric about the Georgia-Ossetian conflict was actually not because of Georgia, but because of the new rising power and will of Russia. She was sure that if Georgia had been located somewhere else geograph­ ically, for example in Siberia, no one would have cared about it. She argued that the “anti-Russian policy of Georgia is necessary for the West” (Ovchinnikov, 2008). Georgia was supposed to serve the purposes of “squeezing Russia out” as a main military player in the Black Sea area (Ovchinnikov, 2008). Stanislav Belkovskii, director of the Institute for National Strat­ egy, argued that Saakashvili had achieved two of his three goals. First, the total destruction of the infrastructure of South Ossetia and its capital, Tskhinvali, was achieved as well as “cleansing” the territory of Ossetians by making them flee. Second, Russia was deprived of the exclusive status of a peacekeeper in the re­ gion, which was his long-term goal. He failed only in his third goal, which was to take full control of the territory of South Ossetia. Belkovskii further argued that accepting the SarkozyMedvedev plan would mean a full defeat for Russia. Recognis­ ing South Ossetia and Abkhazia was the only winning strategy which would allow Russia to legitimise its economic and mili­ tary presence in the region. What seemed probable in the im­ mediate aftermath of the crisis – depriving Russia of the right to hold the Olympic Games in Sochi – is no longer relevant. The reasoning is that the Olympic Games are at first instance a commercial project and involve many partners, including foreign ones. Alexandr Konovalov, president of the Institute of Strategic Evaluation, considered that the events might have sped up the accession of Georgia into NATO, but without the two regions https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Digol, Debate in Russia on Conflict in Georgia of South Ossetia and Abkhazia (Mekhtiev, 2008). He doubted that a change of leadership in Georgia was one of Russia’s aims in this conflict and argued that in Georgia “there is no strong political leader who is also pro-Russian”(Mekhtiev, 2008). Moreover, the conflict influenced the whole of Georgian antiRussian sentiments. The main outcome for Russia, according to him, is that the USA and Western Europe understood that they “should take into account the position of Russia, in particular in matters related to the Caucasus” (Mekhtiev, 2008). He also said that Russia stopped at the right moment, not crossing the line after which a new “Cold War” would have been unavoid­ able (Mekhtiev, 2008). 3.2 Russian intellectuals Vladimir Posner, a Russian journalist and president of the Russian Academy of TV (until October 2008), might be known to a Western reader from the first talk shows and TV bridges between the Soviet Union and USA, which he initiated in the 1980s. He was the one who suggested in 2007 TV-bridges be­ tween Russia and Georgia to talk about the conflict among all parties and look together for ways to solve it. According to him, “every person who sees a bit further than his own nose should have understood that after the recognition of Kosovo, Russia would, inevitably – with 98 per cent probability – recognise South Ossetia and Abkhazia” (Posner, 2008). According to Pos­ ner, Putin, at that time still president, was talking about it very clearly. To a Russian, the situation in Kosovo is not different from the one in Abkhazia and South Ossetia. It was clear that if Georgia would not change its policy and attitude toward these entities (South Ossetia and Abkhazia), then this would be an outcome. He continued by arguing that it is difficult now to im­ agine that South Ossetia and Abkhazia would ever again wish to become a part of Georgia. The Georgians have humiliated and mistreated Abkhazians since Soviet times. This attitude was exacerbated during the rule of former Georgian president Zviad Gamsakhurdia and has continued during the rule of Saakash­ vili. Although not easy, recognition of these entities was the right thing to do from the point of view of Russian state interest (Posner, 2008). With respect to consequences for Russia, these events indeed placed Russia in a position of international isolation. There is, however, mutual interdependence between Russia and the West, thus Posner believed that Russia would not be excluded from the “G8”, no new war was imminent and no return to the Iron Curtain was possible. However, the image of Russia was certainly damaged. Posner suspected American involvement as well; he made particular reference to partisan coverage of the crisis on American TV by which he felt deeply disgusted (Posner, 2008). A famous Soviet and Russian science fiction writer, Boris Stru­ gatskii mentioned philosophically that he “cannot think of any politician, any political power which is not guilty either through its action or through its inaction” (Strugatskii, 2008). This might have created the impression that he approved of the  He had his own political TV-show “Vremena” which, once a week, on Sunday evening, replaced the official evening news. | BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG actions taken by Russia. However, he continued by describing Russia as a totalitarian regime which thrives on short victorious wars. He further stated that the Caucasus has been balancing between peace and war since the collapse of the USSR. One of the reasons is that Russia and Russian elites will never agree to let Georgia out of its sphere of interest. In the dilemma between the “right to self determination” and “territorial integrity”, “power” wins. And only in those cases where “power” is not interested, can the UN be useful somehow (Strugatskii, 2008). Strugatskii compared South Ossetia to Chechnya, and argued that Georgia did not do in South Ossetia anything Russia had not done in Chechnya, even using the same justification of “bringing constitutional order to separatist regions” (Vish­ nevskyi, 2008a). He further blamed Russia for a lack of consist­ ency in its positions. He argued Russia had negatively assessed the actions of the USA in Serbia against the Milosevic regime, but then, in his view, took similar actions in Georgia (Vish­ nevskyi, 2008a). A group of intellectuals from St. Petersburg, including poets, writers, artists, professors, political scientists, and sociologists, wrote an open letter to their colleagues in Georgia. This letter addressed to the Shota Rustaveli Institute of Georgian Litera­ ture in Tbilisi was in essence dedicated to all Georgian intel­ lectuals. The letter containing the words “this is not our war and [these are] not our tanks” (Vishnevskyi, 2008b) and stated that the politicians of two or maybe three states put cultural and human relations between Russia and Georgia at risk. It also recalled that WWII influenced the attitude of Russians towards the Germans. Thus, the authors of the letter were concerned about preventing a similar change in attitude between Rus­ sians and Georgians. The answer from the Georgian side, little expected from the Russian side mainly because of the broken lines of communications, arrived promptly. The letter was full of gratitude for a public expression of opinion and hope that the links between intellectuals would remain untouched and unchanged (Petlyanova, 2008). This was one of the first signs that human relations might be above the politics. The Imperial House of Russia echoed the official position of Russian government. The Head of the House of Romanoff, H.I.H. Grand Duchess Maria Vladimirovna is convinced that “the present conflict in South Ossetia has its origins abroad.” She expressed her profound anguish and “is troubled by the ac­ tions of political leaders, who are only fulfilling the will of for­ eign masters and, because of this, have turned the territory of their own country into a bombing range for foreign weapons, and who have sacrificed their own people for foreign interests” (Imperial House of Russia, 2008). 3.3 Famous Georgians who made a career in the USSR or in Russia This section features various famous persons of Georgian ori­ gin (not necessarily with Georgian citizenship) who are or were active in Russian intellectual or cultural life. Thus, Tina Kan­ delaki, TV star and one of the top television presenters, nick­ named in the Russian press the “Main Georgian of Moscow” (Antonova, 2008), argued in her blog that the Georgian people https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. S+F (27. Jg.) 2/2009 | 115 BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG | Digol, Debate in Russia on Conflict in Georgia were against the Saakashvili regime and were against the war (Kandelaki, 2008). She described Saakashvili as a person with weak nerves but high ambitions and narcissism who compares himself to David the Builder. She argued, “Saakashvili is bound to go down into history of Georgia as Mikheil the Destroyer” (Walker, 2008). She also suspected American involvement in this conflict and suggested that the main interest of Americans in Georgia were not people but strategy: “We are only 40 min­ utes away from Iran” (Walker, 2008). She blamed Saakashvili, however, she believed that Russian-Georgian relations would shortly resume on a normal path again since their cultures have been intertwined for centuries. Nikolai Svanidze, a famous TV journalist, working at the progovernment channel “Russia”, author of Medvedev’s biogra­ phy and creator of the “Historical Chronicles” was one of the most outspoken persons during the days of conflict and im­ mediately afterwards (Svanidze, 2008). He argued that Russia would have scored higher politically if it had not crossed the border between South Ossetia into Georgia. Additionally, he saw the recognition of South Ossetia and Abkhazia in the af­ termath of the military actions as inevitable (Svanidze, 2008). He further argued that these events showed that Russia is not surrounded by a circle of friends (referring to the nearby states), and there are few friends further away (referring to the rest of the world). He is concerned with an intensification of Russia’s isolation. In consequence, he sees that it would be a difficult task to increase Russia’s influence on the international scene (Svanidze, 2008). Otar Kushanashvili, a charismatic journalist and TV presenter, openly named Saakashvili a “recruitee” of the USA and believed that he should be judged by an international criminal tribunal for these events (Russia.Ru, 2008). He further argued that no single life is worth to be lost to enhance the self-esteem of a politician and called for politicians on both sides to overcome their ambitions and start negotiations immediately (Baranets, 2008). Kushanashvili stated that Georgia had no basis for its souvereignty. In his opinion, the only real partner of Georgia is Russia. Thus, Georgia should search for ways to restore its friendship with Russia as soon as possible. As for Saakashvili, the only good move he could still make for Georgia was to vol­ untarily give up his post and ask the Georgians for forgiveness (Russia.Ru, 2008). Vakhtang Kikabidze, a Georgian and Soviet singer, screenwriter, producer, composer and actor who had played in an iconic So­ viet movie “Mimino”, was truly exceptional in his scale of criti­ cism of Russia. He accused Russia of staging a long-planned military and political attack and then occupying Georgia, as­ sessing it as act against Saakashvili and his rule. As a sign of protest against Russian aggression in Georgia he cancelled a scheduled concert in September in St. Petersburg and refused an Order of Honour decoration, offered by the President of Russia, to honour his 70th birthday (Pleshakova, 2008). The translation of his critical interview in the Georgian newspaper “Kvela Siakhle” was widely discussed in Russian mass media. His words concerning the actions Georgia should take against Russia were initially translated from Georgian into Russian, as Georgia “should bring this country [Russia] to an end” or “should put an end to this country [Russia]” (Vorsobin, 2008). 116 | S+F (27 . Jg.) 2/2009 After a wave of disapproval in the Russian mass media of a vari­ ety of persuasions and public calls to ban him indefinitely from entering Russia, he offered his interpretation of his words with the new version sounding like Georgia “should distance itself from Russia” (Sapozhnikova, 2008). He gave further assurances that Georgians are still treating Russians as brothers. Russia is good, its policy is bad would seem a perfect short version of his opinion. Another stand was taken by the principal dancer of the Bolshoi theatre, Nikolai Tsiskaridze, who is Russia’s People’s artist. He was born in Tbilisi and holds Georgian and Russian artistic ti­ tles. He mentioned that while he is, indeed, of Georgian origin, he did not feel any different from those who were bombed and killed in South Ossetia. He continued by suggesting that artists should stay out of politics, and he as artist and Russian artist of Georgian origin would like to do so. For the same reason he disapproved the position and involvement of Kikabidze (Tsiska­ ridze, 2008). The Russian actor and citizen with a Georgian family name, Oleg Bashilashvili, born in 1942, is personally attached to Geor­ gia, including South Ossetia, because this is the place where he spent the early years of his life during the WWII evacuation. He abstained from giving his opinion on the events because he doubted the objectivity of the coverage by Russian media. Nevertheless, he pointed to two issues. First, that Abkhazia and South Ossetia are de iure Georgian territory. Until the interna­ tional community accepts South Ossetia and Abkhazia as inde­ pendent states they should be considered as a part of Georgia. Thus, he continued, “the entrance of our [Russia’s] military is an occupation”(Basilashvili, 2008). Second, he disapproved of Russia’s policy of giving Russian citizenship to the applicants from South Ossetia and Abkhazia (Basilashvili, 2008). Boris Akunin (a pen name of Grigory Chkhartishvili), a phi­ lologist, critic, essayist born in Georgia, was named Russian Writer of the Year in 2000. He defines Russia as not (yet) being a democratic country and strongly disapproves of Putin’s au­ thoritarianism. He argued that, for most Europeans, the Rus­ sian invasion into Georgia beyond the borders of South Ossetia immediately revived the memory of the events in Budapest and Prague several decades ago and this might explain the negative reaction of a number of European countries. With respect to the interstate developments between Russia and Georgia, he believed that relations between Russia and Georgia might only get worse unless the leadership in one country or the other were to change. Further, he believed that in the aftermath of the conflict the efforts of intellectuals alone would not suffice to restore and maintain good relations between Russians and Georgians. This is a moment, he argued, for the political elites to assess once again the actions of Russia and to open a public discussion of the political steps taken by both countries. As far as the consequences are concerned, he saw that Russia found itself isolated, was risking being expelled from the G-8, endan­ gered the Sochi Olympic games and dashed hopes of becoming a member of WTO in the near future. Thus, it was “a small mili­  The People’s Artist of the Union of Soviet Socialist Republics / Russia, also sometimes translated as National Artist of the USSR, is an honorary title granted to citizens of the Soviet Union / Russian Federation. https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Digol, Debate in Russia on Conflict in Georgia tary victory followed by a big political defeat” (Akunin, 2008). Even if it were a part of the big American plan, Russia’s inter­ vention did not alter it significantly (Akunin, 2008). Soso Pavliashvili, a famous pop singer, said in interviews that he felt equally hurt by the sufferings of Georgians and Osse­ tians in this senseless war. He views Ossetians as ethnically the closest to the Georgian nation. He has advocated for Russia to remain an important player in the Caucasus. Hee advised his son to study at the Russian military school in Russia and not at the American one in Georgia. He believed that Georgia was not a place for the American military. “If Russia flourishes, Georgia will flourish”, he concluded (Veligzhanina, 2008). Among the opinions and reactions of yet other famous Geor­ gians are that of Georgi Danelia, a Georgian film director, who condemned the military actions and regretted that he had lived to see a day of war between Georgia and Russia (Veligzhanina, 2008). Tamara Gverdtsiteli, a variety singer and People’s art­ ist of Georgia born in Tbilisi, found these events shocking and could not understand how such horror could happen. Stanis­ lav Sadalskii, an actor and citizen of Georgia, argued that he is “sure that in this case it is not Georgia but America fighting against Russia” (Baranets, 2008). The cancelled performance by Nani Bregvadze, a Georgian People’s artist and famous singer of Russian romances, can be interpreted as a protest against Russia. She postponed her jubilee concert, which was to be performed at the Academic Cappella in St. Petersburg on 26 September 2008 (Bezmenova, 2008). The views of some other famous Georgians were difficult to understand and interpret, as for example the opinion of the President of the Russian Acad­ emy of Arts, Zurab Tsereteli, a controversial Russian-Georgian painter, sculptor and architect. Commenting on these events he said: “Sometimes you need to take a break from love, and that’s what’s happening now. But tomorrow the romance will start again, and it will be passionate!” (Walker, 2008) Some re­ fused to comment on the situation, for example, the director of a singer Valerii Meladze said that the artists would feel hurt to see his name next to any mention of war (Guru Ken Show, 2008). 3.4 Famous Ossetians who made a career in the USSR or in Russia Only a few famous Ossetians could be identified, which is no doubt yet more evidence of how small the Ossetian nation is. A world famous opera director, Valery Gergiev, Ossetian by na­ tionality was full of grief, commenting on the events stressing that for such a small nation as Ossetian to lose 1,5-2 thousand people was a big loss (Beroeva, 2008). The “world most famous Ossetian”, as named by the Russian press (Rozhaeva, 2008), deeply regretted the almost complete destruction of Tskhin­ vali, comparing the ruins of this city to the ruins of Stalingrad  Georgi Danelia originated a series of “sad comedies” as he styles them.  He might be known to a Western reader as the creator of the sculpture “Tear of Grief” officially donated by the Russian government as a memorial to those that died in the September 11th terrorist attacks.  Valery Gergiev is a general director and artistic director of the Mariinsky Theatre, principal conductor of the London Symphony Orchestra and the Rotterdam Philharmonic Orchestra and principal guest conductor of the Met­ ropolitan Opera. | BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG destroyed during WWII (Kireeva, 2008). He strongly believed that it was a campaign carefully planed in advance and organ­ ised by Saakashvili. He promised to carry the message about the initiators of this tragedy on every possible occasion to Europe, the USA or elsewhere. In an interview with the BBC Russian Service, he expressed the gratitude to the Russian army cred­ iting their timely action and support with saving the lives of another 2-3 thousand Ossetians (Beroeva, 2008). Gergiev gave a concert on the ruins of Tskhinvali. He was accompanied by two other famous artists, Dmitri Hvorostovsky and Anna Netrebko (Rozhaeva, 2008). An actor, Bulat Budaev, believed that this was not a war between people, but rather a war provoked by Saakashvili: “I do not know why he is so indebted to the USA” (Veligzhanina, 2008). Another actor, Soslan Fidarov, mentioned in grief that this war took away from him his best friend who died in Tskhinvali. These are his people, he continued, and thus he was suffering with them. The irony of the life, as he put it, was that in his last movie about WWII he, an Ossetian, played a heroic Georgian, while in real life a Georgian took away the life of his best friend (Veligzhanina, 2008). 4. Opinion polls In this section I present the results of opinion polls carried out by two of the most prominent institutions in Russia. The polls included here were conducted in the period from August to Oc­ tober 2008 by the governmental public opinion research centre VCIOM and the non-governmental organization “Levada Ana­ lytical Center” (“Levada-Center”). 4.1 Public Opinion Research Center – VCIOM In the public opinion polls conducted by VCIOM (WCIOM, 2008a, 2008b, 2008c, 2008d), the majority of Russians ap­ proved of the policy of the Russian government and fully sup­ ported Russia’s recognition of the independence of South Os­ setia and Abkhazia. More than 70 per cent of the respondents believed that this conflict touched directly on the interests of Russia; more than 80 per cent of the respondents defined it as a full scale military conflict or war. Three out of four respondents (75%) blamed Georgia and the USA for the conflict. Russia’s involvement in the conflict was supported by more than 80 per cent of respondents; more than 70 per cent believed that Russia should keep its peacekeepers in South Ossetia. Those op­ posing the presence of Russian peacekeepers in South Ossetia argued against new casualties among the Russian population and believed that Georgians and Ossetians should resolve the situation by themselves. Those who supported the continua­ tion of Russia’s military presence in the area argued that this was a way to keep peace, protect the South Ossetian civilian population and prevent further attacks, chaos and genocide. Russians believed that only Russia would be able to protect the civilian population of South Ossetia and Abkhazia (WCIOM, 2008b). Every second inhabitant of Russia became less friend­ ly to Georgians believing that there were more divisive than common features between these two nations (51%) (WCIOM, https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. S+F (27. Jg.) 2/2009 | 117 BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG | Digol, Debate in Russia on Conflict in Georgia 2008d). As regards the future of Russia’s relations with Western countries, Russians are against “cold relations and a hot war” with the West (WCIOM, 2008c). Although expecting a pull­ back in the relations with Western countries in the immedi­ ate aftermath of the conflict, Russians saw it as a temporary phenomenon. Every third respondent saw Germany, the UK, Japan and the USA as partners of Russia and considered Russia a European country (WCIOM, 2008a). 4.2 “Levada Analytical Center” Public opinion surveys carried out by the “Levada-Center” clearly showed that an ever-increasing share of respondents (70%-87%) considered that Russia’s leadership did everything possible to avoid an escalation of the conflict and bloodshed (Levada-Center, 2008a, 2008c, 2008e). As seen by the Russian population, the top three reasons for Russia’s military interven­ tion were maintaining peace, protecting the Ossetian popula­ tion and defending Russian peacekeepers. The majority of Rus­ sians took the side of South Ossetia in this conflict and was in favour of governmental humanitarian assistance to this region (81 % of respondents). With respect to military assistance, half of the respondents were in favour while more that one third was clearly against (Levada-Center, 2008b). Notwithstanding this, the overwhelming majority of the popu­ lation (80%) justified the recognition by Russia of South Osse­ tian and Abkhazian independence. Concerning the future sta­ tus of South Ossetia, 34% were in favour of the independence of South Ossetia from Georgia and 46% for a unification of South Ossetia with Russia (Levada-Center, 2008c). Equal numbers of respondents saw it as a timely act (34%) or overdue (30%), only 12% as a premature act or as wrong (12%) (Levada-Center, 2008d). Among those who disapproved of the act of recogni­ tion, 70 per cent believed that it would increase tensions in the Caucasus, speed up the separatist movements inside Russia and possibly lead to conflict with third countries. Furthermore, the majority of Russians believed that Russia should keep its peacekeepers in the region, encourage the return of Georgian refugees into South Ossetia and Abkhazia, protect them, and finance the rebuilding of the urban and rural areas in South Ossetia (Levada-Center, 2008b). While the overwhelming majority of Russians (74%) believe that the Georgian people have fallen hostage to the geopoliti­ cal ambitions of the USA, they disagree (52%) that the Ossetian people have fallen victim to the conflicting ambitions of Russia and Georgia (Levada-Center, 2008c). The roots of the conflict were seen in the US policy of expand­ ing its influence on Russia’s neighbouring countries (49%) and in the discriminatory policy towards Ossetians and Abkhazians by the Georgian government (32%). It was also seen in Saakash­ vili opting for military action to resolve the territorial conflict in order to facilitate the path towards NATO membership and strengthen his own position so he could remain the country’s president (81%) (Levada-Center, 2008a). Russians also believe that Western countries were supporting Georgia in this conflict because they aim at weakening Russia 118 | S+F (27 . Jg.) 2/2009 and decreasing its role in the Caucasus to the minimum (66%). Nevertheless, almost the same number of respondents (60%) argued in favour of an international resolution of the con­ flict with the participation of the UN and EU (Levada-Center, 2008e). There was some uncertainty concerning future relations of Russia with Western countries. Restoring the status quo in re­ lations was expected by 48%, while another 35% believed in a “new edition” of the Cold War and yet other 18% being in doubt. Every fifth Russian feared Russia’s isolation, every fifth believed in the possibility of a third World War. The majority, however, were of the opinion that the tension would ease in the near future (Levada-Center, 2008d). Every fifth person believed that the USA was the winner in this conflict and almost every third person believed that Russia was the winner. One fourth saw no winners at all (Levada-Center, 2008d). As the results of these polls demonstrate, Russians have over­ whelmingly supported the course of action taken by their gov­ ernment, including the recognition of the independence of South Ossetia and Abkhazia. They have also been concerned about the potential consequences of the crisis for Russia. Rus­ sians strongly believe in a third party involvement in the con­ flict. The majority approved of the continuation of Russia’s involvement in the Caucasus region. 5. Conclusion The objective of this article was to present the debate that took place in Russia in the aftermath of the Russian-Georgian military conflict in August 2008. The article comprises diverse opinions of people ranging from official politicians to political analysts to the public responding to opinion polls. Those who approved the course of the Russian government were united on several issues. They believed that Georgia was the initiator of military action and justified Russia’s military intervention to save the lives of South Ossetians. They also jus­ tified the intervention of the Russian army beyond the borders of South Ossetia on military tactical grounds. They further ap­ proved of the recognition by Russia of South Ossetia and Abkhazia in the aftermath of these events, arguing that never again would Ossetians and Abkhazians be willing to live to­ gether with Georgians in the same state. Moreover, they ap­ proved of Russia acting similarly to the way the international community (in particular, the USA and EU) acted in Kosovo. They suspected the involvement of the USA and they agreed that this conflict has damaged the image of Russia abroad. Those who disapproved of Russia’s actions did so based on one or more points. Some suggested that Georgia responded to constant military provocations from the side of Russia. Others accused Russia of double standards comparing this conflict to the situation in Chechnya, or in imitating the USA, compar­ ing this conflict to the situation in Kosovo. Still others blamed Russia for becoming a party to the conflict long ago, in par­ ticular by awarding Russian citizenship to the South Ossetians and Abkhazians. Yet others were concerned with the negative https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Digol, Debate in Russia on Conflict in Georgia consequences for Russia in the aftermath of the events: the in­ stability of Russia’s borders, political and economical isolation, damaged image, etc. In this analysis those who approved of the actions taken by Rus­ sia outnumbered those who disapproved of these actions. In both camps, there were people of Russian and Georgian origins. No Ossetian or Abkhazian was identified who condemned the actions taken by Russia in South Ossetia. Those who approved mainly focused on the steps taken immediately after the start of the military campaign: intervention in South Ossetia and be­ yond its borders, recognition of the statehood of South Ossetia and Abkhazia. Those who disapproved based their arguments on the potential future negative consequences for Russia: yet more casualties among Russians, Russia’s international image, political isolation and economic consequences. It is possible – even probable – that not all the variations of the opinions of diverse actors were presented and certainly not all those actors directly or indirectly involved were given a voice in this article. I believe, nevertheless, that mainstream opinions of various persuasions were presented here and might serve as a point of reference for an informed and interested reader. | BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG Kasparov, G. (2008b, 10 September). Self-sufficiency of econo­ my - is an illusion. Kasparov. Ru. Kasparov, G. (2008c, 11 August). War or “peace enforcement”? Kasparov. Ru. Kireeva, I. (2008, 22 August 2008). Valery Gergiev and the art­ ists of Mariinsky theatre gave a concert in Tskhinvali. Komsomolskaya Pravda. Levada-Center. (2008a). 10.09.2008. Russians on Abkhazia and South Ossetia. Levada-Center. (2008b). 12.08.2008. Russia –Georgia conflict: new data. Levada-Center. (2008c). 21.08.2008. Opinions of Russians on conflict in Georgia: opinion poll on 15-18 August. Levada-Center. (2008d). 22.09.2008. Russians on Abkhazia and South Ossetia Levada-Center. (2008e). 27.08.2008. Inhabitants of largest Russia’s cities on Georgia-Ossetia conflict Medvedev, D. (2008a, 31 August 2008). Concerning the situa­ tion in South Ossetia, Address presented in Kremlin, Moscow, Russia. Medvedev, D. (2008b, 26 August 2008). Interview with Al­ jazeera. Bibliography Akunin, B. (2008). Russia and Georgia: do we have a common future? [Recorded by X. Larina]. On Kulturnyi Shok [Radio Inter­ view]. Russia: Ekho Moskvy. Antonova, I. (2008, 14 August 2008). Tina Kandelaki: I hope and pray that this is the last sacrifice of the Georgian people. Trud. Baranets, V. (2008, 11 August 2008). Saakashvili has palmed off the under-fulfilled plan of an attack. . Komsomolskaya Pravda. Basilashvili, O. (2008). Russia and Georgia: do we have a com­ mon future? [Recorded by X. Larina]. On Kulturnyi Shok [Radio Interview]. Russia: Ekho Moskvy. Beroeva, N. (2008, 21 August 2008). Valery Gergiev: “To shoot with tanks against children is a shameful and criminal act”. Komsomolskaya Pravda. Medvedev, D. (2008c, 26 August 2008). Interview with BBC. Medvedev, D. (2008d, 26 August 2008). Interview with CNN. Medvedev, D. (2008e, 31 August 2008). Interview with Rossiya, Pervyi, NTV. Medvedev, D. (2008f, 26 August 2008). Interview with Russia Today. Medvedev, D. (2008g, 26 August 2008). Interview with TF1 Tel­ evision. Medvedev, D. (2008h, 2 September 2008). Interview with TV channel “Euronews”. Mekhtiev, A. (2008, 13 August 2008). Stopped in time. Strana. Ru. Bezmenova, A. (2008, 29 August 2008). Nani Bregvadze can­ celled the concert in St. Petersburg. Komsomolskaya Pravda. Mironov, S. (2008a). President of Georgia Michael Saakashvili launched a war against its people. Official site of the party „A Just Russia“. Guru Ken Show. (2008). War with Georgia and show business. Retrieved 30 October, 2008, from http://www.guruken.ru/gu­ ruken/Voina_s_Gruziei_i_shou_biznes Mironov, S. (2008b, 6 September). Saakashvili has signed a death sentence for himself. “Expressen”. As cited at the Official site of the party “A Just Russia”. Imperial House of Russia. (2008). The Head of the House of Romanoff laments the events in South Ossetia. Retrieved 3 October, 2008, from http://www.imperialhouse.ru/eng/dynastynews/ news/2008/828.html Mitrokhin, S. (2008a). Stop the war! Official site of the party „Yabloko“ Kandelaki, T. (2008). Official Site. Retrieved 4 October, 2008, from http://kandelaki.com/ Ovchinnikov, A. (2008, 13 October 2008). The West is again stealing our victories. Komsomolskaya Pravda. Kasparov, G. (2008a, 27 August). Military political “adventure”. Kasparov. Ru. Peace enforcement: war chronicle. (2008, 10 August 2008). Komsomolskaya Pravda. Mitrokhin, S. (2008b, 11 August 2008). Use of force should be limited. Official site of the party „Yabloko“ https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. S+F (27. Jg.) 2/2009 | 119 BEITRÄGE AUS SICHERHEITSPOLITIK UND FRIEDENSFORSCHUNG | Digol, Debate in Russia on Conflict in Georgia Petlyanova, N. (2008, 8 -10 September 2008). Public diplomacy. Novaya Gazeta in St. Petersburg. Pleshakova, A. (2008, 13 August 2008). Vakhtang Kikabidze. The war will stop, the pain in the heart will remain. Komsomolskaya Pravda. Ponomarev, I. (2008). There will be no negative economic con­ sequences for Russia in the aftermath of the military conflict in South Ossetia, unlike widely believed. Posner, V. (2008). Osoboie Mnenie [Recorded by S. Buntman]. On Ekho Moskvy [Radio Interview]. Russia: Ekho Moskvy. Putin, V. (2008a, 13 September 2008). Interview with “Le Fi­ garo”. Putin, V. (2008b, 28 August 2008). Interview with Matthew Chance. CNN. Putin, V. (2008c, 28 August 2008). Interview with Thomas Roth. ARD. Putin, V. (2008d, 6 September 2008). Interview. Vesti. TV chan­ nel Rossiya. Rozhaeva, E. (2008, 21 August 2008). Gergiev and “Mariinsky”: “To you - alive and dead. To you - South Ossetia”. Komsomolskaya Pravda. Russia.Ru. (2008). TV anchor and journalist Otar Kushanash­ vili: “Saakashvili has sold Georgia!” Sapozhnikova, G. (2008, 6 September 2008). Vakhtang Kik­ abidze: Today Mimimo would have became an instructor of military helicopter pilots. Komsomolskaya Pravda. Stack, M. K. (2008, 10 September). Russian opposition leader Mikhail Kasyanov calls for foreign policy ‘built on common values’. Los Angeles Times Strugatskii, B. (2008). I have no illusions about “ottepely”. Gazeta.Spb. Svanidze, N. (2008). Osoboie Mnenie [Recorded by A. Vorobiev]. On Ekho Moskvy [Radio Interview]. Russia: Ekho Moskvy. Tsiskaridze, N. (2008, 1 October 2008). I am similar to those citizens that were bombed. Izvestya. Veligzhanina, A. (2008, 14 August 2008). Calling Georgians... Komsomolskaya Pravda. Vishnevskyi, B. (2008a). Boris Strugatskyi. Ahead is a big in­ crease in state power and decisive militarisation. Novaya Gazeta. SPB.ru(3). Vishnevskyi, B. (2008b, 4 - 7 September 2008). This is not our war and these are not our tanks! Novaya Gazeta in St. Petersburg. WCIOM. (2008a). 100 days of Dmitry Medvedev as a president. WCIOM. (2008b, 25 September 2008). Russia’s assistance to South Ossetia and Abkhazia: only as emergency aid, not as a permanent measure. WCIOM Press release. WCIOM. (2008c, 25 September 2008). “Russians are against cold relations and a hot war “. WCIOM Press release. WCIOM. (2008d, 29 September 2008). “Saakashvili’s aggres­ sions ruined the reputation of Georgians in Russia“. WCIOM Press release. Zhirinovsky, V. (2008a, 8 August). Deputy Speaker of the State Duma addressed the issue of recognition of South Ossetia and Abkhazia. Zhirinovsky, V. (2008b, 8 August). Urgent to all mass media! Zhirinovsky, V. (2008c, 7 September). Zhirinovsky suggests bombing Georgia. News-Russia.Info. Zyuganov, G. (2008a, 10 August). Don’t stop half way. Address of the KPRF to the president and prime minister of the Russian federation to immediately recognise South Ossetia and Abk­ hazia, conclude full-fledged agreements with them and take them under [our] protection. Zyuganov, G. (2008b, 8 August). Russia should stop the colonial war of Saakashvili. Statement of the Central Committee of the Communist Party of the Russian Federation. Zyuganov, G. (2008c, 25 August 2008). To recognise independ­ ence! Address to the State Duma. 7ajk[bb[D[k[hiY^[_dkd] C[diY^[dh[Y^jb_Y^[IjWdZWhZi _dZ[hI_Y^[h^[_jifeb_j_a 8[_jh][pkhh[Y^jiijWWjieh_[dj_[hj[d;lWbk_[hkd] ledI_Y^[h^[_ji][i[jp[d >[hWki][][X[dledCWh_ed7bX[hi kdZHkj^M[_dp_[hb (&&/"YW$'+&I$"YW$(/"ÅÐ" ?I8D/-.#)#.)(/#*))-#& ;hiY^[_djYW$@kb_(&&/ 7X m[bY^[c Fkdaj jWd]_[hj Z_[ I_Y^[h^[_jifeb_j_a ]hkdZ# b[][dZ[H[Y^j[5:[hlec:[kjiY^[d?dij_jkj\”hC[diY^[d# h[Y^j[ _d 8[hb_d c_j ^[hWki][][X[d[ 8WdZ ”X[hfh”\j Z_[ I_Y^[h^[_ji][i[jpj[ ^_di_Y^jb_Y^ _^h[h c[diY^[dh[Y^jb_Y^[d IjWdZWhZi$ Vladov, P. (2008, 14 August). Protests in Moscow at the embas­ sies of the USA and Georgia. Pravda. Vorsobin, V. (2008, 27 August 2008). Vakhtang Kikabidze is no longer interested in Larissa Ivanovna. Komsomolskaya Pravda. Walker, S. (2008, 22 September). Tina Kandelaki: From Georgia with loathing. The Independent. 120 | S+F (27 . Jg.) 2/2009 8_jj[X[ij[bb[dI_[_c8kY^^WdZ[beZ[h l[hiWdZaeij[d\h[_kdj[h mmm$decei#i^ef$Z[ https://doi.org/10.5771/0175-274x-2009-2-112 Generiert durch IP '51.159.168.146', am 13.07.2024, 22:51:42. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig.
https://openalex.org/W3022059855
https://scindeks-clanci.ceon.rs/data/pdf/1451-2092/2020/1451-20922001075I.pdf
Kirghiz, Kyrgyz
null
Assessment of negative influence of manganese in welding fumes on welder's health and ways to reduce it
FME Transactions
2,020
cc-by
5,508
1. INTRODUCTION component), and the disperse medium is formed by gas mixture (welding fumes gaseous component). Welding fumes solid-state component is especially hazardous not only due to its toxic level, but also due to microscopic dust particles with low sedimentation rate (no more than 0.08m/s), and are equally distributed in the airspace at the breathing level in a radius of 5 m, inevitably getting into the body of the welder or any human nearby [6, 7]. Welding works are known to be dangerous for human health as far as a certain amount of welding fumes is emitted into the working area alongside with noise and light irradiation. Apart from gases contained in the welding fumes, a great amount of welding dust resulted from oxidation of the evaporated metal as well as shielding gas, alloyed metals and flux reach environ- ment. Chemical composition of the solid part in the welding fumes primarily depends on the type of filling materials. When the welding arc contacts the metal, evaporations emitted into the air above the working area are gradually oxidized producing fine dust [1-3]. Excessive amount of welding fumes metals in a body causes negative health conditions [8, 9]. Penetration of nano- and micro-dust particles of iron, manganese and silicon oxides into a human body triggers natural protective mechanisms, which in turn can not only activate macrophages, but also lead to inflammatory processes and consequently develop a number of diseases [10-12]. In 2013 American Conference of Governmental and Industrial Hygienists (ACGIH) reconsidered its recom- mendations on quantitative composition of welding fumes and suggested reducing limiting concentrations [4]. This might become a regulation, and make welding materials manufactures follow new standards. These changes are caused by the problem of harmful influence of welding fumes on people’s health. In many countries, particularly in the USA, there are lawsuits between welders who suffer from occupational diseases and manufactures of welding materials [5]. Particles up to 10 μm and more are retained on the surface of the bronchial tree mucosa, while small solid particles (up to 5 μm) penetrate much deeper. While passing through the alveoli membranes, small-sized particles can get into blood which is of the utmost danger [13-16]. When passing from one medium to another, heavy metals can change their chemical structure and cause deficiency of essential elements replacing them in metal-containing protein structures. D.A. Chinakhov D.A. Chinakhov Director, Yurga Institute of Technology National Research Tomsk Polytechnic University Russia The article presents the results of a theoretical study (literature review of publications), which allowed to establish the negative impact of welding aerosol (manganese and other elements) on the human body: you can use special welder protection equipment (ventilation and individual welder protection); reduce the quantitative and qualitative content of manganese welding aerosols (welding technology, power sources, modern welding materials); reduce the content of manganese in the human body, removing it with medicines. E.D. Chinakhova Researcher, Medical Faculty, Department of Anatomy, Siberian State Medical University Russia E.D. Chinakhova Researcher, Medical Faculty, Department of Anatomy, Siberian State Medical University Russia Experimental studies have shown that the use of an inverter power source, compared with a diode rectifier, contributes: to ensuring the drop-by-drop transfer of electrode metal to reduce the time of their formation by 46% and the transition by 28%; ensures the transition of alloying elements from welding materials to the weld metal by 6% and reduces its losses from the fusion line by 6% and HAZ by 3%; to reduce the intensity of education (g / min) SA and their components by 23%; to reduce the specific allocation of CA and their components by 23%. K.Yu. Kirichenko Researcher, SEC Nanotechnology FEFU School of Engineering, Far Eastern Federal University Russia E.V. Verkhoturova Researcher, Research Department, Irkutsk National Research Technical University Russia Keywords: welding, welding fumes, welder’s health, welding process optimization, inverter power supplies. © Faculty of Mechanical Engineering, Belgrade. All rights reserved Received: May 2019, Accepted: July 2019 Correspondence to: Dmitry P. Il’yaschenko, Yurga Institute of Technology National Research Tomsk Polytechnic University, Russia, E-mail: mita8@rambler.ru doi doi:10.5937/fmet2001075I D.P. Il’yaschenko Associate Professor, Industrial Technology Department, Yurga Institute of Technology National Research Tomsk Polytechnic University, Russia D.A. Chinakhov Director, Yurga Institute of Technology National Research Tomsk Polytechnic University Russia E.D. Chinakhova Researcher, Medical Faculty, Department of Anatomy, Siberian State Medical University Russia K.Yu. Kirichenko Researcher, SEC Nanotechnology FEFU School of Engineering, Far Eastern Federal University Russia E.V. Verkhoturova Researcher, Research Department, Irkutsk National Research Technical University Russia D.P. Il’yaschenko Assessment of Negative Influence of Manganese in Welding Fumes on Welder’s Health and Ways to Reduce it Associate Professor, Industrial Technology Department, Yurga Institute of Technology National Research Tomsk Polytechnic University, Russia Keywords: welding, welding fumes, welder’s health, welding process optimization, inverter power supplies. Keywords: welding, welding fumes, welder’s health, welding process optimization, inverter power supplies. 1. INTRODUCTION It increases glutomate content in blood and the content of γ- aminobutyric acid which is the main inhibitory mediator of the brain decreases accordingly [49, 50]. In addition, another brake mediator – dopamine falls under the excessive influence of manganese. Accele- rating dopamine oxidation (while reducing its con- centration), manganese stimulates accumulation of a ne- urotoxic compound which destruct a specific type of receptor on the surface of nerve cells [23, 49, 51-53]. Manganese is essential for synthesis and exchange of neurotransmitters [23, 29]: it increases excitability of adrenoreactive systems and sensitivity of chemore- ceptors, enhances protective inhibition in the cortex of the cerebral hemispheres. Manganese is also a cofactor for some enzymes (hydrolases, transferase, lyases, glutamine synthetases, etc.). In oxidation states (II) or (III), manganese enters the active site of a types of superoxide dismutase and catalase, which are the enzymes necessary for neutralization of a active oxygen intermediate. Researches by a number of authors [19, 54] demonstrate that divalent metallic manganese can alter stability of prion proteins suggesting that stabilization of prion protein caused by manganese can play a role in the pathogenesis of prion diseases. Manganese stimulates synthesis of cholesterol, fatty acids, vitamin C and insulin. It is believed [27, 30] that manganese competes with iron when interacting with proteins and enzymes that include iron in the active site (for example, mitochondrial complex I and aconitase) and if in excess it replaces magnesium in cellular enzymatic reactions. Thus, [30, 55, 56], bioconcentrarion of manganese can lead to a wide range of occupational diseases in latent and active forms due to disturbance in oxidation- antioxidant and metabolic processes, development of central nervous system specific disorders and other negative influences on workers' health. Based on a literature review we found out that the following can reduce negative influence of manganese and other elements on human body: There is evidence [15, 19, 31] that nano compounds of elements oxides containing manganese found in welders’ lungs induce development of certain types of fibrosis as well as interstitial inflammation. This explains the potential risk to respiratory health caused by repeated exposure to welding fumes. 1. Using special means of protection of respira- tory system of the welder (ventilation and personal protective equipment). 2. Reducing an amount of manganese and its compounds in welding fumes (welding technology, po- wer sources, modern welding materials). 1. INTRODUCTION They can not only enter metabolic cycles but also accumulate in human the body and hair [17-21]. By origin, welding fumes fall into the category of condensation aerosols. They can be described as a disperse system in which the disperse phase is formed by small solid particles (welding fumes solid-state Some metals [22, 23] released during welding are able to associate with functional groups of proteins (SH, NH-, NH2-, COO-), change a configuration of the enzymes active centers thereby violating protein functions and suppressing proteins activity, which also causes health disorders. © Faculty of Mechanical Engineering, Belgrade. All rights reserved Received: May 2019, Accepted: July 2019 Correspondence to: Dmitry P. Il’yaschenko, Yurga Institute of Technology National Research Tomsk Polytechnic University, Russia, E-mail: mita8@rambler.ru doi doi:10.5937/fmet2001075I Manganese oxides are the most harmful chemicals [23-26] released during welding (40% of the total dust). FME Transactions (2019) 48, 75-81 75 They can cause organic damage of human nervous system, disrupt lungs, liver and blood circulatory system. Silicon compounds inhaled by humans also dis- rupt body function and cause silicosis. Chromium com- pounds, although released in smaller quantities, accu- mulate in the body causing headaches, diseases of dig- estive system and anemia, while titanium oxide can cause lung disease. This fact [37, 41, 47-49] can be explained by a high rate of oxidative metabolism in nervous tissue and less active antioxidant protection than in other tissues. Manganese easily penetrates to neurons through the blood-brain barrier inhibiting activity of mitochondrial enzymes and accumulating in mitochondria. This again increases lipid peroxidation and causes damage to neu- ron membrane due to a number of biochemical processes. An influence of manganese (Mn) [27, 28] is worth special attention since it accounts for a large proportion of the entire toxic content of welding fumes solid component. Manganese is an essential microelement that greatly contributes to normal body functioning. It takes part in regulating various types of metabolism in cells and body´s tissues, blood clotting, growing of bones and connective tissue. It also regulates function- ing of reproductive organs and affects spermatogenesis. It is believed that astrocytes regulate the manganese content of in the brain. Being damaged first, these nervous tissue cells may lose some of their functions, in particular, capture of glutamate (which excites amino acid). 1. INTRODUCTION Manganese in oxidation states (+4, +6, +7) is most dangerous for humans [23] because it causes oxidative stress due to oxidation of dopamine and other catec- holamines. There is evidence that the content of iron ca- rriers in the body such as transferrin and ferritin incre- ases significantly with excessive intake of manganese, while the content of transferrin receptor decreases. This can be a signal of iron deficiency [23, 32-34]. 3. Reducing bioconcentration of manganese in human body by removing it with medicines. The research objective is to assess reduction in qualitative and quantitative content of manganese in welding fumes of manual metal arc welding (MMA) with modern inverter power supplies. The research objective is to assess reduction in qualitative and quantitative content of manganese in welding fumes of manual metal arc welding (MMA) with modern inverter power supplies. Changes in iron metabolism can decrease hemoglobin contents and stimulate irritation of the erythrocyte lineage of the bone marrow, which can lead to some changes in red blood cells (eg, microcytosis) [32, 35, 36]. RESULTS AND DISCUSSION 3. e results are shown in Table 1 and in Figure 1. ure 1. Oscillograms of droplet transfer current and tage (3mm in diameter E19.9NbB20 electrodes):  - d e; b – Inverter type An analysis of the oscillograms in Fig. 1 (Tab ows that the droplet transfer time is reduced an The results are shown in Table 1 and in Figure 1. Figure 2. Images obtained by micro-X-ray analysis with INCA (Oxford Instruments) Figure 3. Micro-X-ray spectral analysis graph: 1 - weld metal; 2 – weld fusion line; 3 – eat affected zone Figure 2. Images obtained by micro-X-ray analysis with INCA (Oxford Instruments) Figure 2. Images obtained by micro-X-ray analysis with INCA (Oxford Instruments) Figure 3. Micro-X-ray spectral analysis graph: 1 - weld metal; 2 – weld fusion line; 3 – eat affected zone An analysis of the data in Table 2 shows that the content of alloying elements increases in all areas of the weld when an inverter is used, which can be explained by fine-droplet transfer (Table 1). by fine droplet transfer (Table 1). Samples of the welding fumes for hygienic assessment of electrodes were taken during bead welding of steel plates with X12CrNiTi18-9 electrodes. Diode and inverter rectifiers were used as welding arc power supplies. The welding was performed with constant reverse polarity current in the modes shown in Table 1. Welding fumes were captured by a special cover isolating the welding zone. At the same time, according to technological guidelines (MU 1927-78 1980; MU 4945-88 1990), welding fumes were deposi- ted on filters made from FPP-15-1.5 tissue to evaluate the level of fuming and on AFA-HA-18 filters for the following chemical analysis of the welding fumes samples. The emitted fume and their components were weighed. For this purpose, at least five welding fumes samples were taken. The results of the tests (Tables 4-6) were subjected to statistical processing [11]. Figure 1. Oscillograms of droplet transfer current and voltage (3mm in diameter E19.9NbB20 electrodes):  - diode type; b – Inverter type An analysis of the oscillograms in Fig. 1 (Table 1) shows that the droplet transfer time is reduced and the number of short circuits increases when using a high frequency energy conversion power supply (inverter). In this case the transferred electrode metal droplets are smaller in size which influences the weld metal composition [58]. To confirm this, a microradiography analysis of the composition (Fig. RESULTS AND DISCUSSION 2) of sections 1,2,3 shown in Fig. 3 and Table 2 was carried out. In MMA with E19.9NbB20 coated electrodes, deoxidation (alloying) of weld metal occurs in a combined way – through the electrode coating. VOL. 48, No 1, 2020 ▪ 77 2. EXPERIMENTAL PART Welding stability was assessed by means of statistic analysis of oscillograms of welding current and voltage generated by power supplies with different dynamic characteristics. Manganese [37-40] intensifies the process of cell membranes lipid peroxidation by generating free radicals as a result of binding glutathione sulfhydryl groups and blood (tissues) proteins. It breaks the antioxidant defence of the body which maintains homeostasis. We analyzed the following power supplies: welding rectifier of VD-306E type and inverter power supply of Nebula 315 type. To study their dynamic characteristics we compared current and voltage oscillograms obtained with the help of a digital "AKIP-4122 / 1V" storage oscilloscope, "Pintek Electronics" DP-50 " differential probe, "current probe PR 1030"; and "OWON_ Oscilloscope_2.0.8.26" software. Subsequently, lipid peroxidation products accumu- late leading to disruption of functions and integrity of cell membranes and intracellular metabolism in general. According to many authors [41-46], the main negative effect of the excess intake of manganese is central nervous system damage. 76 ▪ VOL. 48, No 1, 2020 FME Transactions FME Transactions Figure 2. Images obtained by micro-X-ray analysis with INCA (Oxford Instruments) Figure 3. Micro-X-ray spectral analysis graph: 1 - weld metal; 2 – weld fusion line; 3 – eat affected zone FME Transactions Power supply - rectifier Electrode Average welding mode parameters Number of short circuits Arc gap short circuit time ts.c., ms Short circuit time Ts.c., ms Imax, А Imin, А Diode type 12 12±3.76 249±76.3 132.1 ±4.7 54.9 ± 4.75 Inverter type E19.9Nb B20 Current 86±2,7А Voltage 24.5±0,6V Welding speed 0.27 m/min 24 8.7±3.52 140±45.8 123 ± 1.63 78 ± 1.06 Table1 . Welding modes with TsL-11 electrodes from various power supplies and static data of coated electrod droplets transfer [57]. 78 ▪ VOL. 48, No 1, 2020 FME Transactions Diode type 12 12±3.76 249±76.3 132.1 ±4.7 54.9 ± 4.75 Inverter type E19.9Nb B20 Current 86±2,7А Voltage 24.5±0,6V Welding speed 0.27 m/min 24 8.7±3.52 140±45.8 123 ± 1.63 78 ± 1.06 Table 2. Chemical composition of weld metal of X12CrNiTi18-9 steel produced by E19.9NbB20 electrodes. Power Supply Element content Diode rectifier Inverter Weight % Atomic % Weight % Atomic % Weld metal (1) Si K 0.36 0.71 0.38 0.75 Ti K 1.44 1.45 1.90 1.91 Cr K 18.54 19.66 19.21 20.37 Fe K 67.71 66.85 69.45 68.57 Ni K 10.19 9.57 10.81 10.15 Weld fusion line (2) Si K 0.50 0.98 0.56 1.10 Ti K 0.60 0.69 0.65 0.75 Cr K 16.45 17.45 17.40 18.42 Fe K 70.52 69.53 71.44 70.77 Ni K 10.87 10.11 11.01 10.20 Heat affected zone (3) Si K 0.50 0.97 0.53 1.05 Ti K 0.61 0.70 0.76 0.88 Cr K 17.19 18.21 17.41 18.45 Fe K 70.96 70.00 70.70 69.73 Ni K 10.63 9.98 10.71 10.05 Table 3. Emission (g/min) of welding fumes and their components. Electrode type (diameter, mm) Power supply - rectifier Welding fumes CrO3 Cr2O3 Mn Ni F Soluble F Insoluble Diode 0.257 0.012 0.006 0.007 0.006 0.012 0.013 E19.9NbB20 (3) Inverter 0.2 0.09 0.004 0.005 0.0045 0.01 0.01 Table 4. Specific emissions (g / kg) of welding fumes and their components. Electrode type (diameter, mm) Power supply - rectifier CA CrO3 Cr2O3 Mn Ni F Soluble F Insoluble Diode 10.48 0.48 0.26 0.31 0,26 0.50 0.51 E19.9NbB20 Inverter 8.07 0.37 0.20 0.24 020 0.42 0.42 Table 5. Toxic level of welding fumes (TLVf) and air exchange (NHL). Electrode type (diameter, mm) Power supply - rectifier TLVf , mg / m3 NHL, m3 / h Diode 0.15 1480 E19.9NbB20 (3) Inverter 0.1 1000 Table 2. Chemical composition of weld metal of X12CrNiTi18-9 steel produced by E19.9NbB20 electrodes. FME Transactions FME Transactions The results (Table 4-5) of the research showed that inverter rectifiers provide better health conditions for a MMA welder compared with diode rectifiers as far as the amount of emitted hazardous chemicals is reduced (welding fumes from 0.257 to 0.2 (19%), manganese from 0.007 to 0.005 (23%), chromium oxide from 0.012 to 0.09 (24%)) Thus, the risk of toxic poisoning and inflammation of the airways mucous membrane of welders and indirect workers is lower. ( g ( ), g Table1 . Welding modes with TsL-11 electrodes from various power supplies and static data of coated electrodes metal droplets transfer [57]. Power supply - rectifier Electrode Average welding mode parameters Number of short circuits Arc gap short circuit time ts.c., ms Short circuit time Ts.c., ms Imax, А Imin, А Diode type 12 12±3.76 249±76.3 132.1 ±4.7 54.9 ± 4.75 Inverter type E19.9Nb B20 Current 86±2,7А Voltage 24.5±0,6V Welding speed 0.27 m/min 24 8.7±3.52 140±45.8 123 ± 1.63 78 ± 1.06 Table 2. Chemical composition of weld metal of X12CrNiTi18-9 steel produced by E19.9NbB20 electrodes. Power Supply Element content Diode rectifier Inverter Weight % Atomic % Weight % Atomic % Weld metal (1) Si K 0.36 0.71 0.38 0.75 Ti K 1.44 1.45 1.90 1.91 Cr K 18.54 19.66 19.21 20.37 Fe K 67.71 66.85 69.45 68.57 Ni K 10.19 9.57 10.81 10.15 Weld fusion line (2) Si K 0.50 0.98 0.56 1.10 Ti K 0.60 0.69 0.65 0.75 Cr K 16.45 17.45 17.40 18.42 Fe K 70.52 69.53 71.44 70.77 Ni K 10.87 10.11 11.01 10.20 Heat affected zone (3) Si K 0.50 0.97 0.53 1.05 Ti K 0.61 0.70 0.76 0.88 Cr K 17.19 18.21 17.41 18.45 Fe K 70.96 70.00 70.70 69.73 Ni K 10.63 9.98 10.71 10.05 with TsL-11 electrodes from various power supplies and static data of coated electrodes metal Table1 . Welding modes with TsL-11 electrodes from various power supplies and static data of coated electrodes metal droplets transfer [57]. N b f A h t Sh t i it lding modes with TsL-11 electrodes from various power supplies and static data of coated electrodes metal nsfer [57]. Table1 . 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FME Transactions Power Supply Element content Diode rectifier Inverter Weight % Atomic % Weight % Atomic % Weld metal (1) Si K 0.36 0.71 0.38 0.75 Ti K 1.44 1.45 1.90 1.91 Cr K 18.54 19.66 19.21 20.37 Fe K 67.71 66.85 69.45 68.57 Ni K 10.19 9.57 10.81 10.15 Weld fusion line (2) Si K 0.50 0.98 0.56 1.10 Ti K 0.60 0.69 0.65 0.75 Cr K 16.45 17.45 17.40 18.42 Fe K 70.52 69.53 71.44 70.77 Ni K 10.87 10.11 11.01 10.20 Heat affected zone (3) Si K 0.50 0.97 0.53 1.05 Ti K 0.61 0.70 0.76 0.88 Cr K 17.19 18.21 17.41 18.45 Fe K 70.96 70.00 70.70 69.73 Ni K 10.63 9.98 10.71 10.05 ble 2. Chemical composition of weld metal of X12CrNiTi18-9 steel produced by E19.9NbB20 electrodes. Table 3. Emission (g/min) of welding fumes and their components. Electrode type (diameter, mm) Power supply - rectifier Welding fumes CrO3 Cr2O3 Mn Ni F Soluble F Insoluble Diode 0.257 0.012 0.006 0.007 0.006 0.012 0.013 E19.9NbB20 (3) Inverter 0.2 0.09 0.004 0.005 0.0045 0.01 0.01 Table 3. 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Occupational and Environmental Medicine. 2007; 64(3):167-177. doi:10.1136/oem.2006.028761. [32] Gromova, O.A., Torshin I.U., Khadzhidis A.K.: Analysis of molecular mechanisms of iron (II), copper and manganese effect in the pathogenesis of iron deficiency anemia. Clinical pharmacology and pharmacoeconomics. 2010;1: p1-9. [46] Streit W.J., Walter S.A., Pennell N.A. Reactive microgliosis. Prog. Neurobiol. 1999;563-581. [33] Dolgov, V.V., Ametov A.S, Shchetnikovich K.A., Demidova T.Yu., Dolgova A.V.: Laboratory diagnosis of carbohydrate metabolism disorders, diabetes mellitus. M.: Triad. 2002;112 p. [47] Sriram K., Pai K.S., Boyd M.R., Ravindranath V.: Evidence for Generation of Oxidative Stress in Brain by MPTP: In Vitro and in Vivo Studies in Mice. Brain Research. 1997;749(1):44-52. doi:10. 1016/S0006-8993(96)01271-1. [34] Kwik-Uribe, C., Smith D.R. Temporal responses in the disruption of iron regulation by manganese. J. Neurosci. Res. 2006;83(8):1601-1610. FME Transactions FME Transactions 80 ▪ VOL. 48, No 1, 2020 Environmental Science 2018;115 (1): 01204. ПРОЦЕНА НЕГАТИВНОГ УТИЦАЈА КОН- ЦЕНТРАЦИЈЕ МАНГАНА У ГАСОВИМА ОД ЗАВАРИВАЊА НА ЗДРАВЉЕ ЗАВАРИВАЧА И КАКО ДА СЕ ТАЈ УТИЦАЈ СМАЊИ [53] Erikson K.M., Aschner M. Manganese Neuro- toxicity and Glutamate-GABA Interaction. Neuro- chemistry International. 2003;43(4-5):475-480. doi:10.1016/S0197-0186(03)00037-8. Д.П. Илјашенко, Д.А. Чинахов, Е.Д. Чинахова, К.Ј. Кириченко, Е.В. Верхотурова [54] Choi C.J., Anantharam V., Martin D.P., Nicholson E.M., Richt J.A., Kanthasamy A., Kanthasamy A.G.: Manganese Upregulates Cellular Prion Pro- tein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Patho- genesis of Prion Disease. Toxicological Sciences. 2010;115(2):535-546. doi:10.1093/toxsci /kfq049. Рад приказује резултате теоријских истраживања негативног утицаја аеросола од заваривања (мангана и других елемената) на људски организам. Заштита заваривача се може обезбедити специјалном опре- мом (вентилацијом и индивидуалном заштитом), редуковањем квалитативне и квантитативне коли– чине мангана у аеросолу (технологијом заваривања, изворима напајања, савременим материјалима за заваривање), коришћењем медикамената за редуко- вање садржаја мангана у организму. Експериментална истраживања су показала да употреба инвертера извора напајања уместо испра- вљача са диодама омогућава пренос метала са елек- троде кап-по-кап чиме се редукује време формирања шава за 46% а пренос метала за 28%, пренос леги- рајућих елемената на шав за 6% и губитак са линије спајања за 6%, из зоне утицаја топлоте за 3%, интензитет обуке и њених компонената се редукује за 23%, а расподела средстава такође за 23%. Рад приказује резултате теоријских истраживања негативног утицаја аеросола од заваривања (мангана и других елемената) на људски организам. Заштита заваривача се може обезбедити специјалном опре- мом (вентилацијом и индивидуалном заштитом), редуковањем квалитативне и квантитативне коли– чине мангана у аеросолу (технологијом заваривања, изворима напајања, савременим материјалима за заваривање), коришћењем медикамената за редуко- вање садржаја мангана у организму. [55] Onishchenko G.G., Zaitseva N.V., Zemlyanova M.A. Hygienic indication of health consequences for external exposure of chemical factors. Perm: The book format. 2011;532 p. Експериментална истраживања су показала да употреба инвертера извора напајања уместо испра- вљача са диодама омогућава пренос метала са елек- троде кап-по-кап чиме се редукује време формирања шава за 46% а пренос метала за 28%, пренос леги- рајућих елемената на шав за 6% и губитак са линије спајања за 6%, из зоне утицаја топлоте за 3%, интензитет обуке и њених компонената се редукује за 23%, а расподела средстава такође за 23%. [56] Dobson A., Erikson K., Aschner M. Manganese Neurotoxicity. Annals of the New York Academy of Science. 2004;1012:115-128. [57] Il'Yaschenko D.P., Chinakhov D.A., Mamadaliev R.A. Effect of Inverter Power Source Characteris- tics on Welding Stability and Heat Affected Zone Dimensions. IOP Conference Series Earth and VOL. 48, No 1, 2020 ▪ 81 FME Transactions
https://openalex.org/W4383213899
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English
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Optimisation of exposure parameters using a phantom for thoracic spine radiographs in antero-posterior and lateral views
Radiography
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C. Sa dos Reis a, *, M. Caso a, L. Dolenc b, K. Howick c, R. Lemmen d, A. Meira e, F. Shatku a, E. Aymon f, S.S. Ghotra a, g C. Sa dos Reis a, *, M. Caso a, L. Dolenc b, K. Howick c, R. Lemmen d, A. Meira e, F. Shatku E. Aymon f, S.S. Ghotra a, g a School of Health Sciences (HESAV), University of Applied Sciences and Arts Western Switzerland (HES-SO), Lausanne, 1011, Switz b Medical Imaging and Radiotherapy Department, Faculty of Health Sciences, University of Ljubljana, Slovenia c Radiography and Diagnostic Imaging, School of Medicine, University College Dublin, Dublin, Ireland d Department of Medical Imaging and Radiation Therapy, Hanze University of Applied Sciences, Groningen, the Netherlands e Medical Imaging and Radiation Therapy, Lisbon School of Health Technology (ESTeSL)/IPL, Lisbon, Portugal f Department of Radiology, Hospital of Sion, Avenue Du Grand-Champsec 80, 1950, Sion, Switzerland g Department of Radiology, Hospital of Yverdon-les-Bains (eHnv), 1400 Yverdon-les-Bains, Switzerland * Corresponding author. E-mail addresses: claudia.sadosreis@hesav.ch (C. Sa dos Reis), melanie.caso@hesav.ch (M. Caso), dolencl@zf.uni-lj.si (L. Dolenc), kayleigh.howick@ucdconnect.ie (K. Howick), r.s.lemmen@st.hanze.nl (R. Lemmen), 2020493@alunos.estesl.ipl.pt (A. Meira), floi.shatku@hesav.ch (F. Shatku), emmanuel.aymon@hopitalvs.ch (E. Aymon), switindersingh.ghotra@hesav.ch (S.S. Ghotra). Contents lists available at ScienceDirect Contents lists available at ScienceDirect a r t i c l e i n f o Article history: Received 3 April 2023 Received in revised form 19 June 2023 Accepted 20 June 2023 Introduction: To investigate the exposure parameters for thoracic spine/(TS) radiography that allows the image acquisition at the lowest dose possible, while maintaining an adequate image quality/(IQ) to identify all relevant anatomical criteria. Methods: An experimental phantom study was conducted, and 48 different radiographs of TS (24 AP/24 lateral) were acquired. The Automatic Exposure Control/(AEC) with the central sensor was used to select the beam intensity, while Source-to-Detector-Distance/(SDD) (AP:115/125 cm; Lateral:115/150 cm), tube potential (AP:70/81/90 kVp; Lateral: 81/90/102 kVp), use of grid/no grid and focal spot (fine/broad) were manipulated. IQ was assessed by observers with ViewDEX. Effective Dose (ED) was estimated using PCXMC2.0 software. Descriptive statistics paired with intraclass correlation coefficient (ICC) were applied to analyse data. Keywords: Plain radiography Source-to-detector distance Beam energy Beam intensity Radiographers Keywords: Plain radiography Source-to-detector distance Beam energy Beam intensity Radiographers Results: The ED increased with a greater SDD for lateral-view, presenting a significant difference (p ¼ 0.038), however IQ was not affected. For both AP and lateral, the use of grid had a significant effect on ED (p < 0.001). Despite the images acquired without grid had lower IQ scores, the observers considered the IQ adequate for clinical use. A 20% reduction in ED (0.042mSve0.033 mSv) was observed when increasing the beam energy from 70 to 90 kVp for AP grid in. The observers ICC ranged from moderate to good (0.5e0.75) in lateral and good to excellent (0.75e0.9) for AP views. Conclusions: The optimised parameters in this context were 115 cm SDD, 90 kVp with grid for the best IQ and lowest ED. Further studies in clinical setting are necessary to enlarge the context and cover different body habitus and equipment. Implications for practice: The SDD impacts on dose for TS; Higher kVp and grid are necessary to better image quality. © 2023 The Author(s). Published by Elsevier Ltd on behalf of The College of Radiographers. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Radiography 29 (2023) 870e877 Radiography 29 (2023) 870e877 https://doi.org/10.1016/j.radi.2023.06.009 1078-8174/© 2023 The Author(s). Published by Elsevier Ltd on behalf of The College of Radiographers. This is an open access article under the CC BY license (http://creativeco mmons.org/licenses/by/4.0/). * Corresponding author. Introduction the clinical situation, available resources and treatment options.2 Computed tomography (CT) and magnetic resonance imaging (MRI) are sensitive in assessing spinal disorders.3 However, these imaging methods are not always available, MRI is expensive and requires longer acquisition times,2,4 while CT involves typically higher radiation dose compared to plain radiography.5,6 Plain The thoracic spine is often affected by trauma, arthropathy, neoplasms, metabolic diseases and congenital deformities, which require medical imaging examinations to diagnose and to follow up these disorders.1 The choice of the imaging modality depends on 9 hed by Elsevier Ltd on behalf of The College of Radiographers. This is an open access article under the CC BY license (http://creativeco C. Sa dos Reis, M. Caso, L. Dolenc et al. Radiography 29 (2023) 870e877 Figure 1. Phantom positioning during image acquisition for antero-posterior projec- tion at Source-to-Detector Distance of 125 cm. radiography is an advantageous modality for thoracic spine assessment due its accessibility, low cost and low dose required and it is useful in assessing skeletal alignment, trauma and arthropathies.2,7 It is important to highlight that patients who re- quires thoracic spine radiography due to spinal arthropathy sus- picion are typically older (60e70 years old),7 while cases of spinal trauma usually affect the younger populations (20e50 years- old),8,9 which is a concern because the younger population is more radiosensitive. Therefore, it is critical to optimise the image acquisition to ensure that patients receive the lowest possible ra- diation dose, while the relevant anatomical structures are still visible to detect the abnormalities.10 Additionally, the thoracic spine is located near by the breast tissue, and this is one of the most radiosensitive anatomical structures, particularly in the female population. Furthermore, the images must be of adequate quality to allow the detection of fracture lines, particularly in the older pop- ulation with less dense bones and less tissue contrast.11 To achieve this balance between dose and image quality, patient positioning, beam energy and intensity, focal spot, use of a grid, collimation, source-to-detector distance (SDD) and additional filtration must be carefully selected and combined.12 The objective of this study was to identify the ideal exposure parameters (SDD, focal spot, use of grid, and beam energy) for thoracic spine radiography, while optimising the dose and the IQ balance. Effective dose estimation The effective dose (ED) was estimated using the PCXMC 2.0 software, a Monte Carlo based program, and the tissue weighting factors were based on the International Commission on Radio- logical Protection (ICRP) 103.15e18 The estimation was performed considering the different parameters that were manipulated during the acquisition as explained in the above section. These include beam energy (kVp), average beam intensity (average mAs), focal-skin distance, beam size (collimation field), beam placement and inherent filtration (1.5 mm Al þ 1 mm in the collimator), different positioning (AP and Lateral), SDD and “adult” setting.15 All these variables were established using mathematical probabilities of the interactions between photons and the phantom's body, such as the photoelectric effect, coherent scattering and incoherent scattering.17 For the simulations, a maximum energy of 150 keV and 20,000 photons were used. Methodology For this research, an experimental phantom study was con- ducted. The research was performed in 4 phases: 1. Image acqui- sition, 2. Effective dose estimation, 3. Perceptual image quality assessment and 4. Data analysis. Figure 1. Phantom positioning during image acquisition for antero-posterior projec- tion at Source-to-Detector Distance of 125 cm. Ethical approval was not required by the University of Ljubljana to carry out this study as it was conducted with a phantom in a controlled environment and not in a clinical setting with real pa- tients. For the third phase (perceptual image assessment), all par- ticipants signed an informed consent form prior to taking part in the assessment. All responses were anonymous, and submissions were in compliance with the General Data Protection Regulation (GDPR). to observe the consistency of the system. The central sensor of the Automatic Exposure Control (AEC) was used for the acquisition of all radiographs (Table 1; Fig.1). The exposure parameters were selected according to the published literature,13e15 namely for the selection of beam energy range (see Fig. 2). Image acquisition An anthropomorphic phantom PBU 60 (Kyotokagaku Co., Ltd, Japan), which represents a human of 160 cm in height and 50 kg in weight, was used to produce anteroposterior (AP) and lateral thoracic spine radiographs (Fig. 1). The images were acquired on a Siemens Multix/Vertix (Siemens, Germany) unit. Prior to the study, quality control (QC) tests of tube voltage accuracy, reproducibility, half-value layer, current time product linearity, tube output, total filtration and the dose area-product (DAP) were performed. In the study, a fine focal spot of 0.6 mm and a broad focal spot of 1.0 mm were used. Total filtration of the beam was 2.5 mm of Aluminium. Images were obtained using two different grids, one focused for SDD 115 cm, with a ratio of 12:1, 40 lines per centimetre and one focused for SDD 150 cm with a ratio 13:1 and 70 lines per centimetre. The digital detector was composed by Amorphous Silicon with a thin- film transistor (TFT) array and a pixel matrix of 2560  3072 with pitch of 140 mm. A constant collimation for the AP projection (43.0  13.9 cm) was applied. Due to the diverging X-ray beam, the collimation had to be adjusted for the lateral projection, using 17.1  43cm with 115 cm SDD and 16.8  41.7 cm with 150 cm SDD. By manipulating different parameters (SDD, kVp, focal spot size, use of grid), 48 different radiographs of thoracic spine (24 AP and 24 lateral) were acquired, and each exposure was repeated three times Table 1 l COM images, this is another reason to choose this program for is research.19,20 Before the assessment, the program was set up ith the images and the criteria used to analyse them (Table 2) The gure 2. Example of radiography used for image quality assessment (acquired in eral position at 115 cm SDD, broad focus, 81 kVp beam energy, with grid in). Protocols used for image acquisition (positioning, beam energy and intensity, focus, grid in/out). Protocols used for image acquisition (positioning, beam energy and intensity, focus, grid in/out). Projection (AP/lateral) SDD (cm) kVp Focus mAs (AEC) Grid AP 115 70 fine 11.7 In 3.13 Out broad 11.9 In 3.14 Out 81 fine 6.12 In 1.72 Out broad 6.25 In 1.7 Out 90 fine 4.26 In 1.27 Out broad 4.34 In 1.24 Out 125 70 fine 14.7 In 3.76 Out broad 1.5 In 3.78 Out 81 fine 7.65 In 2.03 Out broad 7.82 In 2.04 Out 90 fine 5.33 In 1.48 Out broad 5.45 In 1.47 Out Lateral 115 81 fine 4.14 In 1.57 Out broad 4.13 In 1.52 Out 90 fine 3.31 In 1.16 Out broad 3.34 In 1.1 Out 1.24 Out 102 fine 2.17 In 0.87 Out broad 2.18 In 0.8 Out 150 81 fine 6.27 In 2.56 Out broad 7.39 In 2.56 Out 90 fine 5.03 In 1.82 Out broad 4.31 In 1.81 Out 102 fine 3.22 In 1.25 Out broad 3.25 In 1.23 Out Protocols used for image acquisition (positioning, beam energy and intensity, focus, grid in/out). Lateral observers decide if the evaluated image was clinically acceptable (Table 2). Criteria used for VGA are developed based on the assumption that the capacity to reproduce normal anatomical structures is correlated with the possibility of clinically detecting a lesion in radiography, which is normally evaluated with the Receiver Operating Characteristics (ROC) study.23 However, VGA represents a much easier and faster method to evaluate IQ. Figure 2. Example of radiography used for image quality assessment (acquired in lateral position at 115 cm SDD, broad focus, 81 kVp beam energy, with grid in). Three experienced radiographers and three radiography stu- dents were recruited as observers for the image assessment. These observers were chosen instead of other medical professionals as in a clinical setting, the radiographer is the professional responsible for deciding if an image is diagnostically acceptable by ensuring the clinical question posed in the request can be answered. Image quality assessment Perceptual IQ assessment was performed using the software “Viewer for digital evaluation of X-ray images” (ViewDEX).19e21 ViewDEX is an image viewer that can be used for different image evaluation tasks and, in this study, it was used for Visual Grading Analysis (VGA).19,20 The program was developed to support studies for protocol's optimisation. ViewDEX supports 871 Radiography 29 (2023) 870e877 C. Sa dos Reis, M. Caso, L. Dolenc et al. Figure 2. Example of radiography used for image quality assessment (acquired in lateral position at 115 cm SDD, broad focus, 81 kVp beam energy, with grid in). Table 2 energies was calculated using KruskaleWallis in SPSS. IBM SPSS was used to calculate the IQ assessment score from the ViewDEX program. The Intraclass Correlation Coefficient (ICC) was calculated to verify the performance of the observers26 (Table 3). program. Participants were asked to focus on the middle region of the thoracic spine during the assessment because that was where IQ was homogeneous to ensured consistency across the observers with regard to the area of assessment. All participants were required to sign a consent form to inform them about the study objective, the rules and asking their permission to use the data for research purposes. During the assessment, four computers with the same diagnostic monitor model (DELL P2414H) were used. Both ambient lighting (10 lux)24 and resolution (1920  1080 pixels) conditions were kept constant to simulate clinical conditions and to ensure consistency between observers. Participants were not able to zoom in or out of the images and they were not able to change the window level or width to prevent variability in the study. y p ( ) Visual Grading Characteristics (VGC) analysis were also per- formed to compare the answers given by the observers for each image acquired with a different protocol, to identify which one was the best regarding quality, being considered a non-parametric rank-invariant statistical method.28,29 With the VGC Analyser software,30e33 data was analysed and VGC curves were created. These curves show the comparison of the IQ rating for two different parameters manipulated. In this study, the curves compared the images acquired at a SDD of 115 and 125 cm for the AP views and 115 and 150 cm for the lateral views. Additionally, the curves compare the two distances using Area Under the VGC Curve (AUC). If the AUCVGC 95% Interval Confidence (IC) contains 0.5, it means that the two distances are showing similar outcomes regarding IQ. When the 95% IC AUCVGC does not include 0.5, then the distances are statistically different. In this test, an AUC below 0.5 suggests preference for SDD of 115 cm.34 It was required that all participants sit in front of the monitor approximately 1 m away.25 The observers assessed 50 images (each radiograph once and one image repeated three times, to observe the consistency of the observer). They were informed that they could take a break when they reached 30 min to help reduce the influence of fatigue on their evaluation. Table 1 l This is done by assessing overall image quality and the visibility of all relevant anatomical structures.15 DICOM images, this is another reason to choose this program for this research.19,20 Before the assessment, the program was set up with the images and the criteria used to analyse them (Table 2). The criteria were based on previous studies15,17,22 and a 4-Likert scale was selected to score the images, which was adapted to the four groups of details analysed (anatomy, noise, overall image quality and contrast). Finally, a close-ended question was used to let DICOM images, this is another reason to choose this program for this research.19,20 Before the assessment, the program was set up with the images and the criteria used to analyse them (Table 2). The criteria were based on previous studies15,17,22 and a 4-Likert scale was selected to score the images, which was adapted to the four groups of details analysed (anatomy, noise, overall image quality and contrast). Finally, a close-ended question was used to let Before starting the task, the observers were asked about their eye's health and their experience in radiography. They were also 872 C. Sa dos Reis, M. Caso, L. Dolenc et al. Radiography 29 (2023) 870e877 informed and coached about how the assessment would be carried out, the assessment method and the functionalities of ViewDEX informed and coached about how the assessment would be carried out, the assessment method and the functionalities of ViewDEX values for the distance, grid in/out and focus were calculated using the ManneWhitney U test in SPSS. The p-value for the different Table 2 Observer study assessment criteria for thoracic spine with a 4 points Likert-scale. Table 2 Observer study assessment criteria for thoracic spine with a 4 points Likert-scale. Table 2 Observer study assessment criteria for thoracic spine with a 4 points Likert-scale. Anatomical details 1. Superior endplates of thoracic vertebrae are: 1. Not defined 2. Slightly defined 3. Defined 4. Clearly defined 2. Inferior endplates of thoracic vertebrae are: 1. Not defined 2. Slightly defined 3. Defined 4. Clearly defined 3. Spinous process of thoracic vertebrae are: 1. Not defined 2. Slightly defined 3. Defined 4. Clearly defined 4. Pedicles of thoracic vertebrae are: 1. Not defined 2.Slightly defined 3. Defined 4. Clearly defined 5. Intervertebral disc spaces are: 1. Not defined 2. Slightly defined 3. Defined 4. Clearly defined Noise 6. Results The results chapter will be divided into two different sections - effective dose and image quality assessment. Table 3 ICC score interpretation applied in this study.27 < 0.5 Poor reliability 0.5e0.75 Moderate reliability 0.75e0.9 Good reliability >0.9 Excellent reliability Table 2 A pilot was conducted prior to the real assessment to ensure the software setup worked and the questions were clear and understandable, while also answering the objective of the study. The feedback and suggestions to improve the assessment were integrated. During the real assessment, the ob- servers were supported by two members of the research team if necessary. Once the study was completed, the results were saved in a log file and used for the statistical analysis, respecting the data security.20 Table 1 l How would you estimate the noise on this radiograph in general? A. Excessive noise B. Prominent image noise C. Some image noise D. Limited perceptual image noise Overall image quality 7. How would you estimate the image quality in general? a. Very poor b. Poor c. Adequate d. Very good Contrast 8. How would you estimate the contrast in general? a. Very poor b. Poor c. Adequate d. Very good Clinical acceptance 9. Would you accept the radiograph? Yes No Effective dose (ED) Regarding the impact of SDD on ED for AP views, it was observed that the average dose was 0.023 for a SDD 115 cm and 0.024 mSv for a SDD 125 cm, which is not statistically significant different (p ¼ 0.453), while for lateral views the difference was significant (p ¼ 0.038), with an increased ED from 0.007 (SDD 115 cm) to 0.009 mSv (SDD 150 cm). The AP and Lateral projections anatomical criterion analysis by the VGC showed that AUCVGC were approximately 0.5 for the AP and 0.51, which means that they are similar and neither SSD overrides the other. In addition, AUCVGC 95% confidence interval ([0.400856, 0.546690] for the AP and [0.449097, 0.589907] for the lateral) of the two views includes the 0.5 value, which means that they are not statistically different (Fig. 3). The p-value was estimated as 0.478 for the AP and 0.910 for the Lateral views which is statistically no significant. In a similar way, for the IQ criterion of the AP and Lateral views, the results of the AUCVGC curves were estimated at 0.523704 (AP) and 0.467222 (LAT), near to 0.5. As for the anatomical criteria, the IQ criteria values are inside the 95% confidence interval ([0.347802, 0.593452] for the AP [0.443890, 0.652503] for the Lateral). The two results are similar. The p-value corresponds to 0.593 for the AP and 0.337 for the Lateral one, which means that the result is statistically no significant due to the value being above 0.05 for a 95% confidence interval (Fig. 4). ( ) For the AP projection with SDD 115 cm and grid in, the ED lied between 0.033 and 0.042 mSv with an average dose of 0.037 mSv. Similarly, the ED for a SDD 125 cm and grid in lied between 0.043 and 0.034 mSv. Without grid at 115 cm SDD, the average ED was 0.010 mSv varying between 0.010 and 0.009 mSv. For AP with SDD 125 cm without a grid, the ED ranged from 0.011 to 0.009 mSv. For AP grid in and grid out there was a significant difference (p < 0.001). The grid removal for image acquisition promoted a decrease of 74% in ED (Fig. 3). For the lateral projection with SDD 115 cm and a grid in, the ED varied between 0.011 and 0.010 mSv; while for a SDD of 150 cm, the ED ranged between 0.013 and 0.012 mSv. Discussion The objective of this study was to find a set of exposure pa- rameters (SDD, focus, use of grid and beam energy) for thoracic spine radiography that allows the acquisition of images with the lowest dose possible, while keeping an IQ that shows all relevant information to facilitate an adequate diagnosis and/or follow up. Regarding the impact of focal spot-on ED, for the AP and Lateral projections, no significant difference was observed when comparing broad with fine focal spot (p ¼ 0.977 and p ¼ 0.954, respectively). The results of this study showed that for AP projection, the ED remained almost the same (increase of 0.001 mSv) when increasing the SDD to 125 cm, presenting no significant difference (p ¼ 0.453), which is expectable considering that SDD did not largely varied (115 cme125 cm). For the Lateral projection, the difference be- tween the two SDD was larger, 115 and 150 cm, conducting to a significant difference (p ¼ 0.038) of ED. The ED increased from 0.010 mSv (at 115 cm SDD) to 0.013 mSv (at 150 cm SDD) when the Effective dose (ED) Without a grid at 115 cm SDD and 150 cm SDD, the ED was constant (0.004 mSv and 0.005 mSv, respectively). The ED increased with the distance but decreased by 61% when the grid was removed (Table 4). For the lateral grid in and grid out there was also a significant difference (p < 0.001). The ICC values ranged from poor (0.151) to excellent ICC (0.906) (Table 6), with higher values observed to the AP view, while for Lateral views the ICC was always inferior, being worst for the criteria 1 and 2 (see Table 7). The observers ranked IQ from 3.490 to 3.504 for AP views ac- quired with the grid and 2.587 to 2.729 when the grid was removed. For the Lateral views, the scores were lower and ranged from 3.462 to 3.569 when the grid was in and between 3.073 and 3.087 when the grid was removed (see Table 7). Increasing the beam energy while keeping the same distance, reduced the ED. The ED for the AP view, with grid in and SDD 115 cm decreased from 0.042 mSv to 0.033 mSv (20% reduction) when the beam energy was increased from 70 to 90 kVp; but the ED difference for the AP view, was not significant (p ¼ 0.145). For the lateral view, with the SDD 115 cm and grid in, the ED increased from 0.010 to 0.011 mSv when manipulating the kVp. Although, with grid out, the ED remained constant (0.004 mSv). For SDD 150 cm and grid in, the ED decreased from 0.013 mSv to 0.012 mSv when the energy was manipulated from 81 to 102 kVp. For lateral views, there was not observed a significant difference when beam energy was changed (p ¼ 0.983). Most of the observers accepted most of the images acquired (40 out of 48 images) for clinical purposes (Fig. 5). Data analysis Descriptive statistics analysis was performed using MS Excel program for dose estimation. IBM SPSS statistics version 28.0.1.0 was used to calculate the mean and standard deviation. The p- 873 C. Sa dos Reis, M. Caso, L. Dolenc et al. Radiography 29 (2023) 870e877 to 30 years. The participant observers have previously had their eyes tested, being capable of evaluating the images. Table 4 BF 0.035 0.036 0.035 0.010 0.012 0.011 SF 0.035 0.036 0.036 0.010 0.014 0.012 90 Average 0.033 0.035 0.034 0.011 0.013 0.012 BF 0.033 0.034 0.034 0.011 0.013 0.012 SF 0.034 0.035 0.034 0.011 0.013 0.012 102 Average e e e 0.010 0.012 0.011 BF e e e 0.010 0.012 0.011 SF e e e 0.010 0.013 0.011 Out Average OUT 0.010 0.010 0.010 0.004 0.005 0.004 70 Average 0.010 0.011 0.011 e e e BF 0.010 0.011 0.010 e e e SF 0.011 0.011 0.011 e e e 81 Average 0.009 0.009 0.009 0.004 0.005 0.004 BF 0.009 0.009 0.009 0.004 0.005 0.004 SF 0.009 0.009 0.009 0.004 0.005 0.004 90 Average 0.009 0.009 0.009 0.004 0.005 0.004 BF 0.010 0.010 0.010 0.004 0.005 0.004 SF 0.009 0.009 0.009 0.004 0.005 0.004 102 Average e e e 0.004 0.005 0.005 BF e e e 0.004 0.005 0.005 SF e e e 0.004 0.005 0.004 Total Average 0.023 0.024 0.023 0.007 0.009 0.008 Table 4 Table 4 Estimated effective dose (mSv) for all parameters. ( ) p Grid Energy (kVp) Focus Antero-Posterior Lateral SDD 115 cm SDD 125 cm Average (mSv) SDD 115 cm SDD 150 cm Average (mSv) In Average IN 0.037 0.038 0.037 0.010 0.013 0.012 70 Average 0.042 0.043 0.042 e e e BF 0.041 0.043 0.042 e e e SF 0.042 0.043 0.042 e e e 81 Average 0.035 0.036 0.035 0.010 0.013 0.012 BF 0.035 0.036 0.035 0.010 0.012 0.011 SF 0.035 0.036 0.036 0.010 0.014 0.012 90 Average 0.033 0.035 0.034 0.011 0.013 0.012 BF 0.033 0.034 0.034 0.011 0.013 0.012 SF 0.034 0.035 0.034 0.011 0.013 0.012 102 Average e e e 0.010 0.012 0.011 BF e e e 0.010 0.012 0.011 SF e e e 0.010 0.013 0.011 Out Average OUT 0.010 0.010 0.010 0.004 0.005 0.004 70 Average 0.010 0.011 0.011 e e e BF 0.010 0.011 0.010 e e e SF 0.011 0.011 0.011 e e e 81 Average 0.009 0.009 0.009 0.004 0.005 0.004 BF 0.009 0.009 0.009 0.004 0.005 0.004 SF 0.009 0.009 0.009 0.004 0.005 0.004 90 Average 0.009 0.009 0.009 0.004 0.005 0.004 BF 0.010 0.010 0.010 0.004 0.005 0.004 SF 0.009 0.009 0.009 0.004 0.005 0.004 102 Average e e e 0.004 0.005 0.005 BF e e e 0.004 0.005 0.005 SF e e e 0.004 0.005 0.004 Total Average 0.023 0.024 0.023 0.007 0.009 0.008 Table 5 Observer questionnaire results. Type of observer How many years of experience in the field of radiography do you have (since beginning your studies)? (years) Have you ever had your eyes checked? Radiographers 18 Yes 30 Yes 25 Yes Radiography Students 2 Yes 1.5 Yes 2 Yes Figure 4. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD 150 cm) for Lateral projections. Image quality assessment The image assessment was completed by the six observers (Table 5). The experience of the observers ranged from 1.5 years up Figure 3. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD125cm) for AP projections. 874 Figure 3. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD125cm) for AP projections Figure 3. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD125cm) for AP projections. 874 874 Radiography 29 (2023) 870e877 C. Sa dos Reis, M. Caso, L. Dolenc et al. distance increased. No previous studies were done, considering the expected considering the inverse square law,12,35 even if the AEC Table 5 Observer questionnaire results. Type of observer How many years of experience in the field of radiography do you have (since beginning your studies)? (years) Have you ever had your eyes checked? Radiographers 18 Yes 30 Yes 25 Yes Radiography Students 2 Yes 1.5 Yes 2 Yes Figure 4. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD 150 cm) for Lateral projections. Table 4 Estimated effective dose (mSv) for all parameters. Image quality assessment Grid Energy (kVp) Focus Antero-Posterior Lateral SDD 115 cm SDD 125 cm Average (mSv) SDD 115 cm SDD 150 cm Average (mSv) In Average IN 0.037 0.038 0.037 0.010 0.013 0.012 70 Average 0.042 0.043 0.042 e e e BF 0.041 0.043 0.042 e e e SF 0.042 0.043 0.042 e e e 81 Average 0.035 0.036 0.035 0.010 0.013 0.012 BF 0.035 0.036 0.035 0.010 0.012 0.011 SF 0.035 0.036 0.036 0.010 0.014 0.012 90 Average 0.033 0.035 0.034 0.011 0.013 0.012 BF 0.033 0.034 0.034 0.011 0.013 0.012 SF 0.034 0.035 0.034 0.011 0.013 0.012 102 Average e e e 0.010 0.012 0.011 BF e e e 0.010 0.012 0.011 SF e e e 0.010 0.013 0.011 Out Average OUT 0.010 0.010 0.010 0.004 0.005 0.004 70 Average 0.010 0.011 0.011 e e e BF 0.010 0.011 0.010 e e e SF 0.011 0.011 0.011 e e e 81 Average 0.009 0.009 0.009 0.004 0.005 0.004 BF 0.009 0.009 0.009 0.004 0.005 0.004 SF 0.009 0.009 0.009 0.004 0.005 0.004 90 Average 0.009 0.009 0.009 0.004 0.005 0.004 BF 0.010 0.010 0.010 0.004 0.005 0.004 SF 0.009 0.009 0.009 0.004 0.005 0.004 102 Average e e e 0.004 0.005 0.005 BF e e e 0.004 0.005 0.005 SF e e e 0.004 0.005 0.004 Total Average 0.023 0.024 0.023 0.007 0.009 0.008 Table 4 Estimated effective dose (mSv) for all parameters. Table 7 Table 7 Observer study - mean scores for questions 1e8 (anatomy, overall IQ, noise, contrast). SDD (cm) Grid In Grid Out IQ score mean ± STD ED (mSv) IQ score mean ± STD ED (mSv) AP 115 3.504 ± 0.666 0.037 2.729 ± 0.571 0.010 125 3.490 ± 0.678 0.038 2.587 ± 0.667 0.010 Lateral 115 3.462 ± 0.672 0.010 3.087 ± 0.748 0.005 150 3.569 ± 0.598 0.013 3.073 ± 0.692 0.005 This study faced several limitations. Firstly, the use of an anthropomorphic phantom restricted the simulation of diverse body types, such as individuals with higher Body Mass Index or paediatric patients. The simulation solely focused on the radiation absorption properties and scatter production of a standard adult body habitus. Consequently, the impact of these factors on image quality could not be accurately assessed. Furthermore, the anthropomorphic phantom did not incorporate any pathological features and in the majority of contexts, not only the anatomy is assessed but also the low contrast lesions that can be present on the images. To address these limitations, it is recommended to include real patient images featuring different types of lesions (low contrast details). Additionally, conducting receiver operating characteristic analysis can help determine the system's capabilities and limita- tions in detecting major pathologies. The X-ray equipment utilised in the study was not “ceiling mounted,” which limited the ability to manipulate different SDDs. Only two focused grids (115 cm and 150 cm) were available for use, preventing the evaluation of the influence of different grid prop- erties (such as type, grid frequency, interspace materials, and grid ratio). Another constraint is the observers’ study, as it was con- ducted in a controlled “reading lab” environment. This setting is not fully representative of the working conditions in a real radiography department. Figure 5. Answers provided by the observers when asked if they accept the image for clinical purposes. GO corresponds to Grid Out and GI to Grid In; AP e Antero-Posterior and LAT e Lateral Views. IQ. This scatter radiation adds to the patient's dose but does not contribute to the signal to form the radiographic image. The higher the energy used, the higher the amount of radiation that scatters. Therefore, it was expected that the use of a grid had a significant impact on IQ (P < 0.001 for AP and lateral). Table 7 In the AP projection, the dose decreased by 74% when there was no grid being used, while for the lateral projection, the decrease was 61%. IQ. This scatter radiation adds to the patient's dose but does not contribute to the signal to form the radiographic image. The higher the energy used, the higher the amount of radiation that scatters. Therefore, it was expected that the use of a grid had a significant impact on IQ (P < 0.001 for AP and lateral). In the AP projection, the dose decreased by 74% when there was no grid being used, while for the lateral projection, the decrease was 61%. Table 5 SDD (cm) Grid In Grid Out IQ score mean ± STD ED (mSv) IQ score mean ± STD ED (mSv) AP 115 3.504 ± 0.666 0.037 2.729 ± 0.571 0.010 125 3.490 ± 0.678 0.038 2.587 ± 0.667 0.010 Lateral 115 3.462 ± 0.672 0.010 3.087 ± 0.748 0.005 150 3.569 ± 0.598 0.013 3.073 ± 0.692 0.005 Table 6 ICC values for each criterion assessed during image quality evaluation.27 Criteria View ICC Confidence interval ICC Meaning Question 1 AP view 0.894 0.815 0.946 good to excellent LAT view 0.466 0.050 0.739 poor to moderate Question 2 AP view 0.885 0.800 0.942 good to excellent LAT view 0.407 0.056 0.710 poor to moderate Question 3 AP view 0.906 0.836 0.952 good to excellent LAT view 0.742 0.540 0.874 moderate to good Question 4 AP view 0.872 0.777 0.935 good to excellent LAT view 0.673 0.417 0.840 poor to good Question 5 AP view 0.857 0.751 0.928 good to excellent LAT view 0.504 0.118 0.757 poor to good Question 6 AP view 0.894 0.815 0.946 good to excellent LAT view 0.588 0.267 0.799 poor to good Question 7 AP view 0.846 0.732 0.922 moderate to excellent LAT view 0.676 0.423 0.841 poor to good Question 8 AP view 0.682 0.446 0.839 poor to good LAT view 0.610 0.307 0.809 poor to good Figure 5. Answers provided by the observers when asked if they accept the image for clinical purposes. GO corresponds to Grid Out and GI to Grid In; AP e Antero-Posterior and LAT e Lateral Views. Table 6 The ICC values showed mostly good to excellent reliability (0.75e0.9) in the AP images, but in the lateral images the reliability of the ob- servers was mostly poor to moderate (0.5e0.75). This may be caused by the difficulty in clear visualization of structures on the lateral images, as they overlap,37 making it difficult for observers to assess the image quality or the difference reference point used to evaluate the images.38 Another reason for the moderate reliability among observers could be the differences in clinical experience and knowledge amongst the recruited participants in this study.39 The AP and Lateral projection AUCvgc curve values were 0.523704 and 0.467222 respectively, meaning that the images are similar in quality even if the distance has been changed. These results are similar to other studies in literature.40 Their study found that the increased SDD from 100 to 130 cm reduced the radiation dose significantly with no significant effect on image quality. Tug- well and co-workers (2014)41 demonstrated in their study of AP pelvis imaging that there is no significant difference between IQ when increasing source to image distance (SID) from 110 to 140 cm. Table 7 Observer study - mean scores for questions 1e8 (anatomy, overall IQ, noise, contrast). SDD (cm) Grid In Grid Out IQ score mean ± STD ED (mSv) IQ score mean ± STD ED (mSv) AP 115 3.504 ± 0.666 0.037 2.729 ± 0.571 0.010 125 3.490 ± 0.678 0.038 2.587 ± 0.667 0.010 Lateral 115 3.462 ± 0.672 0.010 3.087 ± 0.748 0.005 150 3.569 ± 0.598 0.013 3.073 ± 0.692 0.005 Figure 5. Answers provided by the observers when asked if they accept the image for clinical purposes. GO corresponds to Grid Out and GI to Grid In; AP e Antero-Posterior and LAT e Lateral Views. Regarding the impact of the grid, the average score of IQ without the grid was lower (2.73) than the score given for the images with the grid (3.50). However, the result of question 9 (do you accept the image for clinical purpose?) showed that in the case of the radio- graphs without the grid, most of the images (40/48) were accepted by the observers even if the quality was not as good as the one with the grid. Table 5 Table 6 ICC values for each criterion assessed during image quality evaluation.27 Criteria View ICC Confidence interval ICC Meaning Question 1 AP view 0.894 0.815 0.946 good to excellent LAT view 0.466 0.050 0.739 poor to moderate Question 2 AP view 0.885 0.800 0.942 good to excellent LAT view 0.407 0.056 0.710 poor to moderate Question 3 AP view 0.906 0.836 0.952 good to excellent LAT view 0.742 0.540 0.874 moderate to good Question 4 AP view 0.872 0.777 0.935 good to excellent LAT view 0.673 0.417 0.840 poor to good Question 5 AP view 0.857 0.751 0.928 good to excellent LAT view 0.504 0.118 0.757 poor to good Question 6 AP view 0.894 0.815 0.946 good to excellent LAT view 0.588 0.267 0.799 poor to good Question 7 AP view 0.846 0.732 0.922 moderate to excellent LAT view 0.676 0.423 0.841 poor to good Question 8 AP view 0.682 0.446 0.839 poor to good LAT view 0.610 0.307 0.809 poor to good Table 6 ICC values for each criterion assessed during image quality evaluation.27 Table 6 This result shows that, according to the observers, even if the image is scored lower, it could still be used for clinical practice.42 Table 7 Observer study - mean scores for questions 1e8 (anatomy, overall IQ, noise, contrast). SDD (cm) Grid In Grid Out IQ score mean ± STD ED (mSv) IQ score mean ± STD ED (mSv) AP 115 3.504 ± 0.666 0.037 2.729 ± 0.571 0.010 125 3.490 ± 0.678 0.038 2.587 ± 0.667 0.010 Lateral 115 3.462 ± 0.672 0.010 3.087 ± 0.748 0.005 150 3.569 ± 0.598 0.013 3.073 ± 0.692 0.005 Figure 5. Answers provided by the observers when asked if they accept the image for clinical purposes. GO corresponds to Grid Out and GI to Grid In; AP e Antero-Posterior and LAT e Lateral Views. Table 7 Observer study - mean scores for questions 1e8 (anatomy, overall IQ, noise, contrast). Table 5 Table 5 Observer questionnaire results. Type of observer How many years of experience in the field of radiography do you have (since beginning your studies)? (years) Have you ever had your eyes checked? Radiographers 18 Yes 30 Yes 25 Yes Radiography Students 2 Yes 1.5 Yes 2 Yes Table 5 Observer questionnaire results. Type of observer How many years of experience in the field of radiography do you have (since beginning your studies)? (years) Have you ever had your eyes checked? Radiographers 18 Yes 30 Yes 25 Yes Radiography Students 2 Yes 1.5 Yes 2 Yes Figure 4. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD 150 cm) for Lateral projections. Figure 4. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD 150 cm) for Lateral projections. Figure 4. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD 150 cm) for Lateral projections. Figure 4. AUCVGC curves comparing a reference condition (SDD 115 cm) to a test condition (SDD 150 cm) for Lateral projections. DD 115 cm) to a test condition (SDD 150 cm) for Lateral projections distance increased. No previous studies were done, considering the ideal SDD for thoracic spine radiography, only for lumbar spine radiography,15 but considering the results for ED, the shorter SDD (115 cm) seems more adequate for radioprotection than a higher SDD. The increasing ED is probably due to a small collimation variation which could explain these results. The increasing ED is not expected considering the inverse square law,12,35 even if the AEC system must adjust the exposure parameters to keep the same signal level on the detector.36 Scatter radiation is a known contributor to reducing contrast which impacts the detection of low contrast lesions such as frac- tures in elderly populations and a grid should be used to improve 875 C. Sa dos Reis, M. Caso, L. Dolenc et al. Radiography 29 (2023) 870e877 IQ Thi tt di ti dd t th ti t' d b t d t Table 7 Observer study - mean scores for questions 1e8 (anatomy, overall IQ, noise, contrast). References J J g ( ) 25. Goyal N, Jain N, Rachapalli V. 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Theranostics [Internet] 2013;3(10):774e86. Available from: http://www.thno.org/v03p0774.htm. p 15. Conclusion The objective of this study was to investigate the ideal exposure parameters for thoracic spine radiography for optimal image quality and dose, with a focus on the SDD. It was shown that dose increases when the SDD increases, however, image quality does not degrade when the SDD increased. It can therefore be concluded that the preferable SDD for thoracic spine radiography is 115 cm as the image quality is the same and the patient dose is lower than with an SDD of 125 cm or 150 cm. In this study, when the kVp was increased, while maintaining the same distance and grid in, there was no significant difference (p ¼ 0.145 for APand p ¼ 0.983 for Lateral) in the effective dose, which means that to select the ideal parameters IQ assessment is critical, being necessary to choose the images with higher contrast to be able to detect fractures. Concerning the IQ data analysis with VGC analy- ser, it was shown that there were no significant differences when the distances were changed in lateral (p ¼ 0.593) and in AP (p ¼ 0.337). Future research should consider analysing real patient data, including different body habitus mainly obese, different pathol- ogies, and applying real clinical conditions for a more realistic assessment. 876 C. Sa dos Reis, M. Caso, L. Dolenc et al. Radiography 29 (2023) 870e877 Acknowledgements 20. B€orjesson S, Håkansson M, Båth M, Kheddache S, Svensson S, Tingberg A, et al. A software tool for increased efficiency in observer performance studies in radiology. Radiat Prot Dosimetry 2005;114(1e3):45e52. https://doi.org/10. 1093/rpd/nch550. PMID: 15933080. The authors would like to thank the support from all students and tutors that participated in our study, namely to the observers, to Prof. Nejs Mekis for his availability during the Optimax summer school and to the University of Ljubljana for making this summer school possible. 21. Svensson S, Zachrisson S, Svalkvist A, Ba M, Ha M, Radiology D, et al. ViewDEX: an efficient and easy-to-use software for observer performance studies. Radiat Protect Dosim 2010;139(1):42e51. ( ) 22. Gorham S, Brennan PC. Impact of focal spot size on radiologic image quality: a visual grading analysis. Radiography [Internet] 2010;16(4):304e13. https:// doi.org/10.1016/j.radi.2010.02.007. g/ /j 23. Boita J, van Engen RE, Mackenzie A, Tingberg A, Bosmans H, Bolejko A, et al. Validation of a candidate instrument to assess image quality in digital mammography using ROC analysis. Eur J Radiol [Internet 2021;139(March): 109686. https://doi.org/10.1016/j.ejrad.2021.109686. Conflict of interest statement None of the authors has a conflict of interest. g p y g y J [ 109686. https://doi.org/10.1016/j.ejrad.2021.109686. 24. Brennan PC, McEntee M, Evanoff M, Phillips P, O'Connor WT, Manning DJ. Ambient lighting: effect of illumination on soft-copy viewing of radiographs of the wrist. AJR Am J Roentgenol 2007;188(2):177e80. Claudia Sa dos Reis (on behalf of all the authors). References Lai ZH, Sa dos Reis C, Sun Z. Effective dose and image optimisation of lateral lumbar spine radiography: a phantom study. Eur Radiol Exp 2020;4(1). 40. Karami V, Zabihzadeh M, Shams N, Gilavand A. Optimization of radiological protection in pediatric patients undergoing common conventional radiological procedures: effectiveness of increasing the film to focus distance (FFD). Int J Pediatr 2017;5(4):4771e82. 16. Tapiovaara M, Siiskonen T. PCXMC A Monte Carlo program for calculating patient doses in medical x-ray examinations [Internet]. 2nded. 2008 Availablefrom:.https:// www.julkari.fi/bitstream/handle/10024/124342/stuk-a231.pdf?sequence¼1. 41. Tugwell J, Everton C, Kingma A, Oomkens DM, Pereira GA, Pimentinha DB, et al. Increasing source to image distance for AP pelvis imaging e impact on radiation dose and image quality. Radiography [Internet] 2014;20(4): 351e5. Available from: https://linkinghub.elsevier.com/retrieve/pii/ S1078817414000674. 17. Pazanin A, Skrk D, Zalokar N, Mekis N. Proper collimation effect on radiation dose and image quality in thoracic spine radiography. Nucl Technol Radiat Protect 2020;35(2):181e8. 18. Valentin J, editor. The 2007 recommendations of the international commission on radiological protection [internet]; 2007. Available from: http://www.elsevier. com/wps/find/bookdescription.cws_home/713998/ description#descriptionhttp://www.elsevier.com/wps/find/bookdescription. cws_home/714371/description%23description. 42. Moore CS, Wood TJ, Avery G, Balcam S, Needler L, Smith A, et al. Investigating the use of an antiscatter grid in chest radiography for average adults with a computed radiography imaging system. Br J Radiol [Internet] 2015;88(1047):20140613. Available from: http://www.birpublications.org/doi/10.1259/bjr.20140613. / p / / p / / description#descriptionhttp://www.elsevier.com/wps/find/bookdescription. cws_home/714371/description%23description. 19. Svalkvist A, Svensson S, Håkansson M, Bath M, Mansson LG. ViewDEX : a status report, vol 169; 2016. p. 38e45 (1). 877
https://openalex.org/W2889959090
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Sport Courage, Worry and Fear in Relation to Success of Alpine Ski Learning
Sports
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cc-by
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Received: 5 August 2018; Accepted: 6 September 2018; Published: 14 September 2018 Abstract: (1) Background: Individuals’ psychological traits can influence not just success in sport but also the ability to learn new motor skills. We investigated whether sport courage, worry and fear differ between alpine ski-naive and basic level skiers and how they affect ski learning. (2): A total of 337 students (249 ski-naive and 88 basic level) participated in research consisting of a four-part questionnaire and structured skiing program. (3) Results: For beginners, lower fear (r = −0.30, p < 0.01) and higher Self-efficiency (r = 0.28, p < 0.05) and mastery (r = 0.20, p < 0.01) were associated with better performance; reducing fear and increasing self-efficiency and worry increased performance. Experienced skiers were better in determination, mastery, and self-efficiency (all p < 0.05). In case of lower score in worry (r = −0.28, p < 0.01) and higher in self-efficiency (r = 0.22, p < 0.05) performance was better. Males scored higher in sport courage scale-31 (all p < 0.05). In particular, self-efficiency was associated with better (r = 0.39, p < 0.01), and higher fear with poorer performance (r = −0.33, p < 0.01). Moreover, self-efficiency was a predictor of ski success (p < 0.001). On the other hand, females like ski beginners scored higher in fear (p < 0.001). In females, determination, mastery and self-efficiency had a positive correlation with skiing (r = 0.21, p < 0.05, r = 0.28, p < 0.01, and r = 0.33, p < 0.01, respectively), while association between Fear and skiing (r = −0.46, p < 0.01) was negative, and fear (p < 0.001) was inversely related to success. (4): Conclusions: Psychological factors and gender differences need to be considered during learning phases of alpine skiing. There is a positive association between self-efficiency and performance of male ski beginners, and negative association between fear and achieved results in basic alpine ski school in case of female ski beginners. Keywords: sport courage; self-efficiency; worry; fear; skiing performance Article Vjekoslav Cigrovski 1,*, Ivan Radman 1 , Erkut Konter 2, Mateja Oˇci´c 1 and Lana Ruži´c 1 1 Faculty of Kinesiology Horvacanski zavoj, University of Zagreb, Zagreb 10000, Croatia; ivan.radman@kif.hr (I.R.); mateja.ocic@kif.hr (M.O.); lana.ruzic@kif.hr (L.R.) 2 Sport Science and Technology High School, Dokuz Eylül University, Izmir 35220, Turkey; erkut.konter@gmail.com Vjekoslav Cigrovski 1,*, Ivan Radman 1 , Erkut Konter 2, Mateja Oˇci´c 1 and Lana Ru 1 Faculty of Kinesiology Horvacanski zavoj, University of Zagreb, Zagreb 10000, Croatia; ivan.radman@kif.hr (I.R.); mateja.ocic@kif.hr (M.O.); lana.ruzic@kif.hr (L.R.) 2 Sport Science and Technology High School, Dokuz Eylül University, Izmir 35220, Turkey; erkut.konter@gmail.com * Correspondence: vjekoslav.cigrovski@kif.hr; Tel.: +38-591-544-2202 sports sports sports Sports 2018, 6, 96; doi:10.3390/sports6030096 sports sports 1. Introduction Courage, self-efficiency, worry, anxiety, and fear have clinical importance and can be associated with different medical conditions, alter every-day life, but can also greatly influence individual’s success in sport [1]. The latter has received much attention in sport literature, primarily related to investigation of factors influencing success on a professional level. The literature suggests fear, anxiety and worry usually have detrimental influence on performance, while self-efficiency and courage, historically perceived as virtue, are nowadays treated as skills and instruments that can have a dramatically positive effect on competitive sport [2–6]. Moreover, the term sport courage relates to the ability of a person to be competent, determined, assertive, and make sacrifice on voluntary basis and in challenging circumstances to achieve a sport success/result [7]. www.mdpi.com/journal/sports www.mdpi.com/journal/sports Sports 2018, 6, 96; doi:10.3390/sports6030096 2 of 13 Sports 2018, 6, 96 Sport courage is a dynamic process, influenced by numerous factors such as situations (e.g., danger, fear, risk), type of sport, personal traits, previous experience, and type of the task the athlete is confronted with [8]. Currently, sports psychology focuses on different psychological variables influencing athletes’ performance, with the intention of maximizing efficiency [6]. Psychological outcomes have social foundations that are sometimes gender-related, which means that gender can have an important role in linking psychological factors and performance. According to this view, boys are taught to be more competitive and encouraged to take risks; also they are more self-confident and thus conditioned to be efficacious [9]. Teachers and coaches may contribute to mentioned factors by having different expectations from females and males, and by providing different practice opportunities, which finally can lead to men being more confident in their sport abilities and prone to participate in activities promoting development of motor skills associated with sports. Moreover, boys and men can be more encouraged to take part in adventurous and extreme sports [2,10,11]. From recreational aspect, alpine skiing is one of the most popular winter sports with millions of people participating world-wide [12,13]. Contemporary research literature is mainly related to investigation of physical and physiological characteristics of elite alpine skiers, while research in the field of psychology is scarce, especially in case of recreational alpine skiing [14]. In the present study we examined the influence of fear, worry, self-efficiency and courage on learning alpine skiing in students of kinesiology. 1. Introduction It is a popular sport in Croatia, but due to specific environment and weather conditions it is generally considered demanding and challenging [15,16]. Therefore, fear related to new activity and worry about the mentioned exogenous factors can become a psychological barrier that prevents successful learning. Furthermore, in case of inexperienced learners, the mentioned factors can reduce motivation to learn and lead to withdrawal from the activity at an early stage. Finally, fear of injury may cause a behavioral block with refusal to perform specific elements of a ski technique. Therefore, it is important to address different variables such as fear, anxiety, motivation and courage when studying factors influencing sport success and performance [6]. We hypothesized that self-efficiency and courage would help students to better acquire new knowledge of alpine skiing. In order to test the psychological factors it is important to have reliable testing possibilities. We therefore used questionnaires with previously quantified metric characteristics in different populations and different countries and adopted them to the Croatian language. For this research, we also specifically developed an instrument Alpine skiing fear inventory [17–19]. We tested the metric characteristics of used instruments on young physically capable people, Croatian students of kinesiology with no or only basic previous knowledge of alpine skiing. 2.1. Design and Participants The present study included a total of 340 participants (252 alpine ski naive students and 88 students with existing basic knowledge of skiing). They were all third-year students of Faculty of Kinesiology at the University of Zagreb; 106 (31.2%) were females and 234 (68.8%) males (mean age 21.98 ± 1.41 years). We collected data during three consecutive academic years. An absence of any experience in alpine skiing was a definition for an alpine ski naive person. Prior to being enrolled in the study, all participants were informed about the study protocol and aim and gave their written consent to participation. The study was approved by the Ethics Committee of Faculty of Kinesiology. 2.2. Variables Alpine ski knowledge was assessed through demonstration of eight previously selected elements of alpine ski technique. Upon completion of structured alpine ski school program, the following elements were graded: traversing left (TL), traversing right (TR), uphill turn to the left (UTL), uphill turn to the right (UTR), snow-plough turn (SPT), basic turn (BT), parallel turn (PT), and short turn (ST). Grades ranged from 1 to 5; where 1 meant an unacceptable level of knowledge and 5 was Sports 2018, 6, 96 3 of 13 given for a superb demonstration of an element of the ski technique. Each participant received eight grades and this represented overall knowledge of alpine skiing. Cronbach alpha for skiing performance was 0.91. Moreover, each eligible participant fulfilled a four-part questionnaire consisting of 65 items. The questionnaire tested the way courage, worry, self-efficiency and fear influence the success in alpine ski learning. Each item was rated on a Likert scale. The questionnaire was not anonymous due to the need for comparison of its results with acquired knowledge of alpine skiing. The questionnaire consisted of the following parts: (1) Sports Courage Scale (SCS-31) [19]. SCS-31 is a 31-item self-report instrument with 5 subscales titled “Determination, Assertiveness, Mastery, Venturesome and Sacrificial Behavior”. Each item could be answered on a 5-point Likert based scale graded by 1 (I strongly disagree) to 5 (I strongly agree). SCS-31 scale showed adequate validity and reliability in previous research [19]. (2) Penn State Worry Questionnaire (PSWQ) [17]. PSWQ is a 16-item instrument measuring participants’ worry in particular situation. Each item is rated on a four-point scale, ranging from 1 (little) to 4 (strong). The questionnaire has proven validity and reliability in previous research [17]. Cronbach alpha for this research 0.87. (3) Self-Efficiency in Physical Activity and Alpine Skiing (S-EFPA) [18]. S-EFPA is constructed to assess participants’ self-efficiency. It consists of ten items. Each item is rated on a four-point scale; ranging from 1 (not at all) to 4 (strongly). The questionnaire has a satisfactory validity, but there is a need for more similar studies for its further validation [18]. Cronbach alpha for this research 0.84. (4) Alpine skiing fear inventory (F & S). F & S is a 9-item instrument used to test fear of alpine skiing. Each item is rated on a 4-point scale ranging from 1 (not at all) to 4 (strongly). Cronbach alpha for this research 0.85. 2.2. Variables (4) Alpine skiing fear inventory (F & S). F & S is a 9-item instrument used to test fear of alpine skiing. Each item is rated on a 4-point scale ranging from 1 (not at all) to 4 (strongly). Cronbach alpha for this research 0.85. Since all questionnaires were originally in English, they were translated into Croatian and then translated back to English by a bilingual professional. Moreover, this was the first use of SCS-31 in Croatian population. For this reason, in addition to double translation, we tested the questionnaire on a large sample of participants with similar characteristics (all young, healthy kinesiology students of similar age). We also performed CFA to test the latent structure is in line with original model of SCS-31 (more details available under sections statistical methods and results). Sport Courage Scale (SCS-31) questionnaire was used for the first time in Croatian population. In order to further explore the factor loadings, exploratory principal axis factoring analysis with oblimin rotation was used. Kaiser–Meyer–Olkin measure of adequacy suggested that the data matrix was suitable for extraction (KMO = 0.926), with Bartlett’s Test of Sphericity Approx. Chi-square = 3900.46; df = 465, p < 0.001. Five factors were extracted, explaining a total of 50.4% of variance of SCS; data presented in Figure 1. 4 of 13 4 of 13 Sports 2018, 6, 96 S 2018 6 Figure 1. Five factor model of SCS score (sc-mastery; dt-determination; ve-venturesome; as- assertiveness; aa-sacrificial behavior). The first factor explains the largest proportion of the variance (31.1%), and items loaded ar l f h b l ( “I f l h I h h h b f l Figure 1. Five factor model of SCS score (sc-mastery; dt-determination; ve-venturesome; as-assertiveness; aa-sacrificial behavior). The first factor explains the largest proportion of the variance (31.1%), and items loaded are ostly from the Determination subscale (e.g., “I feel that I have the strength to be successful in difficult Figure 1. Five factor model of SCS score (sc-mastery; dt-determination; ve-venturesome; as- assertiveness; aa-sacrificial behavior). Figure 1. Five factor model of SCS score (sc-mastery; dt-determination; ve-venturesome; as-assertiveness; aa-sacrificial behavior). 2.2. Variables The first factor explains the largest proportion of the variance (31.1%), and items loaded are mostly from the Determination subscale (e.g., “I feel that I have the strength to be successful in difficult conditions”), but also some items form Assertiveness are loading on this factor (“I like to take initiative in the face of difficulties”), indicating less than clear factor structure. The first factor explains the largest proportion of the variance (31.1%), and items loaded are mostly from the Determination subscale (e.g., “I feel that I have the strength to be successful in difficult conditions”), but also some items form Assertiveness are loading on this factor (“I like to take initiative in the face of difficulties”), indicating less than clear factor structure. Sports 2018, 6, 96 5 of 13 The second factor explains 6.6% of variance, and it consists of items regarding mastery (e.g., “I have limited success because I get frightened”, with reverse coding). The third factor explains additional 4.7% of variance and it should consist with Assertiveness items (“I have no problems responding to opponent’s sudden attacks”), but it has only 4 items loaded on this factor. The fourth factor explained additional 4% and contains majority of Venturesome items (e.g., “I would take any type of risks to become successful”), but additional items load on this factor (like Assertiveness “I assert myself even when facing hazards”, Sacrificial Behavior “I compete even if I have much more to lose than to gain”). The fifth factor also additionally explains 4% of variance, but it is a combination of items from Assertiveness “I continue to compete without panicking even when faced with a danger”, Venturesome “Even when facing the possibility of injury, I perform to the best of my ability” and Determination “I perform to the best of my ability no matter how negative the current conditions”. This unclear factor structure indicates the need for further validation of this scale in Croatian athletes. Since the sample is limited to athletes involved in skiing, and not various other sports as in the original validation procedure, and due to possible cultural differences, the original model of latent structure is used in this research and the subscales were formed based on the original factors extracted by the authors of the scale. Factors analysis if SPS-31 questionnaire is presented in Table 1. Table 1. Factor analysis of SPS-31 questionnaire. 2.2. Variables Questionnaire Factor 1 2 3 4 5 scs8 0.720 scs29 0.619 scs17 0.617 scs18 0.552 scs30 0.541 scs25 0.528 −0.223 scs28 0.506 −0.239 scs7 0.374 scs15 scs16 0.694 scs6 0.664 scs21 0.645 scs1 0.523 scs24 −0.319 0.474 scs11 0.399 scs22 −0.343 0.235 −0.263 scs13 −0.254 0.452 −0.212 scs2 0.381 scs3 −0.208 0.283 −0.234 scs27 0.266 scs4 −0.677 scs9 −0.539 0.231 scs14 −0.502 0.259 scs10 −0.493 scs23 0.298 −0.451 scs31 −0.398 scs5 0.306 −0.382 scs19 −0.267 0.555 scs26 0.370 0.385 scs12 0.219 −0.201 0.365 scs20 −0.230 0.351 Table 1. Factor analysis of SPS-31 questionnaire. Questionnaire Factor 1 2 3 4 5 scs8 0.720 scs29 0.619 scs17 0.617 scs18 0.552 scs30 0.541 scs25 0.528 −0.223 scs28 0.506 −0.239 scs7 0.374 scs15 scs16 0.694 scs6 0.664 scs21 0.645 scs1 0.523 scs24 −0.319 0.474 scs11 0.399 scs22 −0.343 0.235 −0.263 scs13 −0.254 0.452 −0.212 scs2 0.381 scs3 −0.208 0.283 −0.234 scs27 0.266 scs4 −0.677 scs9 −0.539 0.231 scs14 −0.502 0.259 scs10 −0.493 scs23 0.298 −0.451 scs31 −0.398 scs5 0.306 −0.382 scs19 −0.267 0.555 scs26 0.370 0.385 scs12 0.219 −0.201 0.365 scs20 −0.230 0.351 Table 1. Factor analysis of SPS-31 questionnaire. Table 1. Factor analysis of SPS-31 questionnaire. Table 1. Factor analysis of SPS-31 questionnaire. Sports 2018, 6, 96 6 of 13 Confirmatory factor analysis (CFA) conducted on data from the current research to verify the latent structure shows rather good model fit (24.6% of variance explained, CFI (confirmatory factor analysis) = 0.965, RMSA (root mean square error for approximation) = 0.06), justifying the use of originally defined 5 subscales. Both confirmatory and exploratory factor analysis show that one item (“I can take criticism of my principles or believes”) is not saturated on any of the five factors and is excluded from the analysis because it decreases overall subscale internal consistency. Therefore, all except one subscale show very good internal consistency: Cronbach alpha for determination, assertiveness, mastery, venturesome are 0.82, 0.80, 0.77, 0.75, respectively. Only subscale sacrificial behavior shows poor Cronbach alpha 0.47 (only 3 items after item 15 is removed). Sacrificial behavior should be considered and used with caution. Moreover, Cronbach alpha for PSWQ, SEFPA, F & S and skiing performance were as follows 0.87, 0.84, 0.85 and 0.91. 2.4. Statistical Analysis Data was analyzed using SPSS software (IBM SPSS Statistics for Windows, Version 24.0. IBM Corp., Armonk, NY, USA). Descriptive statistics was used to describe the basic features of the sample in this study: proportions for categorical data, and mean+/−standard deviation for normally distributed continuous variables, or median and interquartile range for variables deviating from normal distribution. Spearman’s coefficient of correlation was used within subsamples. Respecting statistical significance of Levene’s homogeneity of variance test, T-test for independent samples was used to test the significance of differences between two independent groups. Reliability analysis for scales used in the research was done using Cronbach alpha indicator, while factor structure for SCS-31 was verified using factor analysis: to further explore the factor loadings, exploratory principal axis factoring analysis with oblimin rotation was used. Additionally, confirmatory factor analysis (CFA) was conducted on data to verify the latent structure corresponding to five expected subscales defined by the authors of the SCS-31. Regression analysis was calculated to find independent predictors of skiing performance. 2.3. Investigational Protocol Research was conducted during three consecutive academic years. Participants were included in a 10-day structured program of alpine skiing school and then filled in the four-part questionnaire. Alpine ski school program was identical for all participants; conducted in the same ski center on identical ski slopes. During the ski school, participants were in groups of ten, and learned alpine skiing 6 h each day. Participants rented ski equipment of similar quality, adjusted to specific morphological characteristics. After completing the program of alpine ski school, participants’ knowledge was graded by independent judges. Grades were given for the demonstration of previously selected elements of the alpine ski technique according to the detailed instructions of demonstration layout. Each grade presented the knowledge of demonstrated element of the ski technique and was given by judges with many years of experience in evaluation of ski knowledge. Judges’ objectivity and homogeneity were determined and allowed the use of obtained grades for the assessment of alpine ski knowledge [20]. Each participant received in total eight grades, which was the basis for overall level of acquired alpine ski knowledge. 3. Results Prior to data analysis, descriptive statistics were calculated for all measures used, together with indicators of normality of data distribution. Descriptive statistics for used measures is shown in Table 2. 7 of 13 Sports 2018, 6, 96 Table 2. Mean values and standard deviations, median and interquartile range, and normality of distribution indicator are shown for each scale and subscale. Questionnaire M SD Min Max Median Percentile 25 Percentile 75 P (Kolmogorov- Smirnov Z) SCS-31 Determination 34.62 4.76 19 45 35 32 38 <0.001 Mastery 22.77 4.78 10 34 23 19 26 <0.001 Venturesome 13.21 3.06 4 20 13 11 15 <0.001 Assertiveness 26.12 3.71 14 35 26 24 28 <0.001 Sacrificial Behavior 11.50 1.84 5 15 12 10 13 <0.001 PSWQ 45.24 9.69 22 72 44 39 51 <0.001 SEFPA 38.64 5.89 22 50 39 35 43 <0.001 F & S 14.55 5.40 8 39 13 11 16 <0.001 Skiing Performance 29.77 5.17 18 40 30 26 33.50 <0.05 SCS-31-Sports courage scale; PSWQ: Penn State Worry Questionnaire; SEFPA: Self-efficacy in physical activity and alpine skiing; F & S: Alpine skiing fear inventory; Skiing performance: knowledge of demonstrated element of ski technique. SCS-31-Sports courage scale; PSWQ: Penn State Worry Questionnaire; SEFPA: Self-efficacy in physical activity and alpine skiing; F & S: Alpine skiing fear inventory; Skiing performance: knowledge of demonstrated element of ski technique Although data are not normally distributed (all p < 0.05), due to large sample size and data measured on an interval scale, parametric statistic T-test for independent samples was used to test the significance of differences between males and females, and between beginners and skiers. When Levene’s test of equality of variances is statistically significant, option “variances not assumed” was used to determine statistically significant differences between given groups. Data are shown in Table 3. Table 3. Differences in skiing performance and psychological variables regarding gender and previous skiing experience. Table 3. Differences in skiing performance and psychological variables regarding gender and previous skiing experience. Gender n Mean Std. 3. Results Deviation p Determination male 234 35.20 4.36 0.002 * female 105 33.31 5.34 Mastery male 234 23.20 4.52 0.019 * female 106 21.81 5.22 Venturesome male 234 13.60 2.91 <0.001 * female 105 12.34 3.22 Assertiveness male 234 26.53 3.42 0.005 * female 106 25.22 4.15 Sacrificial Behavior male 234 11.63 1.76 0.054 female 106 11.22 1.99 PSWQ male 230 44.92 9.57 0.378 female 104 45.93 9.94 SEFPA male 230 39.57 5.53 <0.001 * female 106 36.60 6.15 F & S male 232 13.40 4.27 <0.001 * female 106 17.08 6.64 Skiing Performance male 238 29.70 5.03 0.690 female 106 29.94 5.49 8 of 13 Sports 2018, 6, 96 Table 3. Cont. Skiing Previously n Mean Std. Deviation p Determination beginners 249 34.31 5.06 0.017 * skiers 88 35.52 3.66 Mastery beginners 250 22.41 5.02 0.012 * skiers 88 23.75 3.97 Venturesome beginners 250 13.34 3.15 0.217 skiers 87 12.87 2.75 Assertiveness beginners 250 25.98 3.84 0.192 skiers 88 26.58 3.28 Sacrificial Behavior beginners 250 11.55 1.90 0.511 skiers 88 11.40 1.66 PSWQ beginners 248 45.33 9.96 0.791 skiers 85 45.01 8.95 SEFPA beginners 246 37.68 5.76 <0.001 * skiers 88 41.23 5.37 F & S beginners 248 15.36 5.82 <0.001 * skiers 88 12.33 3.11 Skiing Performance beginners 250 28.35 4.62 <0.001 * skiers 89 34.00 4.22 PSWQ—Penn State Worry Questionnaire; SEFPA—Self-efficacy in physical activity and alpine skiing; F & S—Alpine skiing fear inventory; Skiing performance—knowledge of demonstrated element of ski technique. * p < 0.05. There are statistically significant differences between males and females on several variables. Males score higher on average in Determination, Mastery, Venturesome, Assertiveness and Self-efficiency (all p < 0.05). Females have higher average scores in fear inventory (p < 0.001). There are statistically significant differences between beginners and skiers on several variables. Experienced skiers score higher on average inn determination, mastery, self-efficiency and skiing performance (all p < 0.05), while beginners score higher in fear inventory (p < 0.001). Since there are statistically significant differences between males and females and between beginners and skiers, correlations between variables are calculated separately for those subgroups. Data are shown in Table 4. Spearman’s coefficient of correlation was used because it is more appropriate when data is not normally distributed or the relationship between variables is not linear. 3. Results Data in Table 5 show correlations between psychological traits and skiing performance in skiers and ski novices. Data in Table 5 show correlations between psychological traits and skiing performance in skiers and ski novices. Table 5. Spearman’s coefficient of correlation between variables for skiers (n = 88) and ski beginners (n = 249). Skiers (n = 88) Mastery Venturesome Assertiveness Sacrificial Behavior PSWQ SEFPA Fear Skiing Performance Determination 0.42 ** 0.47 ** 0.76 ** 0.34 ** −0.17 0.47 ** −0.21 * 0.15 Mastery 0.24 * 0.42 ** 0.15 −0.35 ** 0.52 ** −0.17 0.05 Venturesome 0.56 ** 0.42 ** −0.08 0.26 * −0.19 0.05 Assertiveness 0.38 ** −0.32 ** 0.44 ** −0.19 0.09 Sacrificial Behavior −0.08 0.22 * −0.12 0 PSWQ −0.32 ** 0.28 * −0.28 ** SEFPA −0.32 ** 0.22 * F & S −0.13 Ski Beginners (n = 249) Mastery Venturesome Assertiveness Sacrificial Behavior PSWQ SEFPA Fear Skiing Performance Determination 0.58 ** 0.59 ** 0.81 ** 0.55 ** −0.24 ** 0.49 ** −0.46 ** 0.103 Mastery 0.40 ** 0.59 ** 0.28 ** −0.51 ** 0.40 ** −0.51 ** 0.20 ** Venturesome 0.57 ** 0.52 ** −0.12 0.27 ** −0.38 ** 0.03 Assertiveness 0.44 ** −0.26 ** 0.41 ** −0.43 ** 0.07 Sacrificial Behavior −0.07 0.26 ** −0.27 ** −0.07 PSWQ −0.31 ** 0.30 ** −0.02 SEFPA −0.48 ** 0.28 ** F & S −0.30 ** PSWQ: Penn State Worry Questionnaire; SEFPA: Self-efficacy in physical activity and alpine skiing; F & S: Alpine skiing fear inventory; Skiing performance: knowledge of demonstrated element of ski technique. ** p < 0.01, * p < 0.05. ble 5. Spearman’s coefficient of correlation between variables for skiers (n = 88) and ski beginners 249) Table 5. Spearman’s coefficient of correlation between variables for skiers (n = 88) and ski beginners (n = 249). PSWQ: Penn State Worry Questionnaire; SEFPA: Self-efficacy in physical activity and alpine skiing; F & S: Alpine skiing fear inventory; Skiing performance: knowledge of demonstrated element of ski technique. ** p < 0.01, * p < 0 05 For skiers, lower score in PSWQ (r = −0.28, p < 0.01) and higher score in Self-efficiency (r = 0.22, p < 0.05) are weakly associated with better skiing performance. 3. Results In females, Determination and Mastery from SCS-31 have a weak positive correlation with skiing performance (r = 0.21, p < 0.05 and 0.28, p < 0.01, respectively), and so does Self-efficiency (r = 0.33, p < 0.01). Moderate association between Fear Inventory and skiing (r = −0.46, p < 0.01) is negative in direction, suggesting females with higher fear have poorer skiing performance. In males, only Self-efficiency is associated with better skiing performance (weak correlation, r = 0.39, p < 0.01), and as in females, higher fear is related to poorer performance (r = 0.33, p < 0.01). Table 4. Spearman’s coefficient of correlation between variables for female sample (n = 105) and for male sample (n = 235). Table 4. Spearman’s coefficient of correlation between variables for female sample (n = 105) and for male sample (n = 235). Females (n = 105) Mastery Venturesome Assertiveness Sacrificial Behavior PSWQ SEFPA Fear Skiing Performance Determination 0.69 ** 0.57 ** 0.80 ** 0.43 ** −0.29 ** 0.56 ** −0.48 ** 0.21 * Mastery 0.44 ** 0.66 ** 0.29 ** −0.49 ** 0.55 ** −0.46 ** 0.28 ** Venturesome 0.66 ** 0.41 ** −0.16 0.28 ** −0.35 ** 0.07 Assertiveness 0.41 ** −0.38 ** 0.47 ** −0.44 ** 0.13 Sacrificial Behavior −0.05 0.18 −0.18 −0.07 PSWQ −0.34 ** 0.24 * −0.17 SEFPA −0.46 ** 0.33 ** F & S −0.46 ** 9 of 13 Sports 2018, 6, 96 Table 4. Cont. Table 4. Cont. Males (n = 235) Mastery Venturesome Assertiveness Sacrificial Behavior PSWQ SEFPA Fear Skiing Performance Determination 0.45 ** 0.50 ** 0.80 ** 0.52 ** −0.19 ** 0.44 ** −0.36 ** 0.12 Mastery 0.27 ** 0.49 ** 0.22 ** −0.46 ** 0.34 ** −0.44 ** 0.14 * Venturesome 0.49 ** 0.52 ** −0.07 0.18 ** −0.25 ** −0.03 Assertiveness 0.41 ** −0.21 ** 0.36 ** −0.31 ** 0.09 Sacrificial Behavior −0.074 0.23 ** −0.23 ** −0.06 PSWQ −0.27 ** 0.33 ** −0.02 SEFPA −0.47 ** 0.39 ** F & S −0.33 ** PSWQ—Penn State Worry Questionnaire; SEFPA—Self-efficacy in physical activity and alpine skiing; F & S-Alpine skiing fear inventory; Skiing performance- knowledge of demonstrated element of ski technique. ** p < 0.01, * p < 0.05. PSWQ—Penn State Worry Questionnaire; SEFPA—Self-efficacy in physical activity and alpine skiing; F & S-Alpine skiing fear inventory; Skiing performance- knowledge of demonstrated element of ski technique. ** p < 0.01, * p < 0.05. 3. Results For beginners, lower score in Fear (r = −0.30, p < 0.01) and higher score in self-efficiency (r = 0.28, p < 0.05) and in mastery (r = 0.20, p < 0.01) are weakly associated with better skiing performance. Regression analysis was calculated to see which variables are independent predictors of skiing performance. Since correlation matrix differs for subgroups according to gender and previous skiing experiences, series of regressions are done on each subsample. Method stepwise forward was used, with 0.05 level for entry and 0.10 level of significance for removing the variable, variables entered in blocks: first block subscales from SCS-31, and the second block FEAR, SEFPA and PSWQ (as psychological characteristics). Data are presented in Table 6. In both subsamples linear regression analysis shows that entered variables explain significant proportion of variance of the dependent variable, skiing performance (9% of variance in beginners and 12% in skiers, p < 0.01 and p < 0.05, respectively). For beginners, regression model was analyzed in 4 steps, finally including 3 significant predictors. Independent predictors of skiing performance are: SEFPA (p = 0.004), PSWQ (p = 0.014), and Fear score (p = 0.043) reducing fear and increasing scores 10 of 13 Sports 2018, 6, 96 on SEFPA and PSWQ increase skiing performance. For skiers, only one predictor has a role in skiing performance: PSWQ score (p = 0.004), meaning decreasing worry increases skiing performance. Table 6. Regression coefficients for predictors of skiing performance, regarding previous skiing experience (beginners and skiers) and gender. Table 6. Regression coefficients for predictors of skiing performance, regarding previous skiing experience (beginners and skiers) and gender. 3. Results Questionnaire Beginners (n = 244) Skiers (n = 84) Standardized Coefficients (Beta) t p Standardized Coefficients (Beta) t p Mastery 0.120 1.518 0.130 SEFPA 0.213 2.933 0.004 * PSWQ 0.160 2.271 0.024 * −0.313 −2.982 0.004 * F & S −0.156 −2.035 0.043 * R2 = 0.13, R2Adj = 0.12, F = 8.996, p < 0.001 R2 = 0.10, R2Adj = 0.09, F = 8.893, p = 0.004 Males (n = 227) Females (n = 102) Standardized Coefficients (Beta) t p Standardized Coefficients (Beta) t p Mastery 0.021 0.319 0.750 0.079 0.762 0.448 SEFPA 0.402 6.141 <0.001 * F & S −0.396 −3.804 <0.001 * R2 = 0.17, R2Adj = 0.16, F = 22.663, p < 0.001 R2 = 0.20, R2Adj = 0.18, F = 11.986, p < 0.001 * p < 0.05. In gender related subsamples linear regression analysis shows that entered variables explain significant proportion of variance of the dependent variable, skiing performance (16% of variance in males and 18% in females, both p < 0.01). For males, regression model was analyzed in 2 steps, finally including 2 predictors, but only one being statistically significant for predicting skiing performance for males: SEFPA (p < 0.001)—increasing self-efficiency increases skiing performance. For females, regression model was analyzed in 2 steps, finally including 2 predictors, but only one remaining significant independent predictor of skiing performance for females, and that is Fear score (p < 0.001) decreasing scores in fear increase skiing performance. 4. Discussion Alpine skiing, although enjoyed by millions of people worldwide, is perceived as a “high-risk” sport [21], demanding for both learning and teaching [22]. Among different factors that can affect skiing, personality traits are highly important. Up to date, they were more often investigated in competitive level skiers, and recently more widely with respect to skiing-related injuries. Research by Johansson and co-authors (2015) suggests that alpine skiers with previous injuries differ from those not injured in the trait stress susceptibility, which is higher among the latter and thus perceived as a protective trait [16]. It seems that psychological traits defined in male skiers correlate with more risky behavior and therefore higher burden of injury [23]. This broadened the investigation of risk-taking behaviors and characteristics of participants in winter sports to set educational campaigns and reduce accident rates [24]. We believe it is an important area to investigate also from the perspective of future ski instructors, who will set example for their alpine ski school participants on how to learn alpine skiing more efficiently and safely. Our previous research suggests traits such as self-efficiency and confidence can help in learning alpine skiing, especially more complex elements of the ski technique [15]. Others have shown how anxiety diminishes effectiveness of alpine ski instruction [25]. In this research we investigated sport courage defined through determination, mastery, assertiveness, venturesome, altruistic behavior and self-efficiency as positive traits known to influence attitude towards sport and sport success on one hand [7] and on the other hand fear and worry as traits with potential to limit sports performance [6,26]. The study included only kinesiology students, 11 of 13 Sports 2018, 6, 96 with either no previous knowledge or only basic knowledge in alpine skiing. From the aspect of alpine skiing, to be daring, venturesome, and willing to participate in difficult situations could be an important trait to differ skiers with better performance. Similarly, from the aspect of alpine skiing, determination is an important trait because it is a quality that makes one continue in trying, even though it might be difficult, especially during initial phases of ski learning. Mastery is power, control and in skiing it might be control over speed for example, which is important for ski beginners as it can lead to reduction of fear. 4. Discussion Assertiveness is defined as confidence, self-assurance which from the skiing perspective relates to better success in learning process and is also important for advancement. Self-efficiency is closely related to capability of succeeding in a specific task and is important for ski beginners as well as competitors. Fear is an unpleasant emotion caused by being aware of danger, and as skiing is by its nature a sport that tests the boundaries of thrill and fear, it can be under negative influence of fear. When addressing fear in terms of alpine skiing, it can encompass many different things and situations; from fear of speed, fear of injury to fear of crowded ski-slopes or ski-lifts, fear of snow conditions or weather to fear of looking foolish while trying to learn alpine skiing. Worry means being anxious or troubled about actual or potential problems, which again in skiing can be multifactorial. Understanding and overcoming undesirable traits or in case of positive traits their accentuation may help in selection of adequate didactic methods and result in more efficient learning [6]. In our research we found differences in psychological traits between alpine ski naive athletes and those with previous basic knowledge of alpine skiing. Already experienced skiers scored higher in determination, mastery, self-efficiency. At the same time beginners scored higher in fear inventory. The mentioned facts suggest how positive previous experience influences attitude towards new activity and can help in better learning. Research by Kintschera and coworkers showed that ski beginners worry about falling and keeping balance during initial phases of alpine ski learning, which is influenced by fear and lower self-efficiency [27] and may limit the speed and results of ski learning. On the other hand, experienced skiers with positive psychological traits achieve better results during evaluation of skiing performance. p p y g g g p According to published data, one can also expect differences between males and females in psychological traits linked to sport success [18,27]. Literature suggests males to be more self-efficient and confident and ready to take more risks, all of which transfers to success and better sport performance. Gender-related differences are especially notable in traits self-efficiency and fear, which according to Giulianou and co-workers has its roots in cultural stereotypes where males receive more attention and support from family, coaches and trainers than their female counterparts, predisposing them to better sport results and taking part in high-risk sports [28]. 4. Discussion Although our research included young athletes of similar interest in sport and one would not expect such pronounced differences in psychological traits, we did notice statistically significant differences between male and female athletes in sport courage related traits. The mentioned facts were all more pronounced in males, and represent potentially more positive attitude of males towards sport and skiing. Similarly, Konter describes male sport participants to be more determined, assertive and venturesome than female counterparts [29]. Moreover, our female participants expressed more fear than males, which is also noted in other sports [18]. Fear is an important trait which might limit learning a new motor activity [30]. According to our results, female participants with more expressed fear had poorer skiing performance than those who were more determined and had greater scores in mastery, and self-efficiency. Interestingly, in males, only self-efficiency was associated with better skiing performance, but as in females, higher fear was related to poorer performance. Similar, detrimental effects of fear on sport success were also described in other sports [31,32]. Regardless of gender, participants with previous ski experience who scored lower in worry and higher in self-efficiency had better skiing performance. At the same time, ski beginners with lower score in fear and higher score in self-efficiency and mastery were better at alpine ski learning. Independent predictors of skiing performance for ski beginners are self-efficiency and worry which when increased relate to better performance and at the same time fear which when reduced also 12 of 13 Sports 2018, 6, 96 helps skiing performance. For skiers, only worry had an influence on skiing performance, meaning decreasing worry increases skiing performance. For males, significant predictor of skiing performance is self-efficiency which when increased leads to better skiing performance. For females, significant independent predictor of skiing performance was fear, suggesting that decreasing fear might improve skiing performance. Similar results showing positive effects of self-efficiency and confidence on reduction of fear of injury are shown by different studies and can help in learning a new motor activity and motivation [33]. Finally, it is important to mention study limitations, primarily related to a sample consisting of only young motorically capable people. In the future research and for the research to have a more practical impact on a broader population it would be necessary to include participants of different age and motor abilities. References 1. Dugas, M.J.; Gosselin, P.; Ladouceur, R. Intolerance of uncertainty and worry: Investigating specificity in a nonclinical sample. Cogn. Ther. Res. 2001, 25, 551–558. [CrossRef] 1. Dugas, M.J.; Gosselin, P.; Ladouceur, R. Intolerance of uncertainty and worry: Investigating specificity in a nonclinical sample. Cogn. Ther. Res. 2001, 25, 551–558. [CrossRef] 2. Moritz, S.; Feltz, D. The relation of self-efficacy measures to sport performance: A meta-analytic review. Res. Q. Exerc. Sport 2000, 280–288. [CrossRef] [PubMed] 2. Moritz, S.; Feltz, D. The relation of self-efficacy measures to sport performance: A meta-analytic review. Res. Q. Exerc. Sport 2000, 280–288. [CrossRef] [PubMed] 3. Corlett, J. Wirtue lost: Courage in Sport. 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Gender differences across age in motor performance: A meta-analysis. Psychol. Bull. 1985, 98, 260–282. 4. Discussion In conclusion, psychological factors need to be considered during learning phases of alpine skiing. To improve ski learning, teachers and ski instructors would need to help ski beginners increase self-efficiency and reduce fear. Additionally, possible gender differences in psychological traits need to be considered. When approaching male ski beginners self-efficiency is associated with better performance, while in female ski beginners it is important to reduce fear to achieve better results in basics alpine ski school program, as there is a negative association between the two. Mentioned approach could not only improve but also speed up the learning process. Author Contributions: Conceptualization V.C. and Y.Y.; Methodology, V.C. and E.K.; Software, V.C., L.R. and I.R.; Validation, V.C., E.K., and L.R.; Formal Analysis, V.C. and I.R.; Investigation, V.C., I.R. and M.O.; Resources, V.C. and E.K.; Data Curation, V.C. and L.R.; Writing: Original Draft Preparation, V.C., M.O. and I.R.; Writing: Review and Editing, V.C., E.K. and L.R.; Visualization, V.C.; Supervision, V.C. and L.R.; Project Administration, M.O. and I.R. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Author Contributions: Conceptualization V.C. and Y.Y.; Methodology, V.C. and E.K.; Software, V.C., L.R. and I.R.; Validation, V.C., E.K., and L.R.; Formal Analysis, V.C. and I.R.; Investigation, V.C., I.R. and M.O.; Resources, V.C. and E.K.; Data Curation, V.C. and L.R.; Writing: Original Draft Preparation, V.C., M.O. and I.R.; Writing: Review and Editing, V.C., E.K. and L.R.; Visualization, V.C.; Supervision, V.C. and L.R.; Project Administration, M.O. and I.R. References [CrossRef] [PubMed] 11. Cross, C.P.; Cyrenne, D.-L.M.; Brown, G.R. Sex differences in sensation-seeking: A meta-analysis. Sci. Rep. 2013, 3, 2486. [CrossRef] [PubMed] 12. Thiel, C.; Rosenhagen, A.; Roos, L.; Huebscher, M.; Vogt, L.; Banzer, W. Physiologic characteristics of leisure alpine skiing and snowboarding. In Science and Skiing IV; M˝uller, E., Lindinger, S., Stoggl, T., Fastenbauer, V., Eds.; Meyer and Meyer Sport: Salzburg, Austria, 2009; pp. 516–522. 13 of 13 Sports 2018, 6, 96 13. 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Enhancing performance and skill acquisition in novice basketball players with instructional self-talk. Sport Psychol. 2002, 16, 368–383. References [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Advancements in Hydrogel-Based Drug Sustained Release Systems for Bone Tissue Engineering
Frontiers in pharmacology
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cc-by
12,147
Edited by: Jianxun Ding, Chinese Academy of Sciences, China Edited by: Jianxun Ding, Chinese Academy of Sciences, China Bone defects caused by injury, disease, or congenital deformity remain a major health concern, and efficiently regenerating bone is a prominent clinical demand worldwide. However, bone regeneration is an intricate process that requires concerted participation of both cells and bioactive factors. Mimicking physiological bone healing procedures, the sustained release of bioactive molecules plays a vital role in creating an optimal osteogenic microenvironment and achieving promising bone repair outcomes. The utilization of biomaterial scaffolds can positively affect the osteogenesis process by integrating cells with bioactive factors in a proper way. A high water content, tunable physio-mechanical properties, and diverse synthetic strategies make hydrogels ideal cell carriers and controlled drug release reservoirs. Herein, we reviewed the current advancements in hydrogel-based drug sustained release systems that have delivered osteogenesis- inducing peptides, nucleic acids, and other bioactive molecules in bone tissue engineering (BTE). Reviewed by: Juan Wang, Sichuan University, China Dan Wang, Institute of Process Engineering (CAS), China Wenjing Yu, University of Pennsylvania, United States Reviewed by: Juan Wang, Sichuan University, China Dan Wang, Institute of Process Engineering (CAS), China Wenjing Yu, University of Pennsylvania, United States *Correspondence: Tingting Yu tingtingyu@bjmu.edu.cn Yan Wei kqweiyan@bjmu.edu.cn Bing Han kqbinghan@bjmu.edu.cn †These authors have contributed equally to this work and share first authorship *Correspondence: Tingting Yu tingtingyu@bjmu.edu.cn Yan Wei kqweiyan@bjmu.edu.cn Bing Han kqbinghan@bjmu.edu.cn *Correspondence: Tingting Yu tingtingyu@bjmu.edu.cn Yan Wei kqweiyan@bjmu.edu.cn Bing Han kqbinghan@bjmu.edu.cn Advancements in Hydrogel-Based Drug Sustained Release Systems for Bone Tissue Engineering 1 Department of Orthodontics, Peking University School and Hospital of Stomatology & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing, China, 2 Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing, China REVIEW REVIEW published: 06 May 2020 doi: 10.3389/fphar.2020.00622 INTRODUCTION Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Received: 12 March 2020 Accepted: 20 April 2020 Published: 06 May 2020 Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Bone defects may be caused by various events, including trauma, inflammation, neoplasm resection, congenital deformity, and degeneration (Crane et al., 1995; Spicer et al., 2012). Despite numerous solutions being applied to tackle this issue, clinical demands remain unmet. Bone defects may be caused by various events, including trauma, inflammation, neoplasm resection, congenital deformity, and degeneration (Crane et al., 1995; Spicer et al., 2012). Despite numerous solutions being applied to tackle this issue, clinical demands remain unmet. To date, autologous bone grafts are still the gold standard and most considered therapeutic strategy for critical-sized bone defects among all restoration methods due to their remarkable osteoconductive and osteoinductive properties. However, de novo problems might arise, such as a limited amount of donor tissue, an excessive harvest procedure, and the possibility of postoperative infection of the donor site (Langer and Vacanti, 1993; Betz, 2002; Ahlfeld et al., 2019). Allografts or xenografts usually serve as secondary alternatives, as slower incorporation, immune rejection, and pathogen transmission might occur (Crane et al., 1995; Haugen et al., 2019). Utilizing biocompatible scaffold materials, such as mesenchymal stem cells (MSCs) and/or bioactive factors (Meijer et al., 2007), bone tissue engineering can offer more possibilities. Achieving sufficient and qualified bone formation via artificial composites is the grand aim of bone tissue engineering. Keywords: hydrogel, sustained drug release, bone tissue engineering, growth factors, mesenchymal stem cells Citation: Zhang Y, Yu T, Peng L, Sun Q, Wei Y and Han B (2020) Advancements in Hydrogel-Based Drug Sustained Release Systems for Bone Tissue Engineering. Front. Pharmacol. 11:622. doi: 10.3389/fphar.2020.00622 Compared with bone harvest operations, MSCs are relatively easy to obtain. These cells exhibit self-renewal, multipotentiality (Prockop, 1997), and immunomodulatory properties (Keating, 2008), May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org Hydrogel-Based Drug Release for BTE Zhang et al. which are imperative for bone regeneration. In addition, bioactive factors, for example, cytokines and growth factors (GFs), play a crucial role in new bone formation. Bone morphogenetic proteins (BMPs) are a group of GFs that have been substantially investigated. Recombinant human BMP-2 and BMP-7 is commercially available for limited clinical usage (Nauth et al., 2011). However, naked GFs are vulnerable in vivo, and to achieve optimal osteogenic effects, a supraphysiological dose of GFs is required. Paradoxically, diffusion or uncontrolled release of GFs may lead to ectopic bone formation and other complications, including carcinogenicity (Carragee et al., 2011; de Melo Pereira and Habibovic, 2018). Hence, attaining sustained release of bioactive factors is an essential objective for scaffold design to promote the therapeutic efficacy of bone tissue engineering. The scaffold materials not only create a congenial microenvironment to promote MSC biological behaviors but also help to maintain bioactive molecules in situ. To date, the controlled release of bioactive factors in bone tissue engineering has been realized by a wide range of biomaterials of different natures and configurations, which provide diverse release profiles in different treatment scenarios (Lee and Shin, 2007). it was reported that MSC-derived exosomes combining scaffolds achieved preferable osteogenesis outcomes (Li et al., 2018), indicating the promising prospect of exosomes-based cell-free bone regeneration. MSCs from different sources, such as bone marrow and dental tissue, are available for bone tissue engineering. The stem cell niche, 3D microenvironments containing specific biophysical and biochemical signals, maintains the stemness of stem cells in vivo (Scadden, 2006; Jones and Wagers, 2008). However, maintaining the viability and stemness of MSCs as well as controlling stem cell fate is a fairly critical issue in regenerative medicine. Substrate-derived stimuli are able to prolong the stemness of stem cells and guide stem cell fate into specific lineages (Fisher et al., 2010; Marklein and Burdick, 2010; Lee et al., 2015). Citation: Moreover, as the proliferation and differentiation of MSCs may drive into specific lineages depending on different microenvironmental cues, biochemical stimuli, including cytokines and GFs, are used in a spatiotemporal sequence during the complex and continuous reparative procedure (Samorezov and Alsberg, 2015; Farokhi et al., 2016). Successful bone regeneration requires the proper combination of stimuli that can trigger MSC differentiation and matrix deposition. As the scaffold material itself is capable of combining substrate- derived and biochemical stimuli, biomimetic and bioinspired synthetic materials with sustained drug release systems should be designed to facilitate bone tissue regeneration. Due to the constraints of current knowledge in this field, the research is far from sufficient. Hydrogels are a category of highly hydrated 3-dimensional (3D) crosslinked homopolymer, copolymer, or macromer networks that can be cast into different shapes and sizes (Slaughter et al., 2009; Seliktar, 2012). The application of hydrogels in tissue engineering, bone tissue engineering in particular, has been garnering increasing attention. Laden with osteogenic-inducing drugs and sustained release profiles, hydrogels have been suggested to be promising bone tissue engineering biomaterials. In this review, we discuss the progress and limitations of current bone tissue engineering, the advantages of hydrogel-based bone regeneration biomaterials and recent advancements in hydrogel-based drug sustained release systems for bone tissue engineering. Natural bone fracture healing requires the coordinated participation of osteogenesis and angiogenesis (Collin-Osdoby, 1994; Marsell and Einhorn, 2011). Bioactive factors and signal pathway crosstalk, which mediates the interplay between epithelial cells and osteoprogenitors, has been well summarized (Ramasamy et al., 2016). Likewise, vascularization in bone substitutes is vital for successful bone tissue engineering. Insufficient blood supply may result in undernutrition, hypoxia, and inadequate cell recruitment, leading to the failure of bone tissue engineering. Varieties of assessments and solutions have been summarized (Rouwkema et al., 2008; Das and Botchwey, 2011), yet there is no convincing evidence that the strategies are ample to sustain large tissue constructs, encouraging the proposal of more promising methods. THE PRESENT CHALLENGES OF BONE TISSUE ENGINEERING To date, substantial progress has been made in bone regenerative medicine. A variety of biomimetic polymers and inorganic materials with bone-like microarchitecture have been designed with advanced manufacturing methods (Wei et al., 2011; Kim et al., 2017b; Yin et al., 2019), including 3D printing, aiming to achieve superb osteogenic properties as well as accuracy and spatial fitness of critical-sized defects. Light-cured, thermal- setting, pH- or enzyme-sensitive, and other smart biomaterials enable bone tissue engineering to serve in many on-demand circumstances. Varieties of seed cells from different origins including umbilical cord MSCs (UCMSCs), induced pluripotent stem cell-derived MSCs (iPSC-MSCs), and embryonic stem cell-derived MSCs (ESC-MSCs) are successfully applied (Xie et al., 2016; Chen et al., 2018). Multifarious drugs or bioactive factors are delivered in situ with different strategies and tailored release profiles, offering osteogenic-friendly environments for relevant cells. Noteworthy, Frontiers in Pharmacology | www.frontiersin.org BMP With the promoted understanding of the underlying mechanism of osteogenesis (Chen et al., 2012), BMP, as a prominent member of the TGF-b superfamily, has always been a favored candidate for bone tissue engineering applications. Since some hydrogels are believed to possess inferior osteoconductive properties, Olthof et al. (2018) modified an oligo[poly(ethylene glycol) (PEG) fumarate] (OPF) hydrogel with bisphosphate. BMP-2 was encapsulated in poly(lactic-co- glycolic acid) (PLGA) microspheres. The additional BMP-2 and drug-laden PLGA microspheres were entrapped in the hydrogel matrix. The researchers believed that the anionic groups would produce a strong interaction between the matrix and inorganic phase of the bone as well as enhance BMP-2-induced bone formation. The hydrogel matrix could be functionalized by peptides, which might be beneficial to reduce the dose of encapsulated BMP. In addition, nanofibrous mesh-hydrogel hybrid composites have been applied to reach a proper spatiotemporal release profile (Kolambkar et al., 2011). Shekaran et al. (2014) modified a matrix metalloproteinase (MMP)-degradable peptide crosslinked PEG with an a2b1 integrin-specific peptide (GFOGER). The interaction between integrin and collagen I has been proposed to be vital in osteogenic differentiation and mineralization. It was suggested that the modified matrix is able to support cell adhesion and proliferation and upregulate osteogenic gene expression. Laden with the low dose of BMP-2, robust bone healing was achieved. Along with BMP-2, BMP-7 is considered to be a promising GF in bone formation. An injectable chitosan/b-glycerophosphate (CS/ b-GP) hydrogel laden with BMP-7 and antibiotic exhibited preferable reparative effects towards infection-induced bone loss (Zang et al., 2019). Growth differentiation factor-5 (GDF-5), also known as BMP-14, regulates the development of numerous tissue and cell types, including limbs and teeth. Bae et al. (2014) mixed different concentrations of GDF-5 with a light-cured hydrogel matrix. The results showed that GDF-5 improved the osteogenic ability in a dose-dependent manner, as the strongest On the other hand, bioactive molecules play an important role in bone regenerative medicine. During bone formation, numerous cytokines and GFs are orchestrated in a spatiotemporal manner (Farokhi et al., 2016), which would provide a suitable microenvironment for MSC proliferation and differentiation, as well as recruit progenitors from surrounding tissue and peripheral blood for further restoration. Apart from competent cell carriers, hydrogels can also be employed as promising local drug reservoirs. Multiple schemes have been applied to reach desirable and smart drug delivery kinetics (Lee and Shin, 2007; Slaughter et al., 2009). THE PREPONDERANCE OF HYDROGELS IN BONE TISSUE ENGINEERING Ideal bone tissue engineering scaffolds should meet the following criteria: (1) biocompatible, nontoxic and nonimmunogenic; (2) porous-structured; (3) proper mechanical properties, load- bearing ability, and sufficient dimensional stability; and (4) fully degradable, with a degradation rate that matches neotissue formation (Lee and Shin, 2007; Slaughter et al., 2009; Haugen et al., 2019). Numerous inorganic scaffolds, such as metals and bioceramics, have been applied in bone regeneration, yet their lack of cell affinity, unbalanced mechanical properties, May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org 2 Zhang et al. Hydrogel-Based Drug Release for BTE HYDROGEL-BASED DRUG SUSTAINED RELEASE SYSTEMS FOR BONE TISSUE ENGINEERING and rather poor degradation cannot be ignored (Pearlin et al., 2018). According to types of raw materials, hydrogels can be briefly categorized into natural and synthetic. It is usually considered that natural hydrogels are more biocompatible and bioactive, while synthetic ones possess more tunable mechanical and degradation properties. 3D-structured, highly water-containing, and biocompatible hydrogels act as excellent extracellular matrix (ECM) analogs. The porous structure of the hydrogel enables substance exchange and cell entrance at the initial stage as well as vascular ingrowth in the follow-up stage. It has been substantially shown that cells are easily suspended within hydrogels, and the viability of the encapsulated cells is highly preserved (Gao et al., 2020; Paez et al., 2020). Extensive drug and sustained release strategies have been designed for bone tissue engineering. Herein, we introduce studies on hydrogel-based controlled release systems according to the category of bioactive molecule loaded within. Peptides The majority of cytokines, GFs, and hormones that stimulate bone formation are peptides. These biomolecules are produced through the autocrine, paracrine, and endocrine systems, acting concertedly to regulate the complex cascade of bone-related gene expression (Lee and Shin, 2007; Farokhi et al., 2016). Hence, a well-orchestrated sustained release system of these peptides has been pursued in order to present a more biomimetic approach. MSCs are highly sensitive to physical parameters (Higuchi et al., 2013), including viscoelasticity Engler et al. (2006) and topography (Fiedler et al., 2013), in the surrounding milieus. The stiffness (elastic modulus) of the matrix is believed to contribute greatly to determining stem cell fate. As Engler et al. (2006) demonstrated, 2D-cultured MSCs exhibited osteogenic characteristics when the microenvironmental stiffness was relatively rigid, at 20–40 kPa. However, osteogenesis occurred at 11–30 kPa when MSCs were cultivated 3-dimensionally (Huebsch et al., 2010). Due to flexible synthetic strategies and the range of constituents, hydrogels possess tunable physio- mechanical properties, which could match the desirable ranges of material elasticity, porosity, and degradation rate required for bone tissue engineering (Slaughter et al., 2009). Meanwhile, photodegradable (Lunzer et al., 2018), thermal-sensitive, or pH-sensitive (GhavamiNejad et al., 2016) linkages as well as other advantageous materials could be subtly introduced into hydrogels, which may fabricate a versatile and intelligent composite system to fulfill the growing clinical demands. Frontiers in Pharmacology | www.frontiersin.org BMP Non-covalent immobilization strategies are the most commonly used in hydrogel-based drug depots, the drug release rate was mainly determined by parameters such as crosslink density, carrier affinity for drugs, and the matrix degradation profile (Dimatteo et al., 2018). Bioactive factors also could be linked covalently to polymers by which a longer drug retention time would be achieved, and covalent linkages could be broken as reactions of specific external cues, leading an on-demand drug controlled release. Moreover, other sustained release systems like microspheres could be introduced to hydrogel matrix, enabling multiple drug molecules sustained release in sequential or spatiotemporal manners (Chen et al., 2010). May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org 3 Hydrogel-Based Drug Release for BTE Zhang et al. augmentation was achieved by the hydrogel loaded with the highest concentration. properties observed in vitro, significant enhancement by VEGF on new bone formation has not been detected. Amirian et al. (2015) coated VEGF and BMP-2 separately onto gelatin-pectin- biphasic calcium phosphate composites. The results revealed that composites coated with VEGF mainly aided in woven bone formation, and the percent of new bone formation was not greater than those coated with BMP-2. Apart from adsorption or physical entrapment, electrostatic, hydrophobic, or other interactions have been introduced into the systems to prolong the release of BMPs. Heparin was reported to be a strong binder to BMPs, yet the side effects were not negligible. Heparin mimics, which are usually negatively charged, are supposed to be capable of controlling BMP release. Chondroitin sulfate (Anjum et al., 2016), 2-N,6-O- sulfated chitosan (26SCS)-based nanoparticles (Cao et al., 2014), alginate sulfate (Park et al., 2018) were synthesized by researchers, and satisfactory results were achieved both in vitro and in vivo. When higher concentrations of heparin mimics were introduced, the release rate of BMP became slower. Seo et al. (2015, 2017) harnessed the ionic and hydrophobic interactions provided by polyphosphazene nanoparticles. They found that release rate of BMPs were controlled by the types and amounts of pendants. Thus, the optimal release profile and osteogenesis outcomes rely on a reasonable proportion of BMP- tethering molecules. Since exclusive delivery of VEGF performed barely satisfactorily in GF-induced osteogenesis, dual or multidrug delivery is warranted. When accompanied by BMP-2, VEGF exhibited a significant improvement in bone formation compared with hydrogels encapsulating BMP-2 alone. BMP VEGF combined with BMP-2 has been used routinely as a GF formula in bone tissue engineering. Similar loading strategies were applied by Barati et al. (2016) and Kader et al. (2019) for spatiotemporal release of BMP-2 and VEGF. MSCs and BMP- tethered nanoparticles were embedded in the outer space, while endothelial colony-forming cells (ECFCs) and VEGF-tethered nanoparticles were dispersed inside the microchannel-patterned hydrogel, as illustrated in Figure 1. Degradation and GF release could be tuned by altering stoichiometric ratio chain-extended molecules and proteolysis. According to the data, the release of VEGF and BMP-2 could last over 10 days and 21 days, respectively. It was observed that the patterned hydrogel dual delivery system performed significantly better than that of single delivery systems, which was attributed to paracrine crosstalk. During bone repair, VEGF expression peaks appear in the early period, while BMP peaks later. Thus, consisting of a PLGA microsphere-incorporating poly(propylene fumarate) (PPF) rod surrounded by a rapidly degrading gelatin hydrogel, the composite was designed as a GF delivery vehicle (Kempen et al., 2009). VEGF was encapsulated in the hydrogel, whereas BMP-2 was immobilized by microspheres inside the rod in order to achieve an ideal GF sequential release pattern. VEGF exhibited a large initial burst release within the first 3 days, and BMP-2 showed sustained release over 8 weeks. Likewise, although VEGF did not induce neo-bone formation, it significantly enhanced BMP-induced osteogenesis. Organic-inorganic modular scaffolds are able to optimally orchestrate dual GF release and serve as an “anatomy-structure-function” trinity system in regenerating weight-bearing bones (Bao et al., 2017). Mesoporous bioactive glass (MBG) with hollowed channels and hierarchical porous structures was introduced in a controlled release system as a scaffold (Tang et al., 2020). VEGF was carried by hydrogel inside the channel, and BMP-2 was adsorbed by the MBG scaffolds. 26SCS acted as an analog of ECM, which exhibits super-affinity to GFs. In vitro experiments showed that 26SCS promoted the bioactivity of BMP-2 and VEGF. It could be assumed that the VEGF hydrogel column in the hollowed channels might induce chemotaxis of vascular endothelial cells, thus regulating cell migration and vascular infiltration. Moreover, increased type H vessels and neotissue ingrowth were observed. Genetic engineering is another option to obtain long-lasting BMP release. As Lin et al. Vascular Epithelial Growth Factor (VEGF) p ( ) Vascularization plays a crucial role in both bone development and bone regeneration (Collin-Osdoby, 1994; Olsen et al., 2000). Blood vessels do not solely work as substance exchange pathways; they are also regarded as highly active paracrine organs targeting various progenitors during bone formation and reconstruction (Collin-Osdoby, 1994). VEGF, a key angiogenic growth factor (Carmeliet and Jain, 2011), has been widely used in bone tissue engineering. The cooperation between VEGF receptors and integrin adhesion receptors has been elucidated in angiogenic regulation. Garcia et al. (2016) engineered a protease- degradable, GFOGER-modified PEG hydrogel as a VEGF depot. They found that covalently linked VEGF remained highly bioactive during a prolonged release period. Whereas it was shown that a GFOGER hydrogel augmented neovascularization regardless of exogenous VEGF, micro computed tomography (micro-CT) showed delivering exogenous VEGF failed to enhance critical-sized bone repair. Heterogenous material composites are manufactured by which we can juggle both timed drug release and osteoconduction. Composed of a 3D multichannel calcium phosphate cement (CPC) and alginate/gellan gum (AlgGG) hydrogel, the CPC/ AlgGG biphasic scaffold tethers VEGF via the interaction with heparin (Ahlfeld et al., 2019). Despite some remarkable Frontiers in Pharmacology | www.frontiersin.org BMP (2019) described in a manuscript, the BMP gene was transduced into human bone marrow-derived stem cells (BMMSCs), obtaining a continuous (up to 56 days) and economical BMP supply. Using visible light-based projection stereolithography (VL-PSL) technology to encapsulate the transduced cells, the researchers were able to fabricate more structurally and geometrically compatible constructs for individualized bone defects, which would be conducive to achieving tissue fusion and bone tissue engineering long-term success. Fibroblast Growth Factor (FGF) FGF signaling is a dominant regulator during bone development and fracture repair (Bourque et al., 1993; Kronenberg, 2003). However, contradictory results have implied that FGF signaling May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org Frontiers in Pharmacology | www.frontiersin.org 4 Zhang et al. Hydrogel-Based Drug Release for BTE A B FIGURE 1 | Schematic illustration of (A) nanogel (NG) assembly and peptide grafting. (B) Achievement of BMP-2 and VEGF spatiotemporal release profiles via a patterned hydrogel-based sustained release system. Reprinted from a previous article by Barati et al. (2016) with permission. B FIGURE 1 | Schematic illustration of (A) nanogel (NG) assembly and peptide grafting. (B) Achievement of BMP-2 and VEGF spatiotemporal release profiles via a patterned hydrogel-based sustained release system. Reprinted from a previous article by Barati et al. (2016) with permission. to VEGF and FGF9 to obtain a covalent connection with the fibrin hydrogel. BMP-2 was transfected into BMSCs, endowing a greater osteogenic ability and resistance of the osteogenic differentiation inhibition induced by fusion with FGF9. Less bone was formed in the FGF9 groups compared to the groups treated with only VEGF, whereas VEGF/FGF9-loaded composites performed the best among the groups. may exert dual-directional effects on osteogenic procedures, probably in a dose-dependent manner (Kato et al., 1998; Quarto and Longaker, 2006). Thus, sustained release should be achieved when FGF is delivered in bone tissue engineering. Two Japanese groups encapsulated FGF in gelatin hydrogels for controlled release (Kodama et al., 2009; Furuya et al., 2014). A longer FGF release period may improve cell proliferation, the expression levels of osteogenic markers and BMP-2 as well as bone mineral density (BMD) at defect sites. However, these enhancements vanished, and side effects occurred when a high dose of FGF was delivered (Kodama et al., 2009). In order to achieve bone-like biomechanical properties and slower release of FGF, a stiffer hydrogel matrix, poly(2-hydroxyethyl methacrylate) copolymerized with 2-vinyl pyrrolidone, was engineered (Mabilleau et al., 2008). The data suggested that in the first 4 days, the FGF release rate was approximately 1% per day, which was relevant to hydrogel swelling. Unfortunately, no significant difference between the FGF and control groups was noted in bone mass, but the poorly mineralized woven bone area was significantly larger in the FGF group. Other Peptides Other peptides that regulate the bone regeneration cascade, including osteoprotegerin (OPG) (Jayash et al., 2017), stromal cell-derived factor-1a (SDF-1a) (Ratanavaraporn et al., 2011; Cipitria et al., 2017; Mi et al., 2017), platelet-derived growth factor (PDGF) (Wang et al., 2020), and parathyroid hormone (PTH) (Erten Taysi et al., 2019), etc., might also be worthy of an attempt. The selected studies of hydrogel-based peptide sustained release systems for bone regeneration and their findings are concluded in Table 1. Frontiers in Pharmacology | www.frontiersin.org Nucleic Acids (Wang et al., 2020) PTH CS microsphere suspended in poloxamer hydrogel 43% of PTH released in first week, sustained release lasted over 27 days New bone formation was found to be significantly higher compared to other groups after 10 days, but on day 21 a significant difference exists only when compared with the no treatment group (Erten Taysi et al., 2019) (Continu TABLE 1 | Summary of selected studies of hydrogel-based peptide sustained release systems Peptide Carrier material Release pattern BMP-2 PLGA microspheres + bisphosphate modified oligo OPF hydrogel Burst and sustained BMP-2 Nanofibrous mesh + peptide modified alginate hydrogel Spatiotemporal controlled release BMP-2 GFOGER-modified MMP-degradable PEG-maleimide hydrogel More than 20% BMP-2 remained after 14 days BMP-2 VL-PSL-manufactured live-cell hydrogel scaffold Sustained expression for 56 days BMP-7 CS/b-GP hydrogel Release 46% in first 12 h, 84% by the end of 336 h GDF-5 Photo-cured hyaluronic acid (HA) hydrogel Release profiles varies with the initial drug concentration, sustained release period over 25 days BMP-2 + VEGF Acrylate-functionalized lactide-chain- extended star polyethylene glycol (SPELA) hydrogel + gelatin methacryloyl (GelMA) hydrogel + PEG nanogel Release of VEGF and BMP-2 lasted over 10 days and 21 days, respectively (tunable release kinetics) VEGF + BMP-2 PLGA microsphere + PPF rod + gelatin hydrogel A large initial burst was shown in vivo, which changed significantly from ex vivo release profiles VEGF + BMP-2 Hydroxyapatite (HA)/ polycaprolactone (PCL) scaffold + PLGA-PEG-PLGA hydrogel Burst release in first 3 days, sustained release for 3 weeks BMP-2 + VEGF MBG-based matrix + GelMA/26SCS hydrogel The release rates of BMP-2 and VEGF were 24.01% and 34.47% respectively within 24 h, 67.90% and 82.73% respectively in 14 days BMP-2 + bFGF Gelatin microspheres + n-HA/PU40 scaffold + F-127 hydrogel Pronounced burst release occurred in first 24 h, linear release in following 29 days VEGF + FGF9 Nanocalcium sulfate + fibrin hydrogel Addition of the peptide sequence decreased GFs release in an enzyme concentration-dependent manner Osteoprotegerin (OPG) CS hydrogel Lasts 28 days, release profile could be adjusted by CS molecular weight SDF-1a CS/carboxymethyl CS nanoparticles + CS/b-GP hydrogel 20% initial burst release, a cumulative release of 40% over 28 days SDF-1a + BMP- 2 Gelatin hydrogel Large initial burst release of SDF- 1a in first 3 days, which may due to BMP-2 combination SDF-1a RGD-modified alginate hydrogel Sustained release over 42 days PDGF-BB +BMP-9 Sericin hydrogel (genetically incorporated) Almost 48% released within 17 days, intermittent rapid and slow release phases PTH CS microsphere suspended in poloxamer hydrogel 43% of PTH released in first week, sustained release lasted over 27 days . (Kempen et al., 2009) Nucleic Acids Ectopic bone formation in hindlimb ischemia model suggested type H vessels and neobone formation significantly increased (Tang et al., 2020) BMP-2 + bFGF Gelatin microspheres + n-HA/PU40 scaffold + F-127 hydrogel Pronounced burst release occurred in first 24 h, linear release in following 29 days Micro-CT analysis indicated dual-delivery reached significantly higher bone volume (BV). Quantitative histological analysis showed remarkable tissue response (Chen et al., 2016) VEGF + FGF9 Nanocalcium sulfate + fibrin hydrogel Addition of the peptide sequence decreased GFs release in an enzyme concentration-dependent manner Radiographical and quantitative analysis of micro-CT showed the highest BV in dual-delivery hybrid composite. Quantification of blood vessels in explanted tissue suggested more neovessels were obtained (Yuan et al., 2016) Osteoprotegerin (OPG) CS hydrogel Lasts 28 days, release profile could be adjusted by CS molecular weight An almost-complete recovery was observed, osteocalcin and osteopontin were upregulated (Jayash et al., 2017) SDF-1a CS/carboxymethyl CS nanoparticles + CS/b-GP hydrogel 20% initial burst release, a cumulative release of 40% over 28 days Micro-CT showed most new bone formation within the defect area (Mi et al., 201 SDF-1a + BMP- 2 Gelatin hydrogel Large initial burst release of SDF- 1a in first 3 days, which may due to BMP-2 combination Better new bone formation was observed in the dual- delivery group. SDF-1a enhanced BMP-2 osteogenic effects (Ratanavarapo et al., 2011) SDF-1a RGD-modified alginate hydrogel Sustained release over 42 days Improvements induced by SDF-1a or hydrogel stiffness levelled within 8 weeks. Higher number of cells were recruited by SDF-1a, but the difference was not significant in vivo (Cipitria et al., 2017) PDGF-BB +BMP-9 Sericin hydrogel (genetically incorporated) Almost 48% released within 17 days, intermittent rapid and slow release phases Biocompatible compared with other materials and stimulated cell proliferation. Osteogenic markers were significantly upregulated, and greater bone formation when accompanied by BMP-9. Nucleic Acids It is a preferable strategy for other GFs to accompany FGF in order to obtain a promising outcome. Chen et al. (2016) chose gelatin microspheres as BMP-2 and basic FGF (bFGF) carriers, which were further embedded in a commercialized injectable thermal-sensitive hydrogel. The hydrogel was injected into a porous cell-loading scaffold before use. Micro-CT revealed that the dual-loaded composites achieved the best reparative results. As expected, composites loading bFGF alone regenerated less bone and neobone at the margin of the defect areas, while the dual-loaded composites showed much more central area bone formation. FGF9 has been indicated to be a stabilizing factor for neovessels, thus, Yuan et al. (2016) introduced FGF9 as an assistant for VEGF, exerting synergetic effects on angiogenesis in bone tissue engineering. A specific peptide segment was fused Since GFs and cytokines are required for weeks during new bone formation, gene therapy might be a feasible alternative. Delivering DNA or RNA locally to increase or knockdown target gene expression, gene therapy is capable of manipulating the microenvironment and determining cell fate in bone regenerative medicine. Fang et al. (1996) utilized collagen sponges as BMP-4 and PTH plasmid DNA carriers to regenerate nonunion rat femur defects early in 1996. Bonadio et al. (1999) confirmed that non-viral DNA delivery possesses numerous advantages compared with the protein strategy. Hydrophilic nucleic acids and hydrogels could provide stable and sequestered environments for gene delivery. Komatsu et al. (2016) demonstrated that gelatin hydrogels could transduce BMP-2 plasmid DNA efficiently, facilitating local bone regeneration. May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org 5 Hydrogel-Based Drug Release for BTE Zhang et al. TABLE 1 | Summary of selected studies of hydrogel-based peptide sustained release systems. Nucleic Acids Peptide Carrier material Release pattern Findings (ex/in vivo) Reference BMP-2 PLGA microspheres + bisphosphate modified oligo OPF hydrogel Burst and sustained Osteoconductivity and osteoinductivity were significantly improved (Olthof et al., 2018) BMP-2 Nanofibrous mesh + peptide modified alginate hydrogel Spatiotemporal controlled release Micro-CT showed more bone regeneration, superior mechanical properties of neobone were achieved (Kolambkar et al., 2011) BMP-2 GFOGER-modified MMP-degradable PEG-maleimide hydrogel More than 20% BMP-2 remained after 14 days GFOGER-modified hydrogel exhibited intrinsic osteogenic activities, micro-CT demonstrated; improved bone repair (Shekaran et a 2014) BMP-2 VL-PSL-manufactured live-cell hydrogel scaffold Sustained expression for 56 days Micro-CT and histological evidence indicated mature and robust bone formation (Lin et al., 201 BMP-7 CS/b-GP hydrogel Release 46% in first 12 h, 84% by the end of 336 h Radiographical and histological observation suggested better periodontal regeneration (Zang et al., 2019) GDF-5 Photo-cured hyaluronic acid (HA) hydrogel Release profiles varies with the initial drug concentration, sustained release period over 25 days Hydrogel with the highest drug concentration displayed promoted osteogenic potential both in vitro and in vivo (Bae et al., 20 BMP-2 + VEGF Acrylate-functionalized lactide-chain- extended star polyethylene glycol (SPELA) hydrogel + gelatin methacryloyl (GelMA) hydrogel + PEG nanogel Release of VEGF and BMP-2 lasted over 10 days and 21 days, respectively (tunable release kinetics) Patterned constructs significantly increase osteogenic and vasculogenic differentiation of precursors, bFGF expression was upregulated (Barati et al., 2016) VEGF + BMP-2 PLGA microsphere + PPF rod + gelatin hydrogel A large initial burst was shown in vivo, which changed significantly from ex vivo release profiles Micro-CT and histological section demonstrated co- delivery significantly enhanced osteogenesis and angiogenesis ectopically, but it did not reach significant results orthotopically (Kempen et al 2009) VEGF + BMP-2 Hydroxyapatite (HA)/ polycaprolactone (PCL) scaffold + PLGA-PEG-PLGA hydrogel Burst release in first 3 days, sustained release for 3 weeks Micro-CT showed newly-formed callus in co-delivery group almost covered defect areas, histological analysis showed no significant difference between co-delivery group and autologous group (Bao et al., 2017) BMP-2 + VEGF MBG-based matrix + GelMA/26SCS hydrogel The release rates of BMP-2 and VEGF were 24.01% and 34.47% respectively within 24 h, 67.90% and 82.73% respectively in 14 days In vitro osteogenic and angiogenic has been markedly improved. Nucleic Acids (Wang et al., 2020) New bone formation was found to be significantly higher compared to other groups after 10 days, but on day 21 a significant difference exists only when compared with the no treatment group (Erten Taysi et al., 2019) May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org Hydrogel-Based Drug Release for BTE Zhang et al. TABLE 1 | Continued Peptide Carrier material Release pattern Findings (ex/in vivo) Reference Abaloparatide (analog of PTH) Photo-crosslinked methacrylated gelatin hydrogel 25% released within 24 h, remaining was released steadily over next 10 days Drug-loaded hydrogel showed significantly higher rate of bone regeneration (Ning et al., 2019) Oxytocin PLGA microsphere + poloxamer hydrogel + b‐tricalcium phosphate (b- TCP) and hydroxyapatite 42% released in first week, complete release within 32 days 4 weeks after operation, the lowest residual graft and highest BMD and BV was obtained among all groups (Akay et al., 2020) Calcium accumulating peptide (artificially synthesized) Gelatin-derived hydrogel Sustained release over 7 days, collagenase accelerated release Bone formation markers expression levels were enhanced. Micro-CT and histology showed the regenerative effect was superior to that of BMP-2 hydrogels (Jo et al., 2018) Findings (ex/in vivo) sustained delivery of Mg2+ have been applied to hydrogel-based scaffolds. Lin et al. (2018b) coated MgO nanoparticles with PLGA and an alginate hydrogel, constructing a monodisperse core-shell delivery system. The release profile of Mg2+ revealed a significant suppression of the initial burst, and its release rate was stable and programmed. Enhancement of progenitor cell viability and proliferation, upregulation of osteogenic gene expression levels, and increased bone regeneration volume in vivo were attributed to the stable and precise Mg2+ supply. Bisphosphonates (BPs) possess two adjacent phosphonic groups, which are propitiously bind to divalent metal ions. Zhang and colleagues (Zhang et al., 2017) developed acrylated- BP-Mg nanoparticles to deliver Mg2+ as well as strengthen the acellular hydrogel composite, which serves as a matrix for in situ bone formation, via multivalent crosslinked domains. They also utilized Mg2+ to fulfill on-demand intelligent drug release in bone tissue engineering (Zhang et al., 2018). Intriguingly, Mg2+ played multiple roles in this research. First, BP-Mg nanoparticles enabled hydrogel formation and stabilized the prodrug. Second, Mg2+ promoted osteogenic differentiation, resulting in increased alkaline phosphatase (ALP) expression. However, and more importantly, Mg2+ is also a critical cofactor of ALP. Nucleic Acids Findings (ex/in vivo) Reference Osteoconductivity and osteoinductivity were significantly improved (Olthof et al., 2018) Micro-CT showed more bone regeneration, superior mechanical properties of neobone were achieved (Kolambkar et al., 2011) GFOGER-modified hydrogel exhibited intrinsic osteogenic activities, micro-CT demonstrated; improved bone repair (Shekaran et al., 2014) Micro-CT and histological evidence indicated mature and robust bone formation (Lin et al., 2019) Radiographical and histological observation suggested better periodontal regeneration (Zang et al., 2019) Hydrogel with the highest drug concentration displayed promoted osteogenic potential both in vitro and in vivo (Bae et al., 2014) Patterned constructs significantly increase osteogenic and vasculogenic differentiation of precursors, bFGF expression was upregulated (Barati et al., 2016) Micro-CT and histological section demonstrated co- delivery significantly enhanced osteogenesis and angiogenesis ectopically, but it did not reach significant results orthotopically (Kempen et al., 2009) Micro-CT showed newly-formed callus in co-delivery group almost covered defect areas, histological analysis showed no significant difference between co-delivery group and autologous group (Bao et al., 2017) In vitro osteogenic and angiogenic has been markedly improved. Ectopic bone formation in hindlimb ischemia model suggested type H vessels and neobone formation significantly increased (Tang et al., 2020) Micro-CT analysis indicated dual-delivery reached significantly higher bone volume (BV). Quantitative histological analysis showed remarkable tissue response (Chen et al., 2016) Radiographical and quantitative analysis of micro-CT showed the highest BV in dual-delivery hybrid composite. Quantification of blood vessels in explanted tissue suggested more neovessels were obtained (Yuan et al., 2016) An almost-complete recovery was observed, osteocalcin and osteopontin were upregulated (Jayash et al., 2017) Micro-CT showed most new bone formation within the defect area (Mi et al., 2017) Better new bone formation was observed in the dual- delivery group. SDF-1a enhanced BMP-2 osteogenic effects (Ratanavaraporn et al., 2011) Improvements induced by SDF-1a or hydrogel stiffness levelled within 8 weeks. Higher number of cells were recruited by SDF-1a, but the difference was not significant in vivo (Cipitria et al., 2017) Biocompatible compared with other materials and stimulated cell proliferation. Osteogenic markers were significantly upregulated, and greater bone formation when accompanied by BMP-9. Ions or Small Molecules To date, a number of metal ions and artificially synthesized compounds have been found to be beneficial in bone regeneration. Achieving a sustained release pattern and longer duration of drug function may lead to promising therapeutic outcomes. Nucleic Acids ALP enzymatic hydrolysis was promoted; thus, more bioactive drug molecules were generated, which introduced positive feedback (Figure 2). According to the results, this strategy significantly enhanced bone regeneration. CS or polyethyleneimine (PEI) is usually introduced as the carrier due to the electrostatic interaction between the negatively charged nucleic acids and the polycations. It was reported that branched PEI-HA-DNA complexes were entrapped in bilayered OPF hydrogels to restore osteochondral defects (Needham et al., 2014). Moreover, BMP-2 plasmid DNA conjugated with CS nanoparticles exhibited significant augmentation in hydrogel-mediated rat calvaria bone regeneration (Li et al., 2017). Due to the low stability of liposomes and electrostatic disturbance of other charged compounds, calcium phosphate (CaP) can also be used for DNA incorporation and transfection in bone tissue engineering (Krebs et al., 2010). MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are groups of short single-stranded RNA fragments that downregulate target gene expression post-translationally. Various miRNAs associated with bone formation have been reported (Fang et al., 2015), shedding new light on future bone tissue engineering. Nguyen et al. (2014) synthesized an 8-arm PEG in situ-forming hydrogel loaded with siRNA/PEI nanocomplexes. siRNA remained bioactive during the prolonged release period. The in vitro results showed that siNoggin and siNoggin/miRNA-20a sustained release promoted hMSC osteogenic differentiation in 3D hydrogel cultivation. As mentioned previously, a stiffer substrate may lead to MSC osteogenic differentiation. Carthew et al. (2020) incorporated PEG/gelatin norbornene hydrogels with mechanosensitive miRNAs. MSCs encapsulated in hydrogels were transfected in situ, which predominantly enhanced osteogenic gene expression and mineralization. Researchers presumed that the higher transfection efficacy might be ascribed to longer cell exposure times to the transfection agent. Other metal ions, such as strontium ions (Sr2+) and cobalt ions (Co2+), may act synergistically in bone reconstruction. A Sr2+- crosslinked RGD-alginate hydrogel combined with Sr-doped hydroxyapatite microspheres was engineered, showing a sustained release of Sr2+ from two sources (Lourenco et al., 2019). The researchers elaborated that this Sr-hybrid system facilitated MSC osteogenic differentiation, inhibited the functions of osteoclasts and modulated the inflammatory response. As a pro-vasculogenic factor, Co2+ was incorporated into the alginate hydrogel shell, while BMP-2 was laden into the collagen core (Perez et al., 2015). Co2+ released relatively rapidly, as expected. VEGF secretion and qPCR revealed that Co2+ not only stimulated angiogenesis but also elevated osteogenic gene expression. These results indicated an appealing prospect for applying metal ions bone tissue engineering in the future. Small Molecules necrosis factor-a (TNF-a) and interferon-g (IFN-g) pathways (Liu et al., 2011). Statins are inhibitors of a key enzyme of cholesterol synthesis and are widely used to lower serum lipids. Researchers have reported that osteogenesis was enhanced concomitant with promoted BMP-2 expression in bone cells when treating cells and rodents with statins (Mundy et al., 1999). Localized and sustained delivery of these drugs via hydrogels has pointed to a new direction in bone tissue engineering. Many relevant studies are listed and outlined in Table 2. A range of pharmaceutical molecules were designed or discovered to be effective in bone regeneration. Highlighted as chelating agents, BPs are utilized as antiresorptive drugs frequently in clinics. BPs mainly target osteoclasts, impeding the differentiation and maturation of osteoclast progenitors. Increasing evidence has shown that BPs directly or indirectly take part in other bone-forming mechanisms and are capable of targeting various cells (Corrado et al., 2017). Since bone healing and regeneration is known to consist of three consecutive phases of inflammation, repair, and remodeling, a proper scale of immune response is indispensable (Claes et al., 2012). However, excess or aberrant immune activation may jeopardize bone repair procedures (Claes et al., 2012; Gibon et al., 2017). Therefore, immunomodulatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), have been applied in bone tissue engineering. Evidence has shown that aspirin elevates MSC osteogenic potency by inhibiting the tumor Metal Ions Since magnesium ions (Mg2+) play an important role in bone metabolism and mineralization, a variety of strategies for the May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org Zhang et al. Hydrogel-Based Drug Release for BTE A B FIGURE 2 | Schematic illustration of (A) positive feedback mediated by a cofactor-assisted smart hydrogel drug release system and (B) in situ application to promote bone regeneration. Reprinted from a previous article by Zhang et al. (2018) with permission. A A B GURE 2 | Schematic illustration of (A) positive feedback mediated by a cofactor-assisted smart hydrogel drug release system and omote bone regeneration. Reprinted from a previous article by Zhang et al. (2018) with permission. tration of (A) positive feedback mediated by a cofactor-assisted smart hydrogel drug release system and (B) in situ application to Reprinted from a previous article by Zhang et al. (2018) with permission. CONCLUSION AND FUTURE PERSPECTIVES Hydrogel loaded with 10 mg of drug formed the largest area of bone (Tanigo et al., 2010) Rosuvastatin chitosan/chondroitin sulfate nanoparticles+ Pluronic F127/hyaluronic acid hydrogel composite Release rate significantly slower than control groups. 60% released from composite in 48 h Low cytotoxicity, more calcium deposits were observed (Rezazadeh et al., 2019) sustained release systems. Findings (ex/in vivo) Reference Hydrogel containing 10-6 M showed the best augmentation in cell proliferation, osteogenic differentiation, and bone regeneration. (Kim et al., 2017a) Drug bioactivity was preserved by the hydrogel. nSi may contribute to in vivo osteogenesis whereas Dex showed limited effects. (Basu et al., 2018) Percent of mineralized tissue was significantly higher compared to control group. (Fang et al., 2018) Low cytotoxicity, significantly improved expression of osteogenic markers and calvarial defect regeneration. Relatively low local inflammation status might be attributed to being laden with aspirin. (Zhang et al., 2019) Osteoblasts grew and mineralized significantly regardless of drug exhaustion. Osteogenic genes increased over time, while osteogenic suppressing gene expression decreased. (Lin et al., 2018a) More newly-formed bone and blood vessels were observed (Nabavi et al., 2020) Good biocompatibility, stimulated ALP activity and mineralization (Yan et al., 2018) Hydrogel loaded with 10 mg of drug formed the largest area of bone (Tanigo et al., 2010) Findings (ex/in vivo) Released 50% and 90% in 2.5 h and 8 h, respectively Hydrogel loaded with 10 mg of drug formed the largest area of bone (Tanigo et al., 2010) Low cytotoxicity, more calcium deposits were observed (Rezazadeh et al., 2019) and drugs, such as electrostatic interactions and covalent bonds, and other drug reservoirs could be introduced into hydrogels, providing more efficacious drug protection and immobilization. However, negative results have been reported from sustained release systems that did not facilitate bone formation mainly because the carrier exhibited an extremely strong affinity towards the growth factor, resulting in a low level of drug concentration in the surrounding tissue (Hettiaratchi et al., 2017). Thus, optimal drug concentrations should also be determined to achieve a more reasonable and effective release profile. rate, which makes them qualified bone tissue engineering scaffolds. Due to their high water content, chemical inertness and relatively sequestered and stable internal environment, they are also excellent in preserving the viabilities of the laden cells and bioactive factors. CONCLUSION AND FUTURE PERSPECTIVES In this review, we summarized a series of investigations focused on hydrogel-based drug sustained release systems in bone tissue engineering. The hydrogels possess a porous microarchitecture, tunable biophysical parameters, and an adjustable degradation May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org 8 Hydrogel-Based Drug Release for BTE Zhang et al. TABLE 2 | Summary of selected studies on hydrogel-based small bioactive compound sustained release systems. Drug Carrier material Release pattern Findings (ex/in vivo) Reference Alendronate Fibrin hydrogel Steady release rate, cumulative release of approximately 45% over 10 days Hydrogel containing 10-6 M showed the best augmentation in cell proliferation, osteogenic differentiation, and bone regeneration. (Kim et al., 2017a) Dexamethasone (Dex) DNA- 2D silicate nanodisks (nSi) hybrid hydrogel Release rate decreased with higher nSi concentration. Half-time of release was measured from 2.5 to 5.5 days Drug bioactivity was preserved by the hydrogel. nSi may contribute to in vivo osteogenesis whereas Dex showed limited effects. (Basu et al., 2018) Aspirin Thermo-sensitive alginate/ b-TCP hydrogel composite 20% drug released in the first day, 40% in 3 days, slowdown in day 5. Percent of mineralized tissue was significantly higher compared to control group. (Fang et al., 2018) Aspirin Tetra-PEG hydrogel Released approximately 40% in first 2 days, cumulative release of 80% in 14 days Low cytotoxicity, significantly improved expression of osteogenic markers and calvarial defect regeneration. Relatively low local inflammation status might be attributed to being laden with aspirin. (Zhang et al., 2019) Diclofenac CS-coated alginate hydrogel Released 50% and 90% in 2.5 h and 8 h, respectively Osteoblasts grew and mineralized significantly regardless of drug exhaustion. Osteogenic genes increased over time, while osteogenic suppressing gene expression decreased. (Lin et al., 2018a) Tacrolimus Type I collagen hydrogel 21 days release profile remained similar for different concentrations. Steady release rate. More newly-formed bone and blood vessels were observed (Nabavi et al., 2020) Simvastatin Maltodextrin micelle-CHO/ hydrogel composite Slow release profile, exhibiting a slight difference according to different degrees of oxidation Good biocompatibility, stimulated ALP activity and mineralization (Yan et al., 2018) Simvastatin L-lactic acid oligomer (LAo) modified gelatin micelle/ gelatin hydrogel composite Drug released faster as hydrogel crosslinking degree decreased. Release rate showed a good correlation with hydrogel degradation rate. Low cytotoxicity, more calcium deposits were observed (Rezazadeh et al., 2019) Frontiers in Pharmacology | www.frontiersin.org REFERENCES encapsulated mesenchymal stromal cells. Acta Biomater. 101, 249–261. doi: 10.1016/j.actbio.2019.11.016 encapsulated mesenchymal stromal cells. Acta Biomater. 101, 249–261. doi: 10.1016/j.actbio.2019.11.016 Ahlfeld, T., Schuster, F. P., Forster, Y., Quade, M., Akkineni, A. R., Rentsch, C., et al. 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A vast number of GFs and cytokines collaboratively trigger the repair cascade. Extensive studies have already been conducted on multiple bioactive factors controlled release. Spatiotemporal sequence release of bioactive factors might be a better mimic of complex regeneration procedures as well as exert extraordinary synergistic effects on bone regeneration. Various of multiple GFs delivery strategies was coherently summarized (Chen et al., 2010). Nevertheless, controlling dose ratio of drugs to maximize the synergistic effects and manipulating multiple bioactive factors release kinetics to mimic physiological release profile in different phases of bone regeneration are obstacles in nowadays bone tissue regeneration which needs further investigation. Evidence has shown that hydrogel-based drug sustained release systems are highly biocompatible and versatile drug deliverers, obtaining satisfactory osteogenesis results both in vitro and in vivo. The drug release profile varies according to the loading strategy, degradation ability of the matrix and drug concentration. Among these studies, physical entrapment and diffusion are the most applied drug loading and release strategies, respectively. In particular, the dispersion of drugs, ions and small molecules largely depends on hydrogel pore size and crosslinking density. Although it is quite simple and easy to operate, there are difficulties in initial burst release management. Swelling or degradation of hydrogel matrices contributes to polymer mesh size enlargement, resulting in drug release acceleration, especially for macromolecular drugs. Stronger interactions between matrices May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org 9 Hydrogel-Based Drug Release for BTE Zhang et al. FUNDING YZ and TY contributed equally to this review. TY, YW, and BH designed and revised this article. YZ, TY, LP, and QS collected This work was supported by the National Natural Science Foundation of China (51972005, 51672009, 51903003), National Natural Science Foundation of China Youth Fund (81922019), and National Youth Top-notch Talent Support Program (QNBJ2019-3). This work was supported by the National Natural Science Foundation of China (51972005, 51672009, 51903003), National Natural Science Foundation of China Youth Fund (81922019), and National Youth Top-notch Talent Support Program (QNBJ2019-3). the literatures, arranged the outline of collected documents, and wrote the articles. All authors reviewed and commented on the entire manuscript. REFERENCES In Vitro and In Vivo Evaluation of Commercially Available Fibrin Gel as a Carrier of Alendronate for Bone Tissue Engineering. BioMed. Res. Int. 2017, 6434169. doi: 10.1155/2017/6434169 Farokhi, M., Mottaghitalab, F., Shokrgozar, M. A., Ou, K. L., Mao, C., and Hosseinkhani, H. (2016). 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Appl. 99, 919–928. doi: 10.1016/ j.msec.2019.02.024 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Zhang, K., Lin, S., Feng, Q., Dong, C., Yang, Y., Li, G., et al. (2017). Nanocomposite hydrogels stabilized by self-assembled multivalent bisphosphonate-magnesium nanoparticles mediate sustained release of magnesium ion and promote in-situ bone regeneration. Acta Biomater. 64, 389–400. doi: 10.1016/j.actbio.2017.09.039 Copyright © 2020 Zhang, Yu, Peng, Sun, Wei and Han. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Zhang, K., Lin, S., Feng, Q., Dong, C., Yang, Y., Li, G., et al. (2017). Nanocomposite hydrogels stabilized by self-assembled multivalent bisphosphonate-magnesium nanoparticles mediate sustained release of magnesium ion and promote in-situ bone regeneration. Acta Biomater. 64, 389–400. doi: 10.1016/j.actbio.2017.09.039 Zhang, K., Jia, Z., Yang, B., Feng, Q., Xu, X., Yuan, W., et al. (2018). Adaptable Hydrogels Mediate Cofactor-Assisted Activation of Biomarker-Responsive Drug Delivery via Positive Feedback for Enhanced Tissue Regeneration. Adv. Sci. (Weinh) 5 (12), 1800875. doi: 10.1002/advs.201800875 Frontiers in Pharmacology | www.frontiersin.org Zhang, K., Lin, S., Feng, Q., Dong, C., Yang, Y., Li, G., et al. (2017). Nanocomposite hydrogels stabilized by self-assembled multivalent bisphosphonate-magnesium nanoparticles mediate sustained release of magnesium ion and promote in-situ bone regeneration. Acta Biomater. 64, 389–400. doi: 10.1016/j.actbio.2017.09.039 Zhang, K., Jia, Z., Yang, B., Feng, Q., Xu, X., Yuan, W., et al. (2018). Adaptable Hydrogels Mediate Cofactor-Assisted Activation of Biomarker-Responsive Drug Delivery via Positive Feedback for Enhanced Tissue Regeneration. Adv. Sci. (Weinh) 5 (12), 1800875. doi: 10.1002/advs.201800875 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Zhang, K., Jia, Z., Yang, B., Feng, Q., Xu, X., Yuan, W., et al. (2018). Adaptable Hydrogels Mediate Cofactor-Assisted Activation of Biomarker-Responsive Drug Delivery via Positive Feedback for Enhanced Tissue Regeneration. Adv. Sci. (Weinh) 5 (12), 1800875. doi: 10.1002/advs.201800875 May 2020 | Volume 11 | Article 622 Frontiers in Pharmacology | www.frontiersin.org 13
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ANALISIS PENETAPAN HARGA KAYU JATI PLUS PERHUTANI BERDASARKAN STUMPAGE COST DAN WILLINGNESS TO PAY
Risalah kebijakan pertanian dan lingkungan
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ANALISIS PENETAPAN HARGA KAYU JATI PLUS PERHUTANI BERDASARKAN STUMPAGE COST DAN WILLINGNESS TO PAY Arga Pramudita1*, Suryanaji2 1 Kelompok Peneliti Industri dan Pemasaran, Puslitbang Perhutani 2 Biro Perencanaan dan Pengembangan Bisnis, Divisi Regional Jawa Tengah Puslitbang Perhutani, Jl. Wonosari, Batokan, Cepu, 58302 * Email: argapram@gmail.com Arga Pramudita1*, Suryanaji2 1 Kelompok Peneliti Industri dan Pemasaran, Puslitbang Perhutani 2 Biro Perencanaan dan Pengembangan Bisnis, Divisi Regional Jawa Tengah Puslitbang Perhutani, Jl. Wonosari, Batokan, Cepu, 58302 * Email: argapram@gmail.com Kata Kunci: Harga Pokok Produksi, Jati, Konsumen, Market Value, Perhutani. Kata Kunci: Harga Pokok Produksi, Jati, Konsumen, Market Value, Perhutani. Kata Kunci: Harga Pokok Produksi, Jati, Konsumen, Market Value, Perhutani. 2017 seluas ± 200.000 ha. Potensi kayu penjarangan dari luasan tanaman JPP dalam kondisi baik, dengan asumsi hasil penjarangan sebesar 4 m3/ha, dapat mencapai ±170.000 m3. 2017 seluas ± 200.000 ha. Potensi kayu penjarangan dari luasan tanaman JPP dalam kondisi baik, dengan asumsi hasil penjarangan sebesar 4 m3/ha, dapat mencapai ±170.000 m3. Risalah Kebijakan Pertanian dan Lingkungan Vol. 8 No. 2 Agustus 2021: 53-59 ISSN : 2355-6226 E-ISSN : 2477-0299 Risalah Kebijakan Pertanian dan Lingkungan Vol. 8 No. 2 Agustus 2021: 53-59 ISSN : 2355-6226 E-ISSN : 2477-0299 RINGKASAN Jati Plus Perhutani (JPP) sebagai salah satu hasil program pemuliaan pohon memiliki beberapa keunggulan komparatif dibandingkan dengan jenis jati yang dikembangkan melalui perbanyakan konvensional dengan biji. Keunggulan dalam hal keliling batang dan produktivitas yang tinggi dalam jangka waktu yang relatif singkat akan memberikan biaya pembangunan tegakan yang minimal sehingga harga juga tidak terlalu tinggi. Penelitian ini bertujuan untuk mengetahui harga pokok produksi kayu JPP hasil penjarangan melalui pendekatan biaya selama daur dan pendekatan pasar (market value). Metode yang digunakan adalah pendekatan nilai tegakan (stumpage cost) dan metode Willingness to Pay (WTP) yang terdiri dari survei pelanggan dengan kuisioner dan in depth interview dengan sistematik random sampling. Hasil penelitian menunjukkan nilai pembangunan tegakan JPP selama 9 tahun sebesar Rp. 20.024.309,-/ha, dengan harga pokok penjualan sortimen AI sebesar Rp. 642.538,- dan sortimen AII sebesar Rp. 881.214,-. Sedangkan berdasarkan pendekatan pasar, konsumen menginginkan harga kayu JPP tidak jauh berbeda dengan kayu jati asal hutan rakyat atau penurunan harga sebesar 13% - 22%. Rekomendasi kebijakan yang disarankan terhadap penentuan harga kayu JPP adalah perlu pemisahan dan pembedaan harga jual dasar kayu JPP supaya dapat bersaing dengan harga kayu rakyat dan dapat menjadi salah satu sumber pendapatan perusahaan dalam waktu yang tidak terlalu lama. PERNYATAAN KUNCI • Jati Plus Perhutani sebagai produk hasil penelitian dan pengembangan Puslitbang Perhutani dan sudah ditanam secara operasional mulai tahun 2007 sampai dengan tahun 53 53 Rumusan Kajian Strategis Bidang Pertanian dan Lingkungan ISSN : 2355 – 6226 E-ISSN : 2477 – 0299 dan furniture menyebabkan permintaan kayu jati semakin meningkat. Seiring dengan permintaan yang semakin meningkat maka otomatis nilai ekonomi kayu jati juga semakin tinggi. Perum Perhutani yang sampai dengan saat ini masih mengelola hutan jati di Pulau Jawa mempunyai peluang untuk meningkatkan produktivitas hutan jatinya yaitu dengan mengembangkan klon unggul yaitu Jati Plus Perhutani (JPP). • Harga Jual Dasar (HJD) merupakan besaran harga yang ditetapkan oleh Direksi Perum Perhutani untuk kepentingan penjualan kontrak dan penjualan langsung secara retail. Harga ini tertera dalam saluran penjualan di Penjualan Online Toko Perhutani yang merupakan e- commerce Perum Perhutani dengan akses melalui tokopedia.com. Jati Plus Perhutani sebagai produk hasil pemuliaan pohon yang dilakukan oleh Puslitbang Perum Perhutani, memiliki keunggulan di antaranya pertumbuhan tegakan lebih cepat, seragam dan kayu yang berkualitas. Dengan keunggulan tanaman JPP ini maka diharapkan dengan penerapan umur tebang yang lebih awal dan lebih cepat bila dibandingkan umur tebang pada jati konvensional tetap dapat dilakukan tanpa mengabaikan faktor ekologis tanaman. Kebijakan harga jual dasar kayu JPP masih disamakan dengan harga jual kayu jati konvensional sehingga konsumen kayu jati cenderung lebih memilih kayu jati konvensional, sehingga kayu JPP dianggap sebagai produk slow moving karena kurang diminati pasar. REKOMENDASI KEBIJAKAN • Perlu dilakukan penetapan harga khusus terhadap kayu JPP, dengan membedakan terhadap kayu jati konvensional yang ada di Tempat Pengumpulan Kayu. Penentuan harga kayu jati saat ini masih dipengaruhi oleh mekanisme pasar dan belum mencerminkan harga pokok produksinya. Dari hal tersebut maka perlu kiranya mengetahui berapa besar biaya yang dikeluarkan untuk memproduksi kayu JPP dari kegiatan pengelolaan awal hingga pemasaran kayunya sampai dengan umur tertentu. Harga Pokok Produksi kayu JPP ini bisa berbeda-beda antara KPH satu dengan yang lain tergantung karakteristik wilayah dan biaya yang dikeluarkan. Hal ini sesuai seperti yang dijelaskan oleh Davis • Dalam hal perencanaan dan penataan wilayah hutan, perlu dibuat cluster kayu JPP yang ditujukan untuk produksi kayu khusus sortimen AI dan AII untuk memenuhi kebutuhan kayu di sentra industri kecil dan menengah. I. PENDAHULUAN Sifat kayu jati yang banyak diminati masyarakat sebagai bahan baku bangunan 54 Risalah Kebijakan Pertanian dan Lingkungan Vol. 8 No. 2 Agustus 2021 (1954 dan 1987) dalam Andayani (1998) bahwa nilai tegakan (stumpage) di masing- masing lokasi produsen sebenarnya berbeda (situasional) sehingga tidak dapat ditetapkan secara sama (satu harga). Perbedaan tersebut antara lain terletak pada beberapa parameter dan faktor-faktor yang mempengaruhi nilai tegakan yaitu daur ekonomi, keadaan hutan, geografis hutan dan komponen biaya pembentukannya. Penelitian ini bertujuan untuk mengetahui harga pokok produksi kayu JPP hasil penjarangan melalui pendekatan biaya selama daur dan pendekatan pasar (market value). penjarangan pada petak lain, sebagaimana disajikan pada Tabel 1. Tabel 1. Hasil penjarangan perhutanan klon JPP tahun 2017 No Lokasi Realisasi (m3) Produktivitas (m3) Keterangan 1. KPH Ngawi 143,49 30,53 Tahun tanam 2004 2. KPH Tuban 134,17 11,37 3. KPH Cepu 214,12 25,49 Tabel 1. Hasil penjarangan perhutanan klon JPP tahun 2017 Kayu hasil penjarangan di atas di dominasi oleh sortimen AI, kelas diameter 16 – 19 cm, mutu T, panjang 2 meter up. Harga rata-rata berdasarkan harga jual dasar yang ditetapkan oleh Perum Perhutani tahun 2018 adalah sebesar Rp. 1.225.000,- untuk sortimen AI dan Rp. 2.100.000,- untuk sortimen AII (mutu T, kelas diameter 16 – 19 cm dan 24 – 26 cm, panjang 2,00 – 2,90 meter). II. SITUASI TERKINI Jati Plus Perhutani mulai dikembangkan secara luas adalah JPP PHT 1 dan PHT 2 mulai tahun 2007. Sampai dengan tahun 2017, sudah ditanam seluas ± 200.000 ha, di seluruh wilayah Perum Perhutani, sedangkan yang dikelola oleh Puslitbang seluas 446,7 ha. Jati Plus Perhutani klon PHT 1 dan PHT 2 juga telah mendapatkan hak perlindungan varietas tanaman dari Kementerian Pertanian tahun 2009. Uji coba penebangan penjarangan Jati Plus Perhutani dilakukan pada tahun 2013, di petak 61a, RPH Kedunggalar, BKPH Kedunggalar, KPH Ngawi dan menghasilkan ume kayu sebesar 150,5 m3/ha, jumlah pohon rata-rata 597 pohon/ha dengan diameter rata-rata 23,1 cm (Wibowo, 2013). Hasil uji coba III. METODOLOGI Penelitian dilakukan tahun 2017, di wilayah Kesatuan Pemangkuan Hutan (KPH) Ngawi, Divisi Regional Jawa Timur. Harga Pokok Produksi ditentukan dengan melalui pendekatan nilai tegakan (stumpage value) dalam Rp/ha. Mekanisme analisis menggunakan metode perhitungan biaya tetap yang dikeluarkan selama daur. Data sekunder yang digunakan adalah laporan keuangan unit kerja pendukung yaitu Kantor Pusat, Kantor Divisi Regional, Kantor Puslitbang, Kantor Pusdikbang, Kantor Biro Perencanaan, dan Kantor KBM Penjualan. 55 Rumusan Kajian Strategis Bidang Pertanian dan Lingkungan ISSN : 2355 – 6226 E-ISSN : 2477 – 0299 Rumusan Kajian Strategis Bidang Pertanian dan Lingkungan ISSN : 2355 – 6226 E-ISSN : 2477 – 0299 Seluruh biaya pembentukan tegakan JPP dibawa ke satuan hektar (Rp/ha) dan dihitung dengan biaya yang dikeluarkan selama daur tanaman JPP yang sudah dilakukan penebangan yaitu umur 9 tahun. Kaitannya dengan konsep joint cost, besarnya biaya yang dialokasikan untuk JPP dihitung dengan pendekatan luas penggunaan lahan untuk tanaman jati. Selanjutnya Untuk mendapatkan nilai harga pokok produksi kayu bundar tiap sortimen, hasil penghitungan biaya tegakan sebelum ditebang ditambah dengan biaya eksploitasi, biaya pemasaran (Rp/m3) dan Provisi Sumber Daya Hutan tiap sortimen (Anonim, 2009). pengolahan dan pemasaran hasil hutan. Perhitungan untuk mengetahui biaya pembangunan tegakan dilakukan dengan analisis finansial menggunakan compounding factor yang disusun dalam bentuk cashflow selama daur dikaitkan dengan premi produksi kayu yang dihasilkan saat pemanenan hasil hutan. Analisis finansial dilakukan dengan menghimpun biaya per masing-masing kegiatan yang dikeluarkan, berdasarkan pada laporan keuangan di KPH Ngawi. Biaya yang dikeluarkan dibagi dalam 3 kategori yaitu biaya investasi langsung, biaya prasarana fisik dan biaya rutin. Biaya investasi langsung terdiri dari biaya perencanaan keluar setiap 10 tahun sekali, biaya persemaian dan biaya tanaman, keluar 1 kali selama daur, biaya pemeliharaan, keluar mulai tahun pertama sampai tahun ke empat, dan biaya perlindungan yang dikeluarkan setiap tahun sampai dengan umur tebang. Data primer yang digunakan untuk mengetahui preferensi konsumen terhadap harga kayu JPP diperoleh dari hasil wawancara terhadap pembeli yang melakukan pembelian kayu JPP di wilayah KBM penjualan Madiun sebanyak 30 responden. Pengambilan sampel responden dilakukan dengan sistematik random sampling dengan pembagian tipe pembeli berdasarkan jenis pembeli perorangan, perusahaan dan trader/agen. Biaya prasarana fisik terdiri atas biaya pemeliharaan dan penyusutan sarana prasarana yaitu kendaraan, tanah dan bangunan, peralatan kantor dan bengkel instalasi. Biaya rutin terdiri atas biaya yang dikeluarkan secara rutin setiap tahun sampai dengan waktu tebang, yang terdiri atas biaya pajak, biaya umum dan administrasi, biaya gaji, premi produksi dan biaya pendidikan dan penyuluhan. IV. ANALISIS DAN ALTERNATIF SOLUSI Perhitungan HPP Kayu JPP KPH Ngawi Tahun 2017 Biaya selama daur Umur tebang HPP 20.024.309 9 tahun AI AII AIII Harga Pokok Produksi di tegakan 123.987 272.928 - Biaya eksploitasi 250.000 250.000 - Biaya pemasaran 59.043 59.043 - PSDH 72.000 114.000 - Biaya pengujian 9.000 9.000 Sharing PHBM (25%) 128.508 176.243 Jumlah 642.538 881.214 - Jumlah biaya rutin, prasarana fisik dan biaya unit kerja penunjang yang lebih kecil daripada biaya investasi langsung juga disebabkan karena dalam tebangan JPP ini dilakukan pada umur 9 tahun. Tabel 2. Perhitungan HPP Kayu JPP KPH Ngawi Tahun 2017 Biaya selama daur Umur tebang HPP 20.024.309 9 tahun AI AII AIII Harga Pokok Produksi di tegakan 123.987 272.928 - Biaya eksploitasi 250.000 250.000 - Biaya pemasaran 59.043 59.043 - PSDH 72.000 114.000 - Biaya pengujian 9.000 9.000 Sharing PHBM (25%) 128.508 176.243 Jumlah 642.538 881.214 - rakyat. Hal ini karena anggapan konsumen bahwa kayu jati JPP secara mutu hampir sama dengan kayu rakyat. Biaya pembangunan tegakan yang terbentuk untuk tegakan tanaman JPP umur 9 tahun adalah Rp. 20.024.309,-/ha. Untuk mengetahui biaya produksi maka biaya pembangunan tegakan ini dibagi dengan realisasi produksi kayu JPP yang dihasilkan. Perincian perhitungan ditunjukkan pada Tabel 2. Tabel 3. Prosentase penurunan harga kayu JPP di wilayah KBM Jawa Timur Unit kerja Prosentase Penurunan Harga Berdasarkan Keinginan Konsumen AI (%) AII (%) AIII (%) Jawa Timur 15,5 ± 2,34 20,6 ± 2,74 - Tabel 3. Prosentase penurunan harga kayu JPP di wilayah KBM Jawa Timur Unit kerja Prosentase Penurunan Harga Berdasarkan Keinginan Konsumen AI (%) AII (%) AIII (%) Jawa Timur 15,5 ± 2,34 20,6 ± 2,74 - Tabel 3. Prosentase penurunan harga kayu JPP di wilayah KBM Jawa Timur Unit kerja Prosentase Penurunan Harga Berdasarkan Keinginan Konsumen AI (%) AII (%) AIII (%) Jawa Timur 15,5 ± 2,34 20,6 ± 2,74 - Tabel 3. Prosentase penurunan harga kayu JPP di wilayah KBM Jawa Timur Harga Pokok Produksi Kayu JPP di KPH Ngawi hasil tebangan petak 61a, berdasarkan perhitungan diperoleh biaya produksi kayu AI sebesar Rp. 642.538,- dan sortimen AII sebesar Rp. 881.214,-. Nilai ini merupakan harga dimana masih merupakan titik impas, sehingga belum ada keuntungan, sehingga perlu ditambah dengan profit margin yang diinginkan. Dari hasil ini maka dapat memberikan gambaran bahwa harga kayu JPP masih perlu dikaji untuk diturunkan sampai dengan batas tertentu di mana pengusaha/ konsumen masih mampu mendapat membeli dengan perbandingan asal kayu rakyat. IV. ANALISIS DAN ALTERNATIF SOLUSI Komponen biaya pembangunan suatu hutan tanaman industri secara garis besar meliputi kegiatan perencanaan, penanaman, penebangan kayu atau pemungutan, pemeliharaan hutan, 56 Risalah Kebijakan Pertanian dan Lingkungan Vol. 8 No. 2 Agustus 2021 Risalah Kebijakan Pertanian dan Lingkungan Risalah Kebijakan Pertanian dan Lingkungan Vol. 8 No. 2 Agustus 2021 Biaya prasarana fisik dan biaya rutin merupakan biaya bersama atau joint cost. Joint cost adalah biaya yang dikeluarkan untuk d b y y g d Gambar 1. Persentase biaya produksi kayu JPP di KPH Ngawi Gambar 1. Persentase biaya produksi kayu JPP di KPH Ngawi menghasilkan dua macam produk atau lebih yang berlainan. Dalam perhitungan beban biaya untuk tanaman JPP di KPH Ngawi, perhitungan joint cost didasarkan pada kontribusi luas tanaman JPP di KPH Ngawi terhadap luas hutan produksi di KPH Ngawi. unit kerja dihitung berdasarkan kontribusi luas tanaman JPP dibandingkan dengan luas hutan produksi jati di masing-masing unit kerja. Bila dilihat besarnya komponen- komponen penyusun biaya produksi kayu JPP, biaya paling tinggi yaitu di komponen biaya investasi langsung yang terdiri dari biaya penyusunan RPKH, biaya persemaian, biaya tanaman, biaya pemeliharaan dan biaya perlindungan. Untuk lebih jelasnya dalam Gambar 1. Biaya produksi kayu JPP juga merupakan bagian dari biaya prasarana fisik dan rutin yang dikeluarkan oleh unit kerja yang mendukung kegiatan pengelolaan sumber daya hutan di KPH yaitu Kantor Pusat, Puslitbang, Pusdikbang, Divisi Regional Jawa Timur, biro perencanaan dan divisi komersial kayu. Biaya unit kerja pendukung ini juga merupakan joint cost dengan komoditi lainnya. Pembebanan biaya yang dikeluarkan oleh masing-masing Distribusi biaya pembangunan tegakan JPP dimana komposisi biaya investasi langsung yang berkaitan erat dengan kegiatan pengelolaan tanaman JPP sudah cukup baik, karena hampir sebagian besar biaya digunakan untuk tanaman JPP. 57 57 Rumusan Kajian Strategis Bidang Pertanian dan Lingkungan Jumlah biaya rutin, prasarana fisik dan biaya unit kerja penunjang yang lebih kecil daripada biaya investasi langsung juga disebabkan karena dalam tebangan JPP ini dilakukan pada umur 9 tahun. Tabel 2. IV. ANALISIS DAN ALTERNATIF SOLUSI Standar deviasi yang masih relatif kecil disebabkan karena variasi responden tidak terlalu besar, hanya dikerucutkan kepada pembeli kayu JPP yang merupakan segmen pembeli menengah sampai dengan kecil. Untuk mengetahui harga beli maksimum yang masih bisa diterima oleh Hasil survei responden di wilayah penjualan Jawa Timur, menunjukkan hasil bahwa konsumen kayu menginginkan penurunan harga terhadap kayu JPP agar minimal tidak berbeda jauh dengan kayu 58 Vol. 8 No. 2 Agustus 2021 Risalah Kebijakan Pertanian dan Lingkungan pembeli secara riil maka nilai persentase kenaikan harga tersebut dikalikan dengan harga jual dasar pada setiap kelas diameter, kelas panjang dan kelas mutu yang ditetapkan. Asri, M. 1991. Marketing. Unit Penerbit dan Percetakan AMP YKPN. Yogyakarta. Priyanto, R. 2004. Analisis Distribusi Kayu Jati Rakyat di Kabupaten Gunungkidul. Skripsi S1. Fakultas Kehutanan. Universitas Gadjah Mada. Yogyakarta. Sofyan, P., Warsito. 1995. Penaksiran Biaya dan Pendapatan Pengusahaan Hutan Tanaman. Bahan Kuliah Analisis Bisnis Pengusahaan Hutan Program Pasca Sarjana. Universitas Gadjah Mada. Yogyakarta. REFERENSI Andayani, W. 1998. Sistem Distribusi dan Penetapan Harga Kayu Bulat Jati di Jawa. Disertasi S3 Program Pasca Sarjana. Institut Pertanian Bogor. Bogor. Wibowo, A. 2013. Uji Coba Tebangan Kayu JPP Asal Stek Pucuk (Studi kasus petak 61a, BKPH Kedunggalar, KPH Ngawi). Laporan Pengamatan. Cepu. Cepu. Anonim. 2009. Analisis Finansial Penerapan Harga Pokok Produksi Kayu Bundar Jati Perhutani. Laporan Penelitian. Puslitbang Perum Perhutani Cepu. Cepu. 59 59
https://openalex.org/W3187247888
http://ijec.ejournal.id/index.php/counseling/article/download/166/134
Indonesian
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Konseling Religi dalam Upaya Menemukan Kebermaknaan Hidup Remaja Korban Broken Home
Indonesian journal of educational counseling
2,021
cc-by
5,151
1 Corresponding author’s address: Program Studi Interdisciplinary Islamic Studies, Konsentrasi Bimbingan Konseling Islam Pascasarjana, UIN Sunan Kalijaga Yogyakarta, Indonesia. E-mail: 2020001107@student.uin- suka.ac.id ABSTRACT RELIGIOUS COUNSELING IN AN EFFFORT TO FIND A MEANINGFULL LIFE FOR TEENAGER VICTIMS OF BROKEN HOME. The purpose of this research is to explain the phenomenon of teenage victims of broken home in their efforts to find meaning in life through religious counseling services. The type of this research is phenomenological qualitative. Data collection techniques were carried out by observation and interviews. The data analysis technique used intra-case and inter-case analysis. The results showed that the reasons for these adolescents to attend religious counseling in order to find meaning in their lives included; forgetting the disappointments they face, finding wisdom from the problems that occur, finding meaning in life, and preparing for a new life. Religious counseling is carried out through several activities such as reading the Koran, listening to lectures, reading Islamic and motivational books. Received: 28.05.2021 Received in revised form: 01.07.2021 Accepted: 08.07.2021 Available online: 30.07.2021 KEYWORDS:1Broken Home, Counseling, Meaningfull Life, Religion, Teenager. KEYWORDS:1Broken Home, Counseling, Meaningfull Life, Religion, Teenager. DOI: 10.30653/001.202152.166 This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2021 Rois Nafi'ul Umam, Maemonah. Indonesian Journal of Educational Counseling is published by FKIP of Universitas Mathla’ul Anwar Banten Indonesian Journal of Educational Counseling, 2021, Vol. 5 No. 2, Page: 64-74 Open Access | Url: http://ijec.ejournal.id/index.php/counseling/article/view/166 Indonesian Journal of Educational Counseling, 2021, Vol. 5 No. 2, Page: 64-74 Open Access | Url: http://ijec.ejournal.id/index.php/counseling/article/view/166 http://ijec.ejournal.id PENDAHULUAN Masa remaja merupakan bagian dari transisi perjalanan hidup manusia dari masa kanak-kanak menuju masa dewasa (Diananda, 2019). Dalam masa transisi tersebut, terjadi perubahan secara mendasar pada sisi jasmani maupun rohani remaja. Menurut Rice, remaja dapat diartikan dengan masa peralihan individu yang tumbuh dari masa kanak- kana menuju masa dewasa yang penuh dengan kematangan, baik secara fisik maupun psikis (Saputro, 2018). Secara fisik, masa remaja dapat ditandai dengan beberapa ciri seperti mulai matangnya organ reproduksi, mengalami menstruasi/mimpi basah, perubahan suara menjadi lebih berat dan munculnya jerawat pada area wajah. Adapun secara psikis, remaja dapat ditandai dengan ciri yang diantaranya memiliki sikap agresif, senang mencoba hal-hal baru, mengalami emosi yang tidak stabil, mulai mencari identitas diri serta memiliki perasaan yang sensitif (Jannah, 2017). Secara psikologis, masa remaja Indonesian Journal of Educational Counseling is published by FKIP of Universitas Mathla’ul Anwar Banten 64 Rois Nafi'ul Umam, Maemonah dapat dikatakan sebagai masa yang labil, yaitu mereka belum sepenuhnya dapat mengendalikan dirinya sendiri secara emosi dan hanya mengikuti naluri dan keingintahuannya tanpa memikirkan dampak yang akan terjadi setelah melakukan hal tersebut. Oleh karenanya dalam masa perkembangannya, remaja perlu untuk dibimbing dan diarahkan oleh orang tuanya agar terhindar dari sikap dan perilaku yang buruk tanpa membatasi dirinya untuk bisa maju dan berkembang (Verawati, 2017). Salah satu faktor yang berperan penting dalam perkembangan remaja adalah orang tua. Sosok orang tua yang telah melahirkan dan membesarkannya dengan sepenuh hati menjadi pihak yang dapat menentukan perilaku atau arah masa depan yang hendak dicapai oleh remaja tersebut. Keluarga yang bahagia dan harmonis merupakan keluarga yang diidamkan oleh remaja, karena ia dapat merasakan kasih sayang dan perhatian yang selayaknya ia dapatkan dari orang tuanya (Muniriyanto & Suharnan, 2014). Selain itu dalam keluarga yang bahagia dan harmonis, remaja tersebut diperlakukan dengan selayaknya seorang anak yang mendapatkan bimbingan dan pengasuhan yang baik dari orang tua. Untuk mencapai kebahagiaan dan keharmonisan dalam keluarga, setiap anggota keluarga memiliki peran penting untuk mewujudkan hal tersebut, baik itu ayah, ibu maupun anak-anaknya (Arintina & Fauziah, 2015). Peran tersebut dijalankan sebagaimana kedudukan mereka dalam rumah tangga, ayah yang berusaha untuk mencari penghidupan yang layak, seorang ibu yang mengurus rumah tangga dan membimbing anak-anaknya, serta anak-anak yang menjalankan tugas dan tanggung jawab yang diberikan dengan baik dan membahagiakan kedua orang tuanya. PENDAHULUAN Melalui keluarga yang bahagia dan harmonis, setiap anggota keluarga di dalamnya akan merasakan damai dan penuh cinta kasih antar sesama anggota keluarga serta dapat mencapai tujuan hidup yang telah ditetapkan oleh masing-masing anggota keluarga (Isminayah & Supandi, 2016). Akan tetapi tidak semua keluarga berada pada kondisi yang diliputi dengan kebahagiaan dan keharmonisan. Dalam keluarga tersebut terjadi konflik internal yang menyebabkan renggangnya hubungan antar anggota keluarga. Konflik tersebut dapat diakibatkan oleh permasalahan dalam keluarga yang tidak terselesaikan dengan baik (Isminayah & Supandi, 2016). Dari konflik internal dalam keluarga tersebut, dapat menghantarkan keluarga tersebut pada perceraian akibat masalah-masalah yang ada tersebut tidak menemui titik temu solusinya. Akibat dari perceraian tersebut, remaja akan kehilangan salah satu sosok dari orang tuanya karena mereka akan tinggal dengan salah satu diantara keduanya (Dewanti & Ediati, 2016). Secara psikis, remaja tersebut merasa kehilangan orang yang penting dalam hidupnya dan merasakan dampak negatif dari perceraian tersebut. Akibat dari broken home yang terjadi, remaja tersebut akan mengalami gangguan psikologis, seperti merasa insecure, perasaan takut menjadi penyebab perceraian yang dilakukan oleh orang tuanya, merasa dirinya tidak berharga dan depresi yang dapat berujung pada bunuh diri (Ramadhani & Krisnani, 2019). Remaja yang menjadi korban broken home tersebut dapat kehilangan motivasi untuk hidup serta mencari pelarian lain untuk mengekspresikan perasaannya dengan melakukan perilaku yang merugikan dirinya sendiri atau orang lain. Fenomena remaja yang menjadi korban dari broken home dan berdampak secara psikologis terhadap pribadi mereka, menjadi sebuah fenomena yang amat perlu untuk dikaji secara mendalam berkaitan dengan kebermaknaan hidup dari remaja tersebut (Wulandri & Fauziah, 2019). Hal ini dikarenakan para remaja yang menjadi korban broken home tersebut merasa hidupnya hampa, kosong dan tidak bermakna karena kehilangan salah satu sosok orang tua yang selama ini telah mendampinginya dan membimbingnya 65 INDONESIAN JOURNAL OF EDUCATIONAL COUNSELING 2021, 5(1), 64-74 dari kecil. Kehampaan dan ketidak berhargaan dalam diri dapat ditunjukkan dalam bentuk hilangnya semangat untuk menjalani hidup, munculnya perasaan yang absurd, merasa tidak berdaya dan putus asa (Aziz, 2015). Dalam diri remaja tersebut, ia berusaha untuk tetap kuat untuk menghadapi kondisi yang tidak ia harapkan tersebut dan berusaha mencari nilai positif dari kejadian tersebut. Selain itu remaja tersebut akan mencari makna dari setiap perjalanan hidupnya. Akan tetapi tidak semua remaja yang menjadi korban broken home memiliki kemampuan dan menjalani usaha tersebut sehingga ia larut dalam permasalahan yang dapat berdampak secara psikologis terhadap pribadinya tersebut (Hafiza & Mawarpury, 2018). PENDAHULUAN Kebermaknaan hidup merupakan sebuah nilai yang mendorong terbentuknya motivasi yang kuat untuk dapat menjalani hidupnya dengan baik, melakukan kegiatan yang berguna serta memberi manfaat kepada diri sendiri dan orang lain. Kebermaknaan hidup dapat dicapai apabila mencapai beberapa indikator meliputi menemukan arti dalam kehidupan yang cocok dengan dirinya, mampu memberi dan menerima cinta serta memiliki tekad yang kuat untuk menjalani hidup (Siddik, Oclaudya, Ramiza, & Nashori, 2018). Upaya untuk menemukan keberhargan diri dapat dimaknai dengan mengubah kondisi hidup yang tidak bermakna dan mengarahkannya pada hidup yang bermakna. Untuk mencapai kebermaknaan hidup, remaja harus melewati beberapa tahap penting yang diantaranya tahap derita, tahap penerimaan diri, tahap pemaknaan hidup, tahap realisasi makna dan tahap kehidupan bermakna. Kebermaknaan hidup individu dapat dipengaruhi oleh dua faktor yaitu secara internal maupun eksternal. Faktor internal meliputi pola berpikir, sikap, konsep diri, kepercayaan dan kepribadian. Adapun faktor eksternal diantaranya adalah pekerjaan, pengalaman, hubungan dalam keluarga dan kebudayaan lokal (Mazaya & Supradewi, 2020). Dalam konteks remaja yang menjadi korban broken home, hubungan antar anggota keluarga yang tidak harmonis dan berujung pada perceraian membuat remaja korban broken home tidak memiliki kebermaknaan dalam hidup, merasa hampa dan tidak semangat dalam menjalani hidup. Para remaja tersebut menghendaki kehidupan yang berjalan dengan semestinya, akan tetapi hal tersebut tidak ia dapatkan ketika remaja tersebut menghadapi kondisi yang tidak ideal yaitu broken home. Dalam penelitian lainnya yang dilakukan oleh Deviana Citra Dewi Widyawati menunjukkan bahwa tidak semua remaja larut dalam kesedihan dan menjadikan dirinya hidup dengan ketidakbermaknaan ketika mengalami broken home. Mayoritas remaja yang menjadi subjek penelitian tersebut memiliki makna hidup yang baik dan tetap semangat dalam menjalani hidupnya (Widyawati, 2009). Hal ini berbanding terbalik dengan penelitian yang dilakukan oleh Yuniar Asih yang menyatakan bahwa kebermaknaan hidup bagi remaja broken home hanya dirasakan oleh sedikit remaja dan lebih banyak remaja yang merasakan ketidak bermaknaan dalam hidupnya (Mustikarani, 2013). Adapun perbedaan dengan beberapa penelitian diatas, penelitian yang dilakukan oleh peneliti berfokus pada upaya menemukan keberhargaan diri bagi remaja korban broken home melalui konseling religi yang dilaksanakan di rumah konseling mutiara hati. Dalam hal ini remaja yang menjadi subjek penelitian akan berusaha untuk menemukan keberhargaa melalui tahapan-tahapan dalam konseling religi yang dilaksanakan di rumah konseling mutiara hati. Konseling religi merupakan proses pemberian bantuan yang dilakukan oleh konselor kepada konseli yang sedang mengalami permasalahan agar konseli tersebut dapat menyelesaikan masalah yang sedang dihadapinya. METODE Jenis penelitian yang digunakan adalah penelitian lapangan (field research) yang dilaksanakan di rumah konseling keluarga mutiara hati. Adapun metode yang digunakan dalam penelitian ini adalah kualitatif fenomenologis. Hal ini dikarenakan peneliti berfokus untuk mengamati dan menggambarkan dari kebermaknaan hidup yang dikehendaki oleh remaja korban broken home melalui pendekatan kualitatif fenomenologi ini. Teknik pengumpulan data dilakukan dengan observasi dan wawancara secara singkat terhadap subjek penelitian ini yaitu remaja korban broken home. Melalui teknik pengumpulan data tersebut, peneliti dapat mengetahui riwayat pribadi dan permasalahan yang dihadapinya, serta proses yang dihadapi oleh subjek penelitian untuk menemukan kebermaknaan hidup. Jumlah subjek dalam penelitian ini sebanyak 2 orang, yaitu konselor berjumlah 1 orang dan konseli sebanyak 1 orang. Teknik analisis data dilakukan dengan teknik analisis intra kasus dan antar kasus untuk memperoleh gambaran umum mengenai kehidupan pribadi subjek penelitian, permasalahan yang dialami oleh subjek penelitian, dan upaya dalam menemukan kebermaknaan hidup melalui konseling religi yang diikutinya. PENDAHULUAN Dalam konseling religi, nilai- nilai dasar dalam agama dan kepercayaan akan diserap dan diadaptasi dalam layanan 66 Rois Nafi'ul Umam, Maemonah konseling, sehingga dapat membentuk nilai yang dapat dipahami oleh konseli dan dapat mengembangkan dirinya berlandaskan nilai-nilai moral keagamaan (Maulana & Hernisawati, 2017). Melalui konseling religi, diharapkan konseli dapat mengembangkan potensi diri, kemampuan berpikir, keimanan dan ketakwaan untuk mencapai pribadi yang baik berlandaskan pada nilai-nilai keagamaan. Dari latar belakang di atas, penelitian ini akan berfokus pada alasan dari remaja yang menjadi korban broken home tersebut untuk mengikuti konseling religi dalam rangka menemukan kebermaknaan hidup dan proses yang dilalui dalam konseling religi untuk mencapai kebermaknaan hidup. Adapun rumusan masalah dalam penelitian ini adalah apa alasan dari remaja tersebut untuk mengikuti konseling religi dalam mencapai kebermaknaan hidup? Bagaimana proses dalam konseling religi untuk mencapai kebermaknaan hidup? HASIL DAN PEMBAHASAN Broken home merupakan retaknya rumah tangga/keluarga yang telah terbangun yang terjadi akibat anggota keluarga yang tidak dapat menjalankan peran sosial atau menjalankan kewajibannya. Broken home terjadi dalam bentuk tanpa hadirnya salah satu dari orang tua, baik ayah ataupun ibu akibat dari perceraian atau meninggalkan rumah. Broken home dalam keluarga dapat terjadi akibat pertengkaran antar anggota keluarga dan tidak adanya sikap dan bentuk kasih sayang dalam lingkungan keluarga (Novianto, Zakso, & Salim, 2017). Keluarga yang mengalami broken home dapat diidentifikasi dengan beberapa ciri yang diantaranya adalah orang tua bercerai, hubungan antara ayah dan ibu atau antara orangtua dengan anak yang kurang baik, serta suasana dalam rumah yang tegang karena penuh dengan masalah serta tidak ada upaya untuk bersama-sama menyelesaikan masalah tersebut (Muttaqin & Sulistyo, 2019). Kondisi keluarga yang mengalami broken home yang diliputi dengan ketegangan dan konflik antar anggota keluarga berdampak secara psikologis terhadap anggota keluarga lainnnya. Salah satu yang merasakan dampak langsung secara psikologis dari kasus broken home adalah anak/remaja. Seorang remaja yang seharusnya mendapatkan arahan dan bimbingan dari 67 INDONESIAN JOURNAL OF EDUCATIONAL COUNSELING 2021, 5(1), 64-74 orang tuanya, justru dihadapkan dengan kasus broken home yang terjadi pada keluarganya. Dampak negatif yang dirasakan oleh remaja dari kasus broken home adalah menjadi tidak percaya diri, memiliki emosi yang tidak stabil, rentan menjadikannya berperilaku agresif, kehilangan motivasi dan tujuan hidup serta tidak menemukan kebermaknaan dalam hidupnya. Jika hal ini tidak ditangani dengan cepat dan tepat, maka sikap dan perilaku ini akan terbawa hingga dewasa dan membuatnya menjadi individu yang bersikap temperamental serta mencari pelampiasan dengan cara yang buruk (Fitria & Ifdil, 2020). Salah seorang remaja yang menjadi subjek dalam penelitian ini merasakan hal-hal yang dimaksud diatas seperti tidak menemukan kebermaknaan dalam hidupnya. Keluarga dari remaja tersebut baru saja mengalami broken home yang berujung pada perceraian. Akibat dari perceraian tersebut, remaja itu hanya tinggal dengan salah seorang dari orang tuanya dan kehilangan salah satu sosok orang tua yang selama ini telah membimbing dan mengarahkannya dalam perjalanan hidupnya. Remaja tersebut merasa tidak percaya diri dengan kondisinya saat ini, merasa tidak bersemangat untuk menjalani hidup, kehilangan kebermaknaan dalam hidup serta berpikir untuk mengakhiri hidup. Dari wawancara yang dilakukan oleh peneliti dengan subjek penelitian, diketahui bahwa remaja tersebut mengikuti konseling religi atas rekomendasi dari pihak keluarga agar remaja tersebut kembali pulih secara psikologis dan tidak lagi trauma dengan kasus broken home yang terjadi. HASIL DAN PEMBAHASAN Menurut subjek penelitian, kesediaannya untuk mengikuti konseling religi di rumah konseling mutiara hati didasarkan pada keinginan untuk mengalihkan kesedihan yang dirasakannya menjadi pribadi yang lebih baik. Berikut akan dijelaskan alasan kesediaan dari remaja tersebut untuk mengikuti konseling religi yang dilaksanakan di rumah konseling mutiara hati. Menemukan Hikmah dari Masalah yang Terjadi Kasus broken home yang terjadi pada keluarga subjek, menimbulkan dampak secara psikologis terhadap subjek. Subjek merasa bahwa hidupnya telah berubah dan tak akan lagi sama seperti sebelum broken home terjadi. Menurut subjek, ia selalu berpikir negatif atas kasus broken home yang terjadi, baik itu penyebab dari perceraian yang dilakukan orang tuanya maupun masa depannya setelah kasus broken home ini terjadi. Setelah beberapa waktu, ia menyadari bahwa di balik sebuah kejadian yang tidak menyenangkan, terdapat hikmah atau nilai positif yang dapat diambil. Untuk dapat menemukan hikmah dari kejadian tersebut, ia memutuskan untuk mengikuti konseling religi tersebut. Berikut adalah ungkapan dari subjek penelitian: “Ketika orang tua saya memilih untuk bercerai, saya merasa bahwa kehidupan saya akan berbeda antara sebelum dan sesudah perceraian ini. Di masa lalu saya merasakan kasih sayang yang sepenuh hati dari kedua orang tua saya, dan kedepannya mungkin saya tidak merasakan itu lagi, kasih sayang yang saya dapat tidak akan seperti dulu atau saya mungkin tidak akan merasakan kasih sayang itu lagi. Terkadang saya juga berpikir negatif tentang penyebab perceraian orang tua saya, dan saya merasa menjadi penyebab dari perceraian diantara keduanya. Saya kadang menceritakan ini kepada sahabat yang saya percaya dan dia menyarankan agar saya berusaha untuk berpikir positif dan mengambil hikmah positif dari masalah broken home yang terjadi. Setelah ada kesempatan untuk mengikuti konseling, disitu saya sekaligus mengusahakan untuk mencari hikmah positif dari kejadian tersebut dengan konselor yang sangat-sangat baik menurut saya. Setelah mengikuti konseling religi yang dilaksanakan di rumah konseling mutiara hati ini, dengan bantuan dari konselor, saya udah berdamai dengan masa lalu dan siap untuk menjalani hidup yang baru di masa yang akan datang. Selain itu saya sudah menemukan hikmah positif dari kejadian broken home di keluarga saya dan sudah menerima kejadian dan keputusan itu dengan ikhlas” (IN, wawancara 13 April 2021). Melupakan Kekecewaan yang Dihadapinya Rasa kecewa yang dirasakan oleh subjek penelitian berkenaan dengan kasus broken home yang terjadi pada keluarganya membuatnya larut dalam suasana yang kesedihan dan suasana yang tidak menentu. Subjek tersebut belum dapat menerima keputusan yang diambil oleh orang tuanya yang memutuskan untuk bercerai. Subjek merasakan kekecewaan yang menurutnya belum pernah ia rasakan sebelumnya. Akan tetapi kemudian subjek menyadari bahwa ia tidak bisa untuk terus berada dalam kondisi yang penuh dengan kekecewaan. Subjek tersebut memiliki keinginan untuk melakukan konseling agar ia dapat melupakan kekecewaan yang dirasakannya selama ini akibat dari kasus broken home tersebut. Subjek tersebut berkata: “Setelah mengetahui keluarga saya mengalami broken home dan kedua orang tua memutuskan untuk bercerai, disitu saya langsung kecewa sejadi-jadinya dengan orang tua saya yang mengambil keputusan seperti itu. Ketika itu saya masih berharap orang tua saya tidak berpisah, akan tetapi Tuhan berkehendak lain dan akhirnya keluarga saya hancur saat itu juga. Saya khawatir tidak dapat beradaptasi ketika tidak lagi tinggal bersama salah satu dari orang tua saya dan saya takut jika nanti mengalami bullying karena broken home yang terjadi pada keluarga saya. Setelah beberapa lama, saya menyadari bahwa hidup terus berjalan dan saya tidak bisa secara terus menerus berada dalam kondisi yang seperti ini. Saya ingin menjadi lebih baik dan saya perlu dukungan untuk menjadi lebih baik dan dukungan itu yang saya dapatkan di rumah konseling mutiara hati ini” (IN, wawancara 13 April 2021). Menurut subjek tersebut dengan melakukan konseling berbasis religi di rumah konseling mutiara hati, ia merasa lebih tenang dan berusaha untuk melupakan 68 Rois Nafi'ul Umam, Maemonah kekecewaan yang pernah dirasakannya, sebagaimana yang diungkapkan oleh subjek berikut: kekecewaan yang pernah dirasakannya, sebagaimana yang diungkapkan oleh subjek berikut: “saya merasa bahwa konseling religi yang dilaksanakan di rumah konseling mutiara hati ini saya belajar tentang ikhlas, berpikir lebih dewasa dalam menghadapi masalah serta dapat membantu mengatasi masalah saya” (IN, wawancara 13 April 2021). “saya merasa bahwa konseling religi yang dilaksanakan di rumah konseling mutiara hati ini saya belajar tentang ikhlas, berpikir lebih dewasa dalam menghadapi masalah serta dapat membantu mengatasi masalah saya” (IN, wawancara 13 April 2021). Menyiapkan Diri untuk Kehidupan yang Baru Setelah subjek telah menerima masalah broken home yang terjadi pada keluarganya, ia berharap dapat menjalani hidup baru dengan baik, tidak lagi terbawa dengan masalah di masa lalu serta yakin dan optimis bahwa kehidupan berikutnya akan menjadi lebih baik. Dengan dasar keyakinan tersebut, ia mencoba untuk mengikuti konseling religi dengan konselor di rumah konseling mutiara hati.Harapannya adalah, konselor tersebut bisa membantunya untuk menyiapkan diri untuk menjalani kehidupan yang baru dan suasana yang baru. Berikut adalah pemaparan dari subjek penelitian: “Saya berusaha untuk menerima kejadian tersebut dan berusaha untuk beradaptasi dengan kehidupan yang baru setelah terjadi perceraian kedua orang tua saya itu. Saya berusaha untuk mencoba menjalani hidup yang baru dan tidak lagi mengingat- ingat masalah yang pernah terjadi di masa lalu. Akan tetapi rasanya berat untuk menjalani hal itu, dan saya butuh bantuan dan orang yang dapat mengerti perasaan saya dan dapat membantu saya untuk merencanakan dan memulai kehidupan yang baru ini. Oleh karenanya saya kemudian mengikuti konseling disini agar saya kedepannya dapat menjalani hidup dengan baik dan tanpa ada beban pikiran lagi perihal broken home yang pernah terjadi” (IN, wawancara 13 April 2021). “Saya berusaha untuk menerima kejadian tersebut dan berusaha untuk beradaptasi dengan kehidupan yang baru setelah terjadi perceraian kedua orang tua saya itu. Saya berusaha untuk mencoba menjalani hidup yang baru dan tidak lagi mengingat- ingat masalah yang pernah terjadi di masa lalu. Akan tetapi rasanya berat untuk menjalani hal itu, dan saya butuh bantuan dan orang yang dapat mengerti perasaan saya dan dapat membantu saya untuk merencanakan dan memulai kehidupan yang baru ini. Oleh karenanya saya kemudian mengikuti konseling disini agar saya kedepannya dapat menjalani hidup dengan baik dan tanpa ada beban pikiran lagi perihal broken home yang pernah terjadi” (IN, wawancara 13 April 2021). Kebermaknaan hidup sebagai sebuah nilai fundamental bagi setiap manusia dalam menjalani hidupnya agar manusia dapat menemukan arti dan makna dari setiap perjalanan hidupnya serta menikmati hidup yang dijalaninya tersebut. Kebermaknaan dalam hidup dimaknai dengan upaya untuk menjadikan hidup yang lebih berarti dan bermanfaat, baik untuk diri sendiri maupun lingkungan sekitarnya. Melalui kebermaknaan hidup tersebut, individu dapat mencapai tujuan dan motivasi hidup, memberi kebermanfaatan bagi orang lain dan memperoleh kebahagiaan bagi individu tersebut (Andriyan & Rumtianing, 2020). Menemukan Kebermaknaan Hidup Masalah broken home yang terjadi pada keluarga subjek membuatnya kehilangan arah, motivasi dan tujuan hidup serta tidak mengetahui arti dan makna dari kehidupan yang dijalaninya saat ini. Hal ini dikarenakan ketidaksiapan dari subjek dalam menghadapi masalah yang terjadi dan suasana baru yang dirasakan setelah adanya kasus broken home tersebut. Subjek kemudian menyadari bahwa ia harus menjadi lebih baik dan menemukan makna atau arti dalam perjalanan hidupnya. Hal ini dilakukannya agar subjek tidak terjebak dalam suasana kesedihan yang selama ini telah ia jalani. Menurutnya melalui kebermaknaan hidup, ia dapat menghayati setiap perjalanan hidup yang dijalani, mengambil makna dari setiap kejadian, selalu berpikir positif jika ada hal yang tidak dikehendaki untuk mencapai kebahagiaan dalam hidupnya. Hal inilah yang mendorong subjek untuk berusaha menemukan kebermaknaan hidupnya melalui konseling religi yang dilaksanakan di rumah konseling mutiara hati. Ia berharap setelah mengikuti 69 INDONESIAN JOURNAL OF EDUCATIONAL COUNSELING 2021, 5(1), 64-74 konseling religi tersebut, ia dapat menjadi pribadi yang lebih baik, menemukan kebermaknaan dalam hidupnya, serta memiliki motivasi dan tujuan hidup di masa yang akan datang. Berikut adalah pemaparan selengkapnya dari subjek penelitian: “Hidup yang bermakna menurut saya yaitu ketika saya dapat menghayati, meresapi dan mendalami setiap bagian dari perjalanan hidup yang dijalani. Selain itu hidup kita menjadi lebih bermakna ketika kita dapat mengambil sisi positif dari sebuah kejadian selalu meyakini bahwa hal tersebut telah diatur oleh yang maha kuasa dan kita meyakini dibalik hal itu ada nilai-nilai positif yang bisa diambil. Untuk menemukan kebermaknaan hidup, saya memutuskan untuk mengikuti konseling religi ini agar hidup dengan bantuan konselor di rumah konseling mutiara hati ini. Setelah mengikuti konseling religi ini, saya jadi mengerti bahwa kebermaknaan hidup saya dijalankan dengan memberi manfaat untuk diri saya sendiri dan orang lain, dan ketika saya tidak bisa melakukan itu, rasanya seperti menyia-nyiakan kesempatan yang mungkin tidak setiap waktu akan datang. Selain itu saya menyadari bahwa kebermaknaan dalam hidup saya ditunjukkan dengan membuat rencana dan tujuan yang baik dan sesuai dengan kemampuan saya serta dapat mengisi waktu yang luang dengan sesuatu yang bermanfaat seperti mempelajari ilmu agama” (IN, wawancara 13 April 2021). Menyiapkan Diri untuk Kehidupan yang Baru Dalam konteks keagamaan, khususnya dalam Islam, kebermaknaan hidup dapat dicapai ketika manusia dapat mengetahui arti dari kehidupannya, menerima atas segala yang telah ditetapkan dan terjadi padanya serta selalu yakin bahwa dibalik sebuah ujian, akan ada kebahagiaan yang akan diberikan 70 Rois Nafi'ul Umam, Maemonah olehnya sebagai balasan atas kesabaran dan usahanya dalam menjalani ujian yang diberikan oleh Tuhannya tersebut (Ningsih, Reza, & Uyun, 2017). Untuk dapat mencapai kebermaknaan dalam hidupnya, manusia perlu menyadari bahwa hidup yang dijalaninya perlu dimaknai secara mendalam dan senantiasa berusaha untuk selalu berbuat baik dan memberi manfaat. Terkadang manusia menyadari pentingnya kebermaknaan hidup saat ia berada dalam kondisi yang sulit dan diliputi dengan permasalahan yang rumit. Saat mengalami hal tersebut, manusia akan berusaha untuk mencari solusi atas permasalahannya dan juga menemukan kebermaknaan dalam hidupnya ketika ia merasa tak lagi memiliki motivasi dan tujuan dalam hidupnya (Hidayat, 2019). Ketika manusia berada dalam kondisi yang sulit tersebut, ia memerlukan dukungan dan orang lain di sekitarnya yang mampu mengarahkannya untuk menemukan kebermaknaan dalam hidupnya. Salah satu upaya yang dapat dilakukan untuk menemukan kebermaknaan dalam hidup adalah dengan layanan konseling. Konseling merupakan pemberian bantuan yang diberikan oleh konselor kepada konseli dalam rangka untuk membantu penyelesaian masalah yang dihadapi oleh konseli. Dalam layanan konseling, konseli dapat menyampaikan permasalahan yang dihadapinya dan konselor membantu mengarahkan konseli tersebut untuk mencari alternatif penyelesaian masalahnya (Sukma, 2018). Dalam konteks remaja yang menjadi korban broken home diatas, layanan konseling dilakukan untuk membantu dirinya dalam menyelesaikan masalah dan menemukan kebermaknaan dalam hidupnya untuk mencapai kebermanfaatan dan kebahagiaan dalam hidupnya. Adapun jenis konseling yang diikuti oleh subjek penelitian ini yaitu konseling religi yaitu layanan konseling yang menginternalisasi nilai-nilai religiusitas dalam keagamaan. Adapun penggunaan konseling religi ini didasarkan pada kebermaknaan hidup yang dikehendaki tidak terlepas dari nilai-nilai spiritual dan nilai tersebut menjadi pendukung dalam membantu subjek untuk mencapai kebermaknaan dalam hidupnya. Layanan konseling religi ini dijalankan dalam empat tahap dan berikut adalah tahapan-tahapan dari konseling religi: Tahap Awal Dalam tahap awal ini, konselor akan menyambut konseli dan memperkenalkan dirinya kepada konseli. Setelah itu konselor memperkenalkan layanan konseling secara umum seperti tujuan dari konseling, asas konseling, aturan-aturan yang harus ditaati dalam proses konseling serta tujuan yang hendak dicapai dari konseling. Selain itu, konselor juga memperkenalkan mengenai konseling religi yang dilandasi oleh nilai-nilai spiritual dalam Alquran. Untuk dapat membangun hubungan yang bersifat kekeluargaan dan tidak memberikan suasana yang tegang bagi konseli, suasana konseling yang dibangun bersifat santai dan memberikan rasa tenang bagi konseli untuk dapat menceritakan permasalahan yang dihadapinya serta dapat mengikuti layanan konseling dengan perasaan yang nyaman. Setelah terbangun hubungan yang baik antara konselor dan konseli, kemudian keduanya menegosiasikan kontrak perihal waktu, tugas dan kerjasama selama proses konseling. Setelah menandatangani kontrak perjanjian, konseli akan diberi kesempatan untuk menceritakan masalah yang dihadapinya kepada konselor. Setelah konseli menyampaikan masalah yang dihadapinya, konselor kemudian memperjelas masalah yang dihadapi oleh konseli dan mendefinisikan masalah yang disampaikan konseli untuk menentukan langkah-langkah yang akan diambil pada tahap berikutnya. 71 INDONESIAN JOURNAL OF EDUCATIONAL COUNSELING 2021, 5(1), 64-74 Tahap Kegiatan Tahap ini merupakan tahap inti dari layanan konseling religi ini, karena fokus dari konseling yang dijalankan berada pada tahap ini. Setelah mengetahui dan mendalami permasalahan yang dihadapi oleh konseli, kemudian konselor menyampaikan rencana intervensi yang akan dilakukan dalam konseling. Mengingat bahwa konseling yang dilakukan merupakan konseling religi, maka kegiatan yang dilakukan berlandaskan pada nilai-nilai dan ajaran agama, khususnya Islam. Adapun pada tahap ini kegiatan yang dilakukan meliputi membaca Alquran, mendengar ceramah , membaca buku-buku motivasi dan keislaman serta membuat rencana dan tujuan hidup yang dikehendaki ketika konseli telah selesai melakukan layanan konseling. Semua kegiatan yang dilaksanakan tersebut, didampingi konselor untuk memastikan setiap prosesnya dapat berjalan dengan baik. Pada kegiatan membaca Alquran, konseli dihadapkan dengan ayat- ayat yang memiliki makna yang mendalam dan motivasi seperti surah yusuf ayat 87, surah At-Taubah ayat 40, surah Ali Imran ayat 139, surah A-Baqarah ayat 216 dan surah Ath-Thalaq ayat 2-3 yang kemudian dapat diambil hikmahnya oleh konseli tersebut. Kegiatan berikutnya adalah dengan mendengar ceramah yang disampaikan oleh ustadz, dalam hal ini ustadz yang menjadi rujukan bagi konseli dalam belajar agama. Konseli mendengarkan ceramah dengan tema yang sesuai dengan permasalahan yang dihadapinya, upaya dalam menemukan kebermaknaan hidup serta motivasi bagi dirinya untuk dapat menjadi pribadi yang lebih baik. Setelah kegiatan membaca Alquran dan mendengarkan ceramah telah dilakukan, kegiatan berikutnya yang dilakukan pada tahap ini yaitu konseli dapat membaca buku-buku mengenai motivasi dan keislaman. Adapun buku-buku yang dibaca sesuai dengan karakteristik konseli dan mengandung isi yang dapat menguatkan sisi positif konseli dan membantunya dalam mencapai kebermaknaan hidup serta dapat memberikan motivasi bagi konseli untuk menjadi lebih baik. Kemudian masuk pada kegiatan terakhir yaitu menuliskan upayanya untuk berdamai dengan masa lalu, cita-cita tujuan dan arah hidupnya di masa yang akan datang, serta kebermaknaan hidup yang telah ia temukan dari kegiatan yang telah dilaksanakan sebelumnya, yaitu membaca alquran, mendengar ceramah dan membaca buku motivasi dan keislaman. SIMPULAN Masalah broken home yang terjadi pada keluarga, dapat berdampak pada anggota keluarga yang lain, salah satunya adalah remaja dalam keluarga tersebut. Dampak negatif dari broken home berpengaruh terhadap kondisi psikologis remaja yang sedang berada pada masa perkembangan, dan membuatnya kehilangan arah, tujuan, motivasi hidup serta kebermaknaan yang ada dalam hidupnya. Untuk dapat membantu remaja tersebut dalam menemukan kebermaknaan hidupnya, dapat dilakukan melalui konseling religi yang dilandasi oleh nilai-nilai keagamaan. Konseling religi diisi dengan kegiatan seperti membaca Alquran, mendengar ceramah dan membaca buku-buku motivasi dan keislaman yang dapat membantunya dalam menemukan kebermaknaan hidup dan upayanya untuk berdamai dengan masalah broken home yang dihadapinya. Setelah mengikuti konseling religi, remaja tersebut telah menemukan kebermaknaan dalam hidupnya, menerima dengan lapang dada permasalahan broken home yang terjadi dan mengambil sisi positifnya serta menyiapkan diri untuk menjalani kehidupan yang baru dengan rencana yang telah ditetapkan. Tahap Akhir Setelah melalui rangkaian kegiatan yang dilaksanakan pada tahap sebelumnya dalam proses konseling, konseli merasakan perubahan positif yang ada pada dirinya dan menemukan kebermaknaan hidup yang dikehendakinya. Perubahan-perubahan tersebut meliputi kesediaan untuk menerima masalah yang terjadi dan mengambil sisi positif dari masalah tersebut, menemukan kebermaknaan dalam hidupnya yaitu konseli menyadari bahwa setiap bagian dari perjalanan hidupnya merupakan sesuatu yang bernilai dan memiliki makna yang mendalam dan menghayati setiap detik-detik dalam kehidupannya agar menjadikannya sebagai manusia yang bersyukur, memberi manfaat bagi diri sendiri dan orang lain. Adapun rencana dalam kehidupan yang baru menurut konseli diisi dengan kegiatan-kegiatan yang bermanfaat bagi dirinya, memberi manfaat bagi orang lain serta tidak bertentangan dengan ketentuan Agama. Selain itu, konseli telah mempersiapkan rencana untuk membahagiakan orang tuanya walaupun salah satu dari mereka tidak lagi tinggal bersama dengan konseli tersebut. Sebelum konseling diakhiri, konselor dan konseli menyampaikan evaluasi atas konseling yang telah dilakukan dan mengatur pertemuan apabila konseli kembali mengalami kendala berhubungan dengan kebermaknaan dalam hidupnya. 72 Rois Nafi'ul Umam, Maemonah REFERENSI Andriyan, & Rumtianing, I. (2020). Kebermaknaan hidup Lansia (Studi kasus di cabang UPT Pelayanan Sosial Tresna Werdha Magetan Jawa Timur). Rosyada: Islamic Guidance and Counseling, 1(1), 72-89. Arintina, Y. C., & Fauziah, N. (2015). Keharmonisan keluarga dan kecenderungan berperilaku agresif pada siswa SMK. Jurnal Empati, 4(1), 208-212. Aziz, M. (2015). Perilaku sosial anak remaja korban broken home dalam berbagai perspektif (Suatu penelitian di SMPN 18 kota Banda Aceh). Jurnal Al-Ijtimaiyyah, 1(1), 30-50. Dewanti, W., & Ediati, A. (2016). Sikap remaja laki-laki dan perempuan terhadap perceraian: Studi komparasi pada remaja siswa SMA Negeri 6 Semarang. Jurnal Empati, 5(3), 594-597. Diananda, A. (2019). Psikologi remaja dan permasalahannya. Journal ISTIGHNA, 1(1), 116- 133. Fitria, L., & Ifdil. (2020). Kecemasan remaja pada masa pandemi Covid-19. Jurnal EDUCATIO: Jurnal Pendidikan Indonesia, 6(1), 1-4. Hafiza, S., & Mawarpury, M. (2018). Pemaknaan kebahagiaan oleh remaja broken home. Psympathic : Jurnal Ilmiah Psikologi, 5(1), 59-66. Hidayat, V. (2019). Kebermaknaan hidup pada mahasiswa semester akhir. Jurnal Psikologi Integratif, 6(2), 141. Isminayah, A., & Supandi. (2016). Relasi tingkat keharmonisan keluarga dengan konsep diri remaja. Al-Balagh : Jurnal Dakwah dan Komunikasi, 1(2), 233-245. Jannah, M. (2017). Remaja dan tugas-tugas perkembangannya dalam Islam. Psikoislamedia : Jurnal Psikologi, 1(1), 243-256. Maulana, A., & Hernisawati. (2017). Konseling religi untuk mengembangkan karakter konseling. Jurnal Islamic Counseling, 1(2), 55-65. 73 INDONESIAN JOURNAL OF EDUCATIONAL COUNSELING 2021, 5(1), 64-74 Mazaya, K. N., & Supradewi, R. (1970). Konsep diri dan kebermaknaan hidup pada remaja di panti asuhan. Proyeksi, 6(2), 103-112 Mazaya, K. N., & Supradewi, R. (1970). Konsep diri dan kebermaknaan hidup pada remaja di panti asuhan. Proyeksi, 6(2), 103-112 Muniriyanto, & Suharnan. (2014). Keharmonisan keluarga, konsep diri dan kenakalan remaja. Persona: Jurnal Psikologi Indonesia, 3(2), 156-164. Mustikarani, A. Y. (2013). Makna hıdup pada mahasıswa yang mengalamı broken home. Skripsi. Bandung: Universitas Pendidikan Indonesia Muttaqin, I., & Sulistyo, B. (2019). Analisis faktor penyebab dan dampak keluarga broken home. Studi Gender dan Anak, 6, 245-256. Ningsih, D. A., Reza, I. F., & Uyun, M. (2017). Kebermaknaan hidup lansia pemulung yang beragama Islam di tempat pembuangan akhir (TPA) Sukajaya Kecamatan Sukarame Palembang. Psikis: Jurnal Psikologi Islami, 3(1), 52-59. Novianto, R., Zakso, A., & Salim, I. (2017). Analisis dampak broken home terhadap minat belajar siswa SMA Santun Untan Pontıanak. Jurnal Pendidikan dan Pembelajaran Khatulistiwa, 8(3), 1-8. Ramadhani, P. E., & Krisnani, H. (2019). Analisis dampak perceraian orang tua terhadap anak remaja. Focus: Jurnal Pekerjaan Sosial, 2(1), 109-119. Saputro, K. Z. (2018). REFERENSI Memahami ciri dan tugas perkembangan masa remaja. Aplikasia: Jurnal Aplikasi Ilmu-Ilmu Agama, 17(1), 25. Siddik, I. N., Oclaudya, K., Ramiza, K., & Nashori, F. (2018). Hubungan antara ıkhlas dan dukungan sosial dengan kebermaknaan hidup pada orang dengan HIV/AIDS. Psikoislamedia Jurnal Psikologi, 3(1), 98-114. Sukma, D. (2018). Concept and application group guidance and group counseling base on Prayitno’s paradigms. Konselor, 7(2), 49-54. Verawati, I. (2017). Dukungan sosial orangtua dalam mengikutsertakan anaknya berlatih di Krakatau Taekwondo klub Medan. Jurnal Edutech, 3(2), 22-28. Widyawati, D. C. D. (2009). Studi kasus tentang kebermaknaan hidup remaja yang orangtuanya bercerai. Yogyakarta: Universitas Sanata Dharma. Wulandri, D., & Fauziah, N. (2019). Pengalaman remaja korban broken home (Studi kualitatif fenomenologis). Empati, 8(1), 1-9. 74 74
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Robustness of STDP to spike timing jitter
Scientific reports
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To cite this version: Yihui Cui, Ilya Prokin, Alexandre Mendes, Hugues Berry, Laurent Venance. Robustness of STDP to spike timing jitter. Scientific Reports, 2018, 8 (1), pp.8139:1-15. ￿10.1038/s41598-018-26436-y￿. ￿hal-01788826v2￿ Robustness of STDP to spike timing jitter Yihui Cui, Ilya Prokin, Alexandre Mendes, Hugues Berry, Laurent Venance To cite this version: Yihui Cui, Ilya Prokin, Alexandre Mendes, Hugues Berry, Laurent Venance. Robustness of STDP to spike timing jitter. Scientific Reports, 2018, 8 (1), pp.8139:1-15. ￿10.1038/s41598-018-26436-y￿. ￿hal-01788826v2￿ Yihui Cui, Ilya Prokin, Alexandre Mendes, Hugues Berry, Laurent Yihui Cui1, Ilya Prokin2,3, Alexandre Mendes1, Hugues Berry   2,3 & Laurent Venance   1 In Hebbian plasticity, neural circuits adjust their synaptic weights depending on patterned firing. Spike- timing-dependent plasticity (STDP), a synaptic Hebbian learning rule, relies on the order and timing of the paired activities in pre- and postsynaptic neurons. Classically, in ex vivo experiments, STDP is assessed with deterministic (constant) spike timings and time intervals between successive pairings, thus exhibiting a regularity that differs from biological variability. Hence, STDP emergence from noisy inputs as occurring in in vivo-like firing remains unresolved. Here, we used noisy STDP pairings where the spike timing and/or interval between pairings were jittered. We explored with electrophysiology and mathematical modeling, the impact of jitter on three forms of STDP at corticostriatal synapses: NMDAR-LTP, endocannabinoid-LTD and endocannabinoid-LTP. We found that NMDAR-LTP was highly fragile to jitter, whereas endocannabinoid-plasticity appeared more resistant. When the frequency or number of pairings was increased, NMDAR-LTP became more robust and could be expressed despite strong jittering. Our results identify endocannabinoid-plasticity as a robust form of STDP, whereas the sensitivity to jitter of NMDAR-LTP varies with activity frequency. This provides new insights into the mechanisms at play during the different phases of learning and memory and the emergence of Hebbian plasticity in in vivo-like activity. Received: 13 February 2018 Accepted: 9 May 2018 Published: xx xx xxxx Long-term changes in synaptic efficacy between neurons are thought to underlie learning and memory1 and can be assessed with a synaptic Hebbian paradigm such as spike timing-dependent plasticity (STDP)2–8. In STDP, the occurrence of timing-dependent-long-term potentiation (tLTP) or -depression (tLTD) is the result of three factors. The two first ones are the two coincident activity patterns on either side of the synapse, which depends on (1) the relative timing between pre- and postsynaptic spikes (ΔtSTDP)4,8, (2) the dendritic and axonal propagation delays9, (3) the number of paired spikes (Npairings)10–13, (4) the frequency of the paired spikes (Fpairings)6,7,10,11,14 and (5) membrane depolarization14,15; the third factor involves in STDP expression mainly refers to neuromodula- tors16,17 and glial cells18. STDP has been attracting a lot of interest in computational and experimental neurosciences because it relies on spike correlation and has emerged as a candidate mechanism for experience-dependent changes in neural circuits, including map plasticity8,19–23. STDP is classically investigated using regular repetitions of the same spike timing and fixed intervals between successive paired stimulations. HAL Id: hal-01788826 https://inria.hal.science/hal-01788826v2 Submitted on 25 May 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. www.nature.com/scientificreports www.nature.com/scientificreports Robustness of STDP to spike timing jitter OPEN Robustness of STDP to spike timing jitter OPEN Yihui Cui1, Ilya Prokin2,3, Alexandre Mendes1, Hugues Berry   2,3 & Laurent Venance   1 Yihui Cui1, Ilya Prokin2,3, Alexandre Mendes1, Hugues Berry   2,3 & Laurent Venance   1 Yihui Cui1, Ilya Prokin2,3, Alexandre Mendes1, Hugues Berry   2,3 & Laurent Venance   1 A typical experimental protocol consists in pairing pre- and postsynaptic stimulations with a fixed ΔtSTDP (ranging from −30 to +30 ms for plasticity induc- tion); ΔtSTDP < 0 when the postsynaptic stimulation occurs before the paired presynaptic one (post-pre pairings), whereas ΔtSTDP > 0 when the presynaptic stimulation occurs before the postsynaptic one (pre-post pairings). These pairings are then repeated between 15 and 200 times (between 0.1 and 5 Hz) with the spike timing and the time interval between successive pairings being kept constant. Regular stimulation paradigms produce patterns of activity that are likely to differ from the variability expected in in vivo-like firing. In vivo paired natural visual stimulations with postsynaptic action potential lead to Hebbian STDP with specific characteristics when compared to ex vivo STDP (broader membrane potential changes and STDP window), which can be explained by jittered presynaptic inputs occurring in vivo22. A theoreti- cal study has started to explore naturalistic stimulations and showed that when neurons fire irregularly, the impact of spike timing in plasticity becomes weaker than the influence of the firing rate24. However, whether STDP emer- gence and maintenance is robust against biological variability remains to be investigated. To address this question, it is important to take into account various forms of STDP, involving distinct intracellular signal transduction 1Dynamics and Pathophysiology of Neuronal Networks Team, Center for Interdisciplinary Research in Biology (CIRB), College de France, CNRS, INSERM, PSL Research University, Paris, France. 2INRIA, Villeurbanne, France. 3University of Lyon, LIRIS UMR5205, Villeurbanne, France. Yihui Cui and Ilya Prokin contributed equally to this work. Hugues Berry and Laurent Venance jointly supervised this work. Correspondence and requests for materials should be addressed to H.B. (email: hugues.berry@inria.fr) or L.V. (email: laurent.venance@college-de-france.fr) SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 1 www.nature.com/scientificreports/ Figure 1. NMDAR- and eCB-mediated STDP induced with fixed ΔtSTDP. (a) STDP protocol: a single postsynaptic spike was paired with a single cortical stimulation, 10 or 100 times at 1 Hz. (b) Example of 5 successive pairings (#4–8, from c1) exhibiting relatively fixed ΔtSTDP (#4–8: −8.0, −7.8, −7.4, −8.8 and −8.0 ms). Plotting the successive ΔtSTDP illustrates their low variance σΔt (2.6 ms). (c) Corticostriatal NMDAR tLTP induced by 100 post-pre pairings with fixed ΔtSTDP. (c1) Example of tLTP induced by 100 post-pre pairings (mΔt = −9 ms, σΔt = 2.6 ms). Yihui Cui1, Ilya Prokin2,3, Alexandre Mendes1, Hugues Berry   2,3 & Laurent Venance   1 The mean baseline EPSC amplitude, 95 ± 3 pA, increased by 77% to 168 ± 6 pA 45 minutes after pairings. Upper panel, time course of Ri (baseline: 172 ± 2 MΩ and 35–45 min after pairings: 170 ± 3 MΩ; change of 1%) and holding current (Ihold). Inset: distribution of the 100 ΔtSTDP. (c2) Averaged time-courses of tLTP induced by 100 post-pre pairings; this tLTP was NMDAR-mediated because prevented by D-AP5 (50 μM). (c3) Relationship between the STDP magnitude and the STDP jitter for each of the recorded neurons. (d) Corticostriatal eCB-tLTD induced by 100 pre-post pairings with fixed ΔtSTDP. (d1) Example of tLTD induced by 100 pre-post pairings (mΔt = +21 ms, σΔt = 2.1 ms); the mean baseline Figure 1. NMDAR- and eCB-mediated STDP induced with fixed ΔtSTDP. (a) STDP protocol: a single postsynaptic spike was paired with a single cortical stimulation, 10 or 100 times at 1 Hz. (b) Example of 5 successive pairings (#4–8, from c1) exhibiting relatively fixed ΔtSTDP (#4–8: −8.0, −7.8, −7.4, −8.8 and −8.0 ms). Plotting the successive ΔtSTDP illustrates their low variance σΔt (2.6 ms). (c) Corticostriatal NMDAR- tLTP induced by 100 post-pre pairings with fixed ΔtSTDP. (c1) Example of tLTP induced by 100 post-pre pairings (mΔt = −9 ms, σΔt = 2.6 ms). The mean baseline EPSC amplitude, 95 ± 3 pA, increased by 77% to 168 ± 6 pA 45 minutes after pairings. Upper panel, time course of Ri (baseline: 172 ± 2 MΩ and 35–45 min after pairings: 170 ± 3 MΩ; change of 1%) and holding current (Ihold). Inset: distribution of the 100 ΔtSTDP. (c2) Averaged time-courses of tLTP induced by 100 post-pre pairings; this tLTP was NMDAR-mediated because prevented by D-AP5 (50 μM). (c3) Relationship between the STDP magnitude and the STDP jitter for each of the recorded neurons. (d) Corticostriatal eCB-tLTD induced by 100 pre-post pairings with fixed ΔtSTDP. (d1) Example of tLTD induced by 100 pre-post pairings (mΔt = +21 ms, σΔt = 2.1 ms); the mean baseline SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 2 www.nature.com/scientificreports/ EPSC amplitude, 206 ± 4 pA, decreased by 25%, to 154 ± 8 pA, 45 minutes after pairings. Upper panel, time course of Ri (baseline: 195 ± 1 MΩ and 45–55 min after pairings: 196 ± 2 MΩ; change <1%) and Ihold. Inset: distribution of the 100 ΔtSTDP. Results Corticostriatal synapses exhibit a bidirectional STDP in which NMDAR-tLTP, eCB-tLTD29,32–34 or eCB-tLTP12,13 are induced depending on the number of pairings (Npairings) and the spike timing (ΔtSTDP). While STDP is classi- cally investigated using fixed ΔtSTDP, we examine here the effect of noisy ΔtSTDP, closer to in vivo-like firing. Here, we define the Inter-Pairing-Interval (IPI) as the time interval between two consecutive presynaptic stimulations, whereas ΔtSTDP is the time interval between the post- and the presynaptic stimulation times within a given paired stimulation (in a “standard” STDP protocol both IPIs and ΔtSTDP are constant). NMDAR- and eCB-mediated STDP triggered with fixed ΔtSTDP. We observed bidirectional asym- metric STDP in MSNs (Fig. 1a): 100 post-pre pairings (ΔtSTDP < 0) induced tLTP whereas 100 pre-post pairings (ΔtSTDP > 0) induced tLTD. An example of the tLTP induced by 100 post-pre pairings with fixed ΔtSTDP (Fig. 1b) is illustrated in Fig. 1c1. The input resistance Ri remained stable over this period. Although we fixed the values of ΔtSTDP, they slightly vary from one pairing to another (Fig. 1b). This low jitter can be written formally as ΔtSTDP = mΔt + ξΔt, were mΔt is the mean spike timing and ξΔtis a random variable with mean 0 and stand- ard deviation σΔt. Overall, 100 post-pre pairings induced tLTP (mean EPSC amplitude recorded 60 min after protocol induction: 149 ± 13% of baseline, p = 0.0028, n = 13) (Fig. 1c2). σΔt was 1.4 ± 0.2 ms (Fig. 1c3). This tLTP was NMDAR-mediated since prevented by D-AP5 (50 μM), a NMDAR antagonist, (88 ± 12%, p = 0.375, n = 5; p < 0.0001 when compared with tLTP in control) (Fig. 1c2). Conversely, pre-post pairings induced tLTD, as shown in the example in Fig. 1d1. Overall, 100 pre-post pairings induced tLTD (76 ± 7%, p = 0.0070, n = 10) (Fig. 1d2). σΔt was 1.4 ± 0.2 ms (Fig. 1d3). This tLTD was CB1R-mediated since prevented by AM251 (3 μM), a CB1R specific inhibitor (99 ± 2%, p = 0.816, n = 5; p < 0.0001 when compared with tLTD in control) (Fig. 1d2). We thus find an anti-Hebbian polarity for corticostriatal STDP. We previously showed that GABA operates as a Hebbian/anti-Hebbian switch at corticostriatal synapses34,35 and corticostriatal STDP polarity depends on the presence32,33 or the absence12,29,36,37 of GABAA receptor antagonists. Yihui Cui1, Ilya Prokin2,3, Alexandre Mendes1, Hugues Berry   2,3 & Laurent Venance   1 Our results further indicate that the robustness of NMDAR-tLTP to jitter is also strengthened by the irregularity of the spike-train stimulations. Yihui Cui1, Ilya Prokin2,3, Alexandre Mendes1, Hugues Berry   2,3 & Laurent Venance   1 (d2) Averaged time-courses of tLTD induced by 100 pre-post pairings; this tLTD was CB1R-mediated because prevented by AM251 (3 μM). (d3) Relationship between the STDP magnitude and the jitter. (e) Corticostriatal eCB-tLTP induced by 10 post-pre pairings with fixed ΔtSTDP. (e1) Example of tLTP induced by 10 post-pre pairings (mΔt = −11 ms, σΔt = 3.1 ms); the mean baseline EPSC amplitude, 147 ± 3 pA, increased by 43% to 209 ± 9 pA 45 minutes after pairings. Upper panel, time course of Ri (baseline: 227 ± 2 MΩ and 45–55 min after pairings: 239 ± 2 MΩ; change of 5%) and Ihold. Inset: distribution of the 10 ΔtSTDP. (e2) Averaged time-courses of tLTP induced by 10 post-pre pairings; this tLTP was CB1R-mediated because prevented by AM251. (e3) Relationship between the STDP magnitude and the jitter. (c1-d1-e1) Insets: average of 12 successive EPSC amplitude at baseline (black traces) and at 40–50 min after pairings (grey traces). (f) Predictions of the mathematical model in the absence of added jitter. The model expresses the biochemical pathways schematized in (f1). (f2) Changes of Wtotal with various ΔtSTDP and Npairings. (f3) Removing NMDAR/ CaMKII signaling suppresses the tLTP observed for ΔtSTDP > 0 and Npairings > 50. (f4) Removing CB1R suppresses the tLTP observed for ΔtSTDP > 0 and Npairings < 20 and the tLTD observed for ΔtSTDP < 0. pathways, i.e. NMDAR-, mGluR- or endocannabinoid(eCB)-mediated STDP25–29 (for reviews see8,30,31). It is thus expected that those various STDP forms might exhibit different robustness to spike train variability. expected that those various STDP forms might exhibit different robustness to spike train variability. Here, we show with patch-clamp recordings and mathematical modeling that corticostriatal NMDAR- and eCB-STDP do not exhibit the same sensitivity to noisy spike timings: eCB-plasticity (eCB-tLTD and eCB-tLTP) appeared robust to jittering whereas NMDAR-tLTP was fragile. However, increasing the number or frequency of pairings improved NMDAR-tLTP robustness. Our results further indicate that the robustness of NMDAR-tLTP to jitter is also strengthened by the irregularity of the spike-train stimulations. p gf p y Here, we show with patch-clamp recordings and mathematical modeling that corticostriatal NMDAR- and eCB-STDP do not exhibit the same sensitivity to noisy spike timings: eCB-plasticity (eCB-tLTD and eCB-tLTP) appeared robust to jittering whereas NMDAR-tLTP was fragile. However, increasing the number or frequency of pairings improved NMDAR-tLTP robustness. Results eCB-tLTP (red full line) was triggered with 10 pairings of average spike timing mΔt = −15 ms and eCB-tLTD (blue full line) with 100 pairings of mΔt = +20 ms. NMDAR-tLTP (red dashed line) corresponded to 100 pairings of mΔt = −15 ms. eCB-tLTD and eCB-tLTP are weakly affected by the jitter. NMDAR-tLTP is less robust. Full and dashed lines show averages and light swaths show ± 1 sem. (b) Evolution of NMDAR-tLTP, eCB-tLTD and eCB-tLTP domains for σΔt = 0 (b1), 5 (b2) and 10 ms (b3) for the STDP protocol of (a). Note the persistence of the eCB-tLTD domain (blue box) while eCB-tLTP (red box) and NMDAR-tLTP domain (red dashed box) disappeared for 5 and 10 ms, respectively. (c–e) Similar results are obtained with other jitter distributions, including Gaussian-distributed jitter (c) and triangularly-distributed jitter (d). We also considered a combination of triangularly-distributed jitter with random IPIs distributed according to an exponential distribution with refractory period τr = 0.95 s (see Methods) in (e). The plots of the amplitude of NMDAR-tLTP, eCB-tLTD and eCB-tLTP for increasing jitter amplitude σΔt (c2, d2, e2) show ii Figure 2. The mathematical model predicts that NMDAR-tLTP is less robust to jitter than eCB-tLTP and eCB-tLTD. (a) Using a jittered STDP protocol combining uniformly-distributed jitter of the spike timing with deterministic IPIs at 1 Hz (a1), the amplitudes of NMDAR-tLTP, eCB-tLTD and eCB-tLTP decrease with increasing jitter amplitudes σΔt (a2). eCB-tLTP (red full line) was triggered with 10 pairings of average spike timing mΔt = −15 ms and eCB-tLTD (blue full line) with 100 pairings of mΔt = +20 ms. NMDAR-tLTP (red dashed line) corresponded to 100 pairings of mΔt = −15 ms. eCB-tLTD and eCB-tLTP are weakly affected by the jitter. NMDAR-tLTP is less robust. Full and dashed lines show averages and light swaths show ± 1 sem. (b) Evolution of NMDAR-tLTP, eCB-tLTD and eCB-tLTP domains for σΔt = 0 (b1), 5 (b2) and 10 ms (b3) for the STDP protocol of (a). Note the persistence of the eCB-tLTD domain (blue box) while eCB-tLTP (red box) and NMDAR-tLTP domain (red dashed box) disappeared for 5 and 10 ms, respectively. (c–e) Similar results are obtained with other jitter distributions, including Gaussian-distributed jitter (c) and triangularly-distributed jitter (d). Results p p g Besides this bidirectional STDP (NMDAR-tLTP and eCB-tLTD) induced for 100 pairings, we reported that low numbers of pairings (Npairings = 5–15) induce an eCB-tLTP, dependent on the activation of CB1R12,13. Figure 1e1 shows an example of tLTP induced by 10 post-pre pairings. Overall, 10 post-pre STDP pairings induced tLTP (145 ± 9%, p = 0.0005, n = 15), which was prevented by AM251 (3 μM) (76 ± 10%, p = 0.080, n = 4; p < 0.0001 when compared with tLTP in control) (Fig. 1e2) as recently reported12. σΔt for 10 post-pre par- ings STDP was 1.8 ± 0.3 ms (n = 15) (Fig. 1e3). Note that there was no difference between the σΔt measured for NMDAR-tLTP, eCB-tLTD and eCB-tLTP (one way ANOVA, p = 0.4094). A computer model emulates NMDAR- and eCB-mediated STDP. To help interpret our experimental results, we used a mathematical model of the signaling pathways implicated in corticostriatal STDP, including NMDAR- and CB1R-plasticity12,29,32,33 (Fig. 1f1). The model is detailed in S1 Text, with parameter values listed in S1 and S2 Tables. We include in the Supporting Information a thorough description of the mechanisms by which eCB- and NDMAR-plasticity are expressed in the model when STDP protocol (Npairings = 100) is applied (S3 Text). Figure 1f2 shows the value of the total synaptic weight (Wtotal) predicted by the model with various Npairings and ΔtSTDP. In agreement with the experimental data, with small σΔt (3 ms), the model features three main plasticity regions: tLTD was observed for short pre-post pairings (0 < ΔtSTDP < +30 ms), whereas short post-pre pairings (−30 < ΔtSTDP < 0 ms) induced a first tLTP region for low numbers of pairings (3 < Npairings < 25) and a second tLTP region emerging for Npairings > 50. Blocking the NMDAR-CaMKII pathway in the model (Fig. 1f3) suppresses the second tLTP region whereas blocking CB1R activation (Fig. 1f4) prevents the expression of tLTD SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 3 3 www.nature.com/scientificreports/ ntificreports/ Figure 2. The mathematical model predicts that NMDAR-tLTP is less robust to jitter than eCB-tLTP and eCB-tLTD. (a) Using a jittered STDP protocol combining uniformly-distributed jitter of the spike timing with deterministic IPIs at 1 Hz (a1), the amplitudes of NMDAR-tLTP, eCB-tLTD and eCB-tLTP decrease with increasing jitter amplitudes σΔt (a2). Results (b) Example of 5 successive pairings (#5–9, from c1) exhibiting jittered values of ΔtSTDP (#5–9: −10.3, −15.2, −30.5, −20.7 and −17.3 ms); these pairings are indicated with the grey frame within the 100 pairings displaying jittered ΔtSTDP. The distribution of the successive ΔtSTDP illustrate their standard deviation σΔt = 7.9 ms. (c) NMDAR-tLTP is prevented with σΔt > 3 ms. (c1) Example of the absence of plasticity after 100 post-pre pairings with mΔt = −18 ms and σΔt = 7.9 ms; the mean baseline EPSC amplitude, 194 ± 3 pA, did not show significant change 45 minutes after pairing, 186 ± 4 pA. Upper panel, time course of Ri (baseline: 198 ± 2 MΩ and 45–55 min after pairings: 194 ± 2 MΩ; change of 2%) and holding current (Ihold). Inset: distribution of the 100 ΔtSTDP. (c2) Averaged time-courses of the absence of plasticity after 100 post-pre pairings with 3 < σΔt < 10 ms. (c3) Relationship between the STDP magnitude and the jitter. (d) eCB-tLTD is not affected by 3 < σΔt < 10 ms. (d1) Example of tLTD induced by 100 pre-post pairings with mΔt = +18 ms and σΔt = 7.1 ms; the mean baseline EPSC amplitude, 160 ± 3 pA, decreased by 50%, to 81 ± 5 pA, 45 minutes after pairings. Upper panel, time course of Ri (baseline: 112 ± 1 MΩ and 35–45 min after pairings: 118 ± 1 MΩ; change of 5%) and Ihold. Inset: distribution of the 100 ΔtSTDP. (d2) Averaged time-courses of tLTD induced by 100 pre-post pairings with 3 < σΔt < 10 ms. (d3) Relationship between the STDP magnitude and the jitter. (e) eCB-tLTP is not affected by 3 < σΔt < 10 ms. (e1) Example of tLTP induced by 10 post-pre pairings with mΔt = −21 ms and σΔt = 6.4 ms; the mean baseline EPSC amplitude, 113 ± 2 pA, increased by 115% to 243 ± 6 pA 45 minutes after pairings. Upper panel, time course of Ri (baseline: 185 ± 1 MΩ and 45–55 min after pairings: 197 ± 2 MΩ; change of 6%) and Ihold. Inset: distribution of the 10 ΔtSTDP. (e2) Averaged time-courses of Figure 3. Differential effect of jitter on NMDAR- and eCB-mediated plasticity. (a) STDP protocol; ΔtSTDP indicates the time between pre- and postsynaptic stimulations. ΔtSTDP < 0 and ΔtSTDP > 0 refer to post-pre and pre-post pairings, respectively. Results We also considered a combination of triangularly-distributed jitter with random IPIs distributed according to an exponential distribution with refractory period τr = 0.95 s (see Methods) in (e). The plots of the amplitude of NMDAR-tLTP, eCB-tLTD and eCB-tLTP for increasing jitter amplitude σΔt (c2, d2, e2) show similar profiles and are similar to panel (a2), indicating that the model predictions are not qualitatively modified by the probability distribution function of the jitter. SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y ntificreports/ Figure 3. Differential effect of jitter on NMDAR- and eCB-mediated plasticity. (a) STDP protocol; ΔtSTDP indicates the time between pre- and postsynaptic stimulations. ΔtSTDP < 0 and ΔtSTDP > 0 refer to post-pre and pre-post pairings, respectively. (b) Example of 5 successive pairings (#5–9, from c1) exhibiting jittered values of ΔtSTDP (#5–9: −10.3, −15.2, −30.5, −20.7 and −17.3 ms); these pairings are indicated with the grey frame within the 100 pairings displaying jittered ΔtSTDP. The distribution of the successive ΔtSTDP illustrate their standard deviation σΔt = 7.9 ms. (c) NMDAR-tLTP is prevented with σΔt > 3 ms. (c1) Example of the absence of plasticity after 100 post-pre pairings with mΔt = −18 ms and σΔt = 7.9 ms; the mean baseline EPSC amplitude, 194 ± 3 pA, did not show significant change 45 minutes after pairing, 186 ± 4 pA. Upper panel, time course of Ri (baseline: 198 ± 2 MΩ and 45–55 min after pairings: 194 ± 2 MΩ; change of 2%) and holding current (Ihold). Inset: distribution of the 100 ΔtSTDP. (c2) Averaged time-courses of the absence of plasticity after 100 post-pre pairings with 3 < σΔt < 10 ms. (c3) Relationship between the STDP magnitude and the jitter. (d) eCB-tLTD is not affected by 3 < σΔt < 10 ms. (d1) Example of tLTD induced by 100 pre-post pairings with mΔt = +18 ms d 7 1 h b li EPSC li d 160±3 A d d b 50% 81±5 A 45 i www.nature.com/scientificreports/ Figure 3. Differential effect of jitter on NMDAR- and eCB-mediated plasticity. (a) STDP protocol; ΔtSTDP Figure 3. Differential effect of jitter on NMDAR- and eCB-mediated plasticity. (a) STDP protocol; ΔtSTDP indicates the time between pre- and postsynaptic stimulations. ΔtSTDP < 0 and ΔtSTDP > 0 refer to post-pre and pre-post pairings, respectively. Results Insets: average of 12 successive EPSC amplitude at baseline (black trace) and at 40–50 min after STDP pairings (grey trace). Error bars represent the SEM. and of the first tLTP region. Therefore, the model emulates both CB1R-tLTD and -tLTP, in agreement with the eCB-tLTD and eCB-tLTP illustrated in the experimental data of Fig. 1d and e, respectively. The model also faith- fully emulates the appearance of NMDAR-tLTP for Npairings > 50. Predicting the effects of jittered STDP with a mathematical model. The small jitter observed in the above experiments was the result of biological variability inherent to the experimental setup. Our next goal was to extend this study to larger jitters (3 < σΔt < 10 ms) that we controlled experimentally. We first used constant intervals between two consecutive pairings (Inter-Pairing Interval, IPI) and used the same definition as above for jittered ΔtSTDP (Methods): ΔtSTDP = mΔt + ξΔt, where ξΔt is a random variable with zero mean and mΔt is the expected value of the spike timing ΔtSTDP over the Npairings. Next, we adjusted the probability distribution function and the variance of ξΔt to perform a parametric exploration of the impact of jitter. p p p p j In Fig. 2, the jitter ξΔt was sampled from a uniform distribution in [−σΔt, +σΔt] (Fig. 2a1). STDP in the model was globally not robust to jitter, since the three forms of plasticity vanished when σΔt was large enough (Fig. 2a2). However, the model delivered the prediction that the three forms of plasticity do not display the same sensitivity to jitter. In particular, NMDAR-tLTP is predicted to be very fragile when subjected to σΔt: its amplitude decreases as soon as σΔt > 2 ms and completely vanishes for σΔt > 4 ms (Fig. 2a2 and b2). In contrast, eCB-plasticity is much less sensitive: eCB-tLTP is still present for jitters as large as 7–8 ms and eCB-tLTD is still expressed with σΔt = 10 ms (Fig. 2b3). We next evaluated the validity of this prediction when the probability distribution function of the jitter changes. Figure 2 illustrates the results we obtained with Gaussian (Fig. 2c) or triangular (Fig. 2d) distributions of σΔt (see Methods). In both cases, the profiles of the robustness curves (Fig. 2c2 and d2) are almost identical to each other and similar to the robustness curves obtained with a uniform distribution (Fig. 2a2). In Fig. Results 2e, we combined a triangular distribution for σΔt with random IPIs, i.e. in this case, stochasticity is applied not only to the timing between the two stimulations of a given pairing, but also to the time interval between two consecutive pairings: IPI distribution was Poisson with refractory period τr and rate λ (see Methods). Despite this increased stochasticity, we found that eCB-tLTD was again more robust, tolerating jitters up to 6–7 ms (eCB-tLTP) or 10 ms (eCB-tLTD) whereas NMDAR-tLTP was fragile, vanishing for σΔt > 4 ms (Fig. 2e2). Therefore, according to our model, the observation that eCB-STDP is more robust to jitter than NMDAR-tLTP could be a general property of the response of the signaling pathways to noisy paired stimulations. Differential effect of jitter on NMDAR- and eCB-mediated plasticity. Based on the model pre- dictions, we next investigated experimentally the sensitivity to jitter of NMDAR-tLTP induced by 100 post-pre pairings (Fig. 3a), using stochastic ΔtSTDP, distributed according to Fig. 2c1. As aforementioned, NMDAR-tLTP can be induced with a σΔt < 3 ms (Fig. 1c3). Increasing σΔt beyond 3 ms was sufficient to obliterate tLTP for 100 post-pre pairings. As exemplified (Fig. 3b and c1), 100 post-pre pairings with σΔt = 7.9 ms and centered on mΔt = −18 ms failed to induce plasticity. Overall, we did not observe tLTP expression for σΔt > 3 ms (92 ± 2%, p = 0.560, n = 7; p < 0.0001 when compared with tLTP observed without jitter) (Fig. 3c2 and c3) in agreement with the model prediction (Fig. 2c2). p g We next investigated the robustness of the eCB-tLTD to the variability of ΔtSTDP. We observed potent tLTD even with large values of σΔt. As shown in Fig. 3d1, 100 pre-post pairings with σΔt = 7.0 ms centered on mΔt = +18 ms induced a large tLTD. Overall, we observed that tLTD was still induced for 100 pre-post pairings with 3 < σΔt < 10 ms (70 ± 6%, p < 0.0001, n = 14; p = 0.1661 when compared with tLTD observed without jitter) (Fig. 3d2 and d3). The mean value of tLTD, observed for 3 < σΔt < 10 ms, was not different from the one observed in control, i.e. for 0 < σΔt < 3 ms (p = 0.400). Results (b) Example of 5 successive pairings (#5–9, from c1) exhibiting jittered values of ΔtSTDP (#5–9: −10.3, −15.2, −30.5, −20.7 and −17.3 ms); these pairings are indicated with the grey frame within the 100 pairings displaying jittered ΔtSTDP. The distribution of the successive ΔtSTDP illustrate their standard deviation σΔt = 7.9 ms. (c) NMDAR-tLTP is prevented with σΔt > 3 ms. (c1) Example of the absence of plasticity after 100 post-pre pairings with mΔt = −18 ms and σΔt = 7.9 ms; the mean baseline EPSC amplitude, 194 ± 3 pA, did not show significant change 45 minutes after pairing, 186 ± 4 pA. Upper panel, time course of Ri (baseline: 198 ± 2 MΩ and 45–55 min after pairings: 194 ± 2 MΩ; change of 2%) and holding current (Ihold). Inset: distribution of the 100 ΔtSTDP. (c2) Averaged time-courses of the absence of plasticity after 100 post-pre pairings with 3 < σΔt < 10 ms. (c3) Relationship between the STDP magnitude and the jitter. (d) eCB-tLTD is not affected by 3 < σΔt < 10 ms. (d1) Example of tLTD induced by 100 pre-post pairings with mΔt = +18 ms and σΔt = 7.1 ms; the mean baseline EPSC amplitude, 160 ± 3 pA, decreased by 50%, to 81 ± 5 pA, 45 minutes after pairings. Upper panel, time course of Ri (baseline: 112 ± 1 MΩ and 35–45 min after pairings: 118 ± 1 MΩ; change of 5%) and Ihold. Inset: distribution of the 100 ΔtSTDP. (d2) Averaged time-courses of tLTD induced by 100 pre-post pairings with 3 < σΔt < 10 ms. (d3) Relationship between the STDP magnitude and the jitter. (e) eCB-tLTP is not affected by 3 < σΔt < 10 ms. (e1) Example of tLTP induced by 10 post-pre pairings with mΔt = −21 ms and σΔt = 6.4 ms; the mean baseline EPSC amplitude, 113 ± 2 pA, increased by 115% to 243 ± 6 pA 45 minutes after pairings. Upper panel, time course of Ri (baseline: 185 ± 1 MΩ and 45–55 min after pairings: 197 ± 2 MΩ; change of 6%) and Ihold. Inset: distribution of the 10 ΔtSTDP. (e2) Averaged time-courses of SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 5 www.nature.com/scientificreports/ tLTP induced by 10 post-pre pairings with 3 < σΔt < 10 ms. (e3) Relationship between the STDP magnitude and the jitter. Results Finally, we investigated the robustness of the eCB-tLTP to the variability of ΔtSTDP and observed potent tLTP even with large values of jitter, i.e. up to σΔt ~ 8 ms. An example of tLTP induced by 10 post-pre pairings with σΔt = 6.4 ms centered on mΔt = −21 ms is illustrated in Fig. 3e1. In summary, tLTP could be induced even for σΔt = 8 ms (160 ± 12%, p = 0.0021, n = 9; p = 0.0732 when compared with tLTP observed without jitter) (Fig. 3e2 and e3); the mean value of tLTP was not different from the one observed in control (p = 0.1660).f f p We ensured that the mΔt absolute values were not different in control and jittered conditions for 100 post-pre pairings (18 ± 2 ms, n = 13, vs 21 ± 3 ms, n = 7, p = 0.4711), 100 pre-post pairings (21 ± 2 ms, n = 10, vs 21 ± 2 ms, n = 14, p = 0.8984), 10 post-pre pairings (17 ± 2 ms, n = 15, vs 20 ± 2 ms, n = 9, p = 0.2297) or between these dif- ferent STDP protocols (one-way ANOVA: p = 0.4851) (Supplementary Fig. 1). p y p pp y g In conclusion, in agreement with our model prediction, whereas NMDAR-tLTP appears fragile against the variability of ΔtSTDP, eCB-plasticity (eCB-tLTD as well as eCB-tLTP) exhibits a large robustness for the temporal imprecision of ΔtSTDP. Model-based analysis of the effects of jittering. The above experimental results provide us with a validation of our mathematical model. We next used this validated model as a tool to investigate the molecular mechanisms behind these differences of robustness. f We have shown in a previous study that eCB-tLTP was expressed when the amount of eCBs produced was large enough that the fraction of activated CB1R, yCB1R, overcomes a threshold θLTP start13. In the absence of added noise, σΔt = 0, our experimental data shows that as few as 5 pairings at 1 Hz and ΔtSTDP = −15 ms are enough to SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 6 www.nature.com/scientificreports/ Figure 4. Model-based analysis of the effects of jittering. Model-predicted time-courses of (a) CB1R activation yCB1R and (b) the concentration of active CaMKII during a protocol with jitter amplitude σΔt = 0 (a1,b1), 5 (a2,b2) or 1 0 (a3,b3) ms. Results The STDP protocol was the same as Fig. 2a1 i.e. uniformly-distributed jitter of the spike timing and deterministic IPIs at 1 Hz. eCB-tLTP (a, red full line) and NMDAR-tLTP (b, red dashed line) were triggered with average spike timing mΔt = −15 ms and eCB-tLTD (a, blue full line) with mΔt = +20 ms. In (a) the shaded boxes locate the areas where yCB1R triggers eCB-tLTP or eCB-tLTD. Those areas are defined by the thresholds θLTP start and (θLTD start, θLTD stop), respectively (see Methods). For mΔt = −15 ms (red), increasing jitter amplitudes progressively hinders the build up of the yCB1R transients. yCB1R still reaches the LTP area for 5–25 pairings with σΔt = 0 (a1) and 5 ms (a2) thus triggering eCB-tLTP but fails to do so for σΔt = 10 ms (a3). Conversely, eCB-tLTD is robust to jitter because yCB1R remains in the LTD region for most of the pairings with mΔt = +20 ms (blue), for all tested jitter amplitudes. In (b), with mΔt = −15 ms (red) the switch from the low activation to the high activation state of CaMKII is obtained in the absence of jittering σΔt = 0 ms (b1). The progressive build up of activated CaMKII is efficiently suppressed as soon as σΔt = 5 ms (b2) thus effectively suppressing NMDAR-tLTP (b3). Figure 4. Model-based analysis of the effects of jittering. Model-predicted time-courses of (a) CB1R activation yCB1R and (b) the concentration of active CaMKII during a protocol with jitter amplitude σΔt = 0 (a1,b1), 5 (a2,b2) or 1 0 (a3,b3) ms. The STDP protocol was the same as Fig. 2a1 i.e. uniformly-distributed jitter of the spike timing and deterministic IPIs at 1 Hz. eCB-tLTP (a, red full line) and NMDAR-tLTP (b, red dashed line) were triggered with average spike timing mΔt = −15 ms and eCB-tLTD (a, blue full line) with mΔt = +20 ms. In (a) the shaded boxes locate the areas where yCB1R triggers eCB-tLTP or eCB-tLTD. Those areas are defined by the thresholds θLTP start and (θLTD start, θLTD stop), respectively (see Methods). For mΔt = −15 ms (red), increasing jitter amplitudes progressively hinders the build up of the yCB1R transients. yCB1R still reaches the LTP area for 5–25 pairings with σΔt = 0 (a1) and 5 ms (a2) thus triggering eCB-tLTP but fails to do so for σΔt = 10 ms (a3). Results Conversely, eCB-tLTD is robust to jitter because yCB1R remains in the LTD region for most of the pairings with mΔt = +20 ms (blue), for all tested jitter amplitudes. In (b), with mΔt = −15 ms (red) the switch from the low activation to the high activation state of CaMKII is obtained in the absence of jittering σΔt = 0 ms (b1). The progressive build up of activated CaMKII is efficiently suppressed as soon as σΔt = 5 ms (b2) thus effectively suppressing NMDAR-tLTP (b3). trigger eCB-tLTP12. Accordingly, our model produces large amounts of eCBs in the first 5–10 pre-post pairings so that yCB1R reaches θLTP start after as few as 5 pairings (Fig. 4a1). When σΔt = 5 ms jitter was added to mΔt = −15 ms, some of the pairings failed to deliver yCB1R transients of maximal amplitude (Fig. 4a2). But for σΔt < 7–8 ms, a number of yCB1R transients still had sufficient amplitude to overcome θLTP start, which was enough to trigger eCB-tLTP (Fig. 4a2). When σΔt was large though (e.g. 10 ms), the yCB1R transients failed to reach the LTP zone and eCB-tLTP was not expressed (Fig. 4a3). p g In experiments without added jitter, eCB-tLTD progressively accumulates when Npairings increases and starts to be significant for Npairings > 2512. In the model with mΔt = +20 ms and σΔt = 0, the yCB1R transients remain in the LTD area for the most part of the STDP pairings (Fig. 4a3). To account for the progressive accumulation of eCB-tLTP with Npairings, each transient in the model contributes a small decrease of the synaptic weight. Even with large amounts of jitter (see e.g. σΔt = 5 or 10 ms in Fig. 4a2,a3) yCB1R transients remain mostly inside LTD area so eCB-tLTD remains expressed even with large jitter. The robustness of NMDAR-tLTP is predicted to be frequency-dependent. Experimentally, NMDAR-tLTP (mΔt = −15 ms and σΔt = 0) was observed at Fpairings = 1 Hz when Npairings > 50, beyond which its amplitude did not depend much on Npairings 13. To account for this feature, the steady-state concentration of acti- vated CaMKII, that sets Wpost in the model, is bistable: 45 min after the stimulation, CaMKII was either almost fully inactivated (“no plasticity” state) or almost fully activated (“LTP” state)13,38. When the frequency of post-pre pairings (−30 < ΔtSTDP < 0 ms) was large enough (i.e. Results Fpairings ≥ 1 Hz), the IPI was smaller than the decay time of the CaMKII activation transient triggered by each post-pre pairings (Fig. 4b1). As a result, in the absence of added noise (σΔt = 0), the CaMKII activation transients progressively built up on top of each other. The accu- mulated CaMKII activation overcomes the threshold between the “no plasticity” and the “LTP” states only for Npairings > 50 (Fig. 4b1)13. With jitter (e.g. σΔt = 5 ms in Fig. 4b2 or 10 ms in Fig. 4b3), many of the IPIs were either too long or too short to trigger maximal amplitude transients of activated CaMKII. As a result, CaMKII activation never reached the threshold, thus explaining the fragility of NMDAR-tLTP with respect to jittering (at 1 Hz). p g g y p j g A major indication from the above analysis is the importance of the IPI frequency for the robustness of NMDAR-tLTP to jitter. Figure 5 compares the robustness to jitter for constant IPI with Fpairings = 1 Hz (Fig. 5a) and 1.05 Hz (Fig. 5b). The size of the green area that locates eCB-tLTD on Fig. 5a2,b2 was not much altered by the increase of the pairing frequency. Likewise, the size of the red area that locates eCB-tLTP in Fig. 5a1,b1 did not p g g y p j g A major indication from the above analysis is the importance of the IPI frequency for the robustness of NMDAR-tLTP to jitter. Figure 5 compares the robustness to jitter for constant IPI with Fpairings = 1 Hz (Fig. 5a) and 1.05 Hz (Fig. 5b). The size of the green area that locates eCB-tLTD on Fig. 5a2,b2 was not much altered by the increase of the pairing frequency. Likewise, the size of the red area that locates eCB-tLTP in Fig. 5a1,b1 did not SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 7 www.nature.com/scientificreports/ Figure 5. Model-based analysis of the change of robustness with frequency. (a) Summary of the robustness of the model plasticity at 1 Hz. The jittered STDP protocol combined uniformly-distributed jitter of the spike timing and constant IPIs at 1 Hz. Two-dimensional maps show alterations of the plasticity obtained with 10 (a1) or 100 (a2) pairings, when the average spike timing mΔt and the jitter amplitude σΔt are changed. Results vary much when the frequency was increased. However, the size of the red area locating NMDAR-tLTP increased drastically with frequency (Fig. 5a2,b2). Therefore, our model predicts that NMDAR-tLTP should become more robust to jitter at larger frequencies, whereas eCB-tLTP and eCB-tLTD are not significantly changed. Several aspects of glutamate signaling at the corticostriatal synapse are known to display complex frequency-dependence39. Our model is calibrated with experimental data at 1 Hz and features none of the above frequency dependencies. Therefore, we cannot expect a precise quantitative match between experiments and model when frequency is varied below or above 1 Hz. However, the model still yields correct predictions of the main qualitative trends observed in the experiments, since the effects of a small change of Fpairings in the model (1.00 to 1.05 Hz) were similar to the effects of larger changes (1 to 3 Hz) in the experiments13. Therefore, in the experiments, we expect to observe a change of robustness pattern similar to Fig. 5b, around Fpairings = 3 Hz rather than 1.05 Hz. j g q , gi y g Several aspects of glutamate signaling at the corticostriatal synapse are known to display complex frequency-dependence39. Our model is calibrated with experimental data at 1 Hz and features none of the above frequency dependencies. Therefore, we cannot expect a precise quantitative match between experiments and model when frequency is varied below or above 1 Hz. However, the model still yields correct predictions of the main qualitative trends observed in the experiments, since the effects of a small change of Fpairings in the model (1.00 to 1.05 Hz) were similar to the effects of larger changes (1 to 3 Hz) in the experiments13. Therefore, in the experiments, we expect to observe a change of robustness pattern similar to Fig. 5b, around Fpairings = 3 Hz rather than 1.05 Hz. Increasing Fpairings and Npairings stabilized NMDAR-tLTP against jitter. We next tested experimentally whether an increase from 1 to 3 Hz of STDP pairings would protect the NMDAR-tLTP against jitter, as predicted by the model. As shown in Fig. 6a1, 100 post-pre pairings at 3 Hz with σΔt = 9 ms and centered on mΔt = −22 ms induced tLTP. Overall, we observed tLTP for 100 post-pre pairings applied at 3 Hz with 4 < σΔt < 9 ms (181 ± 30%, p = 0.0274, n = 9) (Fig. Results The locations of the plasticity blotches are indicated with horizontal lines: full red for eCB-tLTP, full blue for eCB-tLTD and dashed red for NMDAR-tLTP. The length of each of these blotches along the y-axis reflects their robustness to jitter. (a3) A cross-section along the dashed and full horizontal lines of (a1,a2) illustrates the robustness of NMDAR-tLTP, eCB-tLTD and eCB-tLTP. (b) Robustness of the model with larger frequency (1.05 Hz) and same jittered STDP protocol as in (a). Note the distortion between the pairing frequency in the model and in the experiments. The behavior of the model for Fpairings = 1.05 Hz corresponds to experimental results with Fpairings ≈ 3 Hz13. The two-dimensional maps for 10 (b1) or 100 (b2) pairings as well as the cross-sections (b3) predict that for frequencies larger than 1 Hz, the robustness changes, with NMDAR-tLTP becoming more robust. Figure 5. Model-based analysis of the change of robustness with frequency. (a) Summary of the robustness of the model plasticity at 1 Hz. The jittered STDP protocol combined uniformly-distributed jitter of the spike timing and constant IPIs at 1 Hz. Two-dimensional maps show alterations of the plasticity obtained with 10 (a1) or 100 (a2) pairings, when the average spike timing mΔt and the jitter amplitude σΔt are changed. The locations of the plasticity blotches are indicated with horizontal lines: full red for eCB-tLTP, full blue for eCB-tLTD and dashed red for NMDAR-tLTP. The length of each of these blotches along the y-axis reflects their robustness to jitter. (a3) A cross-section along the dashed and full horizontal lines of (a1,a2) illustrates the robustness of NMDAR-tLTP, eCB-tLTD and eCB-tLTP. (b) Robustness of the model with larger frequency (1.05 Hz) and same jittered STDP protocol as in (a). Note the distortion between the pairing frequency in the model and in the experiments. The behavior of the model for Fpairings = 1.05 Hz corresponds to experimental results with Fpairings ≈ 3 Hz13. The two-dimensional maps for 10 (b1) or 100 (b2) pairings as well as the cross-sections (b3) predict that for frequencies larger than 1 Hz, the robustness changes, with NMDAR-tLTP becoming more robust. vary much when the frequency was increased. However, the size of the red area locating NMDAR-tLTP increased drastically with frequency (Fig. 5a2,b2). Therefore, our model predicts that NMDAR-tLTP should become more robust to jitter at larger frequencies, whereas eCB-tLTP and eCB-tLTD are not significantly changed. Results Increasing frequency and Npairings stabilized NMDAR-tLTP against jitter. (a) 100 post-pre pairings at d d T h h ff d b ( ) l f T d d b We next investigated whether a higher Npairings would secure the expression of NMDAR-tLTP even with σΔt > 3 ms (Fig. 6b). When we doubled Npairings we did not observe expression of significant tLTP. Indeed, 200 post-pre pairings (−30 < mΔt < 0 ms with 3 < σΔt < 10 ms) did not induce plasticity (112 ± 9%, p = 0.236, n = 6; Fig. 6b3) but when we increased Npairings up to 250–300, tLTP was reliably observed with σΔt > 3 ms. Figure 6b1 shows an example of tLTP induced by 250 pairings with σΔt = 5 ms and centered on −20 ms. Overall, 250–300 post-pre STDP pairings (−30 < mΔt < 0 ms with 3 < σΔt < 10 ms) induced tLTP (170 ± 10%, p = 0.0012, n = 6; Fig. 6b2 and b3); the mean values of these tLTP observed for Npairings = 250–300 at 1 Hz with 4 < σΔt < 9 ms were not different compared to tLTP induced with 100 post-pre pairings at 1 Hz with σΔt < 3 ms (p = 0.1952). We observed different synaptic efficacy changes following 100, 200 and 250–300 post-pre pairings (ANOVA, p < 0.0001). Transitions from spike-timing- to frequency-dependent plasticity. We next tested with our mathe- matical model whether the robustness of STDP to jitter of the spike timing depends on the regularity of the spike train. In Fig. 2e, we show an example of the combination of stochastic spike timings (triangular distribution) with stochastic IPIs (Poisson distributed) with a constant and relatively large refractory period τr (see Methods- Mathematical model, Protocol 4). However, when τr decreases, short IPIs are more frequently sampled and the train of presynaptic stimulation becomes more irregular. Therefore, through variations of the refractory period τr, the presynaptic stimulation can be progressively switched from highly irregular to regular spike trains. Figure 7 shows model predictions for the alteration of the robustness curves when τr varies. With high refrac- tory period, i.e. when τr ≈ Fpairings, the IPIs are weakly noisy, so one recovers the robustness curves of Fig. 2e, with eCB-STDP being more robust than NMDAR-tLTP. Results 6a2 and a3); the mean value of these tLTP observed at 3 Hz with 4 < σΔt < 9 ms were not significantly different from the one observed with stimulation-related σΔt < 3 ms at 1 Hz (p = 0.520). When we plotted the magnitude of plasticity against σΔt for 100 post-pre pairings at 3 Hz, we observed that tLTP was still induced even for σΔt = 9 ms (Fig. 6a3). We observed different synaptic efficacy changes following 100 post-pre pairings at 1 Hz with and without jitter and at 3 Hz with jitter (ANOVA, p < 0.0001). The NMDAR-tLTP thus acquires certain robustness to the variability of ΔtSTDP with increasing frequency of pairings. 8 SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y www.nature.com/scientificreports/ Figure 6. Increasing frequency and Npairings stabilized NMDAR-tLTP against jitter. (a) 100 post-pre pairings at 3 Hz induced a tLTP, which is not affected by 3 < σΔt < 10 ms. (a1) Example of tLTP induced by 100 post-pre pairings at 3 Hz with mΔt = −22 ms and σΔt = 9 ms; the mean baseline EPSC amplitude, 194 ± 4 pA, increased by 212%, to 606 ± 7 pA, 45 min after pairings. Upper panel, time course of Ri (baseline: 123 ± 1 MΩ and 45–55 min after pairings: 138 ± 1 MΩ; change of 12%) and Ihold for this cell. Inset: distribution of ΔtSTDP for the 100 pairings. (a2) Averaged time-courses of tLTP induced by 100 post-pre pairings at 3 Hz with 3 < σΔt < 10 ms. (a3) Relationship between the STDP magnitude and the jitter for each of the recorded neurons after 100 pairings at 1 Hz (n = 7; blue circles) or at 3 Hz (n = 10; red circles). (b) 250–300 post-pre pairings induced tLTP, which is not affected by 3 < σΔt < 10 ms. (b1) Example of tLTP induced by 250 post-pre pairings with mΔt = −20 ms and σΔt = 5 ms; the mean baseline EPSC amplitude, 126 ± 3 pA, increased by 57% to 198 ± 4 pA 45 minutes after pairings. Upper panel, time course of Ri (baseline: 65 ± 1 MΩ and 45–55 min after pairings: 65 ± 2 MΩ; change < 1%) and Ihold. Inset: distribution of ΔtSTDP for the 250 pairings. (b2) Averaged time-courses of tLTP induced by 250–300 post-pre pairings with 3 < σΔt < 10 ms. Results (b3) Relationship between the STDP magnitude and the jitter for each of the recorded neurons after 100 (n = 7; blue circles), 200 (n = 6; black circles) or 250–300 (n = 7; red circles) post-pre pairings. Insets: average of 12 successive EPSC amplitude at baseline (black trace) and at 40–50 min after STDP pairings (grey trace). Error bars represent the SEM. Figure 6. Increasing frequency and Npairings stabilized NMDAR-tLTP against jitter. (a) 100 post-pre pairings at 3 Hz induced a tLTP, which is not affected by 3 < σΔt < 10 ms. (a1) Example of tLTP induced by 100 post-pre pairings at 3 Hz with mΔt = −22 ms and σΔt = 9 ms; the mean baseline EPSC amplitude, 194 ± 4 pA, increased by 212%, to 606 ± 7 pA, 45 min after pairings. Upper panel, time course of Ri (baseline: 123 ± 1 MΩ and 45–55 min after pairings: 138 ± 1 MΩ; change of 12%) and Ihold for this cell. Inset: distribution of ΔtSTDP for the 100 pairings. (a2) Averaged time-courses of tLTP induced by 100 post-pre pairings at 3 Hz with 3 < σΔt < 10 ms. (a3) Relationship between the STDP magnitude and the jitter for each of the recorded neurons after 100 pairings at 1 Hz (n = 7; blue circles) or at 3 Hz (n = 10; red circles). (b) 250–300 post-pre pairings induced tLTP, which is not affected by 3 < σΔt < 10 ms. (b1) Example of tLTP induced by 250 post-pre pairings with mΔt = −20 ms and σΔt = 5 ms; the mean baseline EPSC amplitude, 126 ± 3 pA, increased by 57% to 198 ± 4 pA 45 minutes after pairings. Upper panel, time course of Ri (baseline: 65 ± 1 MΩ and 45–55 min after pairings: 65 ± 2 MΩ; change < 1%) and Ihold. Inset: distribution of ΔtSTDP for the 250 pairings. (b2) Averaged time-courses of tLTP induced by 250–300 post-pre pairings with 3 < σΔt < 10 ms. (b3) Relationship between the STDP magnitude and the jitter for each of the recorded neurons after 100 (n = 7; blue circles), 200 (n = 6; black circles) or 250–300 (n = 7; red circles) post-pre pairings. Insets: average of 12 successive EPSC amplitude at baseline (black trace) and at 40–50 min after STDP pairings (grey trace). Error bars represent the SEM. igure 6. Discussion Here, we have introduced jittered STDP protocols as noisy variants of STDP protocols where the spike tim- ing, i.e. the delay between a presynaptic and paired postsynaptic stimulation (or vice-versa), is a random varia- ble. Our results show that the magnitude of variability that STDP can tolerate varies depending on the pairing frequency and on the STDP form, i.e. NMDAR-tLTP, eCB-tLTP or eCB-tLTD. At 1 Hz pairing frequency, eCB-STDP (eCB-tLTD and eCB-tLTP) is much more robust than NMDAR-dependent tLTP. However, this robustness depends on the number and frequency of pairings. Indeed, increasing the average pairing frequency to 3 Hz improved the robustness of NMDAR-tLTP. We observed a similar improvement of the robustness of NMDAR-tLTP to jitter when the number of pairings increased more than two folds.hih j p g These results have wide-ranging implications for STDP expression in in vivo-like firing. They indicate that eCB-STDP could be more likely responsible for fast learning involving few trials (eCB-tLTP) or for learning at lower activity frequency than NMDAR-STDP. NMDAR-STDP could however take part in learning for larger fre- quencies or when the same spike timing recurs a large number of times during sustained activity. It also implies that in synapses where NMDAR is the main coincidence detector for tLTD or tLTP3,5,6,40–42, a high degree of precision of the spike timing, and/or an increased number or frequency of pairings, are required for the emer- gence of the NMDAR-mediated plasticity. This can be viewed as a mechanism preventing the occurrence of spu- rious plasticity for noisy neural network activity. At synapses in which NMDAR and endocannabinoids are both involved in STDP expression12,13,25–27,33,43 and in which eCB-LTP can occur, as it is the case in the striatum12,13, the hippocampus44–48 or the neocortex49 the emergence of STDP would be possible even in noisy conditions and could thus serve subsequently as a priming for the subsequent expression of NMDAR-tLTP. h d l d h b f T h ff l h k b l q y p g q p In the present study, we explored the robustness of STDP when noise affects not only the spike timing but also the inter-pairing interval (IPI), i.e. the delay between two successive pairings. In the absence of jitter of the spike timing, our model predicts that IPI irregularity tends to consolidate tLTP at the expense of tLTD. Results Hence, when the presynaptic spike train is progressively switched from regular to very irregular spike trains, two main changes occur: (i) the three plasticity forms (NMDAR-tLTP, eCB-tLTD and eCB-tLTP) become increasingly robust to jittering (at the limit of highly irregular spike trains, jittering hardly affects plasticity amplitudes), and (ii) eCB-tLTD is progressively changed to LTP, so that highly irregular paired stimulations with Fpairings = 1 Hz only produce LTP regardless of the sign of the spike timing. τr, NMDAR-tLTP becomes more robust than eCB-tLTP. Moreover, eCB-tLTD (Fpairings = 1 Hz, mΔt = +20 ms, Npairings = 100) progressively stops triggering LTD when τr decreases, even inducing LTP for very low τr. Hence, when the presynaptic spike train is progressively switched from regular to very irregular spike trains, two main changes occur: (i) the three plasticity forms (NMDAR-tLTP, eCB-tLTD and eCB-tLTP) become increasingly robust to jittering (at the limit of highly irregular spike trains, jittering hardly affects plasticity amplitudes), and (ii) eCB-tLTD is progressively changed to LTP, so that highly irregular paired stimulations with Fpairings = 1 Hz only produce LTP regardless of the sign of the spike timing. SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y Results However, when τr is 70% of Fpairings, the robustness of both eCB-tLTP and NMDAR-tLTP increases and both exhibit similar robustness. With even smaller values of SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 9 www.nature.com/scientificreports/ Figure 7. The robustness of plasticity depends on the regularity of the presynaptic spike train. The figure shows the robustness of the model plasticity (eCB-tLTP with red, eCB-tLTD with blue and NMDAR-tLTP with dashed red) with the jittered STDP protocol of Fig. 2e1, i.e. exponentially-distributed IPIs with refractory period τr and triangularly-distributed jitter ξΔt. From right to left, the refractory period τr is gradually decreased. When τr → 1/Fpairings where Fpairings = 1 Hz is the average pairings frequency, one gets regular presynaptic spike trains, similar to classical STDP protocols. In this case (see e.g. τr = 0.95/Fpairings), the robustness pattern is similar to the previous observations at 1 Hz: eCB-STDP is robust, whereas NMDAR-tLTP is fragile. However, when τr → 0, the presynaptic spike train becomes highly irregular. In this case, the robustness patterns changes totally, with NMDAR-LTP becoming the most robust, and eCB-tLTD turning into a LTP. Hence, irregular presynaptic spike trains are predicted to produce robust LTP at the expense of LTD. Figure 7. The robustness of plasticity depends on the regularity of the presynaptic spike train. The figure shows the robustness of the model plasticity (eCB-tLTP with red, eCB-tLTD with blue and NMDAR-tLTP with dashed red) with the jittered STDP protocol of Fig. 2e1, i.e. exponentially-distributed IPIs with refractory period τr and triangularly-distributed jitter ξΔt. From right to left, the refractory period τr is gradually decreased. When τr → 1/Fpairings where Fpairings = 1 Hz is the average pairings frequency, one gets regular presynaptic spike trains, similar to classical STDP protocols. In this case (see e.g. τr = 0.95/Fpairings), the robustness pattern is similar to the previous observations at 1 Hz: eCB-STDP is robust, whereas NMDAR-tLTP is fragile. However, when τr → 0, the presynaptic spike train becomes highly irregular. In this case, the robustness patterns changes totally, with NMDAR-LTP becoming the most robust, and eCB-tLTD turning into a LTP. Hence, irregular presynaptic spike trains are predicted to produce robust LTP at the expense of LTD. τr, NMDAR-tLTP becomes more robust than eCB-tLTP. Moreover, eCB-tLTD (Fpairings = 1 Hz, mΔt = +20 ms, Npairings = 100) progressively stops triggering LTD when τr decreases, even inducing LTP for very low τr. www.nature.com/scientificreports/ plasticity, SDDP) has been reported in the neocortex51, striatum52 and hippocampus53. In the striatum, SDDP dif- fers from STDP on two aspects: (i) SDDP was induced in a larger temporal window (−100 < ΔtSTDP < +100 ms) and (ii) tLTP and tLTD were induced regardless of the spike timing. Therefore, the action potential would not be necessary for plasticity induction, but determinant for the STDP polarity and the width of the ΔtSTDP. The effects of jitter remain to be determined on SDDP, i.e. subthreshold events. j , The polarity of plasticity depends greatly on the frequency of stimulus presentation. Indeed, prolonged firing activity at low and high frequency promote LTD and LTP, respectively, at the same synapses in hip- pocampus54–56. However, alteration of a regular low-frequency stimulation (900 stimulations at 1 Hz) with Poisson-distributed interstimulus intervals (as an approximation of naturalistic patterns) prevents LTD expres- sion in the neocortex57. Interestingly, this LTD induced with regular low-frequency stimulation has been reported to be NMDAR-mediated58, and we show here that NMDAR-plasticity is fragile to jitter especially for low firing rate (1 Hz). It remains thus to investigate in a rate-based paradigm whether Poisson stimulation would erase LTD induced by higher firing rate (3–5 Hz54). Various forms of naturalistic patterns as an attempt to mimic in vivo activity have been experimentally tested for plasticity expression: from single burst of dendritic spikes, which induced NMDAR-LTP in hippocampus59 and NMDAR-LTD in neocortex60, to noisy STDP-like protocols7,19,20,22 and burst-timing-dependent plasticity paradigms61. Based on data harvested in the neocortex, a phenomeno- logical computer modeling showed that for constant frequencies but random spiking, LTP wins over LTD with increasing frequency7. Activity patterns recorded in vivo during specific behavioral tasks have been replayed ex vivo to test their ability to induce plasticity19,56,62. When sequences of spike trains recorded in vivo from visual cortex of the anaesthetized cat in response to natural scenes were replayed ex vivo in layer 2/3 pyramidal cells of rat visual cortex, both LTP and LTD was observed experimentally and modeling suggested the importance of suppressive interactions between spikes19. In the dentate gyrus, in vivo patterns recorded in granule cell during a task of delayed nonmatch-to-sample were able in most of the cases to induce LTP at mossy fibers56. Similarly, when hippocampal place cells activity recorded in vivo were replayed ex vivo, NMDAR-LTP (also dependent on cholin- ergic tone) was observed62. www.nature.com/scientificreports/ Natural stimuli recruit various neuronal circuits leading to presynaptic jitter, which can have important physiological consequences. Indeed, in vivo visually driven presynaptic inputs induce presynaptic jitter leading to broader membrane potential events, and can consequently promote a spatial reorganization of the responses of neurons to neighboring stimuli within a receptive field, as demonstrated in the rat visual cortex22.h p g g pi , The debate about the nature of the neural coding, i.e. rate versus time codes63–65, is prominent when studying STDP. It remains to be tested with mathematical models and experiments whether activity patterns of various vigilance states differently result in the emergence of spike- or rate-coding plasticity. At most of the synapses both rate and time codes co-exist; for example at CA1 synapses, stimuli induced LTD at 2–3 Hz, no plasticity at 10 Hz and LTP at 50 Hz demon- strating a rate code54, and time codes are also supported as illustrated by the expression of various forms of STDP at the same synapse11. An answer to this debate could be that both rate and time codes work together for plasticity expression, as recently shown in the somatosensory cortex66, but their relative contribution in vivo would depend on the ongoing activity (low or high), the cerebral structures, the noise level, the neuromodulatory systems at play and the signaling pathways involved in plasticity expression (as shown in the present study). Briefly, it appears that STDP can emerge form low frequency activity whereas rate codes would take over for higher frequency ranges7,19,20,22. q y y g q y g A major conclusion from our present work is that the robustness of STDP to jitter depends on the underlying signaling pathways. For instance, the robustness of NMDAR-tLTP in our model is dependent on the amplitude of the activated CaMKII transients triggered by each post-pre pairings or on alterations of the ratio between the decay time of these transients and the IPIs. The robustness of STDP to jitter could be similarly controlled by quantitative variations in the underlying pathways. Such variations are expected to occur between two neuronal subtypes or brain regions but also as a result of the activation of a neuromodulatory pathway. www.nature.com/scientificreports/ Therefore, our work suggests that the expression of STDP in in vivo-like firing might appear or disappear as a result of modulations of its robustness to jitter, depending on the properties of the incoming patterns (reflecting for example fast learning or heavy training), but also on the brain region and, more transiently, on the activation of neuromodulatory pathways. Discussion This prediction that irregular IPIs strengthen tLTP when the spike timing is constant confirms the result obtained independently and with different mathematical models7,19,22,24. When IPIs are regular, adding jitter of the spike timing in our model progressively suppresses STDP. However, with more irregular IPIs our model predicts that the three STDP forms should exhibit larger robustness to noise. Therefore our result suggests that jittering of the spike timing has larger impact on STDP when the IPIs are regular, but that jittering has much less consequence with irregular IPIs. Experimental testing of this model prediction would greatly improve our understanding of the induction and maintenance of STDP in in vivo-like firing.h i g Subthreshold postsynaptic depolarization is a key factor in the induction of plasticity50. The existence of plasticity induced by STDP-like protocols without postsynaptic spikes (subthreshold-depolarization-dependent SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 10 www.nature.com/scientificreports/ Animals and brain slice preparation Current- and voltage-clamp recordings were filtered at 5 kHz and sampled at 10 kHz, with the Patchmaster v2 × 32 program (HEKA Elektronik). Spike timing-dependent plasticity protocols: regular and jittered patterns. Electrical stimulations were performed with a concentric bipolar electrode (Phymep, Paris, France) placed in layer 5 of the somatosen- sory cortex. Electrical stimulation was monophasic, at constant current (ISO-Flex stimulator, AMPI, Jerusalem, Israel). Cortical stimulations evoked glutamatergic excitatory postsynaptic currents (EPSCs) (inhibited by CNQX 10 μM and D-AP5 50 μM, n = 6) and not significantly affected by GABAergic events; Indeed, blocking GABAARs with picrotoxin (50 μM) did not significantly affect EPSC amplitude at corticostriatal synapses (123 ± 31 pA before and 118 ± 26 pA after picrotoxin, p = 0.500, n = 5). MSNs were kept at their resting membrane potential (−74.6 ± 0.6 mV, n = 90) and repetitive control stimuli were applied at 0.1 Hz. Currents were adjusted to evoke 50–200 pA EPSCs. STDP protocols consisted of pairings of pre- and postsynaptic stimulations (at 1 Hz) separated by a specific time interval (ΔtSTDP). The paired stimulations were applied at 1 Hz throughout the study except in Fig. 6a1 and a2 in which 3 Hz stimulations were tested. Presynaptic stimulations corresponded to cortical stimu- lations and the postsynaptic stimulation of an action potential evoked by a depolarizing current step (30 ms dura- tion) in MSNs. ΔtSTDP < 0 ms for post-before-pre pairings, and ΔtSTDP > 0 ms for pre-before-post pairings. Neuronal recordings on neurons were made over a period of 10 minutes at baseline, and for at least 60 minutes after the STDP protocols. EPSC baseline stability was assessed by comparing the average of the EPSC magnitude of the two first and the two last minutes of baseline; if the variation of amplitude exceeded 10%, the experiment was discarded4. Recordings were stopped if the injected current was larger than 50 or 100–150 pA during the baseline and after pair- ings, respectively. We individually measured and averaged the amplitude of 60 successive EPSCs from both baseline and 45–55 minutes after STDP protocol, in which the latter was normalized by the former to calculate long-term synaptic efficacy changes. Neuron recordings were made in voltage-clamp mode during baseline and for the 60 min- utes of recording after the STDP protocol, and in current-clamp mode during STDP protocol. Animals and brain slice preparation Animals and brain slice preparation. OFA (Oncins France Strain A) rats (Charles River, L’Arbresle, France) were used at postnatal day 25–32 for brain slice electrophysiology. All experiments were performed in accordance with the guidelines of the local animal welfare committee (Center for Interdisciplinary Research in Biology Ethics Committee) and the EU (directive 2010/63/EU). The experimental protocol (ref. 2017–02 N° 2016120110255176) was approved by the Ethics Committee in Charge of Animal Experimentation (Paris Centre et Sud). Every precaution was taken to minimize stress and the number of animals used in each series of experi- ments. Animals were housed in standard 12 hours light/dark cycles and food and water were available ad libitum. Horizontal brain slices containing the somatosensory cortical area and the corresponding corticostriatal projec- tion field were prepared as previously described36. Corticostriatal connections (between somatosensory cortex layer 5 and the dorso-lateral striatum) are preserved in the horizontal plane. Brain slices (330 µm-thick) were prepared with a vibrating blade microtome (VT1200S, Leica Microsystems, Nussloch, Germany). Brains were sliced in an ice-cold cutting solution (125 mM NaCl, 2.5 mM KCl, 25 mM glucose 25 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, 1 mM pyruvic acid) through which 95% O2/5% CO2 was bubbled. The slices were transferred to the same solution at 34 °C for one hour and then to room temperature. Patch-clamp recordings. Patch-clamp recordings were performed as previously described12,18. Briefly, for whole-cell recordings in borosilicate glass pipettes of 5–7 MΩ resistance were filled with (in mM): 122 K-gluconate, 13 KCl, 10 HEPES, 10 phosphocreatine, 4 Mg-ATP, 0.3 Na-GTP, 0.3 EGTA (adjusted to pH 7.35 with KOH). The composition of the extracellular solution was (mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 11 www.nature.com/scientificreports/ 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 10 μM pyruvic acid bubbled with 95% O2 and 5% CO2. Signals were amplified using EPC10–2 amplifiers (HEKA Elektronik, Lambrecht, Germany). All recordings were performed at 34 °C (Bath-controller V, Luigs&Neumann, Ratingen, Germany) and slices were continuously superfused with extracel- lular solution, at a rate of 2 ml/min. Slices were visualized under microscope (BX51WI Olympus, Rungis, France), with a 4x/0.13 objective for the placement of the stimulating electrode and a 40x/0.80 water-immersion objective for the localization of cells for whole-cell recordings. Animals and brain slice preparation Experiments were excluded if the input resistance (Ri), measured every 10 sec all along the experiment, varied by more than 20% during the very same time period where the EPSC amplitude were measured for plasticity assessment: 10 minutes of baseline and the 10 last minutes of the recording (generally 45–55 minutes after pairings). After recording of 10 min control baseline, drugs were applied in the bath. A new baseline with drugs was recorded after a time lapse of 10 min (to allow the drug to be fully perfused) for 10 min before the STDP protocol. Drugs were present until the end of the recording. All chemicals were purchased from Tocris (Ellisville, MO, USA), except for picrotoxin (Sigma). DL-2-amino-5-phosphono-pentanoic acid (D-AP5; 50 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) were dissolved directly in the extracellular solution and bath applied. N-(piperidin-1-yl)-5-(4-iodophenyl)- 1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; 3 μM) and picrotoxin (50 μM) were dis- solved in ethanol and added to the external solution, such that the final concentration of ethanol was 0.01–0.1%. Δ i For the jittered ΔtSTDP patterns, we used the following algorithm (see Protocol 2, below). We note tprei and tposti the times of the presynaptic and postsynaptic stimulations in the paired stimulation number i, respectively. To set them, we first fixed the times of each presynaptic stimulation using the pairing frequency, i.e. = t i F prei pairings. The postsynaptic times were then chosen randomly according to tposti = tprei + mΔt + ξΔt,i where mΔt is the average spike timing and ξΔt is a random variable with mean 0 and variance σΔt 2 . We defined the Inter-Pairing-Interval (IPI) as the time interval between the presynaptic stimulation times of two consecutive paired stimulations ( = − + t t IPIi pre pre i i 1 ), whereas the spike timing ξt is the time interval between the postsynaptic and the presynaptic stimulation times within a given paired stimulation (Δ = − t t t STDP post pre i i i). In a standard STDP protocol both the spike timing and the IPI are constant. The above algorithm yields constant IPIs and stochastic spike timings. Electrophysiological data analysis. Off-line analysis was performed with Fitmaster (Heka Elektronik) and Igor-Pro 6.0.3 (Wavemetrics). Statistical analysis was performed with Prism 5.02 software. In all cases “n” refers to an experiment on a single cell from a single slice. Animals and brain slice preparation We set ζ = + t i F i pre pre, i pairings and ζ δ = + + − ∆ t m i F t i post post, i pairings , where ζpre and ζpost are i.i.d. random variables with the uniform distribution of eq. (1). The resulting spike timing is ΔtSTDP = mΔt + ξΔt where ξΔt = ζpost − ζpre has a triangular distribution. Note that in this case, one still has ≈ ∀i IPIi F 1 pairings as long as 2|mΔt|Fpairings ≪ 1, which can safely be assumed here since we used |mΔt| < 50 ms and Fpairings < 2 Hz.i y | Δt| pairings (5) Protocol 4 includes both a stochastic spike timing and stochastic IPIs (Fig. 2e1). We first sampled Npairings IPIs according to an exponential distribution with rate λ and refractory period τr: P(I- PI) = Θ (IPI − τr)λ exp (−λ(IPI − τr)), where Θ(x) is the Heaviside function Θ (x) = 1 if x ≥ 0; 0 otherwise. The spike trains defined with protocol 4 are thus Poisson process. We used these IPIs to fix the stimulation times, adding a triangularly distributed jitter as in Protocol 3 above: ζ = ∑ + < t IPI j i j i pre pre, i and ζ δ = ∑ + + − < ∆ t m IPI j i j t i post post, i . To keep the average stimulation frequency at 1/Fpairings, we con- strained the value of the rate λ as λ = (1/Fpairings − τr)−1. y | Δt| pairings (5) Protocol 4 includes both a stochastic spike timing and stochastic IPIs (Fig. 2e1). We first sampled Npairings IPIs according to an exponential distribution with rate λ and refractory period τr: P(I- PI) = Θ (IPI − τr)λ exp (−λ(IPI − τr)), where Θ(x) is the Heaviside function Θ (x) = 1 if x ≥ 0; 0 otherwise. The spike trains defined with protocol 4 are thus Poisson process. We used these IPIs to fix the stimulation times, adding a triangularly distributed jitter as in Protocol 3 above: ζ = ∑ + < t IPI j i j i pre pre, i and ζ δ = ∑ + + − < ∆ t m IPI j i j t i post post, i . Animals and brain slice preparation To simulate Protocol 1, we first set each presynaptic stimulation time using = t i F prei pairings. Postsynaptic times were then fixed by tposti = tprei + mΔt + ξΔt,i − δ. pairings fixed by tposti = tprei + mΔt + ξΔt,i − δ. p i p i (3) Protocol 2 (Fig. 2c1) shared the same definition as Protocol 1, except that the jitter followed a norma distribution with zero mean and variance σΔt 2 :  ξ σ = ∆ ∆ P( ) (0, ) t t 2 . p i p i (3) Protocol 2 (Fig. 2c1) shared the same definition as Protocol 1, except that the jitter followed a normal distribution with zero mean and variance σΔt 2 :  ξ σ = ∆ ∆ P( ) (0, ) t t 2 . i p i (3) Protocol 2 (Fig. 2c1) shared the same definition as Protocol 1, except that the jitter followed a normal distribution with zero mean and variance σΔt 2 :  ξ σ = ∆ ∆ P( ) (0, ) t t 2 . i i (3) Protocol 2 (Fig. 2c1) shared the same definition as Protocol 1, except that the jitter followed a normal distribution with zero mean and variance σΔt 2 :  ξ σ = ∆ ∆ P( ) (0, ) t t 2 . Δt ξ∆ ∆ ( ) ( ) t t (4) For Protocol 3 (Fig. 2d1), we used a triangular distribution of the jitter by adding uniformly distributed jitter to both presynaptic and postsynaptic times. We set ζ = + t i F i pre pre, i pairings and ζ δ = + + − ∆ t m i F t i post post, i pairings , where ζpre and ζpost are i.i.d. random variables with the uniform distribution of eq. (1). The resulting spike timing is ΔtSTDP = mΔt + ξΔt where ξΔt = ζpost − ζpre has a triangular distribution. Note that in this case, one still has ≈ ∀i IPIi F 1 pairings as long as 2|mΔt|Fpairings ≪ 1, which can safely be assumed here since we used |mΔt| < 50 ms and Fpairings < 2 Hz.i ∆t (4) For Protocol 3 (Fig. 2d1), we used a triangular distribution of the jitter by adding uniformly distributed jitter to both presynaptic and postsynaptic times. SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y Animals and brain slice preparation To keep the average stimulation frequency at 1/Fpairings, we con- strained the value of the rate λ as λ = (1/Fpairings − τr)−1. Stimulations. A detailed account of our mathematical model can be found in S1 Text. We modeled glutamate concentration in the synaptic cleft, G(t), as a train of exponentially-decaying impulses triggered by presynaptic stimuli at time tprei. We modeled the electrical response to these stimulations in a postsynaptic element considered as a single isopotential compartment with AMPAR, NMDAR, VSCC and TRPV1 conductances: = − − − − − − − C dV dt g V V I V G t I V G t I V I V AEA I t / ( ) ( , ( )) ( , ( )) ( ) ( , ) ( ) (2) L m L AMPAR NMDAR VSCC TRPV1 action (2) where V is membrane potential; gL and VL are leak conductance and reversal potential respectively; IAMPAR, INMDAR, IVSCC and ITRPV1 are currents through AMPAR, NMDAR, L-type VSCC (v1.3) and TRPV1 respectively. Iaction is the action current resulting from the postsynaptic stimulation (backpropagating action potential on top of a 30 ms depolarization). Details about the analytical expressions of these currents are given in S1 Text. Biochemical signaling. We modeled the kinetics of the biochemical pathways activated by the above electrical stimulations using the model of ref.13. We give below a quick overview of this model and refer to S1 Text for the details. Free cytosolic calcium is one of the main signaling actors in the model. To model its dynamics, we assumed calcium can be transferred from/to two main sources: (i) extracellular calcium, via the plasma membrane channels of eq. (2) above and (ii) the endoplasmic reticulum (ER), via the IP3-dependent calcium-induced calcium release system. These calcium transients activated a network of biochemical pathways that collectively set the synaptic weight. Hence, in this model, the synaptic weight is entirely fixed by the underlying biochemical signaling network13. More precisely, we assumed that the total synaptic weight is the product of a pre- and a postsynaptic contribution Wtotal = WpreWpost. Postsynaptic plasticity was based on the activation by calcium of calmodulin and CaMKII67,68. We assumed that postsynaptic plasticity is directly proportional to the calcium-dependent activation of CaMKII: = + . Animals and brain slice preparation All results are expressed as mean ± SEM in the text and as mean ± SD in the figures. Statistical significance was assessed in unpaired t tests, one way ANOVA, or in one-sample t tests, as appropriate, using the indicated significance threshold (p). Mathematical model. Jittered STDP protocols. In the model, we define the spike timing as ∆ = tSTDPi δ − + t t post pre i i where δ accounts for the time elapsed between the onset of the postsynaptic step current and the action potential it triggers (~3 ms in MSNs). In agreement with the experiment protocol above, we introduced stochasticity of the spike timing by adding a random jitter ξΔt to the spike timing: ΔtSTDP = mΔt + ξΔt, were mΔt is the average spike timing and ξΔt is a random variable whose distribution is given by the STDP protocol. Here, we explored five STDP protocols: (1) Protocol 0 consisted of pairings with no jittering, i.e. = IPIi F 1 pairings and ξΔt = 0. (1) Protocol 0 consisted of pairings with no jittering, i.e. = IPIi F 1 pairings and ξΔt = 0. Fpairings (2) Protocol 1 consisted of deterministic IPIs and uniformly distributed spike timings (Fig. 2a1), i.e. = ∀i IPI , i F 1 pairings , and the probability distribution function of the jitter is given by pairings (2) Protocol 1 consisted of deterministic IPIs and uniformly distributed spike timings (Fi = ∀i IPI , i F 1 pairings , and the probability distribution function of the jitter is given by SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 12 www.nature.com/scientificreports/ ξ ξ σ =     | | < σ Δ Δ Δ Δ P( ) if , 0 otherwise t t t 1 2 t (1) In eq. (1), σΔt defines the maximal jitter amplitude i.e. the level of noise in the protocol. To simulate Protocol 1, we first set each presynaptic stimulation time using = t i F prei pairings. Postsynaptic times were then fixed by tposti = tprei + mΔt + ξΔt,i − δ. In eq. 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SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y Animals and brain slice preparation ⁎ ⁎ W CaMKII CaMKII 1 3 5 (3) post max (3) where CaMKII* and ⁎ CaMKIImax are the current concentration of activated (phosphorylated) CaMKII and its maximum value. Our model also accounts for the biochemical pathways leading to the production of the endocannabinoids 2-arachidonoylglycerol (2-AG) and AEA, and their subsequent activation of CB1R (see S1 Text for further details). In our model, CB1R activation (yCB1R) controls the presynaptic weight Wpre according to the following rule: Ω =      − Θ < < Θ + Θ < y A y A y ( ) 1 if 1 if 1 otherwise (4) CB1R LTD LTD start CB1R LTD stop LTP LTP start CB1R Ω =      − Θ < < Θ + Θ < y A y A y ( ) 1 if 1 if 1 otherwise (4) CB1R LTD LTD start CB1R LTD stop LTP LTP start CB1R (4) and Ω τ = − W dt y W d / ( ( ) )/ W pre CB1R pre pre (5) Details about model implementation and numerics are given in the supplementary information. Details about model implementation and numerics are given in the supplementary information. SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 13 www.nature.com/scientificreports/ Acknowledgements g We thank the L.V. lab members for helpful suggestions and critical comments, in particular Sebastien Valverde for proofreading of the manuscript. This work was supported by grants from INSERM, Collège de France, the Agence Nationale pour la Recherche (Grant Dopaciumcity ANR-14-NEUC-0003), INRIA, CNRS, and the Ecole des Neurosciences de Paris. www.nature.com/scientificreports/ & Le Novère, N. DARPP-32 is a robust integrator of dopamine and glutamate signals. PLoS Comput. Biol. 2, e176 (2006). Author Contributions L.V. and H.B. designed the study; Y.C. and A.M. performed electrophysiological experiments and analysis; H.B. carried out the conception and the design of the mathematical model; H.B. and I.P. performed the acquisition and analysis of data from the mathematical model; H.B. and L.V. wrote the manuscript and all authors have edited and corrected the manuscript; L.V. supervised the whole study. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5. Wang, W. et al. A Primary Cortical Input to Hippocampus Expresses a Pathway-Specific and Endocannabinoid-Dependent Form o Long-Term Potentiation. eNeuro 3, ENEURO.0160–16.2016 (2016). g 6. Zhu, P. J. & Lovinger, D. M. Persistent synaptic activity produces long-lasting enhancement of endocannabinoid modulation and alters long-term synaptic plasticity. J. Neurophysiol. 97, 4386–4389 (2007). g y p p y p y 47. Lin, Q.-S. et al. Hippocampal endocannabinoids play an important role in induction of long-term potentiation and regulation of contextual fear memory formation. Brain Res. Bull. 86, 139–145 (2011). y ( ) 8. Carlson, G., Wang, Y. & Alger, B. E. Endocannabinoids facilitate the induction of LTP in the hippocampus. Nat. Neurosci. 5, 723–724 (2002). ( ) 49. Maglio, L. E., Noriega-Prieto, J. A., Maraver, M. J. & Fernández de Sevilla, D. Endocannabinoid-Dependent Long-Term Potentiation f S ti T i i t R t B l C t C b C t 1 14 htt //d i /10 1093/ /bh 053 (2017) 49. Maglio, L. E., Noriega-Prieto, J. A., Maraver, M. J. & Fernández de Sevilla, D. Endocannabinoid-Dependent Long-Term Potentiation of Synaptic Transmission at Rat Barrel Cortex. Cereb. Cortex 1–14, https://doi.org/10.1093/cercor/bhx053 (2017). 50. Lisman, J. & Spruston, N. Questions about STDP as a General Model of Synaptic Plasticity. Front. Synaptic Neurosci. 2 (2010). 51. Sjöström, P. J., Turrigiano, G. G. & Nelson, S. B. Endocannabinoid-dependent neocortical layer-5 LTD in the absence of postsynaptic spiking. J. Neurophysiol. 92, 3338–3343 (2004). g p y 2. Fino, E., Deniau, J.-M. & Venance, L. Brief subthreshold events can act as Hebbian signals for long-term plasticity. PLoS ONE 4 e6557 (2009).i ( ) 3. Brandalise, F. & Gerber, U. Mossy fiber-evoked subthreshold responses induce timing-dependent plasticity at hippocampal CA3 recurrent synapses. Proc. Natl. Acad. Sci. USA 111, 4303–4308 (2014).f y p 54. Dudek, S. M. & Bear, M. F. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade. Proc. Natl. Acad. Sci. USA 89, 4363–4367 (1992). p 5. Mulkey, R. M. & Malenka, R. C. Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9, 967–975 (1992).ii pp p ( ) 6. Mistry, R., Dennis, S., Frerking, M. & Mellor, J. R. Dentate gyrus granule cell firing patterns can induce mossy fiber long-term potentiation in vitro. Hippocampus 21, 1157–1168 (2011). pp p 57. Perrett, S. P., Dudek, S. M., Eagleman, D., Montague, P. R. www.nature.com/scientificreports/ & Friedlander, M. J. LTD induction in adult visual cortex: role of stimulus timing and inhibition. J. Neurosci. 21, 2308–2319 (2001).f g 58. Massey, P. V. et al. Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long- term depression. J. Neurosci. 24, 7821–7828 (2004). p ( ) 59. Remy, S. & Spruston, N. Dendritic spikes induce single-burst long-term potentiation. PNAS 104, 17192–17197 (2007). l h ff l h k h l h k b l l d d k d l 60. Holthoff, K., Kovalchuk, Y., Yuste, R. & Konnerth, A. Single-shock LTD by local dendritic spikes in pyramidal neurons of mouse visual cortex. J. Physiol. (Lond.) 560, 27–36 (2004). y 1. Butts, D. A., Kanold, P. O. & Shatz, C. J. A burst-based ‘Hebbian’ learning rule at retinogeniculate synapses links retinal waves to activity-dependent refinement. PLoS Biol. 5, e61 (2007).i 61. Butts, D. A., Kanold, P. O. & Shatz, C. J. A burst-based 1. Butts, D. A., Kanold, P. O. & Shatz, C. J. A burst based Hebbian learning rule at retinogeniculate synapses links retinal waves to activity-dependent refinement. PLoS Biol. 5, e61 (2007). 2. Isaac, J. T. R., Buchanan, K. A., Muller, R. U. & Mellor, J. R. Hippocampal place cell firing patterns can induce long-term synaptic activity-dependent refinement. PLoS Biol. 5, e61 (2007) y pi 2. Isaac, J. T. R., Buchanan, K. A., Muller, R. U. & Mellor, J. R. Hippocampal place cell firing patterns can induce long-term synaptic plasticity in vitro. J. Neurosci. 29, 6840–6850 (2009). p y 3. Shmiel, T. et al. Neurons of the cerebral cortex exhibit precise interspike timing in correspondence to behavior. PNAS 102 18655–18657 (2005). 64. 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SCIENTIFIC RePorTS | (2018) 8:8139 | DOI:10.1038/s41598-018-26436-y 14 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26436-y Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Identification and evaluation of circulating small extracellular vesicle microRNAs as diagnostic biomarkers for patients with indeterminate pulmonary nodules
Journal of nanobiotechnology
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Zheng et al. Journal of Nanobiotechnology (2022) 20:172 https://doi.org/10.1186/s12951-022-01366-0 Zheng et al. Journal of Nanobiotechnology (2022) 20:172 https://doi.org/10.1186/s12951-022-01366-0 Journal of Nanobiotechnology Open Access Identification and evaluation of circulating small extracellular vesicle microRNAs as diagnostic biomarkers for patients with indeterminate pulmonary nodules Di Zheng1†, Yuming Zhu2†, Jiyang Zhang3†, Wei Zhang3†, Huizhen Wang3, Hao Chen3, Chunyan Wu4, Jian Ni1, Xiaoya Xu3, Baoning Nian3, Sheng Chen3, Beibei Wang3, Xiaofang Li3, Yanan Zhang3, Jiatao Zhang5, Wenzhao Zhong5, Lei Xiong3, Fugen Li3, Dadong Zhang3*, Jianfang Xu1* and Gening Jiang2* Abstract Background:  The identification of indeterminate pulmonary nodules (IPNs) following a low-dose computed tomog- raphy (LDCT) is a major challenge for early diagnosis of lung cancer. The inadequate assessment of IPNs’ malignancy risk results in a large number of unnecessary surgeries or an increased risk of cancer metastases. However, limited studies on non-invasive diagnosis of IPNs have been reported. Methods:  In this study, we identified and evaluated the diagnostic value of circulating small extracellular vesicle (sEV) microRNAs (miRNAs) in patients with IPNs that had been newly detected using LDCT scanning and were sched- uled for surgery. Out of 459 recruited patients, 109 eligible patients with IPNs were enrolled in the training cohort (n = 47) and the test cohort (n = 62). An external cohort (n = 99) was used for validation. MiRNAs were extracted from plasma sEVs, and assessed using Small RNA sequencing. 490 lung adenocarcinoma samples and follow-up data were used to investigate the role of miRNAs in overall survival. Results:  A circulating sEV miRNA (CirsEV-miR) model was constructed from five differentially expressed miRNAs (DEMs), showing 0.920 AUC in the training cohort (n = 47), and further identified in the test cohort (n = 62) and in an external validation cohort (n = 99). Among five DEMs of the CirsEV-miR model, miR-101-3p and miR-150-5p were significantly associated with better overall survival (p = 0.0001 and p = 0.0069). The CirsEV-miR scores were calcu- lated, which significantly correlated with IPNs diameters (p < 0.05), and were able to discriminate between benign *Correspondence: dadong.zhang@3dmedcare.com; xujianfang63@aliyun. com; geningjiang@tongji.edu.cn †Di Zheng, Yuming Zhu, Jiyang Zhang and Wei Zhang contributed equally to this work 1 Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, No. 507 Zhengmin Road, Yangpu District, Shanghai 200433, China 2 Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, No. 507 Zhengmin Road, Yangpu District, Shanghai 200433, China g 3 3D Medicines Inc, Building 2A, No. 158 Xin Junhuan Road, Pujiang Hi‑tech Park, Shanghai 201114, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 17 Zheng et al. Journal of Nanobiotechnology (2022) 20:172 and malignant PNs (diameter ≤ 1 cm). The expression patterns of sEV miRNAs in the benign, adenocarcinoma in situ/ minimally invasive adenocarcinoma, and invasive adenocarcinoma subgroups were found to gradually change with the increase in aggressiveness for the first time. Among all DEMs of the three subgroups, five miRNAs (miR-30c-5p, miR-30e-5p, miR-500a-3p, miR-125a-5p, and miR-99a-5p) were also significantly associated with overall survival of lung adenocarcinoma patients. Conclusions:  Our results indicate that the CirsEV-miR model could help distinguish between benign and malignant PNs, providing insights into the feasibility of circulating sEV miRNAs in diagnostic biomarker development. Trial registration: Chinese Clinical Trials: ChiCTR1800019877. Registered 05 December 2018, https://​www.​chictr.​org.​cn/​ showp​roj.​aspx?​proj=​31346. Keywords:  Indeterminate pulmonary nodule, Low-dose computed tomography, Small extracellular vesicle, microRNA, Small RNA sequencing Graphical Abstract LDCT to improve its ability of distinguishing between benign and malignant PNs among IPNs is urgently needed. Background The training cohort consisted of 47 patients with IPNs, including 17 benign PNs (benign group) and 30 malig- nant PNs (malignant group) identified using pathological diagnosis; the test cohort consisted of 62 patients with IPNs, including 24 benign PNs and 38 malignant PNs. Benign PNs were used as controls. In addition, an exter- nal validation cohort (n = 99), including 20 patients with benign PNs and 79 patients with malignant PNs, was used to validate the model (Fig. 1 and Table 1), and 11 healthy people were as healthy control. More than 90% of LUAD patients (92.6%, 63/68) were in the early stage (no lymph node metastasis), and 33.0% (36/109) of patients had IPNs with a diameter less than 1 cm (Table 1). The demographic and clinicopathologic characteristics of the patients are shown in Table 1. Benign PNs included cases of atypical adenomatous hyperplasia (AAH), fibrosis, granulomas, hamartoma, organizing pneumonias (OPs), cyst, and other benign subtypes, while malignant PNs included AIS, MIA, and invasive adenocarcinoma. The pathology subtype composition of the three cohorts is shown in (Additional file 1: Figure S2). The representative imaging features of benign and malignant PNs are shown in (Additional file 1: Figure S3). of lung cancer patients have also been found to be signifi- cantly different from those of healthy people, indicating that serum/plasma sEV miRNAs as potential biomarkers of lung cancer [13, 14]. Some studies have attempted to distinguish malignant PNs using plasma sEV miRNAs [15–17], however, these studies included healthy people as controls to develop a diagnostic model, and lacks fur- ther investigation in biological difference between benign and malignant PNs. Zhang and colleagues managed to distinguish malignant ground-glass nodules and benign nodules using plasma sEV miRNAs, but the sample sizes were relatively small [18]. The discrimination between benign and malignant PNs are quite difficult to identify, yet very important for accurate diagnosis of lung cancer after LDCT scanning in clinical practice. g In the present study, we assessed the expression levels of circulating sEV miRNAs using small RNA sequenc- ing to detect the differences between patients with benign PNs and patients with malignant PNs (early- to mid-stage lung adenocarcinoma). We then developed a CirsEV-miR model to differentiate between benign and malignant PNs in a training cohort (n = 47) and further confirmed the model in a test cohort (n = 62) and in an external validation cohort (n = 99). Participants and clinical characteristics Th kfl f d ll The workflow of our study is illustrated in Fig. 1 To explore circulating sEV miRNAs as diagnostic biomarkers in patients with lung IPNs, a total of 199 and 260 patients were recruited in the training phase and the test phase, respectively. After the elimination of hemolysis, non- lung adenocarcinoma (non-LUAD), failure of sequencing Background Lung cancer is the most frequent malignancy with the highest morbidity and mortality worldwide [1]. In the U.S. National Lung Cancer Screening Test (NLST) trial [2], low-dose computed tomography (LDCT) has been proven to be a highly sensitive method to detect early stage lung cancer in patients with a smoking history. LDCT scanning has dramatically improved the abil- ity to detect pulmonary nodules (PNs); however, a total of 96.4% of false positive screening results mandates improvements in the management of indeterminate pul- monary nodules (IPNs), which are difficult to diagnose. The inadequate assessment of IPNs’ malignancy risk is a major cause of misdiagnosis and mistreatment. Benign PNs require no surgical resection, however, the rates of benign PNs undergoing surgical resection have been reported to range from 1 to 30% in suspected lung cancer cases [3]. The invasive thoracic procedure of unnecessary operations performed in patients with benign PNs results in unnecessary costs, societal burden, and morbidity and mortality risk to the patient, with no therapeutic benefit [4, 5]. Hence, a non-invasive auxiliary diagnostic test for Extensive efforts using blood biomarkers, such as DNA, RNA, and proteins to distinguish malignant from benign PNs have yielded novel insights into lung cancer diagnosis [6]. Small extracellular vesicles (sEVs), secreted by a variety of cells into the blood, contain bioactive mol- ecules such as proteins, lipids, and nucleic acids that can mirror the cellular origin and the physiological state, and these molecules are attractive potential biomark- ers representing the “fingerprint” or “signature” of the donor cell [7]. Moreover, the membranous structure of the sEVs protects the luminal contents, avoiding degra- dation by extracellular enzymes. The remarkable stability and activity strengthen the potential of circulating sEVs to be reservoirs for biomarker development [8, 9]. Micro- RNAs (miRNAs) have been found to be the most abun- dant species among plasma-derived sEVs RNAs [10], and are also notably stable under different storage conditions [11]. Lung cancer cells secrete more sEVs into blood than normal tissue cells [12], and miRNAs derived from sEVs Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 3 of 17 Zheng et al. Journal of Nanobiotechnology (2022) 20:172 library construction, and limitation of the ratio of benign and malignant PNs, a total of 109 patients were included in two independent cohorts (Additional file 1: Figure S1). Circulating sEV characterization Circulating sEVs were successfully isolated from patient plasma samples and characterized using western blot (WB) analysis, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). In accordance with the Minimal Information for Studies of Extracellular Vesicles (MISEV) 2018 [19], several protein markers were evaluated using WB in eight representative sEV samples from the patients with benign and malignant PNs. The expression levels of TSG101, CD63, CD9, and Syntenin were detected in the eight sEV samples, while a negative marker, Calnexin, was absent in all eight sEV samples (Fig. 2a). Furthermore, the majority of the isolated sEVs were around 100 nm in diameter, which is the typical size of sEVs (Fig. 2b). The TEM result from a representative sample showed that the isolated sEVs were cup-shaped (Fig. 2c), which is the typical morphology of sEVs. Fur- thermore, we also detected sEV transmembrane proteins (CD63, CD81, CD9) using ExoView platform. As shown in Fig. 2d, CD63, CD81, and CD9 were all detected in plasma sEVs of patients with benign or malignant PNs. Background Furthermore, we cal- culated CirsEV-miR scores and for the first time explored the relationship between CirsEV-miR scores and clini- cal characteristics, including the diameter of IPNs and the PN aggressiveness in the benign, adenocarcinoma in situ (AIS)/minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma. We also found that the circulating sEV miRNA signature was able to discrimi- nate between benign and malignant PNs with the diam- eter ≤ 1 cm, which are otherwise difficult to distinguish in clinical practice. In total, we analyzed circulating sEV miRNAs of 208 patients with IPNs, providing the larg- est sample size among the available studies. Addition- ally, 490 lung adenocarcinoma samples and follow-up data from The Cancer Genome Atlas (TCGA) were used to investigate the role of miRNAs in overall survival. The development of such non-invasive diagnostic test, i.e., the CirsEV-miR model, may complement the highly sensitive but insufficiently specific LDCT and be integrated into the diagnostic algorithm to achieve higher diagnostic accuracy for patients with IPNs. Construction and validation of a circulating sEV miRNA (CirsEV‑miR) model for IPN diagnosis Construction and validation of a circulating sEV miRNA (CirsEV‑miR) model for IPN diagnosis We then extracted the RNA from sEVs and per- formed small RNA sequencing. Raw data of small RNA We then extracted the RNA from sEVs and per- formed small RNA sequencing. Raw data of small RNA Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 4 of 17 Training Cohort (n = 47) Shanghai Pulmonary Hospital 4/2019 - 5/2019 Benign (n = 17) AIS/MIA (n = 13) Invasive (n = 17) Exosome miRNAs small RNA sequencing Candidate miRNAs (6 miRNAs) CirsEV-miR model (5 miRNAs) Target genes function analysis Survival analysis (TCGA) Test Cohort (n = 62) Shanghai Pulmonary Hospital 9/2019 - 10/2019 Benign (n = 24) AIS/MIA (n = 17) Invasive (n = 21) CirsEV-miR model perfomance (ROC analysis) Perfomance in IPNs ≤ 1 cm CirsEV-miR model perfomance (ROC analysis) Discrimination of AIS/MIA (CirsEV-miR score analysis) miRNA expression differences Survival analysis (TCGA) External Validation Cohort (n = 99) Guangdong Provincial People’s Hospital 7/2017 - 3/2018 Benign (n = 20) AIS/MIA (n = 15) Invasive (n = 64) Model perfomance validation (ROC analysis) Model application in IPNs ≤ 1 cm Different combinations Fig. 1  Overall study design and patients in the training, test, and external validation cohorts. AIS adenocarcinoma in situ; MIA minimally invasive carcinoma, ROC receiver operating characteristic Training Cohort (n = 47) Shanghai Pulmonary Hospital 4/2019 - 5/2019 Benign (n = 17) AIS/MIA (n = 13) Invasive (n = 17) Exosome miRNAs small RNA sequencing Candidate miRNAs (6 miRNAs) CirsEV-miR model (5 miRNAs) Target genes function analysis Survival analysis (TCGA) CirsEV-miR model perfomance (ROC analysis) Different combinations Fig. 1  Overall study design and patients in the training, test, and external validation cohorts. AIS adenocarcinoma in situ; MIA minimally invasive carcinoma, ROC receiver operating characteristic Fig. Integration of five miRNAs achieved an even higher AUC of 0.904 (let-7b-3p, miR-125b-5p, miR-197-3p, miR- 150-5p, and miR-3168), while the AUC of the integra- tion of all six miRNAs dropped to 0.794. We then used LASSO-penalized regression to develop a classifier of five miRNAs (let-7b-3p, miR-125b-5p, miR-150-5p, miR- 101-3p, and miR-3168), called the CirsEV-miR model, which exhibited an AUC of 0.920 in the training cohort (Fig. 3d, Additional file  1: Table  S2, Additional file  1: Table  S3), which was the highest AUC among all clas- sifiers. The sensitivity reached 0.900, and the specificity was 0.882. Construction and validation of a circulating sEV miRNA (CirsEV‑miR) model for IPN diagnosis Based on DEMs, CirsEV-miR scores were also generated through LASSO analysis. CirsEV-miR scores of malignant PNs were significantly higher than those of benign PNs in the training cohort (Fig. 3e, p < 0.0001), suggesting that CirsEV-miR scores increase with malig- nancy. The CirsEV-miR model was further confirmed in the test cohort, showing an AUC of 0.763 (Additional file 1: Figure S4b) and significant differences in CirsEV- miR scores between benign and malignant IPNs (Addi- tional file  1: Figure S4c, p = 0.0004). The CirsEV-miR model was also validated in the external cohort con- sisting of 20 patients with benign PNs and 79 patients with malignant PNs; the AUC in that cohort was 0.781 sequencing were filtered and normalized. Unsupervised hierarchical clustering based on the expression levels of circulating sEV miRNAs categorized the samples in a similar pattern as the clinicopathologic classifications did, both in the training cohort (Fig. 3a) and in the test cohort (Additional file  1: Figure S4a), thereby suggest- ing the feasibility of predicting clinicopathologic clas- sifications using circulating sEV miRNA expression. Six DEMs between benign and malignant PNs were identi- fied in the training cohort (Fig. 3b), and these DEMs were selected to construct a model for distinguishing benign PNs from malignant PNs. To verify the potential of miR- NAs as a suitable classifier, receiver operating character- istic (ROC) analysis was performed, and the area under curve (AUC) was calculated (Fig. 3c, Additional file  1: Table  S1, Additional file  1: Table  S2). Let-7b-3p exhib- ited an AUC of 0.875, which was the best performance of an individual miRNA. Next, we integrated two or more miRNAs to further improve the performance. Integration of let-7b-3p and miR-125b-5p led to a slightly improved AUC of 0.886; integrating three miRNAs achieved an AUC of 0.892 (let-7b-3p, miR-125b-5p, and miR-197-3p); and integrating four miRNAs achieved an AUC of 0.882 (let-7b-3p, miR-125b-5p, miR-197-3p, and miR-150-5p). Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 5 of 17 Table 1  The demographic and clinicopathologic characteristics of the patients in three cohorts AIS adenocarcinoma in situ, MIA minimally invasive adenocarcinoma, AAH atypical adenomatous hyperplasia, OP organizing pneumonias, NA not available Characteristic Training cohort (n = 47) Testing cohort (n = 62) External cohort (n = 99) Categories—no. (%)  Benign 17 (36.2) 24 (38.7) 20 (20.2)  Malignant 30 (63.8) 38 (61.3) 79 (79.8)  Age, mean (SD) 58.7 (12.5) 58.1 (10.7) 57.9 (11.9) Age, no. Construction and validation of a circulating sEV miRNA (CirsEV‑miR) model for IPN diagnosis (%)  < 60 24 (51.1) 28 (45.2) 49 (49.5)  ≥ 60 23 (48.9) 34 (54.8) 50 (50.5) Gender, no. (%)  Female 20 (42.6) 37 (59.7) 55 (55.6)  Male 27 (57.4) 25 (40.3) 44 (44.4) Smoking status, no. (%) NA  Yes 12 (25.5) 6 (9.7)  No 35 (74.5) 56 (90.3) Pathology, no. (%)  AIS 3 (6.4) 6 (9.7) 10  MIA 10 (21.3) 11 (17.7) 5  Invasive 17 (36.2) 21 (33.9) 66  AAH 2 (4.3) 2 (3.2) 0  Fibrosis 2 (4.3) 4 (6.5) 0  Granulomas 4 (8.5) 3 (4.8) 0  Hamartoma 2 (4.3) 3 (4.8) 3  OP 4 (8.5) 4 (6.5) 0  Other benign subtypes 3 (6.4) 8 (12.9) 15 Nodule diameter (cm), no. (%)  ≤ 1 12 (25.5) 24 (38.7) 21 (21.2)  > 1 35 (74.5) 38 (61.3) 78 (78.8) Malignant stages, no. (%) NA  0 3 (10.0) 6 (15.8)  IA1 12 (40.0) 13 (34.2)  IA2 5 (16.7) 11 (28.9)  IA3 6 (20.0) 4 (10.5)  IB 1 (3.3) 0  IIA 2 (6.7) 0  IIB 0 1 (2.6)  IIIA 1 (3.3) 3 (7.9) Table 1  The demographic and clinicopathologic characteristics of the patients in three cohorts n situ, MIA minimally invasive adenocarcinoma, AAH atypical adenomatous hyperplasia, OP organizing pneumonias, NA not available (Additional file  1: Figure S4d). We were also interested in finding the difference in the circulating sEV miR- NAs between healthy participants and lung cancer and benign pulmonary nodule patients. Therefore, to exam- ine the discrimination value of CirsEV-miR score, the blood specimens from 11 healthy participants were col- lected and used to analyze the expression levels of the circulating sEV miRNAs using small RNA sequencing. The results showed that healthy controls could be signifi- cantly different from the lung cancer (Additional file 1: Figure S4e, p = 3.07E-06) and benign pulmonary nodule (Additional file  1: Figure S4d). We were also interested in finding the difference in the circulating sEV miR- NAs between healthy participants and lung cancer and benign pulmonary nodule patients. Therefore, to exam- ine the discrimination value of CirsEV-miR score, the blood specimens from 11 healthy participants were col- lected and used to analyze the expression levels of the circulating sEV miRNAs using small RNA sequencing. Construction and validation of a circulating sEV miRNA (CirsEV‑miR) model for IPN diagnosis a Western blotting identified sEV proteins of eight representative samples, including the sEV positive markers TSG101, CD63, CD9, Systenin, and the negative marker Calnexin. b Nanoparticle tracking analysis (NTA) results from representative sEV samples. c Transmission electron microscopic (TEM) images of representative sEVs. d Representative spots and immunofluorescence staining of CD63 (red), CD81 (green), and CD9 (blue) d CD63 CD81 Benign 10 μm 10 μm 10 Merge 10 μm d CD9 10 μm 10 μm 10 μm 10 μm Fig. 2  Characterization of circulating small extracellular vesicles (sEVs). a Western blotting identified sEV proteins of eight representative samples, including the sEV positive markers TSG101, CD63, CD9, Systenin, and the negative marker Calnexin. b Nanoparticle tracking analysis (NTA) results from representative sEV samples. c Transmission electron microscopic (TEM) images of representative sEVs. d Representative spots and immunofluorescence staining of CD63 (red), CD81 (green), and CD9 (blue) Construction and validation of a circulating sEV miRNA (CirsEV‑miR) model for IPN diagnosis The results showed that healthy controls could be signifi- cantly different from the lung cancer (Additional file 1: Figure S4e, p = 3.07E-06) and benign pulmonary nodule patients (Additional file  1: Figure S4e, p = 0.015), indi- cating that the CirsEV-miR score has great capability of discriminating IPNs compared to healthy people. Expres- sion of the five miRNAs of the CirsEV-miR model was also verified by quantitative reverse-transcription PCR in 26 patients with benign or malignant PNs, showing simi- lar patterns with sequencing results (Additional file  1: Figure S4f). These results revealed that the CirsEV-miR model may be a new promising approach to assist in the differential diagnosis of lung IPNs. Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 6 of 17 a 0 100 200 300 400 500 Diameter (nm) 0 0.5 1.0 1.5 2.0 Concentration (particles / ml) 80 115 180 272 428 b c TSG101 44KD Syntenin 32KD CD63 26KD CD9 25KD Calnexin 90KD Benign Malignant 1 2 3 4 1 2 3 4 d 10 μm CD63 Merge CD81 CD9 Benign Malignant 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 200 nm Fig. 2  Characterization of circulating small extracellular vesicles (sEVs). a Western blotting identified sEV proteins of eight representative samples, including the sEV positive markers TSG101, CD63, CD9, Systenin, and the negative marker Calnexin. b Nanoparticle tracking analysis (NTA) results from representative sEV samples. c Transmission electron microscopic (TEM) images of representative sEVs. d Representative spots and immunofluorescence staining of CD63 (red), CD81 (green), and CD9 (blue) a TSG101 44KD Syntenin 32KD CD63 26KD CD9 25KD Calnexin 90KD Benign Malignant 1 2 3 4 1 2 3 4 0 100 200 300 400 500 Diameter (nm) 0 0.5 1.0 1.5 2.0 Concentration (particles / ml) 80 115 180 272 428 b c 200 nm b a c 0 100 200 300 400 500 Diameter (nm) 0 0.5 1.0 1.5 2.0 Concentration (particles / ml) 80 115 180 272 428 TSG101 44KD Syntenin 32KD CD63 26KD CD9 25KD Calnexin 90KD Benign Malignant 1 2 3 4 1 2 3 4 d 10 μm CD63 Merge CD81 CD9 Benign Malignant 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 10 μm 200 nm Fig. 2  Characterization of circulating small extracellular vesicles (sEVs). Biological function enrichment and overall survival analysis of five DEMs in the CirsEV‑miR model Biological function enrichment and overall survival analysis of five DEMs in the CirsEV‑miR model Biological function enrichment and overall survival analysis of five DEMs in the CirsEV‑miR model to tumorigenesis and progression processes, such as MAPK, TGFβ, Hippo, p53 signaling pathways, cell cycle, and adherence junction processes (Fig. 4c, d). Moreover, 490 lung adenocarcinoma samples and their follow-up data from the TCGA showed that patients with high expression of miR-101-3p or miR-150-5p in tumor tissue samples had better overall survival (Fig. 4e, f, p = 0.0001; p = 0.0069). However, the other three miRNAs, let-7b-3p, miR-125b-5p, and miR-3168, were not significantly associated with overall survival (data not shown). The functional enrichment analysis indicated that five miRNAs were involved in tumori- genesis and progression of lung cancer. To evaluate the potential functions of the five miR- NAs used in the CirsEV-miR model, we performed Gene Ontology (GO) and pathway analysis of their target genes (Additional file  1: Table  S4). We found that 2238 mRNAs were targeted by the five miRNAs through the analysis of the miRNA target database. The target genes were enriched in biological processes (BPs), such as cellular nitrogen compound metabolic process, biosynthetic process, and cellular protein modification processes (Fig. 4a), and in molecular functions (MFs), such as ion binding, enzyme bind- ing, and RNA binding (Fig. 4b). In addition, the target genes were significantly enriched in pathways related Page 7 of 17 Zheng et al. Biological function enrichment and overall survival analysis of five DEMs in the CirsEV‑miR model Journal of Nanobiotechnology (2022) 20:172 Cluster Histology Cluster Cluster 1 Cluster 2 Histology Benign AIS/MIA Invasive -2 0 2 Normalized Expression Benign Malignant 0 3 6 9 let-7b-3p Log2CPM **** Benign Malignant 0 6 12 18 miR-101-3p ** Log2CPM Log2CPM Benign Malignant 3 6 9 12 miR-125b-5p *** Benign Malignant 1 4 7 10 miR-150-5p Log2CPM *** Benign Malignant 1 3 5 7 miR-197-3p Log2CPM ** Benign Malignant 1 4 7 10 miR-3168 Log2CPM * Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.886 Two miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.892 Three miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.882 Four miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.904 Five miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.794 Six miRNA classifier a b c d e One miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.875 0.0 0.5 1.0 1.5 Benign Malignant CirsEV miR score **** Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.920 CirsEV-miR model Fig. 3  Classifier development process for IPNs. a Heatmap of sEV-miRNA expression in the training cohort by unsupervised hierarchical clustering. b Expression levels of six identified DEMs in the training cohort. c ROCs of classifiers constructed with different combinations of the six DEMs. Only ROCs with the best diagnostic powers of different miRNA combinations are shown. d ROC curve of the CirsEV-miR model in the training cohort. Biological function enrichment and overall survival analysis of five DEMs in the CirsEV‑miR model e CirsEV-miR scores of benign and malignant PNs in the training cohort Cluster Histology Cluster Cluster 1 Cluster 2 Histology Benign AIS/MIA Invasive -2 0 2 Normalized Expression a Benign Malignant 0 3 6 9 let-7b-3p Log2CPM **** Benign Malignant 0 6 12 18 miR-101-3p ** Log2CPM Log2CPM Benign Malignant 3 6 9 12 miR-125b-5p *** Benign Malignant 1 4 7 10 miR-150-5p Log2CPM *** Benign Malignant 1 3 5 7 miR-197-3p Log2CPM ** Benign Malignant 1 4 7 10 miR-3168 Log2CPM * b d b a 3 6 Log2CPM d c One miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.875 c d d Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.920 CirsEV-miR model Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.892 Three miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.886 Two miRNA classifier c One miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.875 Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.886 Two miRNA classifier Specificity 0.6 0.4 e 0.0 0.5 1.0 1.5 Benign Malignant CirsEV miR score **** Specificity e p y Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.794 Six miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.882 Four miRNA classifier Specificity Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 AUC: 0.904 Five miRNA classifier Six miRNA classifier Fig. 3  Classifier development process for IPNs. a Heatmap of sEV-miRNA expression in the training cohort by unsupervised hierarchical clustering. b Expression levels of six identified DEMs in the training cohort. c ROCs of classifiers constructed with different combinations of the six DEMs. Only ROCs with the best diagnostic powers of different miRNA combinations are shown. d ROC curve of the CirsEV-miR model in the training cohort. e CirsEV-miR scores of benign and malignant PNs in the training cohort The signature of circulating sEV miRNAs discriminated correlation between the nodule size and risk score of the CirsEV-miR model. All 109 eligible samples were recategorized into two subgroups: the IPNs with diameter ≤ 1 cm and IPNs with diameter > 1 cm, and re-ranked according to the CirsEV-miR scores. The CirsEV-miR scores were significantly higher in the subgroup of IPNs with diameter > 1  cm (Fig. 5a, p = 0.0170), suggesting that the CirsEV-miR scores The signature of circulating sEV miRNAs discriminated between benign and malignant PNs with diameter ≤ 1 cm. The size of IPNs is a key factor that is associated with their malignant potential and patients’ long term sur- vival [20, 21]. In clinical practice, IPNs with a diameter > 1  cm are thought to need resection, whereas IPNs with a diameter ≤ 1  cm require repeated scans and a long-term follow-up [22]. Thus, we also analyzed the g g between benign and malignant PNs with diameter ≤ 1 cm.h The size of IPNs is a key factor that is associated with their malignant potential and patients’ long term sur- vival [20, 21]. In clinical practice, IPNs with a diameter > 1  cm are thought to need resection, whereas IPNs with a diameter ≤ 1  cm require repeated scans and a long-term follow-up [22]. Thus, we also analyzed the Zheng et al. The signature of circulating sEV miRNAs discriminated Journal of Nanobiotechnology (2022) 20:172 Page 8 of 17 endosome focal adhesion cytoplasmic stress granule microtubule organizing center cytosol nucleoplasm protein complex SMAD binding small conjugating protein binding virus receptor activity translation factor activity, nucleic acid binding enzyme regulator activity cytoskeletal protein binding poly(A) RNA binding nucleic acid binding transcription factor activity protein binding transcription factor activity RNA binding enzyme binding ion binding 0 25 50 −log10(P−value) Number 200 400 600 Category Biological Process Cellular Component Molecular Function b innate immune response immune system process cell cycle transcription, DNA−templated nucleobase−containing compound catabolic process response to stress cell death catabolic process cellular component assembly small molecule metabolic process neurotrophin TRK receptor signaling pathway cellular protein modification process gene expression biosynthetic process cellular nitrogen compound metabolic process 0 30 60 90 −log10(P−value) tra nuc a b innate immune response immune system process cell cycle transcription, DNA−templated nucleobase−containing compound catabolic process response to stress cell death catabolic process cellular component assembly small molecule metabolic process neurotrophin TRK receptor signaling pathway cellular protein modification process gene expression biosynthetic process cellular nitrogen compound metabolic process 0 30 60 90 −log10(P−value) endosome focal adhesion cytoplasmic stress granule microtubule organizing center cytosol nucleoplasm protein complex SMAD binding small conjugating protein binding virus receptor activity translation factor activity, nucleic acid binding enzyme regulator activity cytoskeletal protein binding poly(A) RNA binding nucleic acid binding transcription factor activity protein binding transcription factor activity RNA binding enzyme binding ion binding 0 25 50 −log10(P−value) Number 200 400 600 Category Biological Process Cellular Component Molecular Function a b Fatty acid metabolism Biosynthesis of unsaturated fatty acids Hippo signaling pathway TGF−beta signaling pathway MAPK signaling pathway Adherens junction Cell cycle Regulation of actin cytoskeleton p53 signaling pathway Endocytosis Focal adhesion mRNA surveillance pathway 12 Fatty acid biosynthesis Lysine degradation 0 6 −log10(P−value) Number 10 25 50 Categrey Genetic Information Processing Cellular Processes Environmental Information Processing Metabolism Viral carcinogenesis Proteoglycans in cancer Transcriptional misregulation in cancer Alcoholism MicroRNAs in cancer Epstein−Barr virus infection Pathways in cancer Herpes simplex infection PI3K−Akt signaling pathway Hippo signaling pathway AMPK signaling pathway FoxO signaling pathway Wnt signaling pathway Rap1 signaling pathway cGMP−PKG signaling pathway MAPK signaling pathway Ras signaling pathway cAMP signaling pathway Adherens junction Cell cycle Regulation of actin cytoskeleton Focal adhesion Signaling pathways regulating pluripotency of stem cells Endocytosis Tight junction Neurotrophin signaling pathway Dopaminergic synapse Adrenergic signaling in cardiomyocytes RNA transport 52 50 37 33 52 31 75 28 65 39 25 27 30 38 29 51 39 26 30 37 49 45 30 41 25 25 27 25 39 0 20 40 60 Annotated Genes Genetic Information Processing Organismal Systems Cellular Processes Environmental Information Processing Human Diseases c d e f +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++ ++++++ + + ++++++++++++++++++++++++++++++++++++++++++++++++++++++ + ++++++ + miR-101-3p 0.00 0.25 0.50 0.75 1.00 0 50 100 150 200 Time (months) Overall survival + Top 25% (n=122) + Bottom 25% (n=122) 122 21 4 3 1 122 15 1 0 0 Bottom 25% Top 25% p = 0.0001 +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++ +++++++ + ++ +++++++++++++++++++++++++++++++++++++++++++++++++++++++++ ++++++++ ++++ 0.00 0.25 0.50 0.75 1.00 0 50 100 150 200 Time (months) 122 17 6 3 1 122 13 0 0 0 Overall survival + Top 25% (n=122) + Bottom 25% (n=122) p = 0.0069 Bottom 25% Top 25% miR-150-5p Fig. The signature of circulating sEV miRNAs discriminated e, f Overall survival analysis of miR-101-3p (e) and miR-150-5p (f) from the TCGA database b a log10(P value) Fatty acid metabolism Biosynthesis of unsaturated fatty acids Hippo signaling pathway TGF−beta signaling pathway MAPK signaling pathway Adherens junction Cell cycle Regulation of actin cytoskeleton p53 signaling pathway Endocytosis Focal adhesion mRNA surveillance pathway 12 Fatty acid biosynthesis Lysine degradation 0 6 −log10(P−value) Number 10 25 Categrey Genetic Information Processing Cellular Processes S c d log10(P value) Fatty acid metabolism Biosynthesis of unsaturated fatty acids Hippo signaling pathway TGF−beta signaling pathway MAPK signaling pathway Adherens junction Cell cycle Regulation of actin cytoskeleton p53 signaling pathway Endocytosis Focal adhesion mRNA surveillance pathway 12 Fatty acid biosynthesis Lysine degradation 0 6 −log10(P−value) Number 10 25 50 Categrey Genetic Information Processing Cellular Processes Environmental Information Processi Metabolism Signa c d e +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ ++++++++++++++++++++++++++++++++++++ miR-101-3p 0.75 1.00 + Top 25% (n=122) + Bottom 25% (n=122) d sing Viral carcinogenesis Proteoglycans in cancer Transcriptional misregulation in cancer Alcoholism MicroRNAs in cancer Epstein−Barr virus infection Pathways in cancer Herpes simplex infection PI3K−Akt signaling pathway Hippo signaling pathway AMPK signaling pathway FoxO signaling pathway Wnt signaling pathway Rap1 signaling pathway cGMP−PKG signaling pathway MAPK signaling pathway Ras signaling pathway cAMP signaling pathway Adherens junction Cell cycle Regulation of actin cytoskeleton Focal adhesion Signaling pathways regulating pluripotency of stem cells Endocytosis Tight junction Neurotrophin signaling pathway Dopaminergic synapse Adrenergic signaling in cardiomyocytes RNA transport 52 50 37 33 52 31 75 28 65 39 25 27 30 38 29 51 39 26 30 37 49 45 30 41 25 25 27 25 39 0 20 40 60 Annotated Genes Genetic Information Processing Organismal Systems Cellular Processes Environmental Information Processing Human Diseases d 25 50 Cellular Processes Environmental Information Processing Metabolism e +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++ ++++++ + + ++++++++++++++++++++++++++++++++++++++++++++++++++++++ + ++++++ + miR-101-3p 0.00 0.25 0.50 0.75 1.00 0 50 100 150 200 Time (months) Overall survival + Top 25% (n=122) + Bottom 25% (n=122) 122 21 4 3 1 122 15 1 0 0 Bottom 25% Top 25% p = 0.0001 +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++ +++++++ + ++ +++++++++++++++++++++++++++++++++++++++++++++++++++++++++ ++++++++ ++++ 0.00 0.25 0.50 0.75 1.00 0 50 100 150 200 Time (months) 122 17 6 3 1 122 13 0 0 0 Overall survival + Top 25% (n=122) + Bottom 25% (n=122) p = 0.0069 Bottom 25% Top 25% miR-150-5p f e Fig. The signature of circulating sEV miRNAs discriminated 4  Target analysis and survival analysis of the five DEMs used for CirsEV-miR model development. A bubble plot of enriched GOs ((Biological process, (a), and (Cellular component and Molecular Function, (b)) of target genes of the five DEMs. c A bubble plot of enriched KEGG pathways of target genes of the five DEMs. d A bar plot showing the number of DEM target genes in each of the KEGG pathways (target genes > 20). Circulating sEV miRNA analysis reveals that AIS/ MIA can alleviate the differentiation between benign and malignant PNs remarkably increase with the diameter of IPNs. More- over, among the patients with small IPNs (diameter ≤ 1 cm), malignant PNs also exhibited higher CirsEV- miR scores than benign PNs (Fig. 5b, p = 0.0088). The AUC of the CirsEV-miR model in patients with small IPNs (diameter ≤ 1 cm) was 0.767 in our cohort (Addi- tional file 1: Figure S5a), and 0.721 in the external vali- dation cohort (Additional file 1: Figure S5b). Moreover, we achieved the diagnostic specificity of 91.7% for the IPNs with diameter ≤ 1  cm in our cohort, and speci- ficity of 75% in the external validation cohort (Fig. 5c). Among the IPNs with diameter ≤ 1  cm, benign and malignant subgroups showed different miRNA expres- sion patterns (Fig. 5d). Twelve DEMs between benign and malignant PNs with diameter ≤ 1 cm were identi- fied (Fig. 5e), and eight of them were involved in lung cancer tumorigenesis and progression, suggesting that even small IPNs were different from benign PNs. These results suggest that even in IPNs with diameter ≤ 1 cm, the circulating sEV miRNA signature can separate benign PNs from malignant PNs. remarkably increase with the diameter of IPNs. More- over, among the patients with small IPNs (diameter ≤ 1 cm), malignant PNs also exhibited higher CirsEV- miR scores than benign PNs (Fig. 5b, p = 0.0088). The AUC of the CirsEV-miR model in patients with small IPNs (diameter ≤ 1 cm) was 0.767 in our cohort (Addi- tional file 1: Figure S5a), and 0.721 in the external vali- dation cohort (Additional file 1: Figure S5b). Moreover, we achieved the diagnostic specificity of 91.7% for the IPNs with diameter ≤ 1  cm in our cohort, and speci- ficity of 75% in the external validation cohort (Fig. 5c). Among the IPNs with diameter ≤ 1  cm, benign and malignant subgroups showed different miRNA expres- sion patterns (Fig. 5d). Twelve DEMs between benign and malignant PNs with diameter ≤ 1 cm were identi- fied (Fig. 5e), and eight of them were involved in lung cancer tumorigenesis and progression, suggesting that even small IPNs were different from benign PNs. These results suggest that even in IPNs with diameter ≤ 1 cm, the circulating sEV miRNA signature can separate benign PNs from malignant PNs. AIS and MIA represent early-stage lung cancer, and both AIS and MIA patients possess superior prognoses to invasive adenocarcinoma [23, 24]. The signature of circulating sEV miRNAs discriminated (b) The CirsEV-miR scores of benign and malignant subgroups of IPNs ≤ 1 cm. c Model sensitivity and specificity of IPNs ≤ 1 cm. d Gene expression heatmap of benign and malignant subgroups of IPNs ≤ 1 cm. e Differentially expressed miRNAs between the benign and malignant subgroups of IPNs ≤ 1 cm The signature of circulating sEV miRNAs discriminated 4  Target analysis and survival analysis of the five DEMs used for CirsEV-miR model development. A bubble plot of enriched GOs ((Biological process, (a), and (Cellular component and Molecular Function, (b)) of target genes of the five DEMs. c A bubble plot of enriched KEGG pathways of target genes of the five DEMs. d A bar plot showing the number of DEM target genes in each of the KEGG pathways (target genes > 20). e, f Overall survival analysis of miR-101-3p (e) and miR-150-5p (f) from the TCGA database Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 9 of 17 Malignant Benign Pathology a b c d e Normalized Expression -2 0 2 0 1 2 3 miR−125a−5p miR−125b−5p miR−181a−3p miR−195−3p miR−197−3p miR−199a−5p miR−328−3p miR−378d miR−4508 miR−450b−5p miR−500a−3p miR−6087 −1.0 −0.5 0.0 0.5 1.0 -Log10P-value Log2FC IPNs ≤1 cm Pathology Predicted Benign Predicted Malignant Sensitivity Specificity Internal Benign (12) 11 1 54.2% 91.7% Malignant (24) 11 13 External Benign (4) 3 1 82.4% 75.0% Malignant (17) 3 14 −0.3 0.0 0.3 0.6 0.9 1.2 <=1 >1 * CirsEV-miR score ** −0.3 0.0 0.3 0.6 0.9 1.2 Benign Malignant CirsEV-miR score Fig. 5  The CirsEV-miR scores increased with the increase in the diameter of IPNs. a CirsEV-miR scores of IPNs ≤ 1 cm and > 1 cm. (b) The CirsEV-miR scores of benign and malignant subgroups of IPNs ≤ 1 cm. c Model sensitivity and specificity of IPNs ≤ 1 cm. d Gene expression heatmap of benign and malignant subgroups of IPNs ≤ 1 cm. e Differentially expressed miRNAs between the benign and malignant subgroups of IPNs ≤ 1 cm c IPNs ≤1 cm Pathology Predicted Benign Predicted Malignant Sensitivity Specificity Internal Benign (12) 11 1 54.2% 91.7% Malignant (24) 11 13 External Benign (4) 3 1 82.4% 75.0% Malignant (17) 3 14 a b −0.3 0.0 0.3 0.6 0.9 1.2 <=1 >1 * CirsEV-miR score ** −0.3 0.0 0.3 0.6 0.9 1.2 Benign Malignant CirsEV-miR score b ** −0.3 0.0 0.3 0.6 0.9 1.2 Benign Malignant CirsEV-miR score b a c e 0 1 2 3 miR−125a−5p miR−125b−5p miR−181a−3p miR−195−3p miR−197−3p miR−199a−5p miR−328−3p miR−378d miR−4508 miR−450b−5p miR−500a−3p miR−6087 −1.0 −0.5 0.0 0.5 1.0 -Log10P-value Log2FC d e e Fig. 5  The CirsEV-miR scores increased with the increase in the diameter of IPNs. a CirsEV-miR scores of IPNs ≤ 1 cm and > 1 cm. Circulating sEV miRNA analysis reveals that AIS/ MIA can alleviate the differentiation between benign and malignant PNs We further compared all DEMs between the benign, AIS/MIA, and invasive adenocarcinoma subgroups (Fig. 6d, e, Addi- tional file 1: Table S5, Additional file 1: Table S6). We only found two overlapping upregulated DEMs (let-7b-3p and miR-125b-5p), which were also elements of our CirsEV- miR model. Thirteen miRNAs (six upregulated and seven downregulated) with repeated emergence were found in Benign_vs_AIS/MIA and Benign_vs_Invasive adeno- carcinoma subgroups, showing some similarity in sEV miRNA profiles of AIS/MIA and invasive adenocarci- noma. Meanwhile, three upregulated miRNAs and four downregulated miRNAs were found in Benign_vs_AIS/ MIA alone, while nine miRNAs showed altered expres- sion in invasive adenocarcinoma compared with AIS/ MIA (three upregulated and six downregulated), sug- gesting difference in sEV-miRNA profiles between AIS/ MIA and invasive adenocarcinoma. Among all DEMs, five miRNAs (miR-30c-5p, miR-30e-5p, miR-500a-3p, miR-125a-5p, and miR-99a-5p) were significantly asso- ciated with overall survival from the TCGA database (Fig. 6f, p = 0.0008; p = 0.0090; p = 0.0110; p = 0.0310, p = 0.0007). Taken together, the results of the circulating sEV miRNA analysis demonstrated that the presence of AIS/MIA could affect the differentiation between benign and malignant PNs, and that AIS/MIA presented the intermediate molecular features of circulating sEV miR- NAs between benign and malignant PNs. we found that this score remarkably increased with the diameter of IPNs, and then gradually changed with the increase in aggressiveness. Moreover, we found that the molecular features of circulating sEV miRNAs of AIS/ MIA were between benign and invasive adenocarcino- mas, meanwhile, AIS/MIA also possessed unique circu- lating sEV miRNA profiles different from the other two subgroups. Furthermore, 490 lung cancer samples and follow-up data from TCGA were used to investigate the role of miRNAs in overall survival. In summary, the CirsEV-miR model developed using circulating sEV miR- NAs could serve as a non-invasive auxiliary test to help physicians to manage the IPNs for early-stage lung can- cer diagnosis. The differentially expressed circulating sEV miRNAs identified in this study might further serve as potential therapeutic targets in future investigations.h This study focused on IPNs and enrolled the patients with highly suspicious malignant PNs undergoing sur- gery (404/459, confirmed by pathology diagnosis after surgery) from the routine work-ups in a tertiary hospital. Unlike previous studies that focused on early lung can- cer screening in low-prevalence populations, our study recruited patients with a high prevalence, and we used benign PNs as controls. (See figure on next page.) Fig. 6  AIS/MIA are intermediate between benign and malignant PNs. a The CirsEV-miR scores of the benign, AIS/MIA, and invasive adenocarcinoma subgroups categorized according to the pathologies. b Gene expression heatmap of all genes in the benign, AIS/MIA and invasive adenocarcinoma subgroups. c The expression levels of the five DEMs in the benign, AIS/MIA, and invasive adenocarcinoma subgroups. d, e Venn diagrams of upregulated (d) or downregulated (e) genes of the benign, AIS/MIA, and IA subgroups. f Overall survival analysis of miR-30c-5p, miR-30e-5p, miR-500a-3p, miR-125a-5p, and miR-99a-5p from the TCGA database. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant Circulating sEV miRNA analysis reveals that AIS/ MIA can alleviate the differentiation between benign and malignant PNs The discrimination between malignant and benign PNs was more difficult than that between the nodule population and non-nodule con- trols [25]. Benign PNs in this study contained a variety of pathological subtypes without selection, reflecting real- world clinical situations. The benign PNs in the exter- nal validation cohort contained different pathological subtypes from those in the training and test cohorts, yet yielding no significant impairment in the model perfor- mance, which indicates that our CirsEV-miR model may have broad clinical application potential. Circulating sEV miRNA analysis reveals that AIS/ MIA can alleviate the differentiation between benign and malignant PNs Therefore, we specu- lated that the possible reason for the relatively low sen- sitivity of the current model was the misprediction of the clinicopathologically equivocal AIS/MIA samples. To test this hypothesis, 109 eligible samples were catego- rized into three subgroups: benign (41, 37.6%), AIS/MIA (30, 27.5%), and invasive adenocarcinoma (38, 34.9%). The proportions of AIS/MIA samples in the training and the test cohorts were 27.7% and 27.4%, respectively (Table 1). For all 109 samples, we calculated the CirsEV- miR scores and ranked each subgroup. The CirsEV-miR scores in the benign, AIS/MIA and invasive adenocar- cinoma subgroups gradually changed with the increase in aggressiveness (Fig. 6a). We also tested the miRNA expression levels; we found that the expression pattern of the AIS/MIA subgroup was intermediate between benign and invasive adenocarcinoma subgroups (Fig. 6b). The expression levels of the five DEMs in the AIS/MIA Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 10 of 17 subgroup were located between the benign and the inva- sive adenocarcinoma subgroups (Fig. 6c). We further compared all DEMs between the benign, AIS/MIA, and invasive adenocarcinoma subgroups (Fig. 6d, e, Addi- tional file 1: Table S5, Additional file 1: Table S6). We only found two overlapping upregulated DEMs (let-7b-3p and miR-125b-5p), which were also elements of our CirsEV- miR model. Thirteen miRNAs (six upregulated and seven downregulated) with repeated emergence were found in Benign_vs_AIS/MIA and Benign_vs_Invasive adeno- carcinoma subgroups, showing some similarity in sEV miRNA profiles of AIS/MIA and invasive adenocarci- noma. Meanwhile, three upregulated miRNAs and four downregulated miRNAs were found in Benign_vs_AIS/ MIA alone, while nine miRNAs showed altered expres- sion in invasive adenocarcinoma compared with AIS/ MIA (three upregulated and six downregulated), sug- gesting difference in sEV-miRNA profiles between AIS/ MIA and invasive adenocarcinoma. Among all DEMs, five miRNAs (miR-30c-5p, miR-30e-5p, miR-500a-3p, miR-125a-5p, and miR-99a-5p) were significantly asso- ciated with overall survival from the TCGA database (Fig. 6f, p = 0.0008; p = 0.0090; p = 0.0110; p = 0.0310, p = 0.0007). Taken together, the results of the circulating sEV miRNA analysis demonstrated that the presence of AIS/MIA could affect the differentiation between benign and malignant PNs, and that AIS/MIA presented the intermediate molecular features of circulating sEV miR- NAs between benign and malignant PNs. subgroup were located between the benign and the inva- sive adenocarcinoma subgroups (Fig. 6c). Discussion With the development of LDCT scanning for early screening and early diagnosis of lung cancer, the number of detected IPNs has dramatically increased. The inad- equate assessment of IPNs’ malignancy risk is a major cause of a large number of unnecessary surgeries or an increased risk of cancer metastases; hence, the accurate diagnosis of IPNs becomes one of the major challenges in clinic. However, a non-invasive diagnostic test to assist LDCT to distinguish benign PNs from malignant PNs in IPN diagnosis is not yet available. In this study, we included 208 patients with IPNs, which is the largest sample size among the available studies; and we devel- oped, tested, and validated the CirsEV-miR model to discriminate between benign and malignant PNs using small RNA sequencing of circulating sEV miRNAs. We also calculated a CirsEV-miR score from the model, and The diameter is an important parameter in assess- ing the malignant potential of an IPN. The possibility of malignancy positively correlates with the diameter. Lung cancer probability was low in IPNs smaller than 5  mm, intermediate in IPNs with a diameter of 5–10  mm, and high in IPNs larger than 10  mm [22]. Clinically, IPNs > 10 mm would be considered to need resection, while IPNs ≤ 10 mm would require repeated scans. We found that CirsEV-miR score was signifi- cantly higher in IPNs > 10 mm than in IPNs ≤ 10 mm. Even in IPNs ≤ 10 mm, CirsEV-miR score of malignant PNs was significantly higher than that of benign PNs (Fig. 5a, b), suggesting that malignancy may increase Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 11 of 17 Fig. 6  (See legend on previous page.) Fig. 6  (See legend on previous page.) Fig. 6 (See legend on previous page ) Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 12 of 17 miRNAs bind to the 3ʹ-untraslated region (UTR) of tar- get mRNA, resulting in post-translational gene silencing either by mRNA degeneration or by inhibition of transla- tion. Thousands of miRNAs have been linked to various human diseases, including cancers [34]. In this study, we identified differentially expressed miRNAs between benign and malignant PNs and constructed the diagnos- tic model composed of five DEMs using LASSO-penal- ized regression. Discussion Functional enrichment analysis revealed that the target genes of the five miRNAs of our diagnostic model were involved in many cancer-related pathways, such as MAPK, TGFβ, Hippo, p53 signaling pathway, focal adhesion, and cell cycle (Fig. 4c, d). We also found sEV-miRNAs enriched in immune system processes and metabolic processes, in accordance with the previous report that intratumor heterogeneity and immunoedit- ing are early phenomena in PNs [29]. Moreover, we found seven miRNAs (miR-101-3p, miR-150-5p, miR-30c-5p, miR-30e-5p, miR-500a-3p, miR-125a-5p, and miR- 99a-5p) that were associated with better overall survival based on the TCGA databank (Figs. 4e, f and 6f). Interest- ingly, these miRNAs have all been reported to function as tumor suppressors [35–40], although some of them were upregulated in tumor tissues [37, 39], and in circulating sEVs of patients with malignant PNs in our study, indicat- ing that further research is required to clarify the molecu- lar mechanism underlying benign and malignant PNs. with IPNs diameter. Our CirsEV-miR model also had good performance in IPNs with a diameter of less than 1 cm, exhibiting AUC of 0.767 and specificity of 91.7% in our cohorts, and AUC of 0.721 and specific- ity of 75.0% in the external validation cohort (Fig. 5c, Additional file  1: Figure S5). Taken together, these findings indicate that our model has the ability to dis- tinguish between benign and malignant PNs, even in IPNs ≤ 10  mm, thereby expanding the applications widely.i According to IASLC/ATS/ERS and 2015 WHO classifi- cation of lung tumors, AIS is a tumor smaller than 3 cm with no invasion, and MIA denotes tumor smaller than 3 cm with invasion less than 0.5 cm [26]. Both AIS and MIA patients achieve 100% DFS with complete resec- tion [27]. It is believed that LUAD develops stepwise from benign AAH to AIS, to MIA, and eventually to overt invasive adenocarcinoma [28]. However, most studies have focused on histopathological subtypes, while little is known about the genomic evolution from preinvasive to invasive adenocarcinoma of LUAD. In 2019, Zhang et al. reported that mutations in EGFR, ERBB2, NRAS, and BRAF, as well as genomic intratumor heterogeneity and immunoediting, are common and early phenomena har- bored by AIS, MIA, and IA, and that mutations in TP53, as well as cell mobility and metastasis-related genes, may be later events during neoplastic progression [29]. Discussion Our results also demonstrated that the expression profiles of circulat- ing sEV miRNAs in patients with AIS/MIA were inter- mediate between the patients with benign PNs and the patients with invasive adenocarcinoma (Fig. 6b), support- ing the stepwise development hypothesis. Yet, we found seven expression-altered miRNAs unique to AIS/MIA compared with benign PNs, and nine miRNAs unique to AIS/MIA compared with invasive adenocarcinoma (Fig. 6d, e, Additional file  1: Table  S5, Additional file  1: Table S6). Among them, miR-30e-5p, a well-known tumor suppressor that suppresses tumorigenesis via the Sirt1/ JAK/STAT3 signaling pathway [30], was downregulated in both AIS/MIA and invasive adenocarcinoma compared with benign PNs. However, miR-25-3p and miR-128-3p, which have been reported to promote metastasis, exhib- ited higher expression in AIS/MIA than in invasive adeno- carcinoma [31, 32]. These findings suggest that AIS/MIA exhibit unique features different from benign PNs or inva- sive adenocarcinoma. It is also proposed that due to a lack of signals in AIS/MIA specimens, the difficulty in distin- guishing benign PNs from AIS/MIA IPNs is an intrinsic feature [33]. This study is the first report that revealed the circulating sEV miRNA signatures of benign, AIS/MIA, and invasive adenocarcinoma, suggesting that the pres- ence of AIS/MIA could impact the differentiation between benign and malignant PNs. Several limitations of this study are worth mention- ing. First, this was a single-center case control study that was performed in a high prevalence population. Thus, the performance of the CirsEV-miR model needs to be explored further in a community where a low prevalence is expected. Second, a prospective validating study was not conducted. Third, the study lacks in  vitro experi- ments to verify the miRNA expression level in lung ade- nocarcinoma tissues. To overcome the above limitations, a prospective multicenter clinical trial in a larger popula- tion-based setting is ongoing, and the expression profiles and biological functions of the identified miRNAs need to be validated in vitro in lung adenocarcinoma tissues or in lung adenocarcinoma cell lines. Conclusion In summary, our study profiled circulating sEV miRNAs in patients with IPNs and provided a diagnostic model, the CirsEV-miR model, based on the measurement of circulating sEV miRNAs to distinguish benign PNs from malignant PNs to assist LDCT scanning for early-stage lung cancer diagnosis. Five circulating sEV miRNAs (let-7b-3p, miR-101-3p, miR-125b-5p, miR-150-5p, and miR-3168) were revealed, and the CirsEV-miR model consisting of these miRNAs was established and vali- dated in 208 patients with IPNs, which is the largest Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 13 of 17 not a malignant disease. Considering the perspective of the pathological progression of lung adenocarcinoma, we still classified AIS as a type of malignant nodule. The pathological subtypes of benign PNs were without selec- tion and comprised more than 10 subtypes. The patho- logical information of all of the samples was obtained from surgically resected tissue sections in accordance with the 2015 WHO Histological Classification of Lung Cancer [26]. The pathological diagnosis of each patient was confirmed by two pathologists. The tumor–node– metastasis (TNM) stage was determined in accordance with the 8th edition International Association for the Study of Lung Cancer (IASLC) lung cancer staging sys- tem [41]. The pathological subtypes of our training and test cohorts, and those of the external validation cohort, are shown in (Additional file 2: Figure S2). sample size so far. Moreover, CirsEV-miR could discrimi- nate between benign and malignant PNs with diam- eter ≤ 1 cm, which are mostly difficult to distinguish in clinical setting. We primarily revealed that the molecular feature of AIS/MIA was intermediate of benign and inva- sive PNs, while exhibiting its own characteristics. These results suggest that circulating sEV miRNAs as diagnos- tic biomarkers could be integrated with LDCT scan to obtain further evaluation work-ups for IPNs. Patient enrollment and study designh This study was approved by the Institutional Review Board in Shanghai Pulmonary Hospital affiliated with Tongji University (K18-199Y) and registered at the Chi- nese Clinical Trial Registry (http://​www.​chictr.​org.​ cn/) with registration number ChiCTR1800019877. All patients were from Shanghai Pulmonary Hospital affili- ated with Tongji University and had signed written con- sents for their blood samples and clinical information to be used in this study. i The accuracy of a diagnostic test is usually measured by its sensitivity and specificity [42]. In this study, sensitivity represented the model’s ability to correctly identify indi- viduals with malignant PNs, and specificity represented the model’s ability to correctly identify individuals with benign PNs. y Patients with IPNs detected using LDCT scanning, who subsequently underwent surgical resections and diagnosed with LUAD (malignant PNs) and various benign PNs, were enrolled in this study. 199 patients with IPNs were recruited in the training phase from April to May 2019, including 20 patients with benign PNs and 179 with malignant PNs diagnosed using pathological examination. 260 patients with IPNs were recruited in the test phase from September to October 2019, includ- ing 35 patients with benign PNs and 225 patients with malignant PNs. Plasma samples had been prospectively collected in a vacutainer with anticoagulant (REF367863; Becton Dickinson, Franklin Lakes, NJ, USA) prior to surgical operation. After the elimination of pathology samples from patients with non-LUAD (n = 65), seri- ous hemolysis (above or equal to grade 5, n = 107), fail- ure to meet the ratio of benign and malignant PNs set at 1:2 (n = 155), and failure in construction of sequencing libraries (n = 23), finally, the training cohort consisted of 47 patients with IPNs (17 benign PNs and 30 malignant PNs), and the test cohort consisted of 62 patients with IPNs (24 benign PNs and 38 malignant PNs). In addition, an external cohort consisting of 20 patients with benign PNs and 79 patients with malignant PNs was used for validation. In this case–control study, a variety of benign PNs without selection served as controls. Additionally, 11 healthy people were enrolled as healthy control.The whole study design and inclusion/exclusion criteria are depicted in Fig. 1 and (Additional file 1: Figure S1). Methods Patient enrollment and study designh Western blot analysish y The isolated sEVs were lysed in 200  μL lysis buffer (P0013B, Beyotime, Shanghai, China); next, the pro- teins were extracted using an isolation reagent (N3525, 3DMed, Shanghai, China). The protein concentration of the sEVs was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). 20 µg of total protein was resolved on a 12% SDS-PAGE gel, electro- transferred onto a PVDF membrane (Millipore, USA). The membranes were blocked in 5% non-fat milk for 60 min, and incubated with anti- CD9 antibody (diluted 1:500; cat. no. ab92726; Abcam, Cambridge, UK), anti- CD63 antibody (1:2000, ab216130; Abcam, Cambridge, UK), anti-Syntenin antibody (diluted 1:500; cat. no. ab19903; Abcam, Cambridge, UK), anti-TSG101 poly- clonal antibody (diluted 1:500; cat. no. abs115706; Absin Bioscience Inc., Shanghai, China), and anti-Calnexin antibody (diluted 1:1000; cat. no. 2679; Cell Signaling Technology, Danvers, MA, USA) primary antibodies overnight at 4  °C. Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies (Beyotime Biotechnology, China) were used as second- ary antibodies. Antibody binding was detected using an enhanced chemilluminescence system according to the manufacturer’s protocol (Tanon-5200 Multi; Tanon Sci- ence & Technology Co. Ltd., Shanghai, China). RNA isolation from sEVs RNA was extracted from sEVs using the miRNeasy Serum/Plasma Kit (217184; QIAGEN, Shanghai, China) in accordance with the manufacturer’s protocol. The miRNA quality, yield, and distribution were analyzed using the Agilent 2100 Bioanalyzer with Small RNA Chips (5067-1548; Agilent, Savage, MD, USA). Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) added to the supernatant; gently inverted and incubated for 30  min at 4  °C and then centrifuged at 4700×g for 30 min at 4 °C. Finally, the supernatant was removed and the pellets containing the total sEVs were re-suspended in 0.2 mL phosphate-buffered saline (PBS). For TEM analysis, plasma sEVs were suspended in PBS prior to fixing in 4% paraformaldehyde and transferred to the carbon-coated electron microscopy grids. They were washed with PBS twice, and the third time with PBS con- taining glycine (50 mM), each for 3 min; then, they were incubated with PBS containing BSA (0.5%) for 10  min. Finally, the grids were stained with 2% uranyl acetate. After the staining, TEM (H-7650, Hitachi High-Tech- nologies, Japan) was used to analyze the morphology of sEVs. ExoView analysis Plasma sEVs were detected using ExoView chips (NanoView Biosciences, Brighton, MA) printed with antibodies against CD63, CD81, CD9, and mouse IgG1 as a negative control. 35 μL samples were dropped onto the chip and incubated for 16  h. After washing, chips were incubated with a fluorescence antibody cocktail of anti-CD9 ­(CF® 488), anti- CD81 ­(CF® 555), and anti- CD63 ­(CF® 647) for 1 h at room temperature. Chips were then imaged in the ExoView R100 Scanner (NanoView Biosciences, Brighton, MA). Data were analysed using NanoViewer Software (NanoView Biosciences, Brighton, MA). Plasma isolation and sEVs isolation Blood samples were collected from patients in 10-mL vacutainer tubes containing an anticoagulant of K2EDTA (REF367863; Becton Dickinson, Franklin Lakes, NJ, USA), mixed by gently inverting several times, stored with the tubes placed upright, and then transported on ice within 1 h after collection. To harvest the plasma, the samples were centrifuged at 1600×g for 10 min at 4 °C, after which the hemolysis level was determined and recorded. Samples with hemolysis grade of no more than 4 were used [43]. The collected supernatant was centri- fuged again at 16,000×g for 15 min at 4 °C, and then the 1  mL supernatant was transferred into a fresh 1.5  mL tube and stored at − 80 °C prior to use. For the sEV isolation from plasma, a polyethylene glycol-based 3D Medicine isolation reagent [18] (L3525; 3DMed, Shanghai, China) was used. This isolation rea- gent has been modified and improved based on the work of Rider [44], and has been registered to the National Medical Products Administration as a Class I medical device (#HMXB20190091), specifically for the isolation of sEVs in the clinical setting. The plasma samples were centrifuged at 12,000×g for 10 min at 4 °C after a static water bath incubation at 37 ℃ for 5 min. The superna- tant was transferred to a 0.45 µm tube filter (CLS8163- 100EA; Costar, Corning, NY, USA), followed by transfer to a 0.22  µm tube filter (CLS8161-100EA; Costar) and then centrifuged at 12,000×g for 5 min at 4 °C. The fil- tered supernatant was transferred to a fresh 1.5 mL tube. One-quarter volume of an isolation reagent (L3525) was i The LUAD consisted of three pathological subtypes, namely, AIS, MIA, and invasive adenocarcinoma. Nine patients were diagnosed with AIS, which is technically Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Page 14 of 17 Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Zheng et al. Journal of Nanobiotechnology (2022) 20:172 Quantitative reverse‑transcription PCR Quantitative reverse‑transcription PCR Total RNA extraction from sEVs were as previously described. miRNA were reverse transcribed using TaqMan™ Advanced miRNA cfDNA Synthesis Kit (A28007, Applied Biosystems™, USA) according to the manufacturer’s protocol. qPCR was performed on Applied Biosystems 7500 Fast Real-Time PCR systems with specific (miR-451a, miR-125b-5p, miR-101-3p, miR-3168, miR-150-5p and let-7b-3p) probes (A25576, Applied Biosystems™, USA). The expression level of miR- 451a were used as control as previously reported [52]. Relative expression were calculated with mean Ct values using ­2−ΔΔCt method. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12951-​022-​01366-0. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12951-​022-​01366-0. Additional file 1: Figure S1. Cohorts’ details and inclusion/exclusion criteria. Figure S2. Pathology subtypes of the training (a), test (b), and external validation (c) cohorts. Figure S3. Representative pathological images of the benign and malignant pulmonary nodule subtypes. Malig- nant nodule subtypes: adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IA); Benign nodule subtypes: granulomas, atypical adenomatous hyperplasia (AAH), hamar- toma, cyst, fibrosis, organizing pneumonias (OP). Magnification, ×400; Formalin Fixed Paraffin-Embedded (FFPE) tissues. Figure S4. CirsEV-miR model performance in the test cohort and the external cohort. (a) Circu- lating sEV miRNA heatmap of the test cohort by unsupervised hierarchical clustering. (b) ROC curve of the CirsEV-miR model in the test cohort. (c) The CirsEV-miR scores of benign and malignant PNs in the test cohort. (d) ROC curve of the CirsEV-miR model in the external validation cohort. (e) The CirsEV-miR scores of healthy people and patients with benign or malignant PNs. (f) Expression level of the five miRNAs used in CirsEV-miR Bioinformatics analysis of small RNA sequencing datah informatics analysis of small RNA sequencing datah The 3′ adaptors of reads were cleaved using a custom program. Subsequently, the reads were aligned to the human genome hg19 assembly (http://​hgdow​nload.​soe.​ ucsc.​edu/​golde​nPath/​hg19/​bigZi​ps/) using BWA 0.7.12 [45]. An individual Small RNA-Seq dataset is required to have a minimum of 5,000,000 reads with minimum mapping rate 80% that mapped with any annotated RNA transcript in the human genome. The annotations were generated from Gencode v25 [46] and miRBase v21 [47] for statistical analysis and to determine expression levels. The annotation includes all small RNAs, such as miR- NAs, rRNAs, tRNAs, and piRNAs, as well as long tran- scripts from GENCODE, which includes both protein coding genes and long non-coding RNAs (lincRNAs). The percentage of reads that mapped to the annotated miRNAs should be greater than 25% (Additional file  1: Table S7). The miRNA expressions were determined by counting the number of reads mapped to the regions annotated by mature miRNAs. The miRNA mapped by at least two reads in each of the samples and with length less than 30 nt was saved for miRNA expression analy- sis. The miRNA expression analysis was performed using the voom function in the limma package [48], with nor- malization by Trimmed Mean of M-values (TMM) via the edgeR package, and the miRNA expression level was converted to log2-counts-per-million (logCPM) [49]. The Empirical Bayes algorithm implemented in ComBat was applied to the training and the test cohort data sets adjusted for batch effects [50, 51]. Statistical analysis was performed using the statistical programming language R (version 3.6). The dendextend package [53] in R was used to perform average linkage hierarchical clustering of genes and cases. The heatmap was constructed using the ComplexHeatmap package [54] in R/Bioconductor. The biological processes of Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment of the experi- mentally validated targets of miRNAs were examined using mirPath v.3, which provided the Expression Analy- sis Systematic Explorer (EASE) score and false-discovery rates using the Fisher’s exact tests and unbiased empirical distributions [55]. The Kaplan–Meier plot analysis of the TCGA data was performed using OncoLnc [56]. Small RNA libraries preparation and sequencing Nanosight NS 300 system (NanoSight Technology, Mal- vern, UK) was used to characterize the number and size of EVs. Isolated sEVs were resuspended in PBS at a con- centration of 5 μg/mL and were further diluted 100- to 1000-fold, to achieve between 20 and 100 objects/frame. Samples were manually injected into the sample cham- ber at ambient temperature. Each sample was config- ured using a 488 nm laser and a high-sensitivity scientific complementary metal-oxide semiconductor camera, and the measurements were performed in triplicate at a cam- era setting of 13 with an acquisition time of 30 s and a detection threshold setting of 7. At least 200 completed tracks were analyzed and obtained per video. Finally, the NTA analytical software (version 2.3) was used to analyze the nanoparticle tracking data of the sEV samples in this study. Small RNA libraries preparation and sequencing To prepare and construct the small RNA sequencing libraries, a NEB Next Multiplex Small RNA Library Prep Set for Illumina (E7300L; New England Biolabs, Ipswich, MA, USA) was used in accordance with the manufac- turer’s protocol. Briefly, the reverse transcription primer was hybridized after 3ʹ adaptor ligation of 100 ng RNA per sample, following 5ʹ adaptor ligation. A total of 18 PCR cycles were performed with Illumina feasible bar- code primers after the first strand cDNA synthesis. The prepared libraries were resolved on NucleoSpin Gel and PCR Clean-up (740609.50; MACHEREY–NAGEL, Ger- many) and recovered in 30  μL DNase- and RNase-free water. The DNA quality, yield, and distribution were analyzed using the ­LabChip® GX Touch™ HT Nucleic Acid Analyzer with DNA High Sensitivity Reagent Kit (CLS760672; PerkinElmer, Waltham, MA, USA) and the DNA Extended Range LabChip (CLS138948; Perki- nElmer). A total of 20–25 libraries were pooled into a Page 15 of 17 Zheng et al. Journal of Nanobiotechnology (2022) 20:172 single sequencing lane and sequenced using an Illumina HiSeq PE150 analyzer. and selection operator (LASSO) in the training cohort. The test cohort and the external cohort were used to test and validate the diagnostic model. We selected the dif- ferentially expressed sEV-miRNAs (DEMs) determined according to the stringent statistical threshold (Student’s t-test p-value ≤ 0.05, 1.5-fold change, and the mean expression CPM ≥ 50) between the benign and malignant PNs. Based on DEMs, the risk scores were generated using LASSO analysis, and the best parameters of the model constructed using LASSO were ultimately selected using tenfold cross-validation. Abbreviations AAH: Atypical adenomatous hyperplasia; AIS: Adenocarcinoma in situ; AUC​ : Area under curve; BPs: Biological functions; CPM: Counts-per-million; DEMs: Differentially expressed miRNAs; GO: Gene ontology; IPNs: Indeterminate pul- monary nodules; ISEV: International Society for Extracellular Vesicles; LASSO: Least absolute shrinkage and selection operator; LDCT: Low-dose computed tomography; LUAD: Lung adenocarcinoma; MFs: Molecular functions; MIA: Minimally invasive adenocarcinoma; miRNAs: MicroRNAs; MISEV: Minimal Information for Studies of Extracellular Vesicles; NTA: Nanoparticle tracking analysis; OPs: Organizing pneumonias; OS: Overall survival; PNs: Pulmonary nodules; ROC: Receiver operating characteristic; sEV: Small extracellular vesi- cles; TCGA​: The Cancer Genome Atlas; TEM: Transmission electron microscopy; UTR​: Untranslated region; WB: Western blot. Funding 11. Ge Q, Zhou Y, Lu J, Bai Y, Xie X, Lu Z. miRNA in plasma exosome is stable under different storage conditions. Molecules. 2014;19(2):1568–75. 11. Ge Q, Zhou Y, Lu J, Bai Y, Xie X, Lu Z. miRNA in plasma exosome is stable under different storage conditions. Molecules. 2014;19(2):1568–75. This work was supported by Grant No. 16CR2016A from the Shanghai Metro- politan Key Program. This work was supported by Grant No. 16CR2016A from the Shanghai Metro- politan Key Program. 12. Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD, Kloecker GH. Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung Cancer. 2009;10(1):42–6. Author details 1 18. Zhang JT, Qin H, Man Cheung FK, Su J, Zhang DD, Liu SY, et al. Plasma extracellular vesicle microRNAs for pulmonary ground-glass nodules. J Extracell Vesicles. 2019;8(1):1663666. 1 Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, No. 507 Zhengmin Road, Yangpu District, Shanghai 200433, China. 2 Department of Thoracic Surgery, Shanghai Pulmo- nary Hospital, Tongji University School of Medicine, No. 507 Zhengmin Road, Yangpu District, Shanghai 200433, China. 3 3D Medicines Inc, Building 2A, No. 158 Xin Junhuan Road, Pujiang Hi‑tech Park, Shanghai 201114, China. 4 Depart- ment of Pathology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai  200433, China. 5 Guangdong Lung Cancer Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China. 19. Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsito- haina R, et al. 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Lung cancer probability in patients with CT-detected pulmonary nodules: a prespecified analysis of data from the NELSON trial of low-dose CT screening. Lancet Oncol. 2014;15(12):1332–41. Statistical analysish The samples in the training and test cohorts in this study and the samples in the external validation cohort from another study were analyzed [18]. The diagnostic model was constructed using least absolute shrinkage Page 16 of 17 Page 16 of 17 Zheng et al. Journal of Nanobiotechnology (2022) 20:172 and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–49. and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–49. and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–49. model. For each group n = 13. All data is presented with mean ± SD, except let-7b-3p which is presented with mean ± SEM. *p < 0.05; **p < 0.01; ***, p < 0.001. Figure S5. CirsEV-miR model performance of IPNs ≤ 1 cm. (a) CirsEV-miR model performance in our cohorts. (b) CirsEV- miR model performance in the external validation cohort. Table S1. Clas- sifiers constructed from the six identified DEMs. Table S2. Performance of classifiers in the training and test cohorts. Table S3. The five sEV-miRNAs and their corresponding coefficients in the CirsEV-miR model. Table S4. Target genes of the five sEV-miRNAs. Table S5. Upregulated DEMs shared between benign PNs, AIS/MIA, and invasive adenocarcinomas. Table S6. Downregulated DEMs shared between benign PNs, AIS/MIA, and invasive adenocarcinomas. Table S7. Quality control of small RNA sequencing. 2. National Lung Screening Trial Research T, Aberle DR, Adams AM, Berg CD, Black WC, Clapp JD, et al. Reduced lung-cancer mortality with low-dose computed tomographic screening. N Engl J Med. 2011;365(5):395–409. 3. Grogan EL, Weinstein JJ, Deppen SA, Putnam JB Jr, Nesbitt JC, Lambright ES, et al. 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Clin Cancer Res. 2017;23(17):5311–9. Authors’ contributions Conceived and designed the experiments: GN-J, D-Z, DD-Z, JF-X. Performed the experiments: YM-Z, CY-W, J-N, XX-X, BN-N, S-C, YN-Z, XF-L. Analyzed the data: JY-Z, HZ-W, W-Z, H-C, CY-W, BB-W, DD-Z, FG-L. Contributed reagents/ materials/analysis: D-Z, L-X, JT-Z, WZ-Z, JF-X, GN-J. All authors read and approved the final manuscript. 9. Boukouris S, Mathivanan S. Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteom Clin Appl. 2015;9(3–4):358–67. 10. Huang X, Yuan T, Tschannen M, Sun Z, Jacob H, Du M, et al. Characteriza- tion of human plasma-derived exosomal RNAs by deep sequencing. BMC Genom. 2013;14:319. Consent for publication Not applicable. Consent for publication Not applicable. Declarations 14. Smolarz M, Widlak P. Serum exosomes and their mirna load-a potential biomarker of lung cancer. Cancers. 2021;13(6):1373. Acknowledgements h h ld l k The authors would like to thank Huiyan Li, and Chao Sun for their assistance with sample collection for this study. The authors appreciate the support and participation of the physicians and patients in this study. The authors would like to thank LetPub (www.​letpub.​com.​cn) for English language editing. 7. Zhang Y, Liu Y, Liu H, Tang WH. Exosomes: biogenesis, biologic function and clinical potential. Cell Biosci. 2019;9:19. 8. Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, et al. Compara- tive proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics. 2013;13(22):3354–64. Competing interestsfi 17. Zhong Y, Ding X, Bian Y, Wang J, Zhou W, Wang X, et al. Discovery and validation of extracellular vesicle-associated miRNAs as noninvasive detection biomarkers for early-stage non-small-cell lung cancer. Mol Oncol. 2021;15(9):2439–52. 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https://www.frontiersin.org/articles/10.3389/fgene.2019.00450/pdf
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Personalized Interpretation and Clinical Translation of Genetic Variants Associated With Cardiomyopathies
Frontiers in genetics
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cc-by
9,737
ORIGINAL RESEARCH published: 15 May 2019 doi: 10.3389/fgene.2019.00450 Citation: Campuzano O, Fernandez-Falgueras A, Sarquella-Brugada G, Cesar S, Arbelo E, García-Álvarez A, Jordà P, Coll M, Fiol V, Iglesias A, Perez-Serra A, Mates J, del Olmo B, Ferrer C, Alcalde M, Puigmulé M, Mademont-Soler I, Pico F, Lopez L, Tiron C, Brugada J and Brugada R (2019) Personalized Interpretation and Clinical Translation of Genetic Variants Associated With Cardiomyopathies. Front. Genet. 10:450. doi: 10.3389/fgene.2019.00450 Keywords: sudden cardiac death, arrhythmias, cardiomyopathies, genetics, genetic counseling Campuzano O, Fernandez-Falgueras A, Sarquella-Brugada G, Cesar S, Arbelo E, García-Álvarez A, Jordà P, Coll M, Fiol V, Iglesias A, Perez-Serra A, Mates J, del Olmo B, Ferrer C, Alcalde M, Puigmulé M, Mademont-Soler I, Pico F, Lopez L, Tiron C, Brugada J and Brugada R (2019) Personalized Interpretation and Clinical Translation of Genetic Variants Associated With Cardiomyopathies. Front. Genet. 10:450. doi: 10.3389/fgene.2019.00450 Reviewed by: Reviewed by: Serena Sanna, University Medical Center Groningen, Netherlands Natasa Djordjevic, University of Kragujevac, Serbia Richard Hauer, ICIN Netherlands Heart Institute (KNAW), Netherlands Reviewed by: Serena Sanna, University Medical Center Groningen, Netherlands Natasa Djordjevic, University of Kragujevac, Serbia Richard Hauer, ICIN Netherlands Heart Institute (KNAW), Netherlands 1 Cardiovascular Genetics Center, Biomedical Research Institute of Girona, University of Girona, Girona, Spain, 2 Department of Medical Science, School of Medicine, University of Girona, Girona, Spain, 3 Centro Investigación Biomédica Red Enfermedades Cardiovasculares, Madrid, Spain, 4 Department of Biochemistry and Molecular Genetics, Hospital Clinic, Barcelona, Spain, 5 Arrhythmias Unit, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain, 6 Arrhythmias Unit, Hospital Clinic, University of Barcelona, Barcelona, Spain, 7 Cardiology Service, Hospital Josep Trueta, University of Girona, Girona, Spain *Correspondence: Oscar Campuzano oscar@brugada.org Ramon Brugada ramon@brugada.org †These authors have contributed equally to this work Cardiomyopathies are a heterogeneous group of inherited cardiac diseases characterized by progressive myocardium abnormalities associated with mechanical and/or electrical dysfunction. Massive genetic sequencing technologies allow a comprehensive genetic analysis to unravel the cause of disease. However, most identified genetic variants remain of unknown clinical significance due to incomplete penetrance and variable expressivity. Therefore, genetic interpretation of variants and translation into clinical practice remain a current challenge. We performed retrospective comprehensive clinical assessment and genetic analysis in six families, four diagnosed with arrhythmogenic cardiomyopathy, and two diagnosed with hypertrophic cardiomyopathy (HCM). Genetic testing identified three rare variants (two non-sense and one small indel inducing a frameshift), each present in two families. Although each variant is currently classified as pathogenic and the cause of the diagnosed cardiomyopathy, the onset and/or clinical course differed in each patient. New genetic technology allows comprehensive yet cost-effective genetic analysis, although genetic interpretation, and clinical translation of identified variants should be carefully done in each family in a personalized manner. Specialty section: This article was submitted to Genomic Medicine, a section of the journal Frontiers in Genetics Received: 13 January 2019 Accepted: 30 April 2019 Published: 15 May 2019 Specialty section: This article was submitted to Genomic Medicine, a section of the journal Frontiers in Genetics Received: 13 January 2019 Accepted: 30 April 2019 Published: 15 May 2019 Edited by: Lili Milani, University of Tartu, Estonia Edited by: Lili Milani, University of Tartu, Estonia Oscar Campuzano1,2,3,4*†, Anna Fernandez-Falgueras1†, Georgia Sarquella-Brugada2,5, Sergi Cesar5, Elena Arbelo3,6, Ana García-Álvarez6, Paloma Jordà6, Monica Coll1, Victoria Fiol5, Anna Iglesias1,3, Alexandra Perez-Serra1,3, Jesus Mates1, Bernat del Olmo1, Carles Ferrer1, Mireia Alcalde1,3, Marta Puigmulé1, Irene Mademont-Soler1, Ferran Pico1, Laura Lopez1, Coloma Tiron7, Josep Brugada3,5,6 and Ramon Brugada1,2,3,7* INTRODUCTION Sudden cardiac death (SCD) is a major cause of death with an incidence ranging from approximately 1:1000 to 1:3000 individuals per year in the general population (Deo and Albert, 2012). In population <35 years old, inherited cardiomyopathies are the leading cause of SCD, mainly hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 1 Cardiomyopathies: Personalized Diagnosis Campuzano et al. arrhythmogenic cardiomyopathy (ACM) (Straus et al., 2004). Cardiomyopathies are characterized by structural heart abnormalities that induce mechanical and/or electrical dysfunction, leading to ventricular arrhythmias and even SCD. Sometimes the first manifestation of disease is SCD, which usually occurs in a healthy young individual while doing exercise. These disorders induce progressive morphological and histological myocardial features, but abnormalities may be minimal or even absent at young ages (Gopinathannair et al., 2015). families. A multidisciplinary group including cardiologists, pediatricians, genetic counselors, molecular geneticists, and pathologists performed personalized genetic interpretation and clinical translation in each family. Families diagnosed with the same cardiomyopathy due to the same mutation were included in our study. Clinical evaluation of index cases and all available relatives included a complete physical examination, 12- lead electrocardiogram (ECG), 2-dimentional echocardiography (ECHO), cardiac magnetic resonance imaging (MRI), exercise stress test, 24-h Holter monitoring, and comprehensive genetic testing. Regarding post-mortem samples, a complete autopsy was performed according to current international regulations, including macroscopic, microscopic, and toxicological analyses (Basso et al., 2008; Sinard, 2013). When macroscopic autopsy was labeled negative, forensic pathologists performed a complete histological and toxicological investigation, and collected a blood sample for genetic investigation. We excluded cases in which the autopsy was labeled as violent death, including death from drug overdose. We also obtained family history of syncope and unexplained deaths. In post-mortem cases, current guidelines recommend molecular autopsy in all cases without a conclusive cause of death and without any cardiac alteration, but also if a cardiac alteration suspicious of cardiomyopathy is identified (Priori and Blomstrom-Lundqvist, 2015). In addition, ongoing clinical surveillance for family members of affected individuals is recommended because some relatives might have inherited the genetic alteration and thus be at risk of SCD. Therefore, establishing a proper pedigree including both clinical and genetic data is crucial to provide a clear view of diagnosed and asymptomatic relatives, focused on adopting preventive therapeutic measures in each individual. Variant Analysis Genomic DNA was obtained from peripheral blood or saliva. DNA analyzed using NGS technology (MiSeq, Illumina). We screened the most prevalent genes involved in cardiomyopathies and other pathologies associated with SCD (ABCC9, ACTC1, ACTN2, AKAP9, ANK2, ANKRD1, BAG3, CACNA1C, CACNA2D1, CACNA1G, CACNA1H, CACNA1I, CACNB2, CALM1, CALM2, CALM3, CALR3, CASQ2, CAV3, CRYAB, CSRP3, CTNNA3, GJA1, CTF1, DES, DMD, DMPK, DPP6, DSC2, DSG2, DSP, DTNA, ECE1, EMD, EN1, EYA4, FHL2, FKTN, FLNA, FLNC, GAA, GJA5, GLA, GPD1L, HCN1, HCN2, HCN4, JPH2, JUP, KCNA5, KCND3, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, KCNH2, KCNJ2, KCNJ5, KCNJ8, KCNQ1, LAMA4, LAMP2, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYL2, MYL3, MYLK2, MYOZ2, MYPN, NEBL, NEXN, NOS1AP, NOTCH1, NPPA, NUP155, PDLIM3, PHOX2A, PHOX2B, PITX2, PKP2, PLN, PRKAG2, RANGRF, RBM20, RYR2, SCN1B, SCN2B, SCN3B, SCN4B, SCN5A, SCN10A, SDHA, SGCD, SLC22A5, SLC6A4, SLC8A1, SLMAP, SLN, SNTA1, TAZ, TCAP, TGFB3, TLX3, TMEM43, TMPO, TNNC1, TNNI3, TNNT2, TP63, TPM1, TRDN, TRIM63, TRPM4, TTN, TTR, and VCL) (Campuzano et al., 2014a). All gene isoforms described in Ensembl 751 that have been linked at least with either a RefSeq code2 or CCDS3 were included. Massive sequencing technologies such as next generation sequencing (NGS) now allow comprehensive genetic analysis of families in a cost-effective way – genetic data can be obtained in reduced time and at low cost in comparison to traditional Sanger sequencing (Morini et al., 2015). The main current challenge is genetic interpretation as well as clinical translation of these results, as most identified genetic variants remain of unknown/ambiguous significance (Richards et al., 2015; Amendola et al., 2016). This means genetic data are not conclusive and do not help clinicians in the diagnosis, risk stratification, or adoption of potential therapeutic measures to prevent malignant arrhythmias. Consequently, genetic data should be carefully translated into clinical practice. Information of family history, segregation of genetic variants, and genotype-phenotype correlations are crucial to elucidate the role of each genetic variant (Campuzano et al., 2015a). Further, the same genetic variant may be associated with incomplete penetrance and variable expressivity, intrafamilial as well as interfamilial, and complicating clinical interpretation (Giudicessi and Ackerman, 2013). INTRODUCTION Hence, incorporation of genetic testing and genetic counseling is now considered standard of care for cardiomyopathies (Marian and Braunwald, 2017). Frontiers in Genetics | www.frontiersin.org 1http://www.ensembl.org 2http://www.ncbi.nlm.nih.gov/refseq 3https://www.ncbi.nlm.nih.gov/CCDS MATERIALS AND METHODS Alignment of DNA sequences for different species was also performed for novel variations using Uniprot database16. Finally, protein structure and domains were consulted at STRING17 and SMART databases18. Finally, each variant was classified in one of the following current recommendations of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) (Richards et al., 2015). This recommendations describe several items of pathogenicity (PVS, evidence of pathogenicity very strong; PS, evidence of pathogenicity strong; PM, evidence of pathogenicity moderate; and PP, evidence of pathogenicity supporting), and benignity (BA, evidence of benign impact stand-alone; BS, evidence of benign impact strong; and BP, evidence of benign impact supporting), enabling a final score and consequent classification of variants as: pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), or benign (B). The PM2 item in the ACMG score was considered fulfilled if MAF ≤0.1% in relevant population databases (Lek et al., 2016). Concerning frequency of disease-causing variants, the majority of pathogenic variants are extremely rare (<0.01%) (Kobayashi et al., 2017). Concerning potential PVS1 variants, it should be only used for variants in genes where loss of function is a previously established disease mechanism19 (Abou Tayoun et al., 2018). In addition, some items of ACMG/AMP may underlie a lack of specificity or ambiguous or contradictory interpretations, so we check the parameters using Sherloc (semiquantitative, hierarchical evidence-based rules for locus interpretation) (Nykamp et al., 2017). Finally, to avoid bias, three authors with PhDs in human genetics independently and comprehensively investigated published genetic data concerning each analyzed variant. In addition, four independent expert cardiologists (including two pediatric cardiologists) comprehensively reviewed available clinical data to reconfirm diagnoses following current guidelines (Priori and Blomstrom-Lundqvist, 2015). All investigators discussed data included in each item of the ACMG and consensus and the final classification of all variants. Regarding copy number variation (CNV), our approach focused on capturing significant differences between expected normalized coverage and obtained normalized coverage for a given sample in a region of interest. Several samples were analyzed to corroborate similar levels of coverage between samples, as already published by our group (Campuzano et al., 2014b). All CNVs were compared with ExAC, including recently added data concerning CNVs. Genetic analysis in relatives was performed using the Sanger method. Only rare genetic variants confirmed in the index case were analyzed in relatives to perform a comprehensive genetic study in each family. MATERIALS AND METHODS Coordinates of sequence data were based on UCSC human genome version hg19 (NCBI GRCh37 built). Biotinylated cRNA probe solution was used as a capture probe (Agilent Technologies, Santa Clara, CA, United States). Bioinformatic processing of fastq files consisted of low-quality base-trimming followed by a mapping step with BWA-MEM. After removal of duplicates and low-quality reads, variant call was performed with SAMtools v.1.2, and an ad hoc pipeline to generate raw This study was approved by the Ethics Committee of the Hospital Josep Trueta (Girona, Spain), following the Helsinki II declaration. Written informed consent was obtained from all relatives included in the study. Alive individuals were clinically evaluated at Hospital Josep Trueta (Girona, Spain), Hospital Clinic of Barcelona (Barcelona, Spain), or Hospital Sant Joan de Deu (Barcelona, Spain). All patients were Caucasian and native from Spain. We analyzed six families diagnosed with cardiomyopathies caused by 3 variants in 6 families. Each variant in two May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 2 Cardiomyopathies: Personalized Diagnosis Campuzano et al. VCF files. The final annotation steps provided information included in public databases. Identified variations were compared with DNA sequences from 500 healthy Spanish individuals (individuals not related to any index case and of the same ethnicity) as control cases in order to identify potential founder mutations. In addition, in order to determinate the frequency of variants identified, they were contrasted with the Human Gene Mutation Database (HGMD4), HapMap5, 1000 Genomes Project6, Exome Variant Server (EVS7), Genome Aggregation Database (gnomAD8), and Exome Aggregation Consortium (ExAC9). Non-common genetic variants [minor allele frequency (MAF) <1%] identified in NGS analysis were confirmed by Sanger sequencing. Exons and exon-intron boundaries of each gene were amplified (Verities PCR, Applied Biosystems, Austin, TX, United States). PCR products were purified (Exosap-IT, Affymetrix Inc., USB Products, Cleveland, OH, United States), and then were directly sequenced in both directions (Big Dye Terminator v3.1 and 3130XL Genetic Analyzer, Applied Biosystems) with posterior SeqScape Software v2.5 analysis (Life Technologies, Carlsbad, CA, United States), comparing obtained results with the hg19 reference sequence. Sequence variants were described following rules outlined by the Human Genome Variation Society (HGVS10), and checked in Mutalyzer11. In silico prediction of pathogenicity of genetic variations was assessed in Functional Annotations for Non Synonymous SNVs (FannsDB12), Mutation Taster13, Protein Variation Effect Analyzer (PROVEAN14), and Polymorphism Phenotyping v2 (PPH215). 19https://www.ncbi.nlm.nih.gov/projects/dbvar/clingen Frontiers in Genetics | www.frontiersin.org RESULTS Individual III.4 was diagnosed with ACM -abundant fibro-fatty infiltration in epicardium and myocardium of right ventricle (residual myocytes <60%), moderate decrease of right ventricular ejection fraction (45%), and mild right ventricle dilatation-, but with a slow clinical evolution during follow-up and negligible symptoms to date. No other relatives were diagnosed with ACM except one family member (II.1 -74 years old-) who showed scattered cardiac fibrosis but not a definite diagnosis of ACM. She remained asymptomatic thus far, without any episode of palpitations, arrhythmia, or syncope. Neither SCD nor syncope episodes occurred in this family. The grandparents (I.1 and I.2) died at old age (>75 years old both), both due to oncologic diseases, and without documented episodes of syncope. Genetic analysis identified the already reported pathogenic variant -PKP2_p.(Leu92∗)- in three cases, II.1, III.1, and III.3. In addition, a rare variant in DSG2 (c.527C > T, p.T176I – p.Thr176Ile) was also identified in the index case (III.1), with the amino acid threonine (polar, uncharged side chain) changed to isoleucine (non-polar, hydrophobic side chain). This variant is reported in global databases with a low frequency (2/249296, MAF: 0.0008023%). However, in silico analysis predicts an ambiguous role, and alignment between species showed no highly conserved domain. The variant in DSG2 was present in the index case (III.1, clinically diagnosed with ACM), her sister (III.3 -44 years old-, not diagnosed with ACM), and her mother (II.1, not diagnosed with ACM but showing scattered cardiac abnormalities). One relative clinically diagnosed with ACM (III.4) did not carry this variant. Therefore, we disregarded DSG2_p.(Thr176Ile) as pathogenic, at least as the cause of ACM in this family. The PKP2_p.(Leu92∗) variant was reported first as a cause of ACM in a cohort of cases (Fressart et al., 2010). Currently, global population databases have reported this variant with very low frequency (2/251424, MAF: 0.0007955%), and in silico analysis predicts this genetic alteration is deleterious. The PKP2_p.(Arg413∗) variant was reported for the first time in 2006 in a cohort of cases with ACM (Syrris et al., 2006). To date, more than 10 reports describe families with ACM due to this pathogenic variant, and in silico, analysis predicts the variant is deleterious. Despite identification of this variant in global databases (rs372827156), the frequency is very low (2/121408 alleles, MAF: 0.001647%). The third variant, MYBPC3_p.(Arg891Ala_fr∗160), is novel. Current global population databases do not include this variant, and in silico analyses predict a deleterious role. RESULTS Our study retrospectively analyzed six families diagnosed with cardiomyopathy after comprehensive clinical assessment – families A, B, C, and D included relatives clinically diagnosed with ACM, and families E and F included relatives diagnosed with HCM. Comprehensive NGS analysis was performed on at least one patient clinically diagnosed with ACM or HCM in each family. Concerning NGS data, all samples showed <4% duplicates of amplified regions; a minimum of 96% reads mapped uniquely and 55% reads of insert. Finally, the median coverage was 300× and call rate at 30× was 99.9%. After genetic interpretation, three pathogenic rare variants – each present in two different families – were identified as the cause of disease. Families A and B carried the variant PKP2_p.(Leu92∗), families C and D carried the variant PKP2_p.(Arg413∗), and families E and F carried the variant MYBPC3_p.(Arg891Ala_fr∗160). Variants PKP2_p.(Leu92∗) and PKP2_p.(Arg413∗) are previously reported non-sense variants that are widely accepted as pathogenic. Both 4http://www.hgmd.cf.ac.uk/ac/index.php 5http://hapmap.ncbi.nlm.nih.gov 6http://www.1000genomes.org 7http://evs.gs.washington.edu/EVS 8http://gnomad.broadinstitute.org 9http://exac.broadinstitute.org 10http://www.hgvs.org 11https://mutalyzer.nl 12http://bg.upf.edu/fannsdb 13www.mutationtaster.org 14http://provean.jcvi.org/index.php 15http://genetics.bwh.harvard.edu/pph2 16http://www.uniprot.org 17https://string-db.org 18https://smart.embl.de May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 3 Cardiomyopathies: Personalized Diagnosis Campuzano et al. FIGURE 1 | Electropherogram of pathogenic variants identified in our families and closest proteins interactions. (A) Families A and B: c.275T > A (TTG > TAG), p.L92X (p.Leu92∗) in the PKP2 gene. Families C and D: c.1237C > T (CGA > TGA) and p.R413X (p.Arg413∗) in the PKP2 gene. Families E and F: c.2670dupG, p.R891A_frX160 (p.Arg891Ala_fs∗160) in the MYBPC3 gene. (B) Network of ten closest proteins to PKP2. (C) Network of ten closest proteins to MYBPC3. FIGURE 1 | Electropherogram of pathogenic variants identified in our families and closest proteins interactions. (A) Families A and B: c.275T > A (TTG > TAG), p.L92X (p.Leu92∗) in the PKP2 gene. Families C and D: c.1237C > T (CGA > TGA) and p.R413X (p.Arg413∗) in the PKP2 gene. Families E and F: c.2670dupG, p.R891A_frX160 (p.Arg891Ala_fs∗160) in the MYBPC3 gene. (B) Network of ten closest proteins to PKP2. (C) Network of ten closest proteins to MYBPC3. genetic abnormalities radically change the protein, resulting in a final protein that is shorter than wildtype PKP2 (Figure 1 and Tables 1, 2). suffered a syncope due to ventricular arrhythmia, and an implantable cardioverter defibrillator (ICD) was implanted. RESULTS It is a small indel that induces a frameshift, resulting in a longer than wildtype protein. Therefore, all parameters analyzed indicate a pathogenic role (Figure 1 and Tables 1, 2). Frontiers in Genetics | www.frontiersin.org Families A and B Families A and B were clinically diagnosed with ACM (Table 1). The cause of the disease was a pathogenic variant (CM102825) in PKP2 (c.275T > A, p.L92X – p.Leu92∗) (Table 2). Our NGS panel included genes encoding the functionally closest PKP2-related proteins, but no abnormalities were identified except in Family A. In addition, no CNVs were identified in either family. Family A included two members clinically diagnosed with ACM, III.1 -48 years old- and III.4 -42 years old- (Figure 2). Individual III.1 had repeated episodes of palpitations. During clinical assessment (right ventricle ejection fraction: 38%; dilated right ventricle: 4.3 cm; no left ventricle affectation), III.1 Family B also carried the same non-sense variant, PKP2_p.(Leu92∗). Relative III.1-39 years old- was definitely Frontiers in Genetics | www.frontiersin.org May 2019 | Volume 10 | Article 450 4 Cardiomyopathies: Personalized Diagnosis Campuzano et al. TABLE 1 | Clinical data. Family Individual Age Gender Gene_Variant Diagnosis Criteria A II.1 74 F PKP2_p.(Leu92∗) No – III.1 48 F PKP2_p.(Leu92∗) ACM Definite (2 major and 2 minor) III.4 42 F PKP2_p.(Leu92∗) ACM Definite (1 major and 2 minor) B II.1 68 F PKP2_p.(Leu92∗) ACM (1 major and 1 minor) III.1 39 F PKP2_p.(Leu92∗) ACM Definite (1 major and 2 minor) C I.1† 44 M ? Families C and D diagnosed with ACM (Figure 3). She suffered syncope during exercise. Clinical assessment showed dyskinesia with a moderate dilation of the right ventricle (39 mm), a minor reduction of ejection fraction (40%), and no criteria of fibro-adipose infiltration in the myocardium (residual myocytes nearly 75% in right ventricle). The left ventricle was not affected. Given the risk of lethal arrhythmias, this individual had an ICD implanted 6 years ago, with three appropriate shocks registered to date. Individual II.1 -68 years old-, despite old age, showed no symptoms during most of her life (punctual palpitations in the past 8 years during moderate physical activity). One relative (grandfather, I.2) died suddenly at a young age -36 years old- while working on a farm. However, neither complete autopsy nor molecular autopsy was performed. No other rare variants were identified in this family. diagnosed with ACM (Figure 3). She suffered syncope during exercise. Clinical assessment showed dyskinesia with a moderate dilation of the right ventricle (39 mm), a minor reduction of ejection fraction (40%), and no criteria of fibro-adipose infiltration in the myocardium (residual myocytes nearly 75% in right ventricle). The left ventricle was not affected. Given the risk of lethal arrhythmias, this individual had an ICD implanted 6 years ago, with three appropriate shocks registered to date. Individual II.1 -68 years old-, despite old age, showed no symptoms during most of her life (punctual palpitations in the past 8 years during moderate physical activity). One relative (grandfather, I.2) died suddenly at a young age -36 years old- while working on a farm. However, neither complete autopsy nor molecular autopsy was performed. No other rare variants were identified in this family. Families C and D Families C and D were also diagnosed with ACM (Table 1), and both carried a previous reported pathogenic variant (CM060431) in PKP2 (c.1237C > T, p.R413X – p.Arg413∗), associated with ACM (Table 2). Our NGS panel included genes encoding the closest PKP2-related proteins, but no abnormalities were identified in these genes. In addition, no CNVs were identified in either family. Family C included six family members diagnosed with ACM, four of which were alive (II.2 -42 years old-, II.4 - 40 years old-, II.6 -37 years old-, and III.2 -9 years old-) and carried the same genetic variant, PKP2_p.(Arg413∗) (Figure 4). Families A and B ACM Possible (2 minor) II.2 42 F PKP2_p.(Arg413∗) ACM Definite (2 major and 2 minor) II.3† 21 M PKP2_p.(Arg413∗) ACM Definite (1 major and 2 minor) II.4 40 M PKP2_p.(Arg413∗) ACM Definite (2 major and 1 minor) II.6 37 M PKP2_p.(Arg413∗) ACM Definite (2 major and 2 minor) III.2 9 M PKP2_p.(Arg413∗) ACM Definite (1 major and 2 minor) D II.1 74 F PKP2_p.(Arg413∗) ACM Definite (2 major and 1 minor) II.5 71 M PKP2_p.(Arg413∗) ACM Definite (2 major and 1 minor) II.7 68 M PKP2_p.(Arg413∗) ACM Definite (1 major and 2 minor) III.2 39 F PKP2_p.(Arg413∗) ACM Definite (1 major and 2 minor) III.4 35 F PKP2_p.(Arg413∗) No – III.6 30 M PKP2_p.(Arg413∗) ACM Definite (2 major and 1 minor) III.8 38 M PKP2_p.(Arg413∗) ACM Definite (2 major and 1 minor) IV.1† 10 M PKP2_p.(Arg413∗) ACM (2 minor) IV.2 11 M PKP2_p.(Arg413∗) No – IV.5 6 F PKP2_p.(Arg413∗) No – E II.2 68 F MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 19 mm II.3 66 M MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 20 mm III.2 39 M MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 17 mm III.4 34 F MYBPC3_p.(Arg891Alafs∗160) No – III.6† 38 M MYBPC3_p.(Arg891Alafs∗160) HCM Myocardial disarray III.8 36 M MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 24 mm III.11 30 M MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 16 mm IV.2 11 M MYBPC3_p.(Arg891Alafs∗160) No – IV.4 8 F MYBPC3_p.(Arg891Alafs∗160) No – IV.6 3 F MYBPC3_p.(Arg891Alafs∗160) No – IV.8 2 F MYBPC3_p.(Arg891Alafs∗160) No – F II.2 73 M MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 25mm III.1 46 F MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 23mm III.2 41 M MYBPC3_p.(Arg891Alafs∗160) HCM LV wall thickness 22mm ACM, arrhythmogenic cardiomyopathy; F, female; HCM, hypertrophic cardiomyopathy; LV, left ventricle; M, male. Sign †means sudden death. ACM, arrhythmogenic cardiomyopathy; F, female; HCM, hypertrophic cardiomyopathy; LV, left ventricle; M, male. Sign †means sudden death. genic cardiomyopathy; F, female; HCM, hypertrophic cardiomyopathy; LV, left ventricle; M, male. Sign †means sudden death. Families C and D Families C and D Concerning diagnosis, inverted T-wave in right precordial leads were observed in three cases (II.2, II.4, and II.6); in addition, sustained ventricular tachycardia were observed in II.4, and II.6. Image analysis in II.2 showed regional RV akinesia and 38% RV May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 5 Cardiomyopathies: Personalized Diagnosis Campuzano et al. ejection fraction. In III.2, regional RV akinesia and PLAX RV outflow tract of 31 mm were identified. The other two relatives died suddenly at a young age and both were diagnosed with ACM post-mortem (both showing residual myocytes <60% in fibro- fatty infiltration); I.1 died working in a building site -44 years old-, and II.3 died at young age during exercise -21 years old- . Molecular autopsy indicated individual II.3 carried the same genetic variant. Unfortunately, no sample was obtained from I.1 to perform genetic analysis. No other rare variants were identified in this family. Family Gene Nucleotide Protein dbSNP EVS ExAC gnomAD PP2 PROVEAN MuTa FansDB HGMD (disease) ClinVar (pathology) ACMG score A PKP2 c.275T > A p.(Leu92∗) rs763639737 NA NA 2/251424 NA NA NA D CM102825 (ACM) P (ACM) P DSG2 c.527C > T p.(Thr176Ile) rs536617217 NA 2/120200 2/28069 B N DC N NA VUS VUS B PKP2 c.275T > A p.(Leu92∗) rs763639737 NA NA 2/251424 NA NA NA D CM102825 (ACM) P (ACM) P C PKP2 c.1237C > T p.(Arg413∗) rs372827156 1/13005 2/121408 4/282766 NA NA NA D CM060431 (ACM) P (ACM) P D PKP2 c.1237C > T p.(Arg413∗) rs372827156 1/13005 2/121408 4/282766 NA NA NA D CM060431 (ACM) P (ACM) P MYH7 c.4772T > A p.(Leu1591Gln) rs730880808 NA 7/121931 10/251476 B N DC N CM1413450 (SCD) VUS VUS TTN c.60754G > C p.(Ala20252Pro) rs72646880 27/12015 236/120342 513/279434 PD D DC N NA VUS LB E MYBPC3 c.2670dupG p.(Arg891Alafs∗160) Novel NA NA NA NA NA DC D NA NA P F MYBPC3 c.2670dupG p.(Arg891Alafs∗160) Novel NA NA NA NA NA DC D NA NA P ACM, arrhythmogenic cardiomyopathy; ACMG, American College of Medical Genetics and Genomics; B, benign; D, deleterious; DC, disease causing; EVS, exome variant server; ExAC, exome aggregation consortium; HGMD, human gene mutation database; MuTa, mutation taster; NA, not available; N, neutral; P, pathogenic; PD, possibly damaging; PP2, PolyPhen2; SCD, sudden cardiac death; VUS, variant uncertain/unknown significance. Families C and D netics | f ontie sin o g y Family D included six alive family members diagnosed with ACM (II.1 -74 years old-, II.5 -71 years old-, II.7 -68 years old-, III.2 -39 years old-, III.6 -30 years old-, and III.8 -38 years old-) (Figure 5). Inverted T-wave in right precordial leads were observed in five relatives (II.1, II.5, III.2, III.6, and III.8). III.7 also showed inverted T-wave in right precordial leads but in the presence of complete right bundle-branch block. Non-sustained ventricular tachycardia of left bundle-branch morphology were observed in III.6, and III.8. Image analysis showed RV ejection fraction of 38% and 39% in II.1 and II.5, respectively. All other relatives (II.7, II.2, III.6, and III.8) showed RV ejection fraction between 40 and 45%. Two relatives (III.3 and IV.1) died at a young age (15 and 10 years old, respectively). The index case, IV.1, died while sleeping and showed minor post- mortem cardiac abnormalities (including myocarditis) with no definite diagnosis of ACM but possible. Neither autopsy nor molecular autopsy was performed in III.3 despite SCD at a young age during exercise. No syncope or cardiac pathology was diagnosed in either young relative who died suddenly. Molecular autopsy of the index case (IV.1) identified three rare genetic variants –PKP2_p.(Arg413∗), MYH7_p.(Leu1591Gln) and TTN_p.(Ala20252Pro)-. All clinically affected relatives (II.1, II.5, II.7, III.2, III.6, and III.8) carried the PKP2_p.(Arg413∗) variant. Three additional relatives (III.4 -35 years old-, IV.2 - 11 years old-, and IV.5 -6 years old-) carried the same genetic variant, but all remain asymptomatic so far. Individual III.4 showed some cardiac abnormalities, although they were not diagnostic of ACM following current guidelines (Marcus et al., 2010). Neither grandparent (I.1 nor I.2) died suddenly or at a young age (both more than 75 years old). Concerning the other two rare variants identified -MYH7_p.(Leu1591Gln) and TTN_p.(Ala20252Pro)-, previous studies published by our group disregard a pathogenic role (Campuzano et al., 2015b). Frontiers in Genetics | www.frontiersin.org Families E and F Question mark indicates no genetic analysis available. The ECG corresponds to index case. The ECG showing negative T waves in V1-3, and prolonged Terminal Activation Duration as major and respectively, minor diagnostic Task Force Criteria for ACM. FIGURE 2 | Pedigree of family (A). Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are show in gray, clinically unaffected patients are show in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (III.1). The genetic carriers of PKP2_p.(Leu92∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. The ECG corresponds to index case. The ECG showing negative T waves in V1-3, and prolonged Terminal Activation Duration as major and respectively, minor diagnostic Task Force Criteria for ACM. FIGURE 3 | Pedigree of family B. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (III.1). The genetic carriers of PKP2_p.(Leu92∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to index case. ECG showing negative T waves in V1-2 (minor ACM criterion). FIGURE 3 | Pedigree of family B. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (III.1). The genetic carriers of PKP2_p.(Leu92∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to index case. ECG showing negative T waves in V1-2 (minor ACM criterion). III.8 -36 years old-, and III.11 -30 years old-) diagnosed with HCM (Figure 6). Only III.8 (left ventricular wall thickness 24 mm; ejection fraction 41%), had syncope and an ICD was implanted. Families E and F Families E and F were diagnosed with HCM (Table 1). The disease was caused by a novel variant in MYBPC3. This variant is a duplication of G (c.2670dupG), inducing a frameshift (p.Arg891Ala_fr∗160) (Table 2). This genetic alteration induces a radical change in fibronectin type 3 (FN3) domains, changing the amino acid sequence of wildtype MYBPC3 starting at amino acid 891. Our NGS panel included genes encoding the closest MYBPC3-related proteins, but no abnormalities were identified. In addition, neither other rare variants nor CNVs were identified in either family. Family E included five living family members (II.2 - 68 years old-, II.3 -66 years old-, III.2 -39 years old-, May 2019 | Volume 10 | Article 450 6 Cardiomyopathies: Personalized Diagnosis Campuzano et al. FIGURE 2 | Pedigree of family (A). Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are show in gray, clinically unaffected patients are show in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (III.1). The genetic carriers of PKP2_p.(Leu92∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. The ECG corresponds to index case. The ECG showing negative T waves in V1-3, and prolonged Terminal Activation Duration as major and respectively, minor diagnostic Task Force Criteria for ACM. FIGURE 3 | Pedigree of family B. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (III.1). The genetic carriers of PKP2_p.(Leu92∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to index case. ECG showing negative T waves in V1-2 (minor ACM criterion). FIGURE 2 | Pedigree of family (A). Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are show in gray, clinically unaffected patients are show in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (III.1). The genetic carriers of PKP2_p.(Leu92∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Frontiers in Genetics | www.frontiersin.org Families E and F FIGURE 4 | Pedigree of family C Generations are indicated in the left side Each individual is identified with a number Clinically affected patients are shown in gray FIGURE 4 | Pedigree of family C. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (II.2). The genetic carriers of PKP2_p.(Arg413∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to index case. Families A and B were diagnosed with ACM due to a pathogenic variant, PKP2_p.(Leu92∗) (CM102825). Our group previously published the index case of Family A (Alcalde et al., 2014), and here we have included family segregation. Hence, Family A showed incomplete penetrance and variable expressivity because one relative carried the pathogenic variant but had no diagnosis of ACM (despite showing some scattered fibrotic heart abnormalities). Intriguingly, the relative without an ACM diagnosis is the progenitor of both clinically affected patients, may be due to age-dependent penetrance age. The phenotype of both diagnosed patients differed despite their similar age—neither had early disease onset, but the evolution was faster and more severe in one patient in contrast to the other. Further, the variant in DSG2 (p.T176I) was not present in a relative clinically diagnosed with ACM (III.4). Therefore, we disregarded DSG2_p.(Thr176Ile) as pathogenic, at least in Family A, remaining the variant PKP2_p.(Leu92∗) is the most plausible cause of disease in Family A. syncope. The three living relatives carried the same genetic variant, MYBPC3_p.(Arg891Ala_fr∗160). Neither grandparent (I.1 nor I.2) died suddenly or at a young age (both >75 years old). Families E and F All other relatives were diagnosed with HCM: II.2 -left ventricular wall thickness 19 mm; ejection fraction 49%-, II.3 -left ventricular wall thickness 20 mm; ejection fraction 45%-, III.2 -left ventricular wall thickness 17 mm; ejection fraction 49%-, and III.11 -left ventricular wall thickness 16 mm; ejection fraction 51%-. They did not show severe heart abnormalities (follow-up showed slow evolution of disease), and no syncope has been documented thus far. All carried the same rare genetic variant, MYBPC3_p.(Arg891Ala_fr∗160). Individual III.6 was diagnosed post-mortem –died at 38 years old-, during an extreme effort at work. Molecular autopsy identified the same variant (p.Arg891Ala_fr∗160) and forensic report described an asymmetric septal hypertrophy and microscopic analysis identified myocardial disarray with interstitial fibrosis. In addition, five relatives (III.4 -34 years old-, IV.2 -11 ears old-, IV.4 -8 years old-, IV.6 -3 years old-, and IV.8 -2 years old-) carried the same genetic variant, but all remained asymptomatic and without any cardiac alteration, so far. Both grandparents (I.1 and I.2) died at old age (both >75 years old) due to oncologic and ischemic episodes, respectively. Neither had documented episodes of arrhythmia or syncope. Family F included three living family members (III.1, III.2, and II.2) diagnosed with HCM (Figure 7). Individual II.2 - 73 years old- had a syncopal episode several years before, and an ICD was implanted. He showed a left ventricular wall thickness of 25 mm, mitral valve regurgitation, and slightly decreased ejection fraction (48%). The other two relatives (III.1 and III.2), despite young ages (46 and 41 years old, respectively), showed enlarged left ventricular wall thickness (23 mm and 22 mm, respectively), moderately decreased ejection fraction (45 and 47%, respectively), and mitral valve regurgitation. Individual III.1 had an ICD implanted due to May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 7 Cardiomyopathies: Personalized Diagnosis Campuzano et al. FIGURE 4 | Pedigree of family C. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (II.2). The genetic carriers of PKP2_p.(Arg413∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to index case. Frontiers in Genetics | www.frontiersin.org DISCUSSION The “five P” characteristics in current medicine – personalized, predictive, preventive, participative, and precision – are an innovative approach to the patient using available technologies to interpret individually all patient data. Recently updated guidance, first published in 2009 for the Heart Failure Society of America and ACMG, supports the use of NGS in genetic testing for cardiomyopathy to assist patient care and management of at- risk family members (Hershberger et al., 2018). In our study, we carried out an individualized interpretation of three pathogenic variants leading to cardiomyopathy in six families showing high expression variability even for the same variant and family due to different onset, evolution, and/or outcome of the disease. May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 8 Cardiomyopathies: Personalized Diagnosis Campuzano et al. FIGURE 5 | Pedigree of family D. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (IV.1). The genetic carriers of PKP2_p.(Arg413∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to IV.1. FIGURE 5 | Pedigree of family D. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (IV.1). The genetic carriers of PKP2_p.(Arg413∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to IV.1. FIGURE 5 | Pedigree of family D. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (IV.1). The genetic carriers of PKP2_p.(Arg413∗) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to IV.1. Frontiers in Genetics | www.frontiersin.org DISCUSSION Family B, despite only two genetic carriers showed incomplete penetrance and variable expressivity. The younger relative (III.1) showed an aggressive phenotype, with often-malignant arrhythmias, and had an ICD implanted. The older relative (II.1) showed a slow evolution and benevolent phenotype due to minimal symptoms throughout her life. Therefore, considering the clinical data in both Families A and B, PKP2_p.(Leu92∗) may be considered the cause of ACM, although other genetic abnormalities may modify onset, evolution, and outcome of the disease. affected relatives carried the non-sense variant in PKP2, and young relatives showed cardiac abnormalities associated with ACM, suggesting an early onset of disease due to this genetic variant. In 2015, our group also published part of Family D (Campuzano et al., 2015b), and here we have included more relatives to confirm the pathogenic role of PKP2_p.(Arg413∗) as cause of disease. Genotype-phenotype correlation suggests that PKP2_p.(Arg413∗) is the cause of disease in these families, whereas additional variants could play a role as phenotype modulators. We observed incomplete penetrance and variable expressivity, as occurs in other reported families affected by ACM (Kannankeril et al., 2006). Asymptomatic patients carrying this genetic variant were young relatives. However, it is well accepted that ACM is a progressive structural disease, so cardiac abnormalities may not yet be evident in early ages. Families C and D were also diagnosed with ACM and both carried a pathogenic variant (CM060431) in PKP2 (c.1237C > A, p.R413X - p.Arg413∗). Our group published on Family C in 2014 (Alcalde et al., 2014). All clinically May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 9 Cardiomyopathies: Personalized Diagnosis Campuzano et al. FIGURE 6 | Pedigree of family E. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (III.2). The genetic carriers of MYBPC3_p.(Arg891Alafs∗160) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to index case. FIGURE 6 | Pedigree of family E. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. DISCUSSION Index case is indicated with an arrow (III.2). The genetic carriers of MYBPC3_p.(Arg891Alafs∗160) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. SD means Sudden Death. The ECG corresponds to index case. Families E and F were diagnosed with HCM due to a novel frameshift alteration in MYBPC3 that results in a different protein than wildtype MYBPC3. All analyses suggest a pathogenic role. Family E showed incomplete penetrance and variable expressivity, with clinically diagnosed relatives carrying the MYBPC3_p.(Arg891Alafs∗160) variant and showing different phenotypic evolution of heart abnormalities. This agrees with several reports showing different phenotypes from one family diagnosed with HCM due to a single pathogenic variant (Wang et al., 2018). In Family E, asymptomatic patients were young relatives, suggesting a delay in the onset of HCM that makes cardiac manifestations not yet evident at younger ages. This agrees with previous reports suggesting that pathogenic variants in genes associated Indeed, a middle-aged relative in Family D showed some cardiac abnormalities that were not diagnostic of ACM following current guidelines (Marcus et al., 2010). Therefore, the same non-sense variant induces pathology in both Families C and D, but the onset in each family is different, especially in young individuals (<40 years old). In Family C, relatives showed evident heart abnormalities at an early age, suggesting a highly aggressive role of the variant; however, in Family D, the abnormalities seemed to occur progressively, suggesting a pathogenic role of the variant but not with the same temporal onset. Therefore, despite PKP2_p.(Arg413∗) being the cause of ACM, other genetic abnormalities (although not in any of the closest related proteins) may modify the onset, evolution, and outcome of ACM. Indeed, a middle-aged relative in Family D showed some cardiac abnormalities that were not diagnostic of ACM following current guidelines (Marcus et al., 2010). Therefore, the same non-sense variant induces pathology in both Families C and D, but the onset in each family is different, especially in young individuals (<40 years old). In Family C, relatives showed evident heart abnormalities at an early age, suggesting a highly aggressive role of the variant; however, in Family D, the abnormalities seemed to occur progressively, suggesting a pathogenic role of the variant but not with the same temporal onset. DISCUSSION Therefore, despite PKP2_p.(Arg413∗) being the cause of ACM, other genetic abnormalities (although not in any of the closest related proteins) may modify the onset, evolution, and outcome of ACM. May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 10 Cardiomyopathies: Personalized Diagnosis Campuzano et al. FIGURE 7 | Pedigree of family F. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (II.2). The genetic carriers of MYBPC3_p.(Arg891Alafs∗160) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. The ECG corresponds to index case. FIGURE 7 | Pedigree of family F. Generations are indicated in the left side. Each individual is identified with a number. Clinically affected patients are shown in gray, clinically unaffected patients are shown in white, and slashes indicate a deceased relative. Index case is indicated with an arrow (II.2). The genetic carriers of MYBPC3_p.(Arg891Alafs∗160) are represented with a plus sign inside rounds/squares. Minus sign indicates non-carriers of the genetic variant. Question mark indicates no genetic analysis available. The ECG corresponds to index case. with HCM do not induce structural abnormalities at early years (Brion et al., 2009, 2012). Family F showed complete penetrance and variable expressivity. All relatives who carried the MYBPC3_p.(Arg891Alafs∗160) variant had a clinical diagnosis of HCM, including young family members. Therefore, MYBPC3_p.(Arg891Alafs∗160) is the likely cause of HCM in both Families E and F, and other genetic abnormalities (although not in the closest related proteins) may modify onset, evolution, and outcome of the disease in each family. with HCM do not induce structural abnormalities at early years (Brion et al., 2009, 2012). Family F showed complete penetrance and variable expressivity. All relatives who carried the MYBPC3_p.(Arg891Alafs∗160) variant had a clinical diagnosis of HCM, including young family members. Therefore, MYBPC3_p.(Arg891Alafs∗160) is the likely cause of HCM in both Families E and F, and other genetic abnormalities (although not in the closest related proteins) may modify onset, evolution, and outcome of the disease in each family. to high risk of lethal arrhythmias, accordingly to recent published manuscripts (Kapplinger and Ackerman, 2016). DISCUSSION In addition, proper genetic counseling is essential for all family members despite the high complexity of genetic heterogeneity, incomplete penetrance, and variable expressivity related to inherited cardiomyopathies (De Bortoli et al., 2017). In this prospective, further genetic studies aiming to identify modifying factors are needed to improve prevention of malignant arrhythmias and guide appropriate treatment in each patient. Autopsy in young individuals who die suddenly may be not conclusive and may not identify cardiac abnormalities. However, potential cardiomyopathy should not be disregarded, as ultramicroscopic changes that are not evident at autopsy may nonetheless induce arrhythmia responsible for SCD (Campuzano et al., 2014a). Molecular autopsy may identify the genetic alteration in a gene associated with cardiomyopathy but without anatomic heart abnormalities. This fact is important in sudden deaths occurring in infants, children, and even young people (Sarquella-Brugada et al., 2016). A recent study states that, carriers of a pathogenic variant may be at 2.5-fold greater risk of SCD compared to non-carriers (Milano et al., 2016). Nonetheless, it should be communicated to the patient that despite being a carrier, the individual may never express the disease phenotype (Kapplinger and Ackerman, 2016). Similar to our study, a 2010 publication of a cohort of 60-year-old patients diagnosed with ACM and carrying a pathogenic radical variant indicates that only 60% of genetic carriers had experienced any symptoms, without differences between genders (van der Zwaag et al., 2010). It is well-known that cardiomyopathies may display a broad range of phenotype variability, from asymptomatic individuals to severe heart alterations; it is also important to remark that sometimes the first manifestation of disease is SCD, which usually occurs in a healthy young individual while doing exercise, main trigger of life-threatening ventricular arrhythmias. Considering all data, we believe that clinical follow-up including periodic tests of risk should be performed in all genetic carriers, despite remaining asymptomatic due In conclusion, genetic testing has been progressively incorporated into clinical diagnosis, mainly due to improved NGS technology. Although genetic interpretation may classify rare variants as pathogenic, translation into clinical practice sometimes shows both interfamilial and intrafamilial phenotypic differences. We report here families carrying the same pathogenic variant associated with cardiomyopathies but with differential onset and/or clinical progression. Therefore, additional factors may modify the disease phenotype in each individual. Nonetheless, all individuals carrying a pathogenic variant, despite being asymptomatic, are at risk of disease therefore, clinical follow-up should be performed. DISCUSSION Clinical translation remains a main challenge for cardiovascular genetic units and should be carefully performed in a personalized manner. Frontiers in Genetics | www.frontiersin.org REFERENCES dysplasia/cardiomyopathy: spectrum of mutations and clinical impact in practice. Europace 12, 861–868. doi: 10.1093/europace/euq104 Abou Tayoun, A. N., Pesaran, T., DiStefano, M. T., Oza, A., Rehm, H. L., Biesecker, L. G., et al. (2018). Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. Hum. Mutat. 39, 1517–1524. doi: 10.1002/ humu.23626 Giudicessi, J. R., and Ackerman, M. J. (2013). Determinants of incomplete penetrance and variable expressivity in heritable cardiac arrhythmia syndromes. Transl. Res. 161, 1–14. doi: 10.1016/j.trsl.2012.08.005 Gopinathannair, R., Etheridge, S. P., Marchlinski, F. E., Spinale, F. G., Lakkireddy, D., and Olshansky, B. (2015). Arrhythmia-Induced cardiomyopathies: mechanisms, recognition, and management. J. Am. Coll. Cardiol. 66, 1714–1728. doi: 10.1016/j.jacc.2015.08.038 Alcalde, M., Campuzano, O., Berne, P., Garcia-Pavia, P., Doltra, A., Arbelo, E., et al. (2014). Stop-gain mutations in PKP2 are associated with a later age of onset of arrhythmogenic right ventricular cardiomyopathy. PLoS One 9:e100560. doi: 10.1371/journal.pone.0100560 Hershberger, R. E., Givertz, M. M., Ho, C. Y., Judge, D. P., Kantor, P. F., McBride, K. L., et al. (2018). Genetic evaluation of cardiomyopathy: a clinical practice resource of the american college of medical genetics and genomics (ACMG). Genet. Med. 20, 899–909. doi: 10.1038/s41436-018-0039-z Amendola, L. M., Jarvik, G. P., Leo, M. C., McLaughlin, H. M., Akkari, Y., Amaral, M. D., et al. (2016). Performance of ACMG-AMP variant-interpretation guidelines among nine laboratories in the clinical sequencing exploratory research consortium. Am. J. Hum. Genet. 98, 1067–1076. doi: 10.1016/j.ajhg. 2016.03.024 Kannankeril, P. J., Bhuiyan, Z. A., Darbar, D., Mannens, M. M., Wilde, A. A., and Roden, D. M. (2006). Arrhythmogenic right ventricular cardiomyopathy due to a novel plakophilin 2 mutation: wide spectrum of disease in mutation carriers within a family. Heart Rhythm 3, 939–944. doi: 10.1016/j.hrthm.2006. 04.028 Basso, C., Burke, M., Fornes, P., Gallagher, P. J., de Gouveia, R. H., Sheppard, M., et al. (2008). Guidelines for autopsy investigation of sudden cardiac death. Virchows Arch. 452, 11–18. doi: 10.1007/s00428-007-0505-5 Brion, M., Allegue, C., Santori, M., Gil, R., Blanco-Verea, A., Haas, C., et al. (2012). Sarcomeric gene mutations in sudden infant death syndrome (SIDS). Forensic. Sci. Int. 219, 278–281. doi: 10.1016/j.forsciint.2012.01.018 Kapplinger, J. D., and Ackerman, M. J. (2016). Founder mutation genotyping and sudden cardiac arrest: the promise of precision medicine fulfilled or the next step into precise uncertainty. Circ. Cardiovasc. Genet. 9, 107–109. doi: 10.1161/CIRCGENETICS.116.001387 Brion, M. A. C., Gil, R., Torres, M., Santori, M., Poster, S., Madea, B., et al. (2009). REFERENCES Involvement of hypertrophic cardiomyopathy genes in sudden infant death syndrome (SIDS). Forensic. Sci. Int. Genet. Suppl. Ser. 2, 495–496. Kobayashi, Y., Yang, S., Nykamp, K., Garcia, J., Lincoln, S. E., and Topper, S. E. (2017). Pathogenic variant burden in the ExAC database: an empirical approach to evaluating population data for clinical variant interpretation. Genome Med. 9:13. doi: 10.1186/s13073-017-0403-7 Campuzano, O., Allegue, C., Fernandez, A., Iglesias, A., and Brugada, R. (2015a). Determining the pathogenicity of genetic variants associated with cardiac channelopathies. Sci. Rep. 5:7953. doi: 10.1038/srep07953 Lek, M., Karczewski, K. J., Minikel, E. V., Samocha, K. E., Banks, E., Fennell, T., et al. (2016). Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291. doi: 10.1038/nature19057 Campuzano, O., Fernandez-Falgueras, A., Sarquella-Brugada, G., Sanchez, O., Cesar, S., Mademont, I., et al. (2015b). A genetically vulnerable myocardium may predispose to myocarditis. J. Am. Coll. Cardiol. 66, 2913–2914. doi: 10. 1016/j.jacc.2015.10.049 Marcus, F. I., McKenna, W. J., Sherrill, D., Basso, C., Bauce, B., Bluemke, D. A., et al. (2010). Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force criteria. Circulation 121, 1533–1541. doi: 10.1161/CIRCULATIONAHA.108.840827 Campuzano, O., Allegue, C., Partemi, S., Iglesias, A., Oliva, A., and Brugada, R. (2014a). Negative autopsy and sudden cardiac death. Int. J. Legal Med. 128, 599–606. doi: 10.1007/s00414-014-0966-4 Marian, A. J., and Braunwald, E. (2017). Hypertrophic Cardiomyopathy: genetics, pathogenesis, clinical manifestations, diagnosis, and therapy. Circ. Res. 121, 749–770. doi: 10.1161/CIRCRESAHA.117.311059 Campuzano, O., Sarquella-Brugada, G., Mademont-Soler, I., Allegue, C., Cesar, S., Ferrer-Costa, C., et al. (2014b). Identification of genetic alterations, as causative genetic defects in long QT syndrome, using next generation sequencing technology. PLoS One 9:e114894. doi: 10.1371/journal.pone.01 14894 Milano, A., Blom, M. T., Lodder, E. M., van Hoeijen, D. A., Barc, J., Koopmann, T. T., et al. (2016). Sudden cardiac arrest and rare genetic variants in the community. Circ. Cardiovasc. Genet. 9, 147–153. doi: 10.1161/ CIRCGENETICS.115.001263 De Bortoli, M., Calore, C., Lorenzon, A., Calore, M., Poloni, G., Mazzotti, E., et al. (2017). Co-inheritance of mutations associated with arrhythmogenic cardiomyopathy and hypertrophic cardiomyopathy. Eur. J. Hum. Genet. 25, 1165–1169. doi: 10.1038/ejhg.2017.109 Morini, E., Sangiuolo, F., Caporossi, D., Novelli, G., and Amati, F. (2015). Application of next generation sequencing for personalized medicine for sudden cardiac death. Front. Genet. 6:55. doi: 10.3389/fgene.2015.00055 j g Deo, R., and Albert, C. M. (2012). Epidemiology and genetics of sudden cardiac death. Circulation 125, 620–637. doi: 10.1161/CIRCULATIONAHA. FUNDING This work was supported by Obra Social “La Caixa Foundation” (ID 100010434), Fondo Investigacion Sanitaria (FIS, PI14/01773 and PI17/01690) from Instituto Salud Carlos III (ISCIII), and “Fundacio Privada Daniel Bravo Andreu.” CIBERCV is an initiative of the ISCIII, Spanish Ministry of Economy and Competitiveness. Limitations A main limitation of this study is the lack of understanding of the reasons implicated in high expression variability even for the same variant and family. Additional analysis of the assessed genes as well as in other genes not included in our NGS custom-panel is necessary to identify additional genetic variants that could modify the phenotype. In addition, other genetic analyses focused on transcriptional/proteomic modifications are necessary to clarify the role of each genetic variant in each individual. It is important to remark the absence of data on exercise, an important contributor to phenotypic manifestation. Finally, in vivo and/or in vitro studies of each genetic variant may help clarify pathophysiological mechanisms associated with the pathology. May 2019 | Volume 10 | Article 450 11 Cardiomyopathies: Personalized Diagnosis Campuzano et al. ETHICS STATEMENT MP, LL, FP, IM-S, and CT acquired, pre-processed, and analyzed the data. OC, AF-F, and GS-B prepared the manuscript. OC, JB, and RB supervised the study. All authors contributed to manuscript revision, read, and approved the submitted version. This study was approved by the Ethics Committee of the Hospital Josep Trueta (Girona, Spain), following the Helsinki II declaration. Written informed consent was obtained from all relatives included in the study. Alive individuals were clinically evaluated at Hospital Josep Trueta (Girona, Spain), Hospital Clinic of Barcelona (Barcelona, Spain), or Hospital Sant Joan de Deu (Barcelona, Spain). This study was approved by the Ethics Committee of the Hospital Josep Trueta (Girona, Spain), following the Helsinki II declaration. Written informed consent was obtained from all relatives included in the study. Alive individuals were clinically evaluated at Hospital Josep Trueta (Girona, Spain), Hospital Clinic of Barcelona (Barcelona, Spain), or Hospital Sant Joan de Deu (Barcelona, Spain). AUTHOR CONTRIBUTIONS OC, AF-F, GS-B, JB, and RB developed the concept. SC, MC, JM, EA, AG-Á, PJ, AP-S, BdO, CF, MA, VF, AI, REFERENCES 111.023838 Nykamp, K., Anderson, M., Powers, M., Garcia, J., Herrera, B., Ho, Y. Y., et al. (2017). Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria. Genet. Med. 19, 1105–1117. doi: 10.1038/gim.2017.37 Fressart, V., Duthoit, G., Donal, E., Probst, V., Deharo, J. C., Chevalier, P., et al. (2010). Desmosomal gene analysis in arrhythmogenic right ventricular Priori, S. G., and Blomstrom-Lundqvist, C. (2015). 2015 European Society of cardiology guidelines for the management of patients with ventricular May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 12 Cardiomyopathies: Personalized Diagnosis Campuzano et al. arrhythmias and the prevention of sudden cardiac death summarized by co-chairs. Eur. Heart J. 36, 2757–2759. doi: 10.1093/eurheartj/ ehv445 van der Zwaag, P. A., Cox, M. G., van der Werf, C., Wiesfeld, A. C., Jongbloed, J. D., Dooijes, D., et al. (2010). Recurrent and founder mutations in the Netherlands : plakophilin-2 p.Arg79X mutation causing arrhythmogenic right ventricular cardiomyopathy/dysplasia. Neth. Heart J. 18, 583–591. doi: 10.1007/s12471- 010-0839-5 Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., et al. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology. Genet. Med. 17, 405–424. doi: 10.1038/gim.2015.30 Wang, J., Wan, K., Sun, J., Li, W., Liu, H., Han, Y., et al. (2018). Phenotypic diversity identified by cardiac magnetic resonance in a large hypertrophic cardiomyopathy family with a single MYH7 mutation. Sci. Rep. 8:973. doi: 10.1038/s41598-018-19372-4 Sarquella-Brugada, G., Campuzano, O., Cesar, S., Iglesias, A., Fernandez, A., Brugada, J., et al. (2016). Sudden infant death syndrome caused by cardiac arrhythmias: only a matter of genes encoding ion channels? Int. J. Legal Med. 130, 415–420. doi: 10.1007/s00414-016-1330-7 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Sinard, J. H. (2013). Accounting for the professional work of pathologists performing autopsies. Arch. Pathol. Lab. Med. 137, 228–232. doi: 10.5858/arpa. 2012-0012-CP Copyright © 2019 Campuzano, Fernandez-Falgueras, Sarquella-Brugada, Cesar, Arbelo, García-Álvarez, Jordà, Coll, Fiol, Iglesias, Perez-Serra, Mates, del Olmo, Ferrer, Alcalde, Puigmulé, Mademont-Soler, Pico, Lopez, Tiron, Brugada and Brugada. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Genetics | www.frontiersin.org REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Straus, S. M., Bleumink, G. S., Dieleman, J. P., van der Lei, J., Stricker, B. H., and Sturkenboom, M. C. (2004). The incidence of sudden cardiac death in the general population. J. Clin. Epidemiol. 57, 98–102. doi: 10.1016/S0895-4356(03) 00210-5 Syrris, P., Ward, D., Asimaki, A., Sen-Chowdhry, S., Ebrahim, H. Y., Evans, A., et al. (2006). Clinical expression of plakophilin-2 mutations in familial arrhythmogenic right ventricular cardiomyopathy. Circulation 113, 356–364. doi: 10.1161/CIRCULATIONAHA.105.561654 May 2019 | Volume 10 | Article 450 Frontiers in Genetics | www.frontiersin.org 13
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Crafting, Crafting, Crafting - Extreme Programming in Classroom?
Informatics in education
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149 149 Informatics in Education, 2008, Vol. 7, No. 2, 149–158 © 2008 Institute of Mathematics and Informatics, Vilnius © 2008 Institute of Mathematics and Informatics, Vilnius Received: April 2008 Abstract. Extreme Programming (XP) shows several interesting approaches which are very attrac- tive for education. It is centered around early and incremental creation of working software. In the following, the chances XP offers for class are shown – especially for use in a class project, but also for practical phases in all lessons where programming is useful. Finally several common problems which can occur with XP will be shown as well as how to deal with them to make the use in class as smooth as possible. Keywords: extreme programming, XP, education, pair programming, project class, independent work. Crafting, Crafting, Crafting – Extreme Programming in Classroom? Marcus BITZL Technical University of Munich, Department of Informatics, Didactics in Informatics Boltzmannstr. 3, D-85748 Garching e-mail: marcus@bitzl.com Marcus BITZL Technical University of Munich, Department of Informatics, Didactics in Informatics Boltzmannstr. 3, D-85748 Garching e-mail: marcus@bitzl.com Marcus BITZL Technical University of Munich, Department of Informatics, Didactics in Informatics Boltzmannstr. 3, D-85748 Garching e-mail: marcus@bitzl.com 1Here we can already see an important point of Extreme Programming: instead of dictating a schedule and forcing its adherence at any price, one rather aims for the best effort estimation with inclusion of the programmers. 1. Introduction Extreme Programming (XP) is an agile software development methodology founded by Kent Beck. On a closer examination it shows several very interesting approaches for use in classroom which will be shown in the following. Because of XP’s educational values, Brüggemann et al. chose it for their projects in professional education in IT companies (Brüggemann et al., 2006). But scientific papers concerning the use in schools are very rare. Thus, XP will be explained below and interesting aspects for the use in school will be especially emphasized. In addition, different applications and solutions for important problems concerning its use in class will be shown. But first: what does “use in school” mean? The term school is cleared at once: where this paper relates to a special type of school, the Bavarian Gymnasium is meant because of its large amount of informatics lessons, but everything in this paper can be easily generalized to other school types. When it comes up to “use”, there are mainly three possibilities: first, one could apply XP in practical phases in a lesson; second, one could use XP in a class project, and finally, one could make an own elective creating software with XP (e.g., an “XP-Team”). The main source for this paper is “Extreme Programming Explained. Embrace Change” from Kent Beck (Beck, 2000), in which he presents XP. All statements on how XP works are from this book. 150 M. Bitzl 2. Roles For XP, Beck defines several roles (Beck, 2000) which are important for its understand- ing: the central role is the one of the programmer and is similar to the one of other programmers, but with slightly shifted priorities. The programmers’ main objective is communication between each other and with all other roles as shown later. The customer is in XP also part of the team – no matter if it is an external customer or the marketing section of the same company. It is crucial that the customers are real users which would really work with the software that is built. Beside these, there are more roles: the tester helps the customer to create function tests and is responsible for the regular execution of these test, if it is not already au- tomated (unit- and integration-tests are part of the development and therefore tasks of the programmers). The time manager is responsible for the project’s schedule. The time manager is kind of the team’s conscience as he (or she) is responsible for adhering the schedule or for its correction if adhering is impossible. He also watches the team’s per- formance to help them making better effort estimations1. The time manager should stay in the background for not to disturb work more than necessary. The Coach is responsible for the overall process. His job is to direct people in a certain direction if necessary. His interferences should be as few as possible. Moreover, it is recommended to prefer an indirect guidance over a direct one, so people (mainly programmers) could find a better way by themselves. As XP programmers are rather rarely specialists in their customers’ area of application such specialists are taken for a certain time as advisors. The Big-Boss is responsible for the team’s motivation, but also for all organizational concerns like staff or scheduling. The team takes its problems to him and expects his help to solve them. If everything works fine, the Big-Boss should stay in the background – his intervention would perturb the team’s productivity. What do these roles mean for use in class? The students get in any case the program- mers’s role, some of the could also be customers instead. The teacher should take the other roles. In a class project or a practice class it would be possible for some roles (e.g., the advisor) to invite other persons for some time. 3. Values and Principles 3.1. Values 3.1. Values 3.1. Values According to (Beck, 2000), Extreme Programming is based on four values which can be found in all aspects: open and honest communication has to take place throughout the whole project, between the pair-programming partners, between all developers, the man- agement and also with the customers. This is necessary to produce high quality software. 151 Crafting, Crafting, Crafting – Extreme Programming in Classroom? All solutions have to follow the principle of simplicity: an implemented solution may be only as complex as necessary to meet the requirements. If an existing solution is too limited a refactoring, that is a change of the code structure, is applied. To ensure high-quality solutions, some feedback is needed: from the customer to meet his requirements, from the other developers to find the best solutions and to learn. And also from the software itself feedback is demanded by tests to ensure that all requirements are fulfilled and to avoid defects. The fourth value is courage: it takes courage to address problems and conflicts frankly and openly. It also takes courage to replace already work- ing parts of the software with new ones when it is necessary. XP always takes courage for change and for experiments, if an appropriate solution is not clear enough, and at last courage to take the responsibility for the different tasks. If one considers these values together with the constitution of the free state of Bavaria, or the profile of the Bavarian Gymnasium, one can find several interesting parallels: the constitution already emphasizes the importance of a sense of responsibility and the dispo- sition to accept responsibility as an educational objective. This is taken up in the profile of the Bavarian Gymnasium and supplemented with skills like the ability to communi- cate, the ability to work in a team, conflict handling skills and the ability to make sound judgements. 3.2. Basic Principles To use the values above practically, Beck derives five basic principles: mainly based on behavioristic learning theories, but also to recognize problems as early as possible, rapid feedback is demanded. Technically, one gets this with automated tests. For people, stand up meetings and personal discussions are used. Second, the developers should assume simplicity, that is solving any task only as it is given at the moment – not as it will be later or even as it could be later. This is exactly what the customer wants and pays for in the next release. Also, this is the easiest solution to understand and to maintain. For class, this is good because of three reasons: the students get solutions in less time, realize an immediate relation between their work and the resulting use (there is nothing that is not used at the moment) and with a latter extension, they can see the limitations of their present solution and learn new approaches. As the project is under constant development, the third principle is incremental change: large and extensive changes are error prone and therefore difficult to control. If large changes are split into sequences of many small changes, every single one could be very well controlled and realized. This is also very useful for school, as it supports students in realizing changes and understanding their consequences. Moreover, they get working versions even before all changes are implemented. This can only work, if everybody encourages change. This should also be visible in the way how solutions are chosen: the best ones are those solving the problem (without violating another principle) and offer the most options for future development. In every single aspect, high quality work is demanded: if the created software is of poor quality, it is no fun for the developers to work on it. This has negative consequences for the quality of their further work and therefore for the success of the whole project. 152 M. Bitzl In school it would surely be the same: software of poor quality (as one would see from many errors and instabilities) would harm the motivation of all participants seriously. 3.3. More Principles Besides the five base principles, there are ten more principles from which some special ones get further explained: software development is always also considered as a learning process. Therefore, teaching to learn is a crucial task for the coach. Systematic experi- ments are also part of the XP’s everyday life helping to understand concepts or to select solutions (thus, learning again). Furthermore, each developer has to take responsibility, a competence already demanded by the Bavarian constitution. Technically, small initial investments are demanded: the project is naturally quite small when it is started (as it didn’t exist before). Therefore, starting with too much re- sources can lead to problems. How to solve this will be shown later. The principle “playing to win” has to be understood in contrast to “playing not to loose”: our goal has to be to develop a project in intensive communication with our customer and delivering it in high quality. Everything else would harm the project and therefore also the developers’ motivation and the effectiveness of software development. This is also very important for school because students expect a well working result at the end of a project. A low quality product would be certainly demotivating, and a failed project would be disastrous, regardless if the rules of a certain process model have always been followed correctly. In every aspect of the development process, we need open and honest communication, also one has to adopt XP to local conditions, use people’s instincts (not work against them) and travel light – that means, not to use any unnecessary processes or software. When using tests, metrics or simply when speaking with developers and customers honest measurement is important: if measurement results or information retrieved from discussions are not “nice”, this is an important hint that something doesn’t work properly or could be done better. 4.1. Activities According to Beck, the four main activities for XP programmers are coding, testing, listening and designing. Coding is the central point in XP, because it quickly leads to measurable results, one can easily quantify implemented features and clean code is suit- able for communication about solutions. The implementation of an idea, an algorithm or a solution can be tested if they meet the requirements. Different solutions can be com- pared. This enables the programmers not only to find the best solutions or to improve the existing ones, but also to learn with every comparison, selection or solution they make. In school, the students can sample theoretical concepts with concrete problems and solu- tions. The short times till students achieve results and the fact that they can easily quantify their work and understand their code, support a positive feeling of success and therefore supports independent work. 153 Crafting, Crafting, Crafting – Extreme Programming in Classroom? Permanent testing ensures that all implementations fulfil their requirements. There- fore, one creates tests before creating the corresponding implementation. The execution of these tests should be automated, so the programmers can execute them easily and of- ten. In addition, Extreme Programming uses these tests as parts of the documentation, because one can clearly see there the expected behaviors. A meta-analysis done by Jef- fries und Melnik (Jeffries and Melnik, 2007) shows the use of this approach: out of 16 studies 11 showed a considerable improvement of software quality. Only one states a negative influence on software quality and only four do not show any effect at all. For school, this approach is interesting for different reasons: first, the students get a positive (or negative) feedback on their implementations immediately. Therefore, their erroneous work is still present and an immediate feedback after they corrected the mistake leads to an increased learning effect according to behavioristic learning theories (see also the principle of immediate feedback). With unit tests, errors get narrowed down to the smallest possible code regions. Feedback from tests also supports the teacher and also encourages the students’ independent activities with concrete information on errors. Moreover, the exact way of working as demanded in the profile of informatics education (see (Fachprofil Informatik G8, 2004)) is supported as tests only accept exact solutions to their requirements. 4.1. Activities The third activity – listening – is based on a basic problem in software engineer- ing: misunderstandings between the customer and the developers. Therefore, developers have to learn how to listen and have to learn listening and how to do this actively in a discussion, to individuals as well as to large groups. Part of this is to ask questions and to develop a basis for common communication. Moreover, developers help the cus- tomers to understand which things are hard and which ones are easy to achieve. In school, this strong focus on communication supports students by improving their communication skills as for example demanded in Bavarian curriculums. These activities alone could help us only for a short time, because if good design is neglected, code soon gets unmaintainable and almost impossible to change. Therefore, the last activity is design. This is not a phase, but a continuous process interrupted by other activities or melted with programming to one unit. This is very useful for class as implementations immediately follow on design, changing in very short cycles. This allows students to experience the consequences of their good (or bad) design immediately and to realize how appropriate design simplifies their solutions. Designing in XP is different from other software engineering methods: first, program- mers implement a test case and a piece of code solving the current task in the simplest possible way. For the next task, they extend the test case and change the existing code to pass the tests. Doing so, they take care that the code doesn’t contain anything which is not necessary at the moment. For the third, fourth and any further tasks they do the same, until all tasks are done. This is also expedient for use in lessons: breaking up the whole problem into many pieces reduces it to a set of small problems which are easy to solve, and every one is independent from the following changes. Therefore, students can focus on their problems without being distracted by much stuff not necessary at the moment and whose purpose is yet hard to understand. Astrachan et. al suggest to give students (in 154 M. Bitzl his case from university, but for this technique this doesn’t really matter) a task for which they should find a solution which is as simple as possible. 4.1. Activities The following tasks demand a gradual extension of the existing solutions leading slowly to a certain, very elegant de- sign. He reports a strongly improved learning success compared to assignments solved with a design the teacher had only presented his students (Astrachan et al., 2003). 4.2. Workflow If the customer already needs some feature for the next release which is not in the plan yet, he can write a story card for this and replace another story card needing the same effort. If the programmers get the impression that the plan might lose touch with reality, the plan has to be estimated again. Therefore, in XP the customer is always a member of the team and has always to be available for developers’ questions. This is called ‘customer in place’, although physical presence is not needed all the time. For the creation of software several techniques are used: a metaphore is used for com- munication, for an intuitive understanding of the project’s aim and also for a common design. For an accounting software it could look for example like the following: “a vir- tual accountant collects and manages all accounting orders, evaluates them and makes nice charts”. The metaphor should support a simple design. In the case of our virtual ac- countant it could be like this: one package for managing transactions (the “accountant” himself), one for analysis and one for making charts. After some time it could prove use- ful to move the “accounts” from our accountant to a new package of its own – refactoring simplifies design even more. If this software would need interfaces to other accounting solutions, the introduction of a “translator” package could solve this. To do refactoring easily without accidentally destroying other features, continuous testing (with unit tests and continuous integration) is necessary. A very central aspect of XP is programming in pairs: at any time, two programmers do the programming together. One of them has the active part and writes code, the other one follows his line of thought, asks questions and tries if he (or she) can find a solution that is simpler. If they are not sure if a solution is good, they give it a try and maybe they also try another one until it is clear which solution is the simplest. The partner helps to identify problems earlier, which saves time and energy. Together, pair programmers might find solutions each of them alone wouldn’t. The partners can switch their roles anytime. For school all these aspects are advantages, from debugging to creative problem solv- ing and, of course, learning from each other. Werner et. al. 4.2. Workflow An XP-project’s workflow consist of two parts: during the planning game, the project plan is created or adapted together with the customers and so the software development is controlled. On the planning game follows the creation of the software. If requirements change we repeat the planning game for the changed requirements (also during a cre- ational phase) and the project plan is adapted. Afterwards, the creation can be continued without any problems. If a new version of the software should be developed, the planning game is done again. How is the extent of a version determined? According to XP, this can only be done with coarse communication with the customers. This happens in the so called planning game, a process consisting of three phases (see Fig. 1) which will be further explained in the following: in the exploration phase the customers create story cards describing the desired features. The developers estimate for every story card, how long it would take to implement the features. If this is not possible, they talk to the customers which will explain their ideas in a more detailed way or split the story card into several smaller ones. Fig. 1. Phases of the planning game. Fig. 2. A story card. Fig. 1. Phases of the planning game. Fig. 1. Phases of the planning game. Fig. 2. A story card. Fig. 2. A story card. Fig. 2. A story card. 155 Crafting, Crafting, Crafting – Extreme Programming in Classroom? In commitment phase all story cards are sorted into three stacks: first, by the customer by value (absolutely necessary, important and nice, but less important), and second, by the developers by risk. This is measured by how closely the necessary time can be calcu- lated (almost certain, closely and hardly to not at all). Afterwards, the developers tell the customer, how fast they can be in ideal development time per month. At last, the steering phase follows: the customers choose from the selected story cards those, which should be implemented in the next iteration (usually a period from one to three weeks). After the first iteration the system has to work, but doesn’t have to be fully developed. If the developers realize that they have overestimated their development speed, they can correct the schedule together with the customers and remove less important features from the plan. M. Bitzl Common responsibility is another instrument for quality assurance and part of a XP working culture. Every developer is responsible for the whole project. Regularly changing the partners of the programming pairs ensures that all programmers get knowledge of the whole project by and by. If one sees something which he (or she) could do simpler or nicer, he (or she) has to make this change (except somebody else is working on the same piece of software). Especially there are no “private” parts of the software which are off-limits for other programmers. Because of this and also because of design through refactoring, it is very common that parts of the software are completely replaced by others within a few weeks. The tests ensure that changes can be performed without any danger of breaking the software. For students, this is, of course, a chance to learn, but also to take responsibility. The rule “40-hour-week” demands a project plan which allows the programmers to fulfill it without making overtime regularly. This would reduce their manpower as well as their motivation and therefore harm the project. For school, this is very important as overtime is hardly possible while the schedule is very tight. As many programmers work on the same project, XP demands coding standards. These can help students to develop a clean structure for their code. 4.2. Workflow describe a pair-programming- session with middle school girls and introduce some rules for successful pair program- ming with students (Werner et al., 2004). A survey from McDowell and Werner (Mc- Dowell et al., 2003) examines the effects of pair programming in programming courses for beginners. The participants using pair programming showed better results than those who did not. 156 M. Bitzl 5. Problems and Solutions Several circumstances at school make the use of Extreme Programming difficult: the group size, the available amount of time, the experience the developers have, the customer in place and the immediate assignment of all developers. The group size has an important role in XP, because the integration process is a bottle neck. With estimated four minutes for each integration process, ten pairs (twenty pupils) would need 40 minutes to integrate their results. Smaller groups and projects with low interference between the developer pairs can speed this up. The elective solution has one crucial advantage: perhaps the group size one can expect is in acceptable limits, and if not, one could control these factors from the outset. Another critical factor is time: from the 45 minutes of a lesson, a certain amount of time is already lost for other activities (pupils have to come to the computer room, take their places and make pairs, there might be organizational work to do from the school, etc.). In the remaining time the pupils have to take a story card, extract their tasks, imple- ment their tests and solutions and finally integrate their solutions. The teacher as coach supports them when there are problems, but otherwise stay in the background. Here it becomes apparent that the working techniques cannot be introduced only for a class project, but have to be familiar to the students at least partly. For this, there are different approaches: in practical phases, the complexity can be easily controlled, for the used working techniques as well as for the tasks. In class projects, this is more difficult, but with an appropriate control of the planning game it is possible in certain limits. An- other solution is to split the class and give together with another teacher of this class two double periods at the same time. This way, one could get double time and half group size. To use this method, it has to be checked how to deal with the teachers’ overtime. 157 Crafting, Crafting, Crafting – Extreme Programming in Classroom? The pupils’ lack of experience could be faced with appropriate tasks and a stepwise introduction of the XP-techniques. Before pupils can work independent in a class project, they should have made first experiences with pair programming and writing software tests in practical phases in class. 5. Problems and Solutions In an elective, they could make first experiences with software tests together with the teacher and first experiences with pair-programming directly. For the introduction of pair programming, a version of pair programming proposed by Astra- chan et al. (Astrachan et al., 2003) would be appropriate. In this version, the teacher works as active and the whole class together as passive programmer in one programming pair. The class would take part in the solution with ideas, proposals, but also with questions. The customer in place could be replaced with students taking the customer’s role. Doing so, it is also possible to make pairs with an odd number of students and perhaps to slightly lower the number of developer pairs (probably by one or two pairs). It also gives a chance to assign certain, more appropriate tasks to some students (e.g., for motivational purposes). An immediate assignment of all “developers” is hard to avoid in school as we always have to deal with whole classes (or at least large parts of them if they are split), where all students have to be present. Obviously, each of them (or rather, each pair of them) needs an expedient task. One solution might be to give a small code basis to the students at the beginning of the project to avoid that they would disturb each other. Part of this code basis could be developed in practical phases of past lessons or through the teacher-class- pair-programming described above. It would also be possible to start with a part of the students programming, while others do some investigation work (or other not interfering tasks). At last, projects with low coupling between its parts are better. References Astrachan, O., Duvall, R.C. and Wallingford, E. (2003). Bringing extreme programming to the classroom. In M. Marchesi, G. Succi, D. Wells and L. Williams (Eds.), Extreme Programming Perspectives. Addison Wesley, Boston, MA, 237–250. Beck, K. (2000). Extreme Programming Explained. Embrace Change. Addison-Wesley, Reading, Mass. Brüggemann, A., Rohs, M. and Schwill, A. (2006). Extreme programming – extreme learning? Erfahrungen aus dem Grenzbereich zwischen Arbeiten und Lernen. In C. Schulte and M. Thomas (Eds.), Proceedings Didaktik der Informatik, 3. Workshop der GI-Fachgruppe “Didaktik der Informatik”. 19–20 Juni 2006. Kölle, Potsdam, 7–16. Cleland, S. and Mann, S. (2003). Agility in the classroom: using agile development methods to foster team work and adaptability amongst undergraduate programmers. In A. Williamson (Ed.), Proceedings of the 16th Annual NACCQ, 49–52. Fachprofil Informatik G8 (2004). http://www.isb-gym8-lehrplan.de/contentserv/3.1/g8. de/index.php?StoryID=26380 Jeffries, R. and Melnik, G. (2007). TDD: The art of fearless programming. IEEE Software, 24(3), 24–30. McDowell, C., Werner, L., Bullock, H.E. and Fernald, J. (2003). The impact of pair programming on student performance, perception and persistence. In Proceedings of the 25th International Conference on Software Engineering, 602–607. Schulartprofil Gymnasium: Das Gymnasium in Bayern (2004). http://www.isb-gym8-lehrplan.de/ contentserv/3.1/g8.de/index.php?StoryID=26350 Verfassung des Freistaates Bayern (1946). Verfassung des Freistaates Bayern (1946). http://www.verfassungen.de/de/by/bayern46-index.htm http://www.verfassungen.de/de/by/bayern46-index.htm http://www.verfassungen.de/de/by/bayern46-index.htm p g y y Werner, L., Denner, J. and Bean, S. (2004). Pair programming strategies for middle school girls. In Computers and Advanced Technology in Education, 301–305. Werner, L., Denner, J. and Bean, S. (2004). Pair programming strategies for middle school girls. In Computers and Advanced Technology in Education, 301–305. Werner, L., Denner, J. and Bean, S. (2004). Pair programming strategies for middle school girls. In Computers and Advanced Technology in Education, 301–305. M. Bitzl is a teacher student at the Technical University of Munich. He has studied com- puter science, physics and educational science and is now doing his first state examination for qualifying as a teacher at Bavarian Gymnasium. His main research interests include the implementation of independent learning and ontologies both for e-learning and se- mantic web. 6. Conclusion Examining the values, the principles, the basic working techniques up to the whole project flow, it is obvious that Extreme Programming offers many interesting aspects for class. XP emphasizes learning as an important part of the principles and practices in many points and therefore is very appropriate for use in schools. Above three basic scenarios have been presented and will now be summarized shortly. The use in practical phases in a lesson takes the least time and gives the teacher the strongest control. Here, the students can learn the practices together with their teacher. With design through refactoring, students can learn the advantages of certain designs, and programming in pairs supports the students in finding good solutions. The creation of tests supports them in recognizing their mistakes. For the use in a class project, one needs more time and the students have to know the working techniques. The teacher’s control is much smaller and in many cases limited to support at problems. Software tests ensure always a working version of the program. Small iterations allow a fine risk-control and a smooth change of the project plan, so one can ensure that the students will have working software. An XP-elective offers several other opportunities: because it has longer period than a class project and smaller group sizes, the techniques can be learned more intensively and also can be applied much better. This allows larger projects. 158 M. Bitzl No matter in which form Extreme Programming is used – the student’s activities will always be the most important aspect. Acknowledgments. I would like to thank Daniela Drexler and Elisabeth Rothe for their valuable comments on a manuscript of this paper, and also Prof. Dr. Peter Hubwieser for guiding me to research. Marcus BITZL Ekstreminis (kraštutinis) programavimas (angl u k. santrumpa XP) turi idomi u savybi u, ypaˇc tinkam u mokymui. Šis programavimas iš esm˙es susij es su programin˙es irangos, k¯urimo darbais. Straipsnyje aptariami b¯udai, kaip taikyti ekstremini programavim a mokant klas˙eje, ypaˇc atliekant klas˙es projektus, taˇciau tuo neapsiribojama – parodoma, kaip panaudoti ir kitose pamokose, kur tik programavimas gal˙et u b¯uti naudingas. Aptariamos kelios svarbiausios problemos, kurios gali iškilti naudojant ekstremini programavim a, taip pat pateikiami ši u problem u sprendimo b¯udai.
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Hepatitis B virus reactivation associated with CAR T-cell therapy
Holistic integrative oncology
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REVIEW Open Access Abstract Patients with hematological malignancies who also have a hepatitis B virus (HBV) infection need to be aware of the potential risk of HBV reactivation when undergoing anti-cancer treatments. Among these treatments, CAR T-cell therapy has gained significant attention as a promising option, but it also raises concerns regarding HBV reac- tivation. This review aims to provide an overview of published reports on HBV reactivation during CAR T-cell therapy, along with an assessment of the effectiveness of prophylactic antiviral therapy. Additionally, we propose a systematic approach for monitoring and managing HBV reactivation during CAR T-cell therapy to enhance the safety of this treat- ment for patients with HBV infection. Keywords  Hepatitis B virus, Reactivation, CAR T-cell therapy, Hematological malignancies Haolong Lin1,2, Zigang Dai1,2, Liang Huang1,2 and Xiaoxi Zhou1,2* Haolong Lin1,2, Zigang Dai1,2, Liang Huang1,2 and Xiaoxi Zhou1,2* Holistic Integrative Oncology Holistic Integrative Oncology Lin et al. Holistic Integrative Oncology (2024) 3:16 https://doi.org/10.1007/s44178-024-00082-z 1  Background to proactively address the potential challenge of HBV reactivation and its associated, potentially life-threaten- ing complications. This endeavor is grounded in the lat- est advancements in research and clinical experience in this domain. In recent times, a body of research has emerged indicat- ing the potential occurrence of hepatitis B virus (HBV) reactivation during chimeric antigen receptor (CAR) T-cell therapy, which, fortunately, can be effectively miti- gated through the administration of nucleotide analogs (NAs). It is noteworthy that China is situated within regions of high HBV endemicity, and individuals afflicted with HBV infection are at an increased risk of developing non-Hodgkin lymphoma (NHL) and diffuse large B-cell lymphoma (DLBCL). 1.1  HBV infection and B‑cell malignancies Hepatitis B virus (HBV) is a global health concern, with a pervasive presence across the world. Approximately 257 million individuals are currently affected by HBV on a global scale, with a staggering 68% of these cases con- centrated in the regions of Africa and the West Pacific [2]. In China, HBV presents a significant public health challenge, contributing to approximately one-third of the total global burden of HBV infections. Despite wide- spread vaccination efforts, the national prevalence of hepatitis B surface antigen (HBsAg) has declined to 5.2%. However, HBV continues to impact a substantial popu- lation, affecting roughly 70 million individuals annually, with 20–30 million cases evolving into chronic infections and tragically resulting in more than 880,000 deaths each year [3, 4]. Studies have shown that chronic HBV infec- tion significantly increases the risk of NHL and DLBCL [5]. DLBCL patients with concomitant HBV infec- tion have distinct clinical, genomic, and transcriptomic Drawing upon insights from prior clinical investiga- tions and expert consensus, our objective is to provide an actionable framework for healthcare professionals, ena- bling them to judiciously administer CAR T-cell therapy to patients grappling with hematological malignancies concurrent with HBV infection [1]. Our ultimate aim is *Correspondence: Xiaoxi Zhou cello316@163.com 1 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China 2 Immunotherapy Research Center for Hematologic Diseases of Hubei Province, Wuhan 430030, Hubei, China *Correspondence: Xiaoxi Zhou cello316@163.com 1 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China 2 Immunotherapy Research Center for Hematologic Diseases of Hubei Province, Wuhan 430030, Hubei, China *Correspondence: Xiaoxi Zhou cello316@163.com 1 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China 2 Immunotherapy Research Center for Hematologic Diseases of Hubei Province, Wuhan 430030, Hubei, China *Correspondence: Xiaoxi Zhou cello316@163.com 1 Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China 2 Immunotherapy Research Center for Hematologic Diseases of Hubei Province, Wuhan 430030, Hubei, China Lin et al. Holistic Integrative Oncology (2024) 3:16 Page 2 of 7 Lin et al. Holistic Integrative Oncology patients with chronic HBV infection are at high risk of HBV reactivation when they receive CAR T-cell therapy targeting B cells or plasma cells. characteristics, indicating that HBV infection plays a contributory role in the development of B-cell malignan- cies [6]. In China, 9%-30.2% of NHL patients and approx- imately 25% of DLBCL patients harbor chronic HBV infections, and about 20%-44% of DLBCL patients have resolved HBV infections [7, 8]. Given the prevalence of HBV infection among patients with NHL or DLBCL, it is essential to recognize that the risk of HBV reactivation significantly escalates following exposure to immunosup- pressive or cytotoxic agents [9].h While in the patients who had resolved HBV infec- tion, no cases of HBV reactivation were reported during NAs administration, even in case of HBV DNA-positive. However, 2 independent studies each reported 1 patient (3.4–12.5%), who were HBV DNA-negative and did not receive prophylactic NAs, experienced asymptomatic HBV reactivation at 2 months and 6 months after CAR T-cell infusion, respectively [16, 21]. HBV reactivation in these 2 patients presented as an increase in HBV DNA copy number or a reverse seroconversion from HBsAg negativity to positivity and was promptly controlled by the preemptive use of NAs. These data demonstrate that patients with resolved HBV infection might be also at high risk of HBV reactivation after CAR T-cell infusion, and the administration of NAs can prevent HBV reac- tivation in these patients, regardless of the HBV DNA status. The risk of HBV reactivation varies for each patient, depending on their HBV serological status as well as the specific anti-tumor treatment they receive. Because of the persistent B-cell aplasia induced by chemoimmuno- therapy, the incidence of HBV reactivation ranges from 21 to 60% in patients with chronic HBV infection and varies from 2 to 25% in patients with resolved HBV infec- tions [10, 11]. The risk of reactivation can be considered as high risk if the incidence of HBV reactivation reaches or exceeds 10%. Therefore, patients with either chronic or resolved HBV infection are at high risk of HBV reactiva- tion when receiving hematopoietic stem cell transplan- tation or immunochemotherapy (including rituximab and glucocorticoid chemotherapy) [12, 13]. However, randomized controlled clinical studies have shown that the prophylactic use of NAs can reduce the risk of HBV reactivation in this patient cohort. Similarly, since CAR T cells targeting B cells or plasma cells can lead to long- lasting B-cell depletion and humoral immunodeficiency, patients with contaminant HBV infection might also face a high risk of HBV reactivation when undergoing CAR T-cell therapy. Furthermore, HBV can be activated in these high-risk patients after improper withdrawal of prophylactic NAs. Four patients, involving 2 with chronic HBV infection and 2 with resolved HBV infection, experienced HBV reactivation after self-discontinuation of ETV [15, 17, 19, 26]. At the time of HBV reactivation, B-cell depletion or complete remission of disease was still sustained. One patient with chronic HBV infection self-discontinued the use of ETV only 1 month after CAR T-cell infusion, leading to a tragic outcome as the patient succumbed to fulminant hepatic failure just two months later [26]. This mournful incident underscores the critical importance prophylactic NAs are essential for high-risk patients. Similarly, another patient with resolved HBV infection ceased antiviral treatment five months after the CAR T-cell infusion, resulting in HBV reactivation just one month later. Despite prompt intervention with entecavir and tenofovir, the patient eventually succumbed to com- plications of hepatitis and cerebral hemorrhage [19]. The remaining two patients had been on ETV prophylaxis for more than 1 year before the discontinuation of ETV. They presented asymptomatic elevation of HBV DNA titer or hepatitis flare. After prompt recognition of these signs and the subsequent resumption of ETV treatments, HBV reactivation was successfully resolved in both patients [15, 17]. These outcomes provide compelling evidence that prophylactic NAs therapy may serve as a protective measure against HBV reactivation in patients undergo- ing CAR T-cell therapy, whether they have chronic HBV infection or resolved HBV infection. 1.2  HBV reactivation in the setting of CAR T‑cell therapy Recently, HBV reactivation has been described in patients with chronic HBV infection or resolved HBV infection after anti-CD19, CD20, CD22, or B-cell matu- ration antigen (BCMA) CAR T-cell therapy (Table 1). In a study including 19 patients with B-cell malignancies and concomitant chronic HBV infection, HBV reactiva- tion and HBV-related severe hepatitis were detected in 1 (5.3%) patient at 4 months after anti-CD19 CAR T-cell infusion, although they had received entecavir (ETV) [14]. Holistic Integrative Oncology Page 3 of 7 Table 1  Summary of HBV reactivation cases in CAR T-cell therapy Study Disease Targets of CAR T-cell therapy HBV characteristic at baseline Number reactivat total Strati P 2019 [17] R/R-DLBCL CD19 Resolved HBV infec- tion 1/3 Yang C 2020 [15] R/R-DLBCL CD19 Chronic HBV infection 3/15 Lai P 2020 [18] R/R B-ALL CD19 Chronic HBV infection 0/3 Cui R 2020 [19] 7 R/R B-ALL and 13 R/R DLBCL CD19 Chronic HBV infection 0/5 Liu W 2020 [20] R/R B-cell lymphoma CD19 Chronic HBV infection 0/6 Resolved HBV infec- tion 0/11 Han L 2020 [21] R/R MM BCMA Chronic HBV infection 0/1 Resolved HBV infec- tion 1/8 Wang Y 2020 [16] advanced B-cell cancers CD19, CD20 or BCMA Chronic HBV infection 2/12 Resolved HBV infec- tion 1/29 Cao W 2020 [14] R/R B-ALL or DLBCL CD19 and CD22 CAR T cocktail Chronic HBV infection 1/19 Resolved HBV infec- tion 0/37 Ma Y 2021 [22] One stomach lymph- oblastic lymphoma and one R/R DLBCL CD19 and CD22 CAR T cocktail Chronic HBV infection 2/2 Li P 2021 [23] R/R B-cell malignan- cies CD19 Resolved HBV infec- tion 2/30 Kong D 2023 [24] R/R DLBCL CD19, CD19 com- bined with CD20 or CD22, tandem CD19/CD22 Chronic HBV infection 1/6 Resolved HBV infec- tion 1/25 Fu S 2023 [25] R/R MM BCMA Chronic HBV infection 1/7 Resolved HBV infec- tion 3/43 Table 1  Summary of HBV reactivation cases in CAR T-cell therapy Study Disease Targets of CAR T-cell therapy HBV characteristic at baseline Numbers of reactivation/ total Strati P 2019 [17] R/R-DLBCL CD19 Resolved HBV infec- tion 1/3 Yang C 2020 [15] R/R-DLBCL CD19 Chronic HBV infection 3/15 Lai P 2020 [18] R/R B-ALL CD19 Chronic HBV infection 0/3 Cui R 2020 [19] 7 R/R B-ALL and 13 R/R DLBCL CD19 Chronic HBV infection 0/5 Liu W 2020 [20] R/R B-cell lymphoma CD19 Chronic HBV infection 0/6 Resolved HBV infec- tion 0/11 Han L 2020 [21] R/R MM BCMA Chronic HBV infection 0/1 Resolved HBV infec- tion 1/8 Wang Y 2020 [16] advanced B-cell cancers CD19, CD20 or BCMA Chronic HBV infection 2/12 Resolved HBV infec- tion 1/29 Cao W 2020 [14] R/R B-ALL or DLBCL CD19 and CD22 CAR T cocktail Chronic HBV infection 1/19 Resolved HBV infec- tion 0/37 Ma Y 2021 [22] One stomach lymph- oblastic lymphoma and one R/R DLBCL CD19 and CD22 CAR T cocktail Chronic HBV infection 2/2 Li P 2021 [23] R/R B-cell malignan- cies CD19 Resolved HBV infec- tion 2/30 Kong D 2023 [24] R/R DLBCL CD19, CD19 com- bined with CD20 or CD22, tandem CD19/CD22 Chronic HBV infection 1/6 Resolved HBV infec- tion 1/25 Fu S 2023 [25] R/R MM BCMA Chronic HBV infection 1/7 Resolved HBV infec- tion 3/43 Table 1  Summary of HBV reactivation cases in CAR T-cell therapy Table 1  Summary of HBV reactivation cases in CAR T-cell therapy R/R Relapsed/refractory HBV Hepatitis B virus CAR T Chimeric antigen receptor T cell DLBCL Diffuse large B cell lymphoma B ALL B cell acute lymphoblastic leukemia Study Disease Targets of CAR T-cell therapy HBV characteristic at baseline Numbers of reactivation/ total Prophylactic antiviral therapy Clinical manifestations and outcomes Strati P 2019 [17] R/R-DLBCL CD19 Resolved HBV infec- tion 1/3 All received entecavir Successfully treated with entecavir Yang C 2020 [15] R/R-DLBCL CD19 Chronic HBV infection 3/15 All received entecavir or Lamivudine Asymptomatic HBV reactivation Lai P 2020 [18] R/R B-ALL CD19 Chronic HBV infection 0/3 All received entecavir or tenofovir No HBV reactivation Cui R 2020 [19] 7 R/R B-ALL and 13 R/R DLBCL CD19 Chronic HBV infection 0/5 All received pro- phylactic entecavir or tenofovir No HBV reactivation Liu W 2020 [20] R/R B-cell lymphoma CD19 Chronic HBV infection 0/6 All 6 patients were on prophylactic entecavir No HBV reactivation Resolved HBV infec- tion 0/11 Only 2 of the 11 were on prophylactic ente- cavir or lamivudine No HBV reactivation Han L 2020 [21] R/R MM BCMA Chronic HBV infection 0/1 All received entecavir or lamivudine No HBV reactivation Resolved HBV infec- tion 1/8 Only 2 of the 8 were on prophylactic ente- cavir or lamivudine One asymptomatic HBV reactivation in no pro- phylactic group Wang Y 2020 [16] advanced B-cell cancers CD19, CD20 or BCMA Chronic HBV infection 2/12 All received entecavir, tenofovir disoproxil, or lamivudine Asymptomatic HBV reactivation Resolved HBV infec- tion 1/29 No Asymptomatic HBV reactivation Cao W 2020 [14] R/R B-ALL or DLBCL CD19 and CD22 CAR T cocktail Chronic HBV infection 1/19 All received prophy- lactic entecavir Severe hepatitis and died of septic shock Resolved HBV infec- tion 0/37 Only 2 of the 37 received prophylactic entecavir No HBV reactivation Ma Y 2021 [22] One stomach lymph- oblastic lymphoma and one R/R DLBCL CD19 and CD22 CAR T cocktail Chronic HBV infection 2/2 All received prophy- lactic entecavir Both successfully treated with entecavir and tenofovir Li P 2021 [23] R/R B-cell malignan- cies CD19 Resolved HBV infec- tion 2/30 No antiviral prophy- laxis One asymptomatic HBV reactivation and one severe hepa- titis, both successfully treated with entecavir Kong D 2023 [24] R/R DLBCL CD19, CD19 com- bined with CD20 or CD22, tandem CD19/CD22 Chronic HBV infection 1/6 All received pro- phylactic entecavir or adefovir combined with entecavir Hepatic malignant tumor Resolved HBV infec- tion 1/25 19 received prophy- lactic entecavir One asymptomatic HBV reactivation in no pro- phylactic group Fu S 2023 [25] R/R MM BCMA Chronic HBV infection 1/7 6 patients received entecavir and 1 received tenofovir One developed to liver failure. R/R Relapsed/refractory, HBV Hepatitis B virus, CAR T Chimeric antigen receptor T-cell, DLBCL Diffuse large B-cell lymphoma, B-ALL B-cell acute lymphoblastic leukemia, MM Multiple myeloma, BCMA B cell maturation antigen Similarly, in another 2 studies which included 15 and 12 patients with B-cell or plasma-cell malignancies and concomitant chronic HBV infection, respectively, 3 (20%) and 2 (16.7%) patients experienced HBV reactiva- tion [15, 16]. Even with the continuous administration of NAs, HBV reactivation in these patients occurred mainly within 6  months after CAR T-cell infusion and mani- fested asymptomatically as an increase in HBV DNA copy number while without hepatitis flare. Because of the high incidence of HBV reactivation in these studies, It remains uncharted territory to fully understand the impact of CAR T-cell therapy on HBV reactivation in T-cell malignancies. A noteworthy case involves a patient Lin et al. Holistic Integrative Oncology (2024) 3:16 Lin et al. then died, Resolved HBV infec- tion 3/43 No One hepatitis flare and successfully treated with entecavir, and two asymptomatic HBV reactivation Kong D 2023 [24] R/R DLBCL CD19, CD19 com- bined with CD20 or CD22, tandem CD19/CD22 Chronic HBV infection 1/6 Resolved HBV infec- tion 1/25 Fu S 2023 [25] R/R MM BCMA Chronic HBV infection 1/7 Resolved HBV infec- tion 3/43 Lin et al. Holistic Integrative Oncology (2024) 3:16 Page 4 of 7 resolved HBV infection who test positive for HBV DNA should receive prophylactic NAs therapy before initiat- ing CAR T-cell therapy. It’s essential to recognize that even HBV DNA-negative patients with resolved HBV infection must adopt a preemptive treatment approach, as lethal HBV reactivation can still occur following CAR T-cell infusion [19]. In patients with resolved HBV infection, baseline HBsAb seronegativity may serve as a potential risk factor for HBV reactivation, particularly in the absence of antiviral prophylaxis [23]. Addition- ally, an HBsAb titer of 56.48  IU/ml has been identified as a critical threshold for predicting HBV reactivation, a finding validated in both rituximab-containing chemo- therapy and CAR T-cell therapy [20, 28]. HBcAb repre- sents another important biomarker, with a cutoff value of 6.41 IU/ml serving as a cautionary indicator of poten- tial HBV recurrence [29]. Furthermore, the presence of HBeAg positivity is considered a risk factor for reactiva- tion and is associated with elevated viral loads and infec- tivity levels [15]. with T-cell acute lymphoblastic leukemia (T-ALL) who exhibited high HBV DNA levels. This patient underwent treatment with donor-derived anti-CD7 CAR T-cells, fol- lowed by allogeneic hematopoietic stem cell transplanta- tion while receiving continuous antiviral therapy. There was a transient surge in HBV DNA levels observed two weeks following CAR T-cell infusion. However, these levels quickly diminished to a safe range. Impressively, by the 10-month mark following CAR T-cell reinfusion (which was 8 months after transplantation), the patient achieved complete remission with undetectable HBV DNA levels [27]. This case underscores the significance of not overlooking the possibility of HBV reactivation in the context of CAR T-cell therapy for different malignancies beyond B-cells or plasma cells. In addition, although higher serum interleukin-6 had been observed in patients with chronic HBV infection when receiving CAR T-cell therapy, there is no clear evi- dence that HBV infection affects the safety or efficacy of CAR T-cell therapy [17]. 2.2  Prophylactic NA therapyh y y The aforementioned populations with a high risk of HBV reactivation should receive highly potent and low-resist- ance prophylactic NAs before undergoing CAR T-cell therapy. These NAs include entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide fumarate tablets (TAF). Commencement of NA treat- ment typically initiates one week before, or at the latest, concurrently with prechemotherapy procedures. Cur- rently, most guidelines recommend that patients with hematologic malignancies should receive prophylactic NAs for 12–18  months after immunochemotherapy. Because the sustained duration of B lymphocyte deple- tion is not consistent between different individuals after receiving a CAR T-cell infusion, the course of prophy- lactic NAs should be determined by the duration of B lymphocyte depletion in patients [30]. We recommend monitoring the levels of peripheral blood lymphocyte subpopulations and serum immunoglobulins (IgG, IgM, and IgA) in patients with prophylactic NAs. Prophylactic NAs should be given at least 12–18 months after the lev- els of peripheral blood B lymphocytes and serum immu- noglobulins are restored. fi Taken together, these findings collectively underscore the indispensable and efficacious role of NAs prophylaxis in averting HBV reactivation among patients with con- comitant chronic HBV infection or resolved HBV infec- tion when undergoing CAR T-cell therapy. Importantly, it appears that the presence of chronic or resolved HBV infection does not impair the safety or efficacy of CAR T-cell therapy. Given the retrospective nature and limited scale of these studies, validation in multicenter prospec- tive trials is warranted. Both animal studies and clinical studies have shown that HBV infection did not increase the incidence or severity of hepatotoxicity, cytokine release syndrome (CRS), or neurotoxicity during CAR T-cell therapy and did not compromised the activity and clinical efficacy of CAR T-cells [14, 18, 24, 25].i 2  Prevention and Treatments of HBV reactivation during CAR T‑cell therapy 2.1  Identification of high‑risk populations HBV serological markers including HBsAg, anti-hep- atitis B surface antigen (HBsAb) and anti-hepatitis B core antigen (HBcAb), HBV DNA, and liver function indexes (alanine aminotransferase, aspartate aminotrans- ferase, bilirubin, albumin) should be routinely assessed in patients with hematologic malignancies who receive CAR T-cell therapy. To effectively mitigate the risk of HBV reactivation in these patients, a comprehensive evaluation of risk factors is crucial, guiding the adop- tion of appropriate prevention and treatment strategies (Fig. 1). Notably, both HBsAg positivity and HBV DNA positivity represent virological risk factors for HBV reac- tivation. Patients with either chronic HBV infection or 2.3  Monitoring and treatment of HBV reactivation While undergoing NAs treatment, it is crucial to main- tain regular monitoring of HBV DNA levels, HBV sero- logical markers, and liver function. Monitoring intervals should occur at least every one to three months, ensur- ing early intervention when necessary. HBV reactivation often occurs within 6 months after CAR T-cell infusion, Page 5 of 7 Lin et al. Holistic Integrative Oncology (2024) 3:16 Lin et al. Holistic Integrative Oncology Fig. 1  Prevention and management strategies of HBV reactivation in CAR T-cell therapy. The necessity for prophylactic Nucleotide Analogue (NAs) treatment hinges upon the baseline HBV status of patients prior to CAR T-cell reinfusion. Patients with chronic HBV infection (HBsAg +) or resolved HBV infection (HBsAg- HBcAb +) who exhibit high-risk characteristics (such as a low HBsAb titer or the presence of positive HBeAg) should continue prophylactic NAs until 12–18 months after the restoration of peripheral blood B lymphocytes and serum immunoglobulin levels. HBV status should be monitored regularly until more than one year after NAs withdrawal, with monthly HBV monitoring in the first 6 months and once every 3 months thereafter. In the event of HBV recurrence, immediate initiation of antiviral therapy with NAs that do not share cross-resistance is paramount. CAR T-cell: chimeric antigen receptor T-cell; NAs: nucleotide analogs. HBsAg: hepatitis B surface antigen; HBsAb: anti-hepatitis B surface antigen; HBcAb: anti-hepatitis B core antigen; HBeAg: hepatitis B e antigen; HBV: hepatitis B virus Fig. 1  Prevention and management strategies of HBV reactivation in CAR T-cell therapy. The necessity for prophylactic Nucleotide Analogue (NAs) treatment hinges upon the baseline HBV status of patients prior to CAR T-cell reinfusion. Patients with chronic HBV infection (HBsAg +) or resolved HBV infection (HBsAg- HBcAb +) who exhibit high-risk characteristics (such as a low HBsAb titer or the presence of positive HBeAg) should continue prophylactic NAs until 12–18 months after the restoration of peripheral blood B lymphocytes and serum immunoglobulin levels. HBV status should be monitored regularly until more than one year after NAs withdrawal, with monthly HBV monitoring in the first 6 months and once every 3 months thereafter. In the event of HBV recurrence, immediate initiation of antiviral therapy with NAs that do not share cross-resistance is paramount. CAR T-cell: chimeric antigen receptor T-cell; NAs: nucleotide analogs. 2.3  Monitoring and treatment of HBV reactivation HBsAg: hepatitis B surface antigen; HBsAb: anti-hepatitis B surface antigen; HBcAb: anti-hepatitis B core antigen; HBeAg: hepatitis B e antigen; HBV: hepatitis B virus viral resistance mutations should be considered, particu- larly in patients with lamivudine resistance who are more susceptible to entecavir resistance mutations [32, 33]. Alongside drug-resistance gene testing, adjustments to antiviral treatment regimens should be made, including switching to or adding NAs drugs with no cross-resist- ance, such as ETV, TDF and TAF. However, potential adverse reactions associated with NAs should be moni- tored. Multidisciplinary therapy may be initiated if nec- essary. Additionally, based on the patient’s liver function, hepatoprotective drugs and supportive treatments should be administered accordingly. during which the above indicators should be monitored monthly and once every three months thereafter. Because HBV reactivation can still recur after discontinuation of NAs, the above indicators should continue to be moni- tored for 12 months after NA withdrawal, at a frequency of once every one to three months, especially within six months. For patients with resolved HBV infection who receive preemptive treatment, a similar monitor- ing regimen is advised [18, 31]. This vigilant monitoring approach is essential for maintaining patient safety and addressing any potential HBV reactivation promptly. If HBV reactivation is suspected, prompt assessment and treatment protocols should be initiated to prevent the occurrence of fatal acute liver failure. For patients who have not received prophylactic NAs, immediate initiation of NAs therapy, even before confirmation of HBV reactivation, is essential to prevent further increase in HBV DNA copies. In cases where HBV reactivation occurs despite prophylactic NAs use, the possibility of Funding 17. Strati P, Nastoupil LJ, Fayad LE, Samaniego F, Adkins S, Neelapu SS. Safety of CAR T-cell therapy in patients with B-cell lymphoma and chronic hepatitis B or C virus infection. Blood. 2019;133(26):2800–2. 18. Lai P, Chen X, Qin L, et al. The efficacy and safety of CAR-T cell therapy in patients with refractory ALL and concomitant HBV infection. Leukemia. 2020;34(10):2790–3. 3  Conclusion CAR T-cell therapy represents a groundbreaking advancement in the treatment of hematological malig- nancies and is gaining increasing traction. However, it is essential to conduct meticulous evaluations of the safety and efficacy of CAR T-cell therapy, especially in specific Lin et al. Holistic Integrative Oncology (2024) 3:16 Page 6 of 7 patient subgroups. A substantial portion of patients with hematologic malignancies presents concurrent chronic or resolved HBV infection. Consequently, when administering CAR T-cell therapy to such individu- als, a heightened awareness of the risk of HBV reactiva- tion is paramount. The continuous prophylactic use of NAs has demonstrated its effectiveness in curbing HBV recurrence. Equally important is the early identifica- tion of high-risk populations and the implementation of long-term, regular monitoring of HBV DNA levels. In addition, based on several independent clinical stud- ies, we concluded that the infection status of HBV does not compromise the safety and efficacy of CAR T-cell therapy. Therefore, CAR T-cell therapy for patients with hematological malignancies and concomitant HBV infec- tion holds significant promise. References 1. 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References Abbreviations B-ALL B-cell acute lymphoblastic leukemia BCMA B cell maturation antigen CAR​ Chimeric antigen receptor CRS Cytokine release syndrome DLBCL Diffuse large B-cell lymphoma ETV Entecavir HBcAb Anti-hepatitis B core antigen HBeAg Hepatitis B e antigen HBsAb Anti-hepatitis B surface antigen HBsAg Hepatitis B surface antigen HBV Hepatitis B virus MM Multiple myeloma NAs Nucleotide analogs NHL Non-Hodgkin lymphoma R/R Relapsed/refractory T-ALL T-cell acute lymphoblastic leukemia Acknowledgements Not applicable Authors’ contributions HL and ZD searched the literatures and wrote this manuscript. LH and XZ revised the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 82170167 to Xiaoxi Zhou). Availability of data and materials Not applicable. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. 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Did an ancient chlamydial endosymbiosis facilitate the establishment of primary plastids?
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Did an ancient chlamydial endosymbiosis facilitate the establishment of primary plastids? Jinling Huang*†‡ and Johann Peter Gogarten‡ Addresses: *Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858, USA. †NASA Astrobiology Institute at Marine Biological Laboratory, Woods Hole, MA 02543, USA. ‡Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Road, Storrs, CT 06269-3125, USA. Correspondence: Jinling Huang. Email: huangj@ecu.edu Received: 30 November 2006 Revised: 6 March 2007 Accepted: 4 June 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/6/R99 © 2007 Huang and Gogarten; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Primary plastid evolution <p>Phylogenomic analyses of the red alga <it>Cyanidioschyzon merolae </it>shows that at least 21 genes were transferred between chlamydiae and primary photosynthetic eukaryotes suggesting an ancient chlamydial endosymbiosis with the ancestral primary photosyn- thetic eukaryote </p> Abstract Background: Ancient endosymbioses are responsible for the origins of mitochondria and plastids, and they contribute to the divergence of several major eukaryotic groups. Although chlamydiae, a group of obligate intracellular bacteria, are not found in plants, an unexpected number of chlamydial genes are most similar to plant homologs, which, interestingly, often contain a plastid-targeting signal. This observation has prompted several hypotheses, including gene transfer between chlamydiae and plant-related groups and an ancestral relationship between chlamydiae and cyanobacteria. Results: We conducted phylogenomic analyses of the red alga Cyanidioschyzon merolae to identify genes specifically related to chlamydial homologs. We show that at least 21 genes were transferred between chlamydiae and primary photosynthetic eukaryotes, with the donor most similar to the environmental Protochlamydia. Such an unusually high number of transferred genes suggests an ancient chlamydial endosymbiosis with the ancestral primary photosynthetic eukaryote. We hypothesize that three organisms were involved in establishing the primary photosynthetic lineage: the eukaryotic host cell, the cyanobacterial endosymbiont that provided photosynthetic capability, and a chlamydial endosymbiont or parasite that facilitated the establishment of the cyanobacterial endosymbiont. Conclusion: Our findings provide a glimpse into the complex interactions that were necessary to establish the primary endosymbiotic relationship between plastid and host cytoplasms, and thereby explain the rarity with which long-term successful endosymbiotic relationships between heterotrophs and photoautotrophs were established. Our data also provide strong and independent support for a common origin of all primary photosynthetic eukaryotes and of the plastids they harbor. dria and plastids are derived respectively from an α-proteo- bacterial and a cyanobacterial endosymbiont in early eukaryotes [1]. It also has been suggested that the nucleus, a Open Access Open Access Open Access Results and discussion Chlamydiae are a group of obligate intracellular bacteria of uncertain evolutionary position [13-15]. Many chlamydiae, including Chlamydophila pneumoniae and Chlamydia tra- chomatis, are important pathogens in humans and other ani- mals [16] whereas others such as Protochlamydia, Neochlamydia, and Fritschea are endosymbionts in environ- mental amoebae and insects [17,18]. Although the available evidence suggests increasing chlamydial diversity in free-liv- ing amoebae and in the environment [19], thus far no chlamy- dial species has been reported in photosynthetic eukaryotes or plastid-containing lineages. However, chlamydial genome analyses revealed an unexpected number of genes that are most similar to plant homologs [20,21], which, interestingly, often contain a plastid-targeting signal [13]. This observation has prompted several hypotheses, notably an ancestral evolu- tionary relationship between cyanobacteria (plastids) and chlamydiae [13] and gene transfer between the two groups with the donor being either chlamydiae [22,23] or plant- related groups [21,24,25]. Additionally, it has also been sug- gested that plants might have acquired these genes from mitochondria [26] or through intermediate vectors such as insects [17]. y g The nuclear genome of Cyanidioschyzon contains 4,771 pre- dicted protein-coding genes [32]. Phylogenomic screen and subsequent phylogenetic analyses identified 16 probable chlamydiae-related genes in Cyanidioschyzon, 14 of which were also found in green plants. Five other previously reported genes [13,23] from green plants were also classified as chlamydiae-related after careful re-analyses. The genome sequences of glaucophytes are currently not publicly availa- ble, but the gene encoding ATP/ADP translocase is reportedly present in the glaucophyte Cyanophora paradoxa and the diatom Odontella sinensis [25]. In our search of the Taxo- nomically Broad EST Database (TBestDB) [33], ATP/ADP translocase homologs were also found in another glaucophyte (Glaucocystis nostochinearum), euglenids (Astasia longa and Euglena gracilis), and a haptophyte (Pavlova lutheri). This suggests that chlamydiae-related genes are present in all primary photosynthetic eukaryotic lineages and that the ADP/ATP translocase has been retained in at least some sec- ondary photosynthetic groups (eukaryotic lineages that emerged by engulfing another algal cell as endosymbiont). Therefore, a total of 21 genes from primary photosynthetic eukaryotes are listed here as chlamydiae-related (Table 1). Sequences that are not exclusively related to chlamydial homologs and those that form a monophyletic group with chlamydial homologs but with insufficient bootstrap support (<80%) are not included. These very stringent criteria excluded a large portion (18/37) of previously reported chlamydiae-related plant sequences [13,23]. Background Background Ancient symbioses are responsible for some of the major eukaryotic innovations. It is widely accepted that mitochon- Genome Biology 2007, 8:R99 Genome Biology 2007, 8:R99 R99.2 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.2 Genome Biology 2007, Volume 8, Issue 6, Article R9 http://genomebiology.com/2007/8/6/R99 hallmark of eukaryotic cells, either arose directly from or was mediated by an ancient symbiosis between archaeal and bac- terial partners [2-7]. Additionally, secondary and tertiary symbioses through engulfment of a plastid-containing cell played an important role in the evolution of several major eukaryotic lineages, including heterokonts, apicomplexans, dinoflagellates, euglenids, and others [8-12]. Undoubtedly, the evolution of extant eukaryotes was significantly shaped by past symbioses. chlamydial homologs. Our data suggest a likely ancient sym- biosis (sensu deBary; including mutualism, commensalisms, and parasitism) [31] between a chlamydial endosymbiont and the ancestor of primary photosynthetic eukaryotes. The ancient chlamydial endosymbiont contributed genes to the nuclear genome of primary photosynthetic eukaryotes and might have facilitated the early establishment of plastids. Table 1 Table 1 Chlamydiae-like genes detected in red algae and green plants Gene name or gene product Presence Putative function Phosphoglycerate mutase G Glycolysis CMP-KDO synthetase G Cell envelope formation 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE) R and G Isoprenoid biosynthesis Polynucleotide phosphorylase R and G RNA degradation Aspartate transaminase R and G Amino acid metabolism Tyrosyl-tRNA synthetase R and G Translation Oligoendopeptidase F R Amino acid biosynthesis 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ispD) R and G Isoprenoid biosynthesis Enoyl-ACP reductase (fabI) R and G Fatty acid biosynthesis 23S rRNA (Uracil-5-)-methyltransferase R and G RNA modification Glycerol-3-phosphate acyltransferase R and G Phospholipid biosynthesis ADT/ATP translocase R and G ATP/ADP transport Isoamylase R and G Starch biosynthesis Phosphate transporter G Phosphate transport Hypothetical protein R Unknown β-Ketoacyl-ACP synthase (fabF) R and G Fatty acid biosynthesis Malate dehydrogenase G Energy conversion Sodium:hydrogen antiporter R and G Ion transport 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (gcpE) (GcpE) R and G Isoprenoid biosynthesis Sugar phosphate isomerase G Sugar interconversion Cu-ATPase R and G Ion transport The genome sequences of glaucophytes are not available for comparison, except for the ATP/ADP translocase, which is reportedly present in the glaucophyte Cyanophora paradoxa [25] and was also identified in another glaucophyte Glaucocystis from the Taxonomically Broad EST Database (TBestDB). G, green plants; R, red algae. The genome sequences of glaucophytes are not available for comparison, except for the ATP/ADP translocase, which is reportedly present in the glaucophyte Cyanophora paradoxa [25] and was also identified in another glaucophyte Glaucocystis from the Taxonomically Broad EST Database (TBestDB). G, green plants; R, red algae. The genome sequences of glaucophytes are not available for comparison, except for the ATP/ADP translocase, which is reportedly present in the glaucophyte Cyanophora paradoxa [25] and was also identified in another glaucophyte Glaucocystis from the Taxonomically Broad EST Database (TBestDB). G, green plants; R, red algae. to function in plastids in plants [13]. However, the Arabidop- sis CMP-KDO synthetase homolog (GenBank: NP_175708), although containing a weak plastid-targeting signal (score 0.504 and 0.610 from ChloroP and TargetP, respectively), is believed to be associated with the endomembrane of plant cells [23]. because of insufficient phylogenetic signal as evidenced by low internal bootstrap support, and sometimes because of possible differential gene losses or other evolutionary scenar- ios (for instance, see Figure 2). Results and discussion Reconstructing possible evolutionary scenarios that explain the chlamydial and plant sequence similarity requires an understanding of the taxonomic distribution and the origin of all involved genes. However, available phylogenetic data from chlamydial genome analyses often suffer from small taxo- nomic sample size [20,21]. Most other relevant studies are heavily biased toward the gene encoding ATP/ADP translo- case, which has an uncertain evolutionary origin and a nar- row distribution, mainly in obligate intracellular bacteria (chlamydiae and rickettsiae) and photosynthetic eukaryotes [22,25-28]. The evolutionary history of a single gene, even if correctly interpreted, might not reflect those of others. If a single evolutionary event underlies the current observation of chlamydial and plant sequence similarity, then a compatible evolutionary history of multiple genes should provide more convincing evidence. The chlamydiae-like genes identified in this study does not constitute an accurate list of all chlamydiae-related genes in primary photosynthetic eukaryotes, but rather is an estimate from our phylogenomic analyses. This number is probably an underestimate, because the evolutionary origin of many genes is difficult to ascertain using available phylogenetic algorithms, and because some other chlamydiae-like genes may exist in glaucophytes and other red algae but are not retained in the smaller genome of Cyanidioschyzon. With the exception of the genes encoding sugar-phosphate isomerase and a hypothetical protein, the protein sequences of all other genes listed in Table 1 contain a plastid-targeting signal as predicted by ChloroP [34] or TargetP [35], or experimentally determined to be plastid localized; this is consistent with the previous report that chlamydiae-related gene products tend Given the common belief that all primary photosynthetic eukaryotes, including glaucophytes, red algae, and green plants, share a common ancestry [11,29,30], we undertook a phylogenomic analysis of Cyanidioschyzon merolae (the only red alga whose complete genome sequence is currently avail- able) to search for genes that are evolutionarily related to Genome Biology 2007, 8:R99 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.3 http://genomebiology.com/2007/8/6/R99 Table 1 These chlamydial and primary photosynthetic eukaryotic sequences also do not appear to be particularly related to homologs from other eukaryotes (Fig- ures 1 and 2, and Additional data file 1). The chlamydial and plant sequence similarities were previ- ously suggested to be an indication of gene transfer from plants or plant-related groups to chlamydiae [21,24,25,28]. However, the genes listed in Table 1 are predominantly dis- tributed in bacteria, indicating a likely bacterial origin (Fig- ures 1 and 2, and Additional data file 1). In all cases, sequences from primary photosynthetic eukaryotes and sometimes from other plastid-containing lineages form a well supported monophyletic group with chlamydial homologs. In most cases they are more closely related to homologs of Can- didatus Protochlamydia amoebophila UWE25 (a chlamydial species that is found in free-living acanthamoebae and envi- ronmental samples and was previously classified as a member of Parachlamydia or Parachlamydia-related [36]) than to chlamydial sequences as a whole (Figures 1 and 2, and Addi- tional data file 1). However, the sequence relationships among primary photosynthetic eukaryotes vary slightly and differ from the expected organismal relationship, mostly The bacterial nature of chlamydiae-like genes in primary pho- tosynthetic eukaryotes suggests that they were transferred either from chlamydiae to these eukaryotes or from plastids to chlamydiae. The latter scenario (plastid-to-chlamydiae transfer) implies a plastidic (cyanobacterial) origin for the transferred genes listed (Table 1). For many of the genes, this scenario can be rejected because it does not account for the specific relationship of the chlamydiae-like genes in primary photosynthetic eukaryotes to the Protochlamydia homologs (Figure 1a-d, Figure 2a, and Additional data file 1), and because it is incompatible with the cyanobacterial homologs forming a well supported group that is distinct from the chlamydial homologs (Figures 1 and 2, and Additional data file 1). After all, plastids evolved from a cyanobacterial ances- tor, and therefore any gene acquired by chlamydiae from plastids should also be more closely related to cyanobacterial than to other bacterial sequences. Is there an ancestral relationship between chlamydiae and cyanobacteria? The relationship between chlamydiae and other bacterial groups remains largely unresolved. Phylogenetic analyses of 16S rRNA suggested that chlamydiae form a sister group with either planctomycetes and verrucomicrobia [14,15,20,37] or cyanobacteria [13,38], without significant support. An ances- tral relationship between chlamydiae and cyanobacteria was hypothesized by Brinkman and coworkers [13], largely based on the possession of a predicted plastid-targeting signal in chlamydiae-like plant sequences. Those authors explicitly excluded the possibility of horizontal gene transfer between chlamydiae and their hosts, and assumed that these plant plastid-targeted sequences were of cyanobacterial origin. According to Brinkman and coworkers, these plant sequences are similar to chlamydial homologs because chlamydiae and cyanobacteria (plastids) are evolutionarily related. A few additional characters uniquely shared by cyanobacteria and chlamydiae were identified in the usually structurally con- served ribosomal superoperon [13], including the absence of S10 and S14 genes, which are present in different chromo- somal locations in chlamydiae and cyanobacteria. However, a more detailed phylogenetic and comparative study of S14 sug- gested that this gene was probably independently transferred from α-proteobacteria to cyanobacteria, chlamydiae, and actinomycetes [39]. Therefore, the absence of S14 from the cyanobacterial and chlamydial ribosomal superoperons might be due to relaxed selection to maintain redundant homologs in the genome, rather than an indication of evolu- tionary relatedness between the two groups. In all studies of gene transfer, there are always alternative explanations for each individual gene tree (for example, see the discussion of Figure 2 in section "Further evidence for an ancient chlamydial endosymbiosis with primary photosyn- thetic eukaryotes") [40]. Overall, however, the pattern from our phylogenetic analyses does not support the hypothesis that cyanobacteria (plastids) and chlamydiae share a close ancestral relationship. This consistent phylogenetic signal from multiple genes should not be dismissed lightly as arti- facts of phylogenetic reconstruction, but rather suggests a clear evolutionary link between chlamydiae and primary photosynthetic eukaryotes. Furthermore, given their often specific affinity with environmental Protochlamydia homologs (Figures 1 and 2, and Additional data file 1), if all of these primary photosynthetic eukaryotic sequences were Although the chlamydiae-cyanobacteria hypothesis offers a popular explanation for the sequence similarity between chlamydiae and plants [20,22,27], it has not been rigorously tested. The major shortcoming of this hypothesis is that all plastids certainly are derived from a past cyanobacterial rather than a chlamydial endosymbiont. Table 1 Additionally, five of the Genome Biology 2007, 8:R99 R99.4 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.4 Genome Biology 2007, Volume 8, Issue 6, Article R99 http://genomebiology.com/2007/8/6/R99 gy g g p g gy gure 1 (see legend on next page) 0.2 Rhodopseudomonas Agrobacterium Haemophilus Escherichia Nitrosomonas Streptomyces Frankia Mycobacterium Thermus Deinococcus Thermotoga Anabaena Crocosphaera Gloeobacter Synechococcus Prochlorococcus Pelobacter Syntrophomonas Solibacter Moorella Blastopirellula Aquifex Leptospira Chlamydia Chlamydophila Cyanidioschyzon Protochlamydia Arabidopsis Oryza Staphylococcus Staphylococcus Bacteroides Cytophaga Clostridium Thermoanaerobacter Bacillus Chlorobium Fusobacterium 100/95 99/98 88/93 95/90 76/86 100/100 66/65 91/93 100/100 63/51 100/100 87/86 100/100 56/* 52/53 63/* 88/86 Cyanobacteria Chlamydiae Red algae Chalmydiae Green plants Proteobacteria High G+C gram+ Thermus/Deinococci Delta-proteobacteria Thermotogae Low G+C gram+ Acidobacteria Low G+C gram+ Planctomycetes Aquificae Spirochaetes Low G+C gram+ Low G+C gram+ Chlorobi Bacteroidetes Fusobacteria Rhodopseudomonas Mesorhizobium Deinococcus Nitrosomonas Rubrivivax Pseudomonas Haemophilus Desulfotalea Chlorobium Aquifex Bacteroides Fusobacterium Chlamydomonas Oryza Cyanidioschyzon Protochlamydia Chlamydophila Chlamydia Solibacter Listeria Bacillus Nocardia Streptomyces Synechococcus Prochlorococcus Nostoc Trichodesmium Crocosphaera Gloeobacter Dehalococcoides Blastopirellula Treponema Thermotoga 0.2 94/96 93/78 97/99 86/96 88/100 */53 96/100 62/70 100/100 57/54 99/100 100/100 100/100 89/92 92/98 57/* Chlamydiae Cyanobacteria Red algae Green plants Alpha-proteobacteria Beta, gamma-proteobacteria Deinococci Delta-proteobacteria Chlorobi Aquificae Bacteroidetes Fusobacteria Acidobacteria Low G+C gram+ High G+C gram+ Chloroflexi Planctomycetes Spirochaetes Thermotogae Aedes Homo Neurospora Dictyostelium Arabidopsis Tetrahymena Rhodospirillum Rickettsia Campylobacter Solibacter Solibacter Coxiella Pseudomonas Rubrivivax Treponema Thermus Deinococcus Chlamydia Chlamydophila Arabidopsis Ostreococcus Ostreococcus Arabidopsis Cyanidioschyzon Protochlamydia Brachyspira Chlorobium Cytophaga Flavobacteria Toxoplasma Clostridium Nostoc Nostoc Synechocystis Crocosphaera Rhodopirellula Chloroflexus Chloroflexus Mycobacterium Nocardioides Thermotoga Eukaryotic mt Green plants Red algae Chlamydiae Chlamydiae Cyanobacteria Alpha-proteobacteria Planctomycetes Epsilon-proteobacteria Thermus/Deinococcus Bacteroidetes Gamma, beta-proteobacteria Acidobacteria Apicomplexan High G+C gram+ Low G+C gram+ Chlorobi Spirochaetes Chloroflexi Spirochaetes Thermotogales 0.2 88/88 89/90 95/70 80/86 92/93 100/100 59/* 62/84 67/* 100/100 100/100 100/100 100/100 94/98 94/89 92/94 100/100 100/100 99/100 67/70 79/90 99/100 Pseudomonas Escherichia Ralstonia Chlorobium Campylobacter Rhodospirillum Mesorhizobium Crocosphaera Anabaena Desulfovibrio Methanosarcina Archaeoglobus Aquifex Anabaena Trichodesmium Moorella Dehalococcoides Bacillus Oryza Chloroflexus Pyrococcus Aeropyrum Clostridium Desulfitobacterium Bacteroides Methanothermobacter Geobacter Synechococcus Nostoc Crocosphaera Trichodesmium Prochlorococcus Leptospira Cyanidioschyzon Oryza Arabidopsis Oryza Arabidopsis Protochlamydia Chlamydophila Chlamydia 98/98 100/100 100/100 100/100 100/99 59/59 59/72 60/64 100/100 100/96 100/100 69/86 66/56 100/100 87/89 71/59 65/61 100/100 100/99 100/100 59/7599/100 97/100 55/51 95/91 100/100 Gamma, beta-proteobacteria Chlorobi Alpha, epsilon-proteobacteria Cyanobacteria Cyanobacteria Cyanobacteria Red algae Green plants Green plants Chlamydiae Spirochaetes Delta-proteobacteria Delta-proteobacteria Aquificae Chloroflexi Euryarchaeota Low G+C gram+ Chloroflexi Low G+C gram+ Crenarchaeota Euryarchaeota Low G+C gram+ Bacteroidetes Euryarchaeota 0.2 (a) (b) (c) (d) 0.2 Rhodopseudomonas Agrobacterium Haemophilus Escherichia Nitrosomonas Streptomyces Frankia Mycobacterium Thermus Deinococcus Thermotoga Anabaena Crocosphaera Gloeobacter Synechococcus Prochlorococcus Pelobacter Syntrophomonas Solibacter Moorella Blastopirellula Aquifex Leptospira Chlamydia Chlamydophila Cyanidioschyzon Protochlamydia Arabidopsis Oryza Staphylococcus Staphylococcus Bacteroides Cytophaga Clostridium Thermoanaerobacter Bacillus Chlorobium Fusobacterium 100/95 99/98 88/93 95/90 76/86 100/100 66/65 91/93 100/100 63/51 100/100 87/86 100/100 56/* 52/53 63/* 88/86 Cyanobacteria Chlamydiae Red algae Chalmydiae Green plants Proteobacteria High G+C gram+ Thermus/Deinococci Delta-proteobacteria Thermotogae Low G+C gram+ Acidobacteria Low G+C gram+ Planctomycetes Aquificae Spirochaetes Low G+C gram+ Low G+C gram+ Chlorobi Bacteroidetes Fusobacteria (a) Rhodopseudomonas Mesorhizobium Deinococcus Nitrosomonas Rubrivivax Pseudomonas Haemophilus Desulfotalea Chlorobium Aquifex Bacteroides Fusobacterium Chlamydomonas Oryza Cyanidioschyzon Protochlamydia Chlamydophila Chlamydia Solibacter Listeria Bacillus Nocardia Streptomyces Synechococcus Prochlorococcus Nostoc Trichodesmium Crocosphaera Gloeobacter Dehalococcoides Blastopirellula Treponema Thermotoga 0.2 94/96 93/78 97/99 86/96 88/100 */53 96/100 62/70 100/100 57/54 99/100 100/100 100/100 89/92 92/98 57/* Chlamydiae Cyanobacteria Red algae Green plants Alpha-proteobacteria Beta, gamma-proteobacteria Deinococci Delta-proteobacteria Chlorobi Aquificae Bacteroidetes Fusobacteria Acidobacteria Low G+C gram+ High G+C gram+ Chloroflexi Planctomycetes Spirochaetes Thermotogae (b) (b) Desulfotalea Chlorobium Bacteroides Pseudomonas Escherichia Ralstonia Chlorobium Campylobacter Rhodospirillum Mesorhizobium Crocosphaera Anabaena Desulfovibrio Methanosarcina Archaeoglobus Aquifex Anabaena Trichodesmium Moorella Dehalococcoides Bacillus Oryza Chloroflexus Pyrococcus Aeropyrum Clostridium Desulfitobacterium Bacteroides Methanothermobacter Geobacter Synechococcus Nostoc Crocosphaera Trichodesmium Prochlorococcus Leptospira Cyanidioschyzon Oryza Arabidopsis Oryza Arabidopsis Protochlamydia Chlamydophila Chlamydia 98/98 100/100 100/100 100/100 100/99 59/59 59/72 60/64 100/100 100/96 100/100 69/86 66/56 100/100 87/89 71/59 65/61 100/100 100/99 100/100 59/7599/100 97/100 55/51 95/91 100/100 Gamma, beta-proteobacteria Chlorobi Alpha, epsilon-proteobacteria Cyanobacteria Cyanobacteria Cyanobacteria Red algae Green plants Green plants Chlamydiae Spirochaetes Delta-proteobacteria Delta-proteobacteria Aquificae Chloroflexi Euryarchaeota Low G+C gram+ Chloroflexi Low G+C gram+ Crenarchaeota Euryarchaeota Low G+C gram+ Bacteroidetes Euryarchaeota 0.2 (d) Figure 1 (see legend on next page) Aedes Homo Neurospora Dictyostelium Arabidopsis Tetrahymena Rhodospirillum Rickettsia Campylobacter Solibacter Solibacter Coxiella Pseudomonas Rubrivivax Treponema Thermus Deinococcus Chlamydia Chlamydophila Arabidopsis Ostreococcus Ostreococcus Arabidopsis Cyanidioschyzon Protochlamydia Brachyspira Chlorobium Cytophaga Flavobacteria Toxoplasma Clostridium Nostoc Nostoc Synechocystis Crocosphaera Rhodopirellula Chloroflexus Chloroflexus Mycobacterium Nocardioides Thermotoga Eukaryotic mt Green plants Red algae Chlamydiae Chlamydiae Cyanobacteria Alpha-proteobacteria Planctomycetes Epsilon-proteobacteria Thermus/Deinococcus Bacteroidetes Gamma, beta-proteobacteria Acidobacteria Apicomplexan High G+C gram+ Low G+C gram+ Chlorobi Spirochaetes Chloroflexi Spirochaetes Thermotogales 0.2 88/88 89/90 95/70 80/86 92/93 100/100 59/* 62/84 67/* 100/100 100/100 100/100 100/100 94/98 94/89 92/94 100/100 100/100 99/100 67/70 79/90 99/100 Pseudomonas Escherichia Ralstonia Chlorobium Campylobacter Rhodospirillum Mesorhizobium Crocosphaera Anabaena Desulfovibrio Methanosarcina Archaeoglobus Aquifex Anabaena Trichodesmium Moorella Dehalococcoides Bacillus Oryza Chloroflexus Pyrococcus Aeropyrum Clostridium Desulfitobacterium Bacteroides Methanothermobacter Geobacter Synechococcus Nostoc Crocosphaera Trichodesmium Prochlorococcus Leptospira Cyanidioschyzon Oryza Arabidopsis Oryza Arabidopsis Protochlamydia Chlamydophila Chlamydia 98/98 100/100 100/100 100/100 100/99 59/59 59/72 60/64 100/100 100/96 100/100 69/86 66/56 100/100 87/89 71/59 65/61 100/100 100/99 100/100 59/7599/100 97/100 55/51 95/91 100/100 Gamma, beta-proteobacteria Chlorobi Alpha, epsilon-proteobacteria Cyanobacteria Cyanobacteria Cyanobacteria Red algae Green plants Green plants Chlamydiae Spirochaetes Delta-proteobacteria Delta-proteobacteria Aquificae Chloroflexi Euryarchaeota Low G+C gram+ Chloroflexi Low G+C gram+ Crenarchaeota Euryarchaeota Low G+C gram+ Bacteroidetes Euryarchaeota 0.2 (c) (d) Figure 1 (see legend on next page) Aedes Homo Neurospora Dictyostelium Arabidopsis Tetrahymena Rhodospirillum Rickettsia Campylobacter Solibacter Solibacter Coxiella Pseudomonas Rubrivivax Treponema Thermus Deinococcus Chlamydia Chlamydophila Arabidopsis Ostreococcus Ostreococcus Arabidopsis Cyanidioschyzon Protochlamydia Brachyspira Chlorobium Cytophaga Flavobacteria Toxoplasma Clostridium Nostoc Nostoc Synechocystis Crocosphaera Rhodopirellula Chloroflexus Chloroflexus Mycobacterium Nocardioides Thermotoga Eukaryotic mt Green plants Red algae Chlamydiae Chlamydiae Cyanobacteria Alpha-proteobacteria Planctomycetes Epsilon-proteobacteria Thermus/Deinococcus Bacteroidetes Gamma, beta-proteobacteria Acidobacteria Apicomplexan High G+C gram+ Low G+C gram+ Chlorobi Spirochaetes Chloroflexi Spirochaetes Thermotogales 0.2 88/88 89/90 95/70 80/86 92/93 100/100 59/* 62/84 67/* 100/100 100/100 100/100 100/100 94/98 94/89 92/94 100/100 100/100 99/100 67/70 79/90 99/100 Pseudomonas Escherichia Ralstonia Chlorobium Campylobacter Rhodospirillum Mesorhizobium Crocosphaera Anabaena Desulfovibrio Methanosarcina Archaeoglobus Aquifex Anabaena Trichodesmium Moorella Dehalococcoides Bacillus Oryza Chloroflexus Pyrococcus Aeropyrum Clostridium Desulfitobacterium Bacteroides Methanothermobacter Geobacter Synechococcus Nostoc Crocosphaera Trichodesmium Prochlorococcus Leptospira Cyanidioschyzon Oryza Arabidopsis Oryza Arabidopsis Protochlamydia Chlamydophila Chlamydia 98/98 100/100 100/100 100/100 100/99 59/59 59/72 60/64 100/100 100/96 100/100 69/86 66/56 100/100 87/89 71/59 65/61 100/100 100/99 100/100 59/7599/100 97/100 55/51 95/91 100/100 Gamma, beta-proteobacteria Chlorobi Alpha, epsilon-proteobacteria Cyanobacteria Cyanobacteria Cyanobacteria Red algae Green plants Green plants Chlamydiae Spirochaetes Delta-proteobacteria Delta-proteobacteria Aquificae Chloroflexi Euryarchaeota Low G+C gram+ Chloroflexi Low G+C gram+ Crenarchaeota Euryarchaeota Low G+C gram+ Bacteroidetes Euryarchaeota 0.2 (c) (d) Aedes Homo Neurospora Dictyostelium Arabidopsis Tetrahymena Rhodospirillum Rickettsia Campylobacter Solibacter Solibacter Coxiella Pseudomonas Rubrivivax Treponema Thermus Deinococcus Chlamydia Chlamydophila Arabidopsis Ostreococcus Ostreococcus Arabidopsis Cyanidioschyzon Protochlamydia Brachyspira Chlorobium Cytophaga Flavobacteria Toxoplasma Clostridium Nostoc Nostoc Synechocystis Crocosphaera Rhodopirellula Chloroflexus Chloroflexus Mycobacterium Nocardioides Thermotoga Eukaryotic mt Green plants Red algae Chlamydiae Chlamydiae Cyanobacteria Alpha-proteobacteria Planctomycetes Epsilon-proteobacteria Thermus/Deinococcus Bacteroidetes Gamma, beta-proteobacteria Acidobacteria Apicomplexan High G+C gram+ Low G+C gram+ Chlorobi Spirochaetes Chloroflexi Spirochaetes Thermotogales 0.2 88/88 89/90 95/70 80/86 92/93 100/100 59/* 62/84 67/* 100/100 100/100 100/100 100/100 94/98 94/89 92/94 100/100 100/100 99/100 67/70 79/90 99/100 (c) (d) (c) 0.2 0.2 Figure 1 (see legend on next page) Figure 1 (see legend on next page) Genome Biology 2007, 8:R99 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.5 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.5 Genome Biology 2007, Volume 8, Issue 6, Article R99 http://genomebiology.com/2007/8/6/R99 Phylogenetic analyses of chlamydiae-like genes in primary photosynthetic eukaryotes Figure 1 (see previous page) Phylogenetic analyses of chlamydiae-like genes in primary photosynthetic eukaryotes. Table 1 Numbers above the branch show bootstrap values for maximum likelihood and distance analyses, respectively. Asterisks indicate values lower than 50%. (a) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ispD). (b) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE). (c) β-Ketoacyl-ACP synthase (fabF). (d) Aspartate transaminase. Note that red algal and green plant sequences form a well supported monophyletic group with environmental Protochlamydia homologs. mt, mitochondrial precursor. Colors represent different phylogenetic affiliations. chlamydiae-like genes in photosynthetic eukaryotes lack identifiable cyanobacterial homologs. (The genes encoding ATP/ADP translocase, glycerol-3-phosphate acyltransferase, oligoendopeptidase F, sodium-hydrogen antiporter, and the malate dehydrogenase chloroplast precursors in green plants lack significant hits to cyanobacterial sequences in GenBank searches.) This suggests that the majority of the genes listed in Table 1 were probably transferred from chlamydiae to pri- mary photosynthetic eukaryotes. cyanobacterial than to chlamydial homologs, unless these sequences diverged so rapidly as to generate long-branch attraction artifacts or lateral gene transfer was involved. Based on this reasoning, we have paid particular attention to the relationships among homologous sequences from chlamydiae, cyanobacteria, and primary photosynthetic eukaryotes in our analyses. All red algal and green plant genes listed in Table 1 clearly are more closely related to chlamydial than to cyanobacterial homologs, with the exception of the genes encoding 4- hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (gcpE) and enoyl-ACP reductase (fabI), which include distinct gene copies in red algae, glaucophytes, and other plastid-contain- ing eukaryotes (Figure 2 and Additional data file 1). Some of the identified chlamydiae-like genes (for instance, that encoding polyribonucleotide phosphorylase) also contain a number of conserved amino acid residues uniquely shared by chlamydiae, red algae, and green plants. In our molecular phylogenetic analyses of 12 of the genes that contain both cyanobacterial and chlamydial homologs, the cyanobacterial sequences form a clade that is clearly distinct from the chlamydial homologs (Figures 1 and 2, and Additional data file 1). Added to this observation is the fact that several chlamydiae-like genes are not found in cyanobacteria and that the gene encoding glycerol-3-phosphate acyltransferase has identifiable homologs (using Arabidopsis [GenBank: NP_849738] and Protochlamydia [GenBank: CAF24042] sequences as queries) only in chlamydiae, red algae, green plants, and the apicomplexan Plasmodium, which also con- tains a nonphotosynthetic plastid. The closer relationship between certain chlamydial and plant sequences was also observed in independent studies [20]. Random horizontal gene transfer versus ancient chlamydial endosymbiosis Conceivably, if horizontal gene transfer occurred from chlamydiae to the earlier cyanobacterial progenitor of plastids, then chlamydial genes could end up in the nuclear genomes of photosynthetic eukaryotes following subsequent intracellular transfer from plastids to the nucleus. Given the number of chlamydiae-like genes detected in our analyses and the fact that most of the original plastidic (cyanobacte- rial) genes were lost in modern photosynthetic eukaryotes [41], this scenario entails massive gene transfers from ancient chlamydiae to the cyanobacterial progenitor of plastids. Although gene transfer indeed occurs frequently in prokaryo- tes [42-44], thus far no chlamydiae-like genes have been reported in any extant cyanobacterium to suggest such mas- sive transfer events. Rickettsia Mesorhizobium Campylobacter Rhodopirellula Chloroflexus Geobacter Solibacter Listeria Clostridium Nostoc Prochlorococcus Cyanidioschyzon Synechococcus Gloeobacter Salmonella Haemophilus Ralstonia Aquifex Ralstonia Symbiobacterium Cytophaga Bacteroides Chlorobium Frankia Mycobacterium Deinococcus Thermus Oryza Arabidopsis Chlamydophila Chlamydia Phaeodactylum Protochlamydia Toxoplasma Bigelowiella 0.2 100/100 97/85 100/89 57/51 95/90 100/100 76/95 100/100 72/63 100/99 100/99 50/64 50/* 60/59 74/94 63/85 Red algae Cyanobacteria Cyanobacteria Green plants Chlamydiae Diatom Apicomplexan Chlamydiae Chlorarachniophyte Thermus/Deinococcus Gamma, beta-proteobacteria Aquificae Beta-proteobacteria Alpha-proteobacteria Epsilon-proteobacteria Chloroflexi Delta-proteobacteria High G+C gram+ Acidobacteria Low G+C gram+ High G+C gram+ Planctomycetes Bacteroidetes Chlorobi (b) Because chlamydiae are found in insects, it has also been sug- gested that plants might have acquired chlamydial genes through insect feeding activities [17]. However, the presence of chlamydiae-like genes in red algae and glaucophytes, which are not a favorable food source for insects, makes this scenario less likely. Furthermore, red algae, glaucophytes, and green plants represent one of the major deep lineages of eukaryotes [45,46]. Such an insect-to-plant transfer scenario would also push the emergence of insects to before the split of primary photosynthetic eukaryotes, which contradicts all available molecular and fossil evidence [47,48]. An unexpectedly high number of chlamydial genes that are most similar to plant homologs has been reported in several independent studies [13,20,21]. For example, most eukary- ote-related sequences of Chlamydia trachomatis tend to group with plant homologs in phylogenetic analyses [21]. In a similarity-based genome survey, sequences of rickettsia, cyanobacteria, and chlamydiae represented only 14% of the analyzed genes, but they accounted for 65% of bacterial genes that were most similar to eukaryotic homologs; these cyano- bacterial and chlamydial sequences disproportionately corre- spond to plant proteins [13]. Is there an ancestral relationship between chlamydiae and cyanobacteria? Rhodospirillum Zymomonas Silicibacter Pseudomonas Erwinia Vibrio Haemophilus Symbiobacterium Helicobacter Fusobacterium Clostridium Bacillus Dehalococcoides Aquifex Frankia Treponema Thermotoga Rhodopirellula Brucella Deinococcus Solibacter Anabaena Trichodesmium Crocosphaera Cyanophora Glaucocystis Cyanidioschyzon Prochlorococcus Synechococcus Bacteroides Porphyromonas Cytophaga Chlorobium Pelodictyon Leptospira Ostreococcus Isochrysis Bigelowiella Chlamydomonas Arabidopsis Plasmodium Protochlamydia Chlamydophila Chlamydia 0.2 100/100 100/97 79/87 69/* 99/64 76/62 97/100 77/* 93/87 100/100 100/100 56/* 83/71 80/84 67/72 90/94 */74 53/86 100/100 100/100 79/87 57/57 76/74 52/* 100/99 100/99 100/100 79/65 100/100 90/72 100/99 Proteobacteria Epsilon-proteobacteria High G+C gram+ High G+C gram+ Low G+C gram+ Planctomycetes Fusobacteria Chloroflexi Acidobacteria Alpha-proteobacteria Deinococci Thermotogales Spirochaetes Aquificae Cyanobacteria Cyanobacteria Red algae Glaucophytes Bacteroidetes Chlorobi Spirochaetes Green plant Green plants Chlamydiae Apicomplexan Chlorarachniophyte Haptophyte Rickettsia Mesorhizobium Campylobacter Rhodopirellula Chloroflexus Geobacter Solibacter Listeria Clostridium Nostoc Prochlorococcus Cyanidioschyzon Synechococcus Gloeobacter Salmonella Haemophilus Ralstonia Aquifex Ralstonia Symbiobacterium Cytophaga Bacteroides Chlorobium Frankia Mycobacterium Deinococcus Thermus Oryza Arabidopsis Chlamydophila Chlamydia Phaeodactylum Protochlamydia Toxoplasma Bigelowiella 0.2 100/100 97/85 100/89 57/51 95/90 100/100 76/95 100/100 72/63 100/99 100/99 50/64 50/* 60/59 74/94 63/85 Red algae Cyanobacteria Cyanobacteria Green plants Chlamydiae Diatom Apicomplexan Chlamydiae Chlorarachniophyte Thermus/Deinococcus Gamma, beta-proteobacteria Aquificae Beta-proteobacteria Alpha-proteobacteria Epsilon-proteobacteria Chloroflexi Delta-proteobacteria High G+C gram+ Acidobacteria Low G+C gram+ High G+C gram+ Planctomycetes Bacteroidetes Chlorobi (a) (b) Rhodospirillum Zymomonas Silicibacter Pseudomonas Erwinia Vibrio Haemophilus Symbiobacterium Helicobacter Fusobacterium Clostridium Bacillus Dehalococcoides Aquifex Frankia Treponema Thermotoga Rhodopirellula Brucella Deinococcus Solibacter Anabaena Trichodesmium Crocosphaera Cyanophora Glaucocystis Cyanidioschyzon Prochlorococcus Synechococcus Bacteroides Porphyromonas Cytophaga Chlorobium Pelodictyon Leptospira Ostreococcus Isochrysis Bigelowiella Chlamydomonas Arabidopsis Plasmodium Protochlamydia Chlamydophila Chlamydia 0.2 100/100 100/97 79/87 69/* 99/64 76/62 97/100 77/* 93/87 100/100 100/100 56/* 83/71 80/84 67/72 90/94 */74 53/86 100/100 100/100 79/87 57/57 76/74 52/* 100/99 100/99 100/100 79/65 100/100 90/72 100/99 Proteobacteria Epsilon-proteobacteria High G+C gram+ High G+C gram+ Low G+C gram+ Planctomycetes Fusobacteria Chloroflexi Acidobacteria Alpha-proteobacteria Deinococci Thermotogales Spirochaetes Aquificae Cyanobacteria Cyanobacteria Red algae Glaucophytes Bacteroidetes Chlorobi Spirochaetes Green plant Green plants Chlamydiae Apicomplexan Chlorarachniophyte Haptophyte (a) ema pirellula Epsilon-proteobacteria High G+C gram+ High G+C gram+ Low G+C gram+ Planctomycetes Fusobacteria Chloroflexi Acidobacteria Alpha-proteobacteria Deinococci Thermotogales Spirochaetes Aquificae Is there an ancestral relationship between chlamydiae and cyanobacteria? Even if chlamydiae and cyanobacteria indeed shared a common ancestry, any sequences of plastidic origin should be more closely related to Genome Biology 2007, 8:R99 R99.6 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.6 Genome Biology 2007, Volume 8, Issue 6, Article R99 http://genomebiology.com/2007/8/6/R99 indeed of plastidic origin (even though they do not group with cyanobacterial homologs), then this would make chlamydiae a paraphyletic group. Such an observation contradicts the common belief that chlamydiae are monophyletic [14,15] and weighs further against the hypothesis that chlamydiae and cyanobacteria are sister taxa. Random horizontal gene transfer versus ancient chlamydial endosymbiosis Although our focus is to elucidate the cause of chlamydial and plant sequence similar- ity rather than to reiterate the previous conclusion, our anal- yses yielded similar findings. For all likely transferred genes in Cyanidioschyzon whose origins can be reliably inferred, chlamydiae-like genes (n = 16) account for the greatest number from any single group other than cyanobacteria (plastids) and α-proteobacteria (mitochondria), and are fol- lowed by five genes from γ-proteobacteria and β-proteobacte- ria. The latter are also the most represented bacterial groups Primary ph and chlamy Figure 2 About 30 genes of plastidic origin (<1% of the nuclear genome) were reported in the human malaria para- site Plasmodium falciparum [55] and only two such genes were identified in Cryptosporidium that probably lost the plastid entirely [49]. None of these intracellularly transferred genes in apicomplexans are related to photosynthesis. A sim- ilar scenario was suggested for Entamoeba, which contains a reduced mitochondrion-derived organelle and appears to have lost most mitochondrial pathways [56]. Therefore, the number of chlamydiae-like genes identified in photosynthetic eukaryotes, albeit being a small fraction of the cyanobacterial genes reported in Arabidopsis and still lower than reported in some heterotrophic apicomplexans, is many times higher than in Cryptosporidium. This lower number of chlamydiae- like genes in primary photosynthetic eukaryotes is in accord- ance with the seeming absence of chlamydial endosymbionts in modern plastid-containing lineages. in GenBank (the taxonomy browser in Entrez of the National Center for Biotechnology Information [NCBI] reported 1,692,357 protein sequences from γ and β-proteobacteria, and only 28,831 from the chlamydiae/verrucomicrobia group as of 4 November 2006). Because of a greater level of stringency, the number of chlamydiae-like genes identified in our phylo- genetic analyses is much lower than previously reported (19 versus the 37 reporrted by Brinkman and coworkers [13] in green plants), but this number is still striking, given that using similar methods only a total of 31 genes acquired from all other sources (including those likely from plants and plas- tids) were identified in the apicomplexan Cryptosporidium parvum [49] and about 50 genes in the kinetoplastid Trypanosoma brucei [50]. The high number of genes transferred between chlamydiae and photosynthetic eukaryotes is probably due to a more sta- ble association of these two groups in the past. In general, such an association could theoretically occur in the form of symbiosis or physical contact between donor and recipient organisms. However, given the distribution of chlamydiae- like genes mainly in primary photosynthetic eukaryotes and the fact that all extant chlamydial species are obligate endo- symbionts, we propose that these genes resulted from an ancient chlamydial endosymbiosis with the ancestor of pri- mary photosynthetic eukaryotes, rather than multiple inde- pendent horizontal transfer events or an ancestral relationship between chlamydiae and cyanobacteria. Presum- ably, the chlamydial symbiotic partner was similar to extant environmental Protochlamydia. Primary ph and chlamy Figure 2 We use the term 'symbiont' (or 'endosymbiont' in the case of chlamydiae) in the sense of deBary [31] to include mutualistic, parasitic, and commensal associations. Such an ancient endosymbiotic association, similar to those giving rise to mitochondria and plastids, would allow ample time for intracellular (or endosymbiotic) gene transfer from the chlamydial endosymbiont to the nucleus of its eukaryotic hosts (either the ancestor of all pri- mary photosynthetic eukaryotes or individual plastid-con- taining lineages such as green plants or red algae) and occasionally between the chlamydial endosymbiont and the plastids. Additionally, because the protein products of these intracellularly transferred genes often target to the original organelles, the co-existence of chlamydial and cyanobacterial endosymbionts within the same eukaryotic cell also led to the targeting of chlamydial gene products into plastids and vice versa. Primary ph and chlamy Figure 2 Primary photosynthetic eukaryotes contain gene copies of both plastidic and chlamydial origin. Numbers above the branch show bootstrap values for maximum likelihood and distance analyses, respectively. Asterisks indicate values lower than 50%. (a) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (gcpE). (b) Enoyl-ACP reductase (fabI). Note that in panel (a) sequences from red algae and glaucophytes are of plastidic origin, whereas those from green plants, apicomplexans, haptophytes, and chlorarachniophytes are of chlamydial origin. Also note that that in panel (b) sequences from green plants, diatoms, chlorarachniophytes, and apicomplexans form a strongly supported group, whereas cyanobacterial and red alga Cyanidioschyzon homologs form another group. Colors represent different phylogenetic affiliations. Genome Biology 2007, 8:R99 Genome Biology 2007, Volume 8, Issue 6, Article R99 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.7 Huang and Gogarten R99.7 http://genomebiology.com/2007/8/6/R99 To answer this question, it should be re-emphasized that all extant chlamydial species are obligate intracellular bacteria. Therefore, if these chlamydiae-like genes in primary photo- synthetic eukaryotes resulted from gene transfer from chlamydiae, then they are probably derived from a chlamy- dial endosymbiont. It should also be noted that although gene transfer from organelles to the nucleus occurs frequently in eukaryotes, the actual scope of transfer might vary among lin- eages. For example, up to 18% of the nuclear genome was interpreted as derived from plastids in Arabidopsis, but a much lower percentage of intracellular gene transfer has been found in the glaucophyte Cyanophora paradoxa (9.1%) [54] and in the red alga Cyanidioschyzon merolae (Huang and Gogarten, unpublished data). Given the relatively smaller genome of Cyanidioschyzon (4,771 predicted protein-coding genes) and a lower level of intracellular gene transfer, it would probably not be possible to identify thousands of genes of any organellar or endosymbiont origin (mitochondrial, plastidic, or chlamydial) in our genome analyses. Most importantly, the retention of transferred genes is often related to the retention and functionality of the organelles in eukaryotic cells [41]. Because the protein products of intrac- ellularly transferred genes often function in the original organelles, loss of certain biochemical functions or even of the organelles themselves will certainly lead to the loss of related transferred genes. For instance, even though thousands of genes were reportedly transferred from chloroplasts in Arabi- dopsis [53], the number of such genes is significantly lower in apicomplexan parasites that harbor a relict, nonphotosyn- thetic plastid. Counting the numbers: can endosymbiosis be inferred from 21 genes? The sec- ond possibility is that independent transfer events occurred from plastids to the nucleus of green plants, apicomplexans, other plastid-containing groups, and the ancestor of extant chlamydiae. In either of the alternative scenarios, a chlamy- dial endosymbiont and its co-existence with plastids in the same host cell would provide a favorable intracellular envi- ronment for transfer between chlamydiae and other organelles. The hypothesis of an ancient chlamydial endosymbiosis is consistent with the available data. For example, the gene encoding ATP/ADP translocase is a key innovation by obli- gate intracellular bacteria (chlamydiae and rickettsiae) that live as energy parasites. Instead of making ATP on their own, these bacterial parasites gain ATP from their host cells and transport ADP back for recycling. Aside from these obligate intracellular bacteria, recognizable homologs of the ATP/ ADP translocase gene are only found in the microsporidial Encephalitozoon (another obligate intracellular parasite) and photosynthetic eukaryotes, where they provide plastids (the original cyanobacterial endosymbiont) with the ATP neces- sary for starch and fatty acid biosynthesis or as an energy sup- plement for carbon dioxide fixation [58-60]. The common origin of plastidic and chlamydial ATP/ADP translocases was confirmed by all available phylogenetic analyses [22,25-28], and various evolutionary scenarios have been proposed [13,22,25-28]. However, gene transfer from a chlamydial endosymbiont to its photosynthetic eukaryotic hosts offers a more logical and parsimonious explanation (also see Schmitz-Esser and coworkers [27] for related discussions). The phylogenies of gcpE and fabI (Figure 2) also are in agree- ment with our hypothesis of an ancient chlamydial endosym- biont in the ancestor of primary photosynthetic eukaryotes. Like the genes encoding 2-C-methyl-D-erythritol 4-phos- phate cytidylyltransferase (ispD) and 4-diphosphocytidyl-2- C-methyl-D-erythritol kinase (ispE; Figure 1a,b), gcpE is related to isoprenoid biosynthesis (see section "Implications for plastid and eukaryotic evolution" for discussion). The gcpE sequences from green plants, apicomplexans, hapto- phytes, and chlorarachniophytes form a strongly supported monophyletic group with chlamydial (in particular Protoch- lamydia) homologs. On the other hand, the gcpE sequences from red algae and glaucophytes form another strongly sup- ported group with cyanobacterial homologs. These two groups are not particularly related (Figure 2a) and it is highly unlikely that the chlamydial gcpE gene was acquired from cyanobacteria or plastids. Counting the numbers: can endosymbiosis be inferred from 21 genes? The residence of plastids and mitochondria within eukaryotic cells led to frequent gene transfers from these organelles to the nucleus [51-53]. Indeed, it has been reported that thou- sands of genes were transferred from chloroplasts to the nucleus in Arabidopsis [53]. Therefore, an apparent question related to our hypothesis is, can we infer the chlamydial endo- symbiosis event based on 21 genes? Because chlamydiae are found in diverse eukaryotes such as acanthamoebae and animals [17,57], it is tempting to specu- late that the chlamydial endosymbiosis might have existed before the split of the primary photosynthetic eukaryotes from other eukaryotic groups. In this study, we searched the GenBank database, which includes genome sequences of Genome Biology 2007, 8:R99 R99.8 Genome Biology 2007, Volume 8, Issue 6, Article R99 R99.8 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten http://genomebiology.com/2007/8/6/R99 probably resulted from independent losses of the plastidic (cyanobacterial) gcpE gene copy in these taxa. many early-branching eukaryotes, and the TBestDB, which covers diverse groups of protists. Chlamydiae-like genes were found to be restricted mainly to primary photosynthetic eukaryotes and other plastid-containing lineages, supporting specifically an association between chlamydiae and the ancestor of primary photosynthetic eukaryotes. An associa- tion with an even earlier eukaryote is not supported. FabI is another chlamydiae-related gene, aside from the gene encoding β-ketoacyl-ACP synthase (fabF; Table 1 and Figure 1c), which is involved in type II fatty acid biosynthesis. Simi- lar to the gcpE gene phylogeny, cyanobacterial and red algal Cyanidioschyzon fabI sequences form one group, whereas homologs from chlamydiae, green plants, apicomplexans, chlorarachniophytes, and diatoms form another group. These two very distinct sequence groups differ in several highly con- served insertions and deletions, but the relationship between them is less certain because of insufficient internal bootstrap support on the gene tree. Therefore, although it is more likely that the two sequence groups in photosynthetic eukaryotes are derived from cyanobacterial and chlamydial endosymbi- onts, respectively, it is also theoretically possible that chlamy- diae acquired their fabI from the plastids of a plant-related group [63]. Because of the distinct sequence difference of the two fabI copies, this second scenario entails one of the two following possibilities. The first is the existence of two fabI paralogs in the cyanobacterial progenitor of plastids and sub- sequent independent losses in extant cyanobacteria, and in red algae and other plastid-containing eukaryotes. Counting the numbers: can endosymbiosis be inferred from 21 genes? The most plausible explanation for this observation is that two distinct gcpE gene copies were originally contributed by chlamydial and cyanobacterial (plastidic) endosymbionts to the nuclear genome of the ancestral primary photosynthetic eukaryote and differentially retained in green plants, red algae, and glaucophytes. The chlorarachniophyte Bigelowiella and apicomplexan Plasmo- dium, and the haptophyte Isochrysis are believed to contain a green and a red algal endosymbiont, respectively [10,61,62]; their proximity in the same chlamydial group (Figure 2a) Implications for plastid and eukaryotic evolution Implications for plastid and eukaryotic evolution Ancient endosymbionts gave rise to organelles, including mitochondria, hydrogenosomes, and plastids [1]. Thus far, no chlamydial endosymbiont has been reported in photosyn- thetic eukaryotes. Whether a relict organelle derived from the proposed ancient chlamydial endosymbiont exists in extant plastid-containing lineages remains to be further investi- gated. On the other hand, it also would not be surprising if the chlamydial endosymbiont had degenerated entirely during the evolution of photosynthetic eukaryotes. As obligate intra- cellular bacterial parasites, chlamydiae depend on their hosts for certain nutrients, and consequently they have a relatively reduced genome [20,21]. Many dispensable genes were prob- ably lost as a result of their parasitic lifestyle. Given the lack of apparent benefit to the host cell, such gene losses in an iso- lated intracellular system could gradually lead to deteriora- tion of the endosymbiont genome and ultimately the endosymbiont itself [64]. Almost all chlamydiae-like genes identified in red algae and green plants (Table 1) contain a predicted plastid-targeting signal, although the example of CMP-KDO synthetase sug- gests that this prediction may not always be reliable. The Genome Biology 2007, 8:R99 Genome Biology 2007, Volume 8, Issue 6, Article R99 H Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.9 http://genomebiology.com/2007/8/6/R99 Huang and Gogarten R99.9 In the first stage, a chlamydial bacterium, similar to the extant Protochlamydia, entered a mitochondrion-containing eukaryote as a bacterial parasite. This chlamydial endosymbi- ont possessed a necessary transport system to gain nutrients and other metabolites from the host cell. At about the same time, a once free-living photoautotrophic cyanobacterium was captured by the eukaryotic host by chance, initially possi- bly as a food source (Figure 3a,b). chlamydiae-like genes are involved in a variety of biochemical activities in plastids, including fatty acid biosynthesis, ion transport, nitrogen metabolism, and RNA processing, among others. Notably, three of these genes (ispD, ispE, and gcpE) are key enzymes of the deoxyxylulose 5-phosphate (DXP) pathway, which leads to the formation of isopentenyl diphos- phate, a major metabolite for isoprenoid biosynthesis in bac- teria and plastids [65-67]. The chlamydial origin of ispD and ispE was also confirmed by independent studies [68], which hypothesized that the DXP pathway in primary photosyn- thetic eukaryotes was probably derived from plastids. Implications for plastid and eukaryotic evolution In the fourth and final stage, once the plastid organelle was fully established in the host cell, the benefits of the chlamydial endosymbiont to the host became less apparent. It is possible that the chlamydial endosymbiont remained in the host cell mostly as a bacterial parasite. Such a parasitic relationship might not be sustained over a long period of time and the chlamydial endosymbiont might have gradually degenerated. It is also possible that the chlamydial endosymbiont was transformed into an organelle yet to be recognized in photo- synthetic eukaryotes (Figure 3e). In the fourth and final stage, once the plastid organelle was fully established in the host cell, the benefits of the chlamydial endosymbiont to the host became less apparent. It is possible that the chlamydial endosymbiont remained in the host cell mostly as a bacterial parasite. Such a parasitic relationship might not be sustained over a long period of time and the chlamydial endosymbiont might have gradually degenerated. It is also possible that the chlamydial endosymbiont was transformed into an organelle yet to be recognized in photo- synthetic eukaryotes (Figure 3e). We suggest that three organisms were involved in establish- ing the primary photosynthetic lineage: the eukaryotic host cell, the cyanobacterial endosymbiont that provided photo- synthetic capability, and a chlamydial endosymbiont or para- site that facilitated the establishment of the cyanobacterial endosymbiont. The coexistence of three partners with differ- ent biological requirements and capabilities might have offered an opportunity for some transient mutualistic inter- actions, and the acquisition of genes such as those encoding ATP/ADP translocase and sodium:hydrogen antiporter from the chlamydial endosymbiont might have facilitated the suc- cessful endosymbiosis of cyanobacteria by allowing energy flux into the protoplastid organelle and effective regulation of ion composition. Specifically, we hypothesize that the origin and the establishment of primary plastids might have involved the following stages. Once a rich repertoire of transporters were in place in pri- mary photosynthetic eukaryotes, transport of photosynthetic products and other metabolites across the photosynthetic organelle might be more easily adapted to the different mem- branes in secondary or tertiary endosymbiotic hosts [70]. This also explains the observation that secondary endosymbi- osis is more frequent than primary endosymbiosis in eukary- otic evolution. The likely ancient chlamydial endosymbiosis with primary photosynthetic eukaryotes has some other important impli- cations for eukaryotic evolution. Implications for plastid and eukaryotic evolution In our phylogenetic analyses of ispD and ispE, the chlamydial and primary photosynthetic eukaryotic sequences form a mono- phyletic group that is distinct from the cyanobacterial homologs (Figure 1a,b), suggesting probable gene displace- ment after the endosymbiotic origin of plastids. In the second stage, gene transfer between the chlamydial endosymbiont and the host cell ensued because of their phys- ical association. As a result, the eukaryotic host acquired transporters from the chlamydial endosymbiont, facilitating its communication with the cyanobacterial captive. At this stage, the relationship between chlamydial endosymbiont and the host cell might be considered transiently mutualistic. Gene transfer between the cyanobacterial captive and the host cell might also have occurred during this stage (Figure 3c). Secondary endosymbioses between photoautotrophic algae and heterotrophic host cells occurred several times during eukaryotic evolution [9-12]. In contrast, the formation of the primary cyanobacterial endosymbiosis appears to have been unique (but also see the report by Marin and coworkers [69]). Our finding of the probable participation of a third symbiotic partner offers an explanation for this rarity. As free-living photoautotrophic cells, in which ATP is generated in the main cytoplasmic compartment, cyanobacteria do not need mech- anisms to transport energy-rich metabolites between mem- brane-enclosed compartments. However, for an enslaved cyanobacterium in a heterotrophic host to transform into a photosynthetic organelle, new transport systems are neces- sary. Therefore, the initial adaptation of a photoautotrophic cyanobacterium toward a photosynthetic organelle was prob- ably a difficult process contingent on the simultaneous pres- ence of suitable transport systems. At least in part these transporters might have evolved in a chlamydial parasite that was present within the same eukaryotic host cell. These trans- porters enabled chlamydiae to parasitize energy and other molecules from the host cell, but also allowed for ATP/ADP equilibration with the cyanobacterium. In the third stage, the cyanobacterial captive was gradually transformed into a photosynthetic organelle (plastid) in the host cell and a stable, mutualistic relationship between the plastid and the host cell was in place. The plastid provided photosynthetic products to the host, whereas the host offered shelter and also transported protein products of the intracel- lularly transferred genes (both from the chlamydial endosym- biont and the cyanobacterial captive) and other necessary metabolites to the plastid organelle (Figure 3d). Hypotheti Figure 3 (d) ? X? X X (e) (d) Implications for plastid and eukaryotic evolution Ancient endosymbiosis events, such as those that gave rise to mitochondria and plas- tids, to a large degree defined the evolution of eukaryotes. Our data suggest that these ancient endosymbiosis events might Genome Biology 2007, 8:R99 Huang and Gogarten R99.10 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.10 Genome Biology 2007, Volume 8, Issue 6, Article R99 http://genomebiology.com/2007/8/6/R99 Figure 3 (a) (b) ? ? (c) ? ? (d) ? X? X X (e) have occurred more frequent been contingent on others. S subsequent intracellular gen evolution of host organisms regardless of whether an org symbionts is retained in exta The finding of this study also primary photosynthetic euk origin of these groups and ported by many studies [29, ticularly those of nuclear gen conflicting results [71,72]. Th biosis at the root of the prim vides strong and independen of all primary photosyntheti they harbor. Conclusion The availability of a comple alga Cyanidioschyzon and ex for diverse deep eukaryotes the distribution and evoluti primary photosynthetic euka ogenomic analyses indicate t are unlikely to have derive gene transfer events or evo chlamydiae and cyanobacter photosynthetic eukaryotes pr endosymbiosis event between primary photosynthetic euka ner being similar to extant e This ancient chlamydial end synthetic eukaryotes might establishment of plastids by p functions and by allowing eff h b i l d b Figure 3 (a) (b) ? ? (c) ? ? (d) ? X? X X (e) Hypothetic stages of plastid origin a Figure 3 Hypothetic stages of plastid origin a discussed in the text) are displayed stage; (d) third stage; and (e) fourt colors show α-proteobacterial (mit cyanobacterial endosymbionts, as w respective origins. Arrows directly symbiotic partner that receives the indicates the degree of benefit. Das intracellular gene transfer, whereas the transferred genes. Crosses indic transfer processes that might not e eukaryotes. Note that chlamydial e parasite in the first stage, but it had with the host cell in the second and degenerated in modern photosynth cyanobacterial endosymbiont was i host cell (panel b), and then receive process facilitated by the chlamydia transformed into a plastid organelle (a) Hypotheti Figure 3 Hypothetic stages of plastid origin and establishment Figure 3 Hypothetic stages of plastid origin and establishment. The stages (as discussed in the text) are displayed as follows: (a,b) first stage; (c) second stage; (d) third stage; and (e) fourth stage. White, yellow, and green colors show α-proteobacterial (mitochondrial), chlamydial, and cyanobacterial endosymbionts, as well as genes and proteins of their respective origins. Arrows directly from the endosymbiont point to the symbiotic partner that receives the benefit, and the thickness of the arrow indicates the degree of benefit. Dashed lines indicate directions of intracellular gene transfer, whereas solid lines show protein targeting of the transferred genes. Crosses indicate chlamydial endosymbiont and gene transfer processes that might not exist in extant photosynthetic eukaryotes. Note that chlamydial endosymbiont was initially a bacterial parasite in the first stage, but it had a transient mutualistic relationship with the host cell in the second and third stages, and then might have degenerated in modern photosynthetic eukaryotes. Note also that the cyanobacterial endosymbiont was initially captured to solely benefit the host cell (panel b), and then received metabolites from the host cell (a process facilitated by the chlamydial endosymbiont) and was gradually transformed into a plastid organelle in the host cell (panels d and e). (a) (b) (b) have occurred more frequently, and some of them might have been contingent on others. Such ancient endosymbioses and subsequent intracellular gene transfers contributed to the evolution of host organisms and their descendent lineages, regardless of whether an organelle derived from past endo- symbionts is retained in extant species. have occurred more frequently, and some of them might have been contingent on others. Such ancient endosymbioses and subsequent intracellular gene transfers contributed to the evolution of host organisms and their descendent lineages, regardless of whether an organelle derived from past endo- symbionts is retained in extant species. ? ? (c) (c) The finding of this study also weighs into the relationship of primary photosynthetic eukaryotes. Although a common origin of these groups and of their plastids has been sup- ported by many studies [29,30], several other analyses, par- ticularly those of nuclear genes, have provided ambiguous or conflicting results [71,72]. The ancient chlamydial endosym- biosis at the root of the primary photosynthetic lineages pro- vides strong and independent evidence for a common origin of all primary photosynthetic eukaryotes and of the plastids they harbor. ? ? (d) ? ? Phylogenomic analyses y g y Phylogenomic analyses of Cyanidioschyzon, which is the only red algal species whose complete genome sequence is yet available, were performed using PhyloGenie [75] and the NCBI nonredundant protein sequence database to identify chlamydiae-related genes. The results were compared with those of previously published analyses and further detailed phylogenetic analyses were performed. For detailed phyloge- netic analyses, sequences were selected from major groups within each domain of life. Multiple protein sequence align- ments were performed using MUSCLE [76] and clustalx [77], followed by cross-comparisons and manual refinement. Only unambiguously aligned sequence portions were used. The alignments are detailed in Additional data file 1. Phylogenetic analyses were performed with a maximum likelihood method using PHYML [78] and a distance method using the program neighbor of PHYLIP version 3.65 [79], with maximum likeli- hood distances calculated using TREE-PUZZLE [80]. Branch lengths and topologies of the trees depicted in all figures (Fig- ures 1 and 2, and Additional data file 1) were calculated with PHYML [78]. All maximum likelihood calculations were based on the JTT substitution matrix and a mixed model of four gamma-distributed rate classes plus invariable sites. Maximum likelihood distances for bootstrap analyses were calculated using TREE-PUZZLE [80] and PUZZLEBOOT v1.03 (ME Holder and AJ Roger; available on the web [81]). The plastid-targeting signal of identified chlamydiae-like pro- tein sequences was predicted using the web-based ChloroP [34] and TargetP [35]. 8. McFadden GI: Mergers and acquisitions: malaria and the great chloroplast heist. Genome Biol 2000, 1:reviews1026.1-1026.4. 8. McFadden GI: Mergers and acquisitions: malaria and the great chloroplast heist. Genome Biol 2000, 1:reviews1026.1-1026.4. 9. Bhattacharya D, Yoon HS, Hackett JD: Photosynthetic eukaryo- tes unite: endosymbiosis connects the dots. Bioessays 2004, 26:50-60. 10. Keeling PJ: Diversity and evolutionary history of plastids and their hosts. Am J Botany 2004, 91:1481-1493. J y 11. Palmer JD: The symbiotic birth and spread of plastids: how many times and whodunit? J Phycol 2003, 39:4-11. y J y 12. Delwiche CF: Tracing the thread of plastid diversity through the tapestry of life. Am Nat 1999, 154:S164-S177. 13. Brinkman FS, Blanchard JL, Cherkasov A, Av-Gay Y, Brunham RC, Fernandez RC, Finlay BB, Otto SP, Ouellette BF, Keeling PJ, et al.: Evi- dence that plant-like genes in Chlamydia species reflect an ancestral relationship between Chlamydiaceae, cyanobacteria, and the chloroplast. Genome Res 2002, 12:1159-1167. 14. Ludwig W, Klenk H-P: Overview: a phylogenetic backbone and taxonomic framework for prokaryotic systematics. Conclusion h l b l Co c us o The availability of a complete genome sequence of the red alga Cyanidioschyzon and expressed sequence tag (EST) data for diverse deep eukaryotes allows a more detailed study of the distribution and evolution of chlamydiae-like genes in primary photosynthetic eukaryotes. Our very stringent phyl- ogenomic analyses indicate that these chlamydiae-like genes are unlikely to have derived from independent horizontal gene transfer events or evolutionary relatedness between chlamydiae and cyanobacteria. The chlamydiae-like genes in photosynthetic eukaryotes probably resulted from an ancient endosymbiosis event between chlamydiae and the ancestor of primary photosynthetic eukaryotes, with the chlamydial part- ner being similar to extant environmental Protochlamydia. This ancient chlamydial endosymbiosis with primary photo- synthetic eukaryotes might also have played a role in the establishment of plastids by providing genes that possess new functions and by allowing effective communications between the cyanobacterial endosymbiont and the eukaryotic host cell. ? X? X X (e) (e) Figure 3 Genome Biology 2007, 8:R99 http://genomebiology.com/2007/8/6/R99 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.11 Genome Biology 2007, Volume 8, Issue 6, Article R99 H Additional data files The following additional data are available with the online version of this paper. Additional data file 1 contains protein sequence alignments used for phylogenetic analyses and resulting phylogenetic trees. Additi l Fil 1 P t i li t d h l ti t Th d t t i t i li t d f h l ti l d lti h l ti t E h i l d G B k GI b f ll d b th i Cli k h f fil 22. 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Eukaryot Cell 2006, 5:609-612. 49. Huang J, Mullapudi N, Lancto CA, Scott M, Abrahamsen MS, Kissinger JC: Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum. Genome Biol 2004, 5:R88. 71. Stiller JW, Hall BD: The origin of red algae: implications for plastid evolution. Proc Natl Acad Sci USA 1997, 94:4520-4525. p 72. Stiller JW, Riley J, Hall BD: Are red algae plants? A critical eval- uation of three key molecular data sets. J Mol Evol 2001, 52:527-539. 50. Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bar- tholomeu DC, Lennard NJ, Caler E, Hamlin NE, Haas B, et al.: The genome of the African trypanosome Trypanosoma brucei. Sci- ence 2005, 309:416-422. 73. Cyanidioschyzon merolae Genome Project [http://mero lae.biol.s.u-tokyo.ac.jp] 51. Archibald JM, Rogers MB, Toop M, Ishida K, Keeling PJ: Lateral gene transfer and the evolution of plastid-targeted proteins in the 74. y jp] 74. Chlamydomonas reinhardtii v2.0 [http://genome.jgi-psf.org/ Genome Biology 2007, 8:R99 Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.13 http://genomebiology.com/2007/8/6/R99 chlre2/chlre2.home.html] ] 75. Frickey T, Lupas AN: PhyloGenie: automated phylome genera- tion and analysis. Nucleic Acids Res 2004, 32:5231-5238. tion and analysis. Nucleic Acids Res 2004, 32:5231-5238. y 76. Genome Biology 2007, Volume 8, Issue 6, Article R99 Huang and Gogarten R99.13 Acknowledgements Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004, 32:1792-1797. 77. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for mul- tiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997, 25:4876-4882. 78. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003, 52:696-704. 79. Felsenstein J: PHYLIP (Phylogeny Inference Package) version 3.65 (distrib- uted by the author) Seattle, Washington: Department of Genome Sci- ences, University of Washington; 2005. y g 80. Schmidt HA, Strimmer K, Vingron M, von Haeseler A: TREE-PUZ- ZLE: maximum likelihood phylogenetic analysis using quar- tets and parallel computing. Bioinformatics 2002, 18:502-504. p p g f 81. TREE-PUZZLE 5.2 [http://www.tree-puzzle.de] Genome Biology 2007, 8:R99
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What is a Fresh Scent in Perfumery? Perceptual Freshness is Correlated with Substantivity
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What is a Fresh Scent in Perfumery? Perceptual Freshness is Correlated with Substantivity Manuel Zarzo Sensors 2013, 13, 463-483; doi:10.3390/s130100463 Sensors 2013, 13, 463-483; doi:10.3390/s130100463 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS sensors ISSN 1424-8220 www.mdpi.com/journal/sensors OPEN ACCESS 1. Introduction Fresh is an odor character descriptor commonly used in perfumery. Actually, it is the one most frequently encountered in a semantic olfactory database of 119 perfume materials reported by Thiboud [1]. In a recent study of this database, it was found that warm, oriental and powdery were the descriptors most dissimilar to fresh (see Figure 6 of [2]). Consistent with this result, fresh and oriental are regarded as opposite families of perfumes in the Fragrance Wheel proposed by Edwards [3]. The fresh category comprises citrus, green, water, and fruity subfamilies. The Odor Effects Diagram is an olfactory representation of perfumery notes based on two basic polarities: (i) erogenous vs. antierogenous (refreshing) and (ii) narcotic vs. stimulating [4]. Citrus, green, watery, and aldehydic are regarded as refreshing scents, while erogenous, animal, musk, vanilla and powdery appear at the opposite pole (see Figure 5 of [2]). Fruity is located in this diagram between floral and fresh, and the same criterion was considered by Edwards [3]. Perfume is a complex mixture of odorants with different volatilities. The parameter that measures the lasting property of a material when applied on the skin is called substantivity or tenacity. It is well known by perfumers that olfactory notes perceived as fresh tend to evaporate quickly, while the opposite applies to those most dissimilar to fresh. Actually, fresh and green are attributes commonly encountered in the description of top notes (i.e., the ones that are perceived firstly when smelling a fragrance) [2]. Light refers to scents with high volatility, while heavy, rich or tenacious is applied to materials with high substantivity. Light fragrances are those perceived as non-sweet with a predominant fresh note that is often associated with citrus, greens or aldehydes [5]. Conversely, the least volatile ingredients such as mosses and animal scents dominate in heavy perfumes [1]. Vapor pressure is the basic factor that determines the volatility of a specific compound [6], but the vapor composition in equilibrium with the liquid is difficult to predict in mixtures due to the complex molecular interactions that occur [7–10]. Sensory ratings on a scale of freshness are difficult to obtain because the fresh dimension of olfactory perception is not well understood yet. Probably for this reason, psychophysical studies aimed at quantifying the relationship between this odor quality and tenacity have not received much attention yet. Manuel Zarzo Department of Applied Statistics, Operations Research and Quality, Universidad Politécnica de Valencia, Cmno. Vera s/n, 46022 Valencia, Spain; E-Mail: mazarcas@eio.upv.es; Tel.: +34-963-877-490; Fax: +34-963-877-499 Received: 26 November 2012; in revised form: 19 December 2012 / Accepted: 20 December 2012 / Published: 28 December 2012 Received: 26 November 2012; in revised form: 19 December 2012 / Accepted: 20 December 2012 / Published: 28 December 2012 Abstract: Perfumes are manufactured by mixing odorous materials with different volatilities. The parameter that measures the lasting property of a material when applied on the skin is called substantivity or tenacity. It is well known by perfumers that citrus and green notes are perceived as fresh and they tend to evaporate quickly, while odors most dissimilar to ‘fresh’ (e.g., oriental, powdery, erogenic and animalic scents) are tenacious. However, studies aimed at quantifying the relationship between fresh odor quality and substantivity have not received much attention. In this work, perceptual olfactory ratings on a fresh scale, estimated in a previous study, were compared with substantivity parameters and antierogenic ratings from the literature. It was found that the correlation between fresh odor character and odorant substantivity is quite strong (r = −0.85). ‘Fresh’ is sometimes interpreted in perfumery as ‘cool’ and the opposite of ‘warm’. This association suggests that odor freshness might be somehow related to temperature. Assuming that odor perception space was shaped throughout evolution in temperate climates, results reported here are consistent with the hypothesis that ‘fresh’ evokes scents typically encountered in the cool season, while ‘warm’ would be evoked by odors found in nature during summer. This hypothesis is rather simplistic but it may provide a new insight to better understand the perceptual space of scents. Keywords: fragrance; tenacity; odor descriptor; perceptual dimension; olfactory map Sensors 2013, 13 Sensors 2013, 13 464 1. Introduction Several sensory maps of scents reported in the literature are investigated in the present work attempting to further understand the psychological aspects involved in the perception of refreshing odor character. The main target of the present work is to study the correlation between this odor quality and odorant substantivity. This relationship is well established in perfumery, but only at descriptive level. 2.1. Classification of Odor Descriptors on a Scale of Perceptual Freshness Hedonic tones of odor descriptors are available from the literature, and they can be used to interpret underlying dimensions of odor profile databases [11]. Similarly, it is possible to assign a refreshing tone to odor descriptors as described ahead. Boelens and Haring [12] asked a panel of six perfumers to smell 309 aroma chemicals and to rate on a 0–9 scale the odor similarity to 30 reference materials. Each one was selected as a standard for a certain odor character descriptor. The resulting sensory dataset, which will be referred to hereafter as B-H database, was analyzed in previous studies using principal components analysis (PCA) [2,11]. The first principal component (PC1) is the linear combination of variables (reference materials) that explains the maximum amount of data variability (17.5% in this case). The contributions of variables in the formation of a given PC are called loadings, being p1 and p2 the loadings corresponding to PC1 and PC2, respectively. PC1 was interpreted as a dimension of freshness because fresh was the descriptor with highest p1 loading. The p1 values can be regarded as (i) indirect assessments of the 30 reference materials on a scale of odor freshness, and (ii) refreshing tones of the odor descriptors associated to the reference materials. Thiboud [1] reproduces a two-dimensional projection of a similarity matrix developed from interviews with consumers in England, USA, Spain, Brazil, and Japan relating to defined perfume bases and to verbal descriptions. The horizontal axis of this fragrance mapping discriminates masculine vs. feminine scents, and fresh is one of the descriptors that determine the vertical axis. The same interpretation was proposed for PC2 and PC1 of the B-H database [2]. Thus, a scatterplot of p2 vs. p1, which is usually referred to as loading plot, was superimposed with Thiboud’s sensory map, after being properly scaled and rotated in order to achieve the best matching between both odor representations. Chastrette et al. [13] analyzed semantic odor profiles of 628 pure odorous substances commonly encountered in perfumery. The database was obtained by Firmenich SA from a team of seven perfumers who assessed each compound and assigned two to four notes chosen among 32 possible descriptors. The three most frequent ones were considered as the odor profile. A transformed matrix derived from this database was analyzed with PCA, and the PC1/PC2 loading plot was proposed as a sensory map of scents. 2. Methods In order to characterize the connection between perceived fresh odor character and substantivity, three stages have been carried out: (i) to estimate the freshness of odorant materials and descriptors on a numeric scale, (ii) to obtain substantivity values from the literature, and (iii) to study the correlation between them by means of simple linear regression. Each one of these stages is detailed below in a different section. In the present work, ‘fresh’ and ‘refreshing’ are used as synonyms. The different connotations of both terms are discussed in Section 4.4. Sensors 2013, 13 Sensors 2013, 13 465 2.1. Classification of Odor Descriptors on a Scale of Perceptual Freshness 2.2. Fresh Odor Quality and Antierogenic Character of Odorant Materials Thiboud [1] compiled semantic odor profiles for 44 natural odorants and 75 synthetic chemicals. Each material was labeled with three or four main odor descriptors and with a set of ancillary attributes ranging from 0 to 14 (average = 6.6). This database was analyzed in a previous study [2] and it was found that freshness was an underlying dimension. Based on a few representative descriptors of this dimension, I obtained estimated scores on a fresh scale for the 44 natural odorants as described below. Jellinek [4] conducted a sensory study attempting to classify perfume materials as erotic or not erotic. Three experienced perfumers assessed the erogenic character of 61 essential oils, 34 extracts and 105 aroma chemicals. Each material was added to four perfume compositions at a level that was just sufficient to create a clearly noticeable difference, and the panel had to decide whether the added material increased or decreased the erotic character of the perfume. From these sensory ratings, an antierogenic index (AI) was derived ranging from −100 to 100. The maximum value 100 indicates that the material was regarded by the panel as clearly antierogenic, while −100 corresponds to a noticeably erogenic scent. Jellinek [4] considered that antierogenic could also be interpreted as refreshing in odor description. To study this issue, I checked the correlation between p1 and AI of reference materials in the B-H database. 2.1. Classification of Odor Descriptors on a Scale of Perceptual Freshness I obtained the projections over the first factorial axis from this plot (Figure 1 of [13]) and compared these projections with p1 loadings of equivalent descriptors in the B-H database. Different sensory maps of scents have been developed by chemical companies that supply perfume raw materials. One of them is the Rosace of Firmenich [14], which is comprised by 13 categories of scents properly arranged according to the opinion of professional perfumers. Citrus and balsamic are located at opposite positions, which suggests that this direction can be interpreted as a dimension of freshness. I obtained the orthogonal projections of all odor classes over this direction. Next, the correlation between these projections and p1 loadings from the B-H database was studied. Another olfactory representation is the Field of Odors, which displays in a semicircle different descriptors and odor categories [15]. It was derived from an odor profile database of perfume materials. Attempting to study if freshness is a salient dimension of this odor map, I obtained the polar coordinates for the different odor classes (i.e., angular position in the semicircle and distance to the center). The angular coordinates were compared with p1 loadings of equivalent attributes in the B-H database. Sensors 2013, 13 Sensors 2013, 13 466 2.2. Fresh Odor Quality and Antierogenic Character of Odorant Materials 2.4. Relationship between Fresh Odor Character and Substantivity The p1 loadings derived from the B-H database can be regarded as assessments of the 30 reference materials on a perceptual scale of odor freshness. These values can also be interpreted as refreshing tones of the odor descriptors associated to reference materials (i.e., the degree of perceptual similarity or dissimilarity of a given odor quality with respect to the fresh odor character). The correlation between p1 loadings and the substantivity index of equivalent descriptors was studied by means of linear regression. The evaporation index and SGS are available for most reference materials in the B-H database. Thus, the correlation between p1, EI and SGS was also checked. CS values are available for the 44 odorants used by Thiboud [1]. I studied the correlation of CS with the scores of freshness derived from odor profiles, as well as with AI and other substantivity parameters. Additional materials commonly used in perfumery were also taken into consideration. 3. Results The comparison of refreshing tones derived from odor profiles and sensory maps yields consistent results, as indicated in the next section. This issue is of interest to further understand the psychological issues involved in the perception of fresh odor character. Section 3.2 describes the correlation between the fresh odor character and substantivity. Sensors 2013, 13 Sensors 2013, 13 467 Another substantivity parameter was reported by Appell [7], who calculated the volatility of 81 essential oils by obtaining the amount of oil evaporated in various time intervals. The quantity (mg) evaporated in one hour from one gram of oil was called evaporation index. Another substantivity parameter was reported by Appell [7], who calculated the volatility of 81 essential oils by obtaining the amount of oil evaporated in various time intervals. The quantity (mg) evaporated in one hour from one gram of oil was called evaporation index. The website of The Good Scents Company (www.thegoodscentscompany.com) provides useful information about suppliers, safety, organoleptic properties, and physical parameters of aroma chemicals and natural perfume materials. Substantivity values, which will be referred as SGS, are available for most odorants. This parameter is measured in hours and ranges from 1 to 400 h. The maximum SGS in fact indicates a censored value higher than 400 (i.e., ≥16.7 days). Actually, certain materials can smell for months [8]. 2.3. Substantivity Parameters of Perfume Materials After conducting an extensive literature review, I found four substantivity parameters of perfume materials as described next. These parameters were not obtained in the same way and may not be accurate, but they are correlated and can be compared with perceptual freshness. Perfumes are described according to their top, middle and base notes, which are also called head, heart and bottom notes, respectively. The top note is the first odor perceived when smelling a fragrance product, and it usually consists of the most volatile portion of the composition [1]. Middle notes, which represent the main body of a blend, are perceived after the top notes fade away [5]. Base notes are basically determined by fixative materials with a very low volatility and great substantivity that yield the characteristic lasting note of any fragrance. The three stages of evaporation are often illustrated with a pyramid [16]. The H&R Fragrance Guide [17] contains the semantic odor description of 820 commercial perfumes (367 men’s and 453 women’s). I counted the total number of times that a given attribute was applied to describe top (Ntop), middle (Nmid) and base notes (Nbase). A substantivity index was calculated according to Equation (1), which takes the value 0, 50 and 100 for attributes that are only applied to describe top, middle and base notes, respectively. Thus, it provides an estimation of the substantivity associated to a given descriptor on a 0-100 scale: Substantivity index = base mid top base mid N N N N N + + ⋅ + ⋅ 100 50 (1) (1) Poucher [18] obtained the duration of evaporation by olfaction for 332 odorous materials. A certain quantity of each odorant was placed on a paper strip, and he determined the time that the smelling strip retained the typical odor note. Based on the results, a coefficient of substantivity (CS) from 1 to 100 was assigned to each material. Sensors 2013, 13 Sensors 2013, 13 468 Regarding the vertical axis of Figure 1, men-husbands appears at the bottom and feminine is found at the opposite side. This result further supports the hypothesis of a dimension influenced by psychological and cultural aspects that discriminates feminine vs. masculine cosmetic odors, as discussed elsewhere [2,19]. Figure 1. Loading plot (p2 vs. p1) of the database obtained by Boelens & Haring [12] (white triangles). Data were mean-centered and scaled to unit variance prior to the PCA. Thiboud’s fragrance map [1] is superimposed (filled diamonds; labels in italics) after being properly scaled and rotated (DIM1 and DIM2 stand for dimension 1 and 2, respectively, in the original publication). Dotted lines group the floral descriptors. Equivalent or related odor attributes located close to each other are joined with dashed lines. Another sensory map of scents was obtained by Chastrette et al. [13]. Most descriptors in this odor map have a direct correspondence with attributes in the B-H database. Herbaceous was paired with vegetable because the reference for the latter smells like herbs [2]. Medicinal was matched with phenolic because a significant similarity between both descriptors was found in a previous study [22]. Amber was paired with erogenic because a mixture of ambergris and costus oil was the reference material for erogenic in the B-H database. Figure 2 compares the projection of olfactory notes over the first factorial axis obtained by Chastrette et al. [13] with p1 loadings of equivalent descriptors in the B-H database. The correlation is statistically significant (r = 0.86, p < 10−4), which reveals that freshness is the most salient dimension. Sensors 2013, 13 green vegetable minty medicinal powdery jasmine tuberose aromatic clinging erogenic aldehyde citrusy sourish night evening oriental fresh spicy narcissus -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 sweet sweet fruity coconut feminine dusty lemons fresh earthy woody smoky spicy balsamic animal honey buttery floral fruity fatty watery lavender coniferous metallic tart (dry) men-husbands fern aftershave sharp-tangy camphoraceous-pine cologne green mountains minty agrestic-herbal oily honey animal autumn carnation winter medicinal woody aldehydic fruity raspberry fruity young wives-mothers orris-violet chypre fruity apple lily-of-the-valley spring morning rose lilac hyacinth gardenia summer afternoon musky anisic p1 p2 DIM1 DIM2 green vegetable minty medicinal powdery jasmine tuberose aromatic clinging erogenic aldehyde citrusy sourish night evening oriental fresh spicy narcissus -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 sweet sweet fruity coconut feminine dusty lemons fresh earthy woody smoky spicy balsamic animal honey buttery floral fruity fatty watery lavender coniferous metallic tart (dry) men-husbands fern aftershave sharp-tangy camphoraceous-pine cologne green mountains minty agrestic-herbal oily honey animal autumn carnation winter medicinal woody aldehydic fruity raspberry fruity young wives-mothers orris-violet chypre fruity apple lily-of-the-valley spring morning rose lilac hyacinth gardenia summer afternoon musky anisic p1 p2 DIM1 DIM2 fruity apple Another sensory map of scents was obtained by Chastrette et al. [13]. Most descriptors in this odor map have a direct correspondence with attributes in the B-H database. Herbaceous was paired with vegetable because the reference for the latter smells like herbs [2]. Medicinal was matched with phenolic because a significant similarity between both descriptors was found in a previous study [22]. Amber was paired with erogenic because a mixture of ambergris and costus oil was the reference material for erogenic in the B-H database. Figure 2 compares the projection of olfactory notes over the first factorial axis obtained by Chastrette et al. [13] with p1 loadings of equivalent descriptors in the B-H database. The correlation is statistically significant (r = 0.86, p < 10−4), which reveals that freshness is the most salient dimension. Another sensory map of scents was obtained by Chastrette et al. [13]. Most descriptors in this odor map have a direct correspondence with attributes in the B-H database. Herbaceous was paired with vegetable because the reference for the latter smells like herbs [2]. 3.1. Freshness as an Underlying Dimension in Odor Maps Figure 1 shows the PC1/PC2 loading plot of the B-H database, superimposed with a rotated fragrance map [1]. Although the position of comparable descriptors is not exactly coincident, both sensory maps are strikingly similar. An odor map based on equivalent dimensions has also been reported [19]. Certain disparity exists in Figure 1 for some attributes (e.g., spicy, aldehydic, honey, or animal) probably due to the lack of consensus in their interpretation. For example, eugenol and cinnamon are frequently chosen by perfumers as a reference for spicy [20], but the latter smells sweeter. Decanal was the reference for aldehydic in the B-H database. It smells antierogenic, but most aldehydes are perceived as erogenic [4], which would explain the disparity of aldehydic. Honey scents are often described in perfumery as sweet-medicinal, and Abe et al. [21] found a similarity between honey and animal, which would clarify the discrepancy of honey in Figure 1. Sensors 2013, 13 Sensors 2013, 13 469 Figure 2. Plot of fitted regression analysis of p[1] (projections on the first factorial axis) from the Firmenich database analyzed by Chastrette et al. [13] vs. p1 of the B-H database. Empty diamonds correspond to pairs of similar descriptors (e.g., ‘acidic—sourish’ are assumed to be equivalent attributes in the Firmenich and the B-H databases, respectively). Figure 3. (left) Olfactory representation adapted from the Rosace of Firmenich [14]. (right) Projections of dots over the solid line (Sfreshness) are compared with p1 loadings of equivalent attributes in the B-H database. -8 -6 -4 -2 0 2 4 6 8 10 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 powdery balsamic green fruity anisic honey minty fatty citrus spicy pyrogenous - smoky phenolic - medicinal animal woody amber - erogenic floral humus - earthy piney - coniferous acidic - sourish herbaceous - vegetable p1 (Boelens & Haring, 1981) p[1] (Chastrette et al., 1991) Figure 3. (left) Olfactory representation adapted from the Rosace of Firmenich [14]. (right) Projections of dots over the solid line (Sfreshness) are compared with p1 loadings of equivalent attributes in the B-H database. powdery -2 0 2 4 6 8 10 12 14 16 18 20 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 balsamic spicy floral fruity earthy animal woody pyrogen. green herbaceous aldehydic citrus p1 (Boelens & Haring, 1981) Sfreshness aldehydic citrus herbaceous green floral spicy fruity pyrogeneous animal balsamic powdery woody moss-earthy Figure 3 shows the 13 odor classes contained in the Rosace of Firmenich [14]. Citrus and aldehydic are located in opposite positions with respect to balsamic and powdery, which suggests that this direction (solid line in Figure 3 left) can be regarded as the fresh dimension. Scores of freshness (Sfreshness) were obtained as the distance between the orthogonal projection of odor classes (dots in the figure) over the solid line and the right end of this line, measured in a metric arbitrary scale. Most of these odor classes can be directly matched with attributes in the B-H database. Herbaceous and powdery -2 0 2 4 6 8 10 12 14 16 18 20 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 balsamic spicy floral fruity earthy animal woody pyrogen. Sensors 2013, 13 Medicinal was matched with phenolic because a significant similarity between both descriptors was found in a previous study [22]. Amber was paired with erogenic because a mixture of ambergris and costus oil was the reference material for erogenic in the B-H database. Figure 2 compares the projection of olfactory notes over the first factorial axis obtained by Chastrette et al. [13] with p1 loadings of equivalent descriptors in the B-H database. The correlation is statistically significant (r = 0.86, p < 10−4), which reveals that freshness is the most salient dimension. Another sensory map of scents was obtained by Chastrette et al. [13]. Most descriptors in this odor map have a direct correspondence with attributes in the B-H database. Herbaceous was paired with vegetable because the reference for the latter smells like herbs [2]. Medicinal was matched with phenolic because a significant similarity between both descriptors was found in a previous study [22]. Amber was paired with erogenic because a mixture of ambergris and costus oil was the reference material for erogenic in the B-H database. Figure 2 compares the projection of olfactory notes over the first factorial axis obtained by Chastrette et al. [13] with p1 loadings of equivalent descriptors in the B-H database. The correlation is statistically significant (r = 0.86, p < 10−4), which reveals that freshness is the most salient dimension. Sensors 2013, 13 green herbaceous aldehydic citrus p1 (Boelens & Haring, 1981) Sfreshness aldehydic citrus herbaceous green floral spicy fruity pyrogeneous animal balsamic powdery woody moss-earthy powdery -2 0 2 4 6 8 10 12 14 16 18 20 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 balsamic spicy floral fruity earthy animal woody pyrogen. green herbaceous aldehydic citrus p1 (Boelens & Haring, 1981) herbaceous Figure 3 shows the 13 odor classes contained in the Rosace of Firmenich [14]. Citrus and aldehydic are located in opposite positions with respect to balsamic and powdery, which suggests that this direction (solid line in Figure 3 left) can be regarded as the fresh dimension. Scores of freshness (Sfreshness) were obtained as the distance between the orthogonal projection of odor classes (dots in the figure) over the solid line and the right end of this line, measured in a metric arbitrary scale. Most of these odor classes can be directly matched with attributes in the B-H database. Herbaceous and 470 Sensors 2013, 13 pyrogeneous were paired with vegetable and smoky, respectively, based on their odor similarity. If Sfreshness is compared with p1 loadings from the B-H database, it turns out that the correlation is statistically significant (r = 0.84, p = 0.0003), which indicates that freshness is an underlying dimension of this odor map. Interestingly, the orthogonal direction to the solid line in Figure 3 is defined by floral vs. earthy, which are also located at opposite positions in Figure 1. However, the position of fruity is arguable taking into account that this descriptor is usually regarded as fresh [3]. The Field of Odors [15] displays different odor classes from citrus in one end to animal at the other, which suggests that categories are arranged according to perceptual freshness. In order to study this issue, I compared the angular position (α) of each class with the p1 loading of equivalent attributes in the B-H database. The descriptors vegetable, earthy, powdery, erogenic, aromatic, coniferous, watery, and sweet from the B-H database were paired with herbaceous, mossy, musk, amber, vanilla, terpenic, marine and caramel, respectively, from the Field of Odors, which refer to similar smells. The correlation between α and p1 is statistically significant (r = 0.82, p < 10−4), which confirms that freshness is also the most salient dimension in the Field of Odors. Curiously, a similar correlation coefficient is obtained in Figure 2 (r = 0.86) and Figure 3 (r = 0.84). It is important to keep in mind that odor descriptors are not always interpreted in the same way, which would partly explain the residual variability observed in Figures 2–4. For example, eugenol is usually regarded as the reference material for spicy, but some perfumers may choose cinnamon [20], which smells sweeter. Figure 4. Plot of fitted regression analysis of αJaubert (angular coordinate of odor classes in the semicircular Field of Odors [15], measured in degrees) vs. p1 loadings of equivalent descriptors in the B-H database. 3.2. Sensors 2013, 13 smoky—leathery [14,21,24]; powdery—warm [2]. The p1 loadings and substantivity index (SI) of these descriptors are shown in Table 1. Table 2 displays all reference materials in the B-H database (except buttery) and indicates p1, AI, SI, SGS, and evaporation index (EI). Strikingly, p1 is tightly correlated with SI (Table 1: r = −0.81, p < 10−4) and with EI0.5 (Table 2: r = 0.70, p = 0.005). The square root transformation (i.e., EI0.5) is required to normalize the data. Moreover, AI is also significantly correlated with p1 (r = 0.75, p < 10−4), SI (r = −0.63, p = 0.001), SGS (r = −0.68, p = 0.0003) and with EI0.5 (r = 0.72, p = 0.006). Taking into account that (i) antierogenic and refreshing are equivalent concepts [4], and (ii) p1 loadings can be interpreted as sensory ratings on a scale of freshness, the observed correlation among p1, AI, SI, SGS, and EI reveals that materials with a low substantivity tend to be perceived as fresh or refreshing. Table 1. Correspondence between attributes/reference materials in the B-H database and descriptors in the H&R Fragrance Guide [17]. A substantivity index (SI) is calculated according to Equation (1) based on the frequency of occurrence of odor descriptors used to describe the top (NT), middle (NM) and base note (NB) of 820 commercial perfumes. a H&R Fragrance Guide H&R Fragrance Guide Attribute a p1 Descriptor NT NM NB SI Attribute a p1 Descriptor NT NM NB SI bergamot oil 0.341 bergamot 722 0 0 0 floral −0.010 floral 86 681 47 48 fresh 0.341 fresh 576 63 8 6 honey −0.064 honey 3 50 38 69 green 0.279 green 240 35 1 7 anisic −0.081 anise 50 11 0 9 watery 0.260 watery 0 1 0 cedarwood oil −0.124 cedarwood 0 159 364 85 lemon oil 0.189 lemon 401 1 0 0 woody −0.124 woody 3 166 305 82 citrusy 0.189 citrusy 57 0 0 0 civet absolute −0.142 civet 0 0 199 100 galbanum res. Sensors 2013, 13 Relationship between Fresh Odor Character and Substantivity buttery 20 40 60 80 100 120 140 160 180 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 floral honey anisic spicy balsamic musk mossy animal woody amber vanilla sweet coniferous lavender minty fruity herbaceous citrus aldehyde marine green fresh p1 (Boelens & Haring) αJaubert buttery 20 40 60 80 100 120 140 160 180 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 floral honey anisic spicy balsamic musk mossy animal woody amber vanilla sweet coniferous lavender minty fruity herbaceous citrus aldehyde marine green fresh p1 (Boelens & Haring) αJaubert 3.2. Relationship between Fresh Odor Character and Substantivity Most reference materials and attributes in the B-H database have a direct correspondence with descriptors used by the H&R guide (Table 1). Some attributes were paired taking into account their similarity in perfumery: erogenic—sensual; animal—castoreum [23]; earthy—mossy [21]; 471 a Reference materials (in italics) and descriptors are listed by decreasing order of p1 (loadings in the formation of the first principal component). The term ‘res.’ stands for resinoid. The correspondence between descriptors and references used by Boelens & Haring [12] is indicated in Table 2. b Peach is the fruity descriptor most frequently encountered in the H&R guide. c Pine and fir trees are conifers and their essential oil smells alike. d Peppermint or spearmint. e Mixture of ambergris and costus oil. f Clove oil contains >85% of eugenol [25]. g Tonka is the second attribute (after vanilla) most frequently associated to sweet in the H&R guide. h Mixture of musk ketone and coumarin. Sensors 2013, 13 Table 2. Substantivity (SI: substantivity index from Table 1; SGS: substantivity value from www.thegoodscentscompany.com; EI: evaporation index [7]) and fresh odor character (p1: loadings in the formation of PC1; AI: antierogenic index [4]) of reference materials used by Boelens & Haring [12]. Values in bold appear as outliers in Figure 5. Freshness Substantivity Reference material Attribute p1 AI SI SGS EI Bergamot oil Fresh 0.341 23 0 450 Methyl 2-octynoate Green 0.279 100 7 240 Styrallyl acetate Sourish 0.277 100 8 Cyclamen aldehyde Watery 0.260 100 72 Lemon oil Citrusy 0.189 83 0 4 800 d Galbanum resinoid Tart (dry) 0.180 100 3 45 Aldehyde C-10 Aldehyde 0.177 50 0 36 10-undecen-1-ol Fatty 0.166 60 152 Bay oil Metallic 0.165 −20 34 364 140 Lavender oil Lavender 0.128 50 6 12 410 Clary sage oil Vegetable 0.126 7 21 36 180 Hexadecanal Fruity 0.085 −100 8 Fir needle oil Coniferous 0.061 100 82 24 580 Peppermint oil Minty 0.045 100 a 3 16 300 Oakmoss resinoid Earthy −0.008 −35 100 400 Jasmine absolute Floral −0.010 −100 50 280 50 Methyl salicylate Medicinal −0.052 −15 8 Ethyl phenylacetate Honey −0.064 69 312 Fennel oil Anisic −0.081 9 24 150 Cedarwood oil Woody −0.124 10 85 388 30 Civet absolute Animal −0.142 −100 100 400 Cade oil Smoky −0.144 −80 b 100 400 10 Ambergris + costus oil Erogenic −0.161 −100 100 316c Eugenol Spicy −0.182 −100 50 52 20 e Patchouli oil Dusty −0.197 −100 83 400 4 Vanillin Aromatic −0.221 −100 100 400 Olibanum resinoid Balsamic −0.249 −100 99 284 Heliotropin Sweet −0.280 −23 88 212 Musk ketone+coumarin Powdery −0.323 −60 100 400 a Value of spearmint (both plants are botanically related). b Value of birch tar oil (both smell smoky and are obtained by destructive distillation of wood). c Value of costus oil (SGS of ambergris is not available). d Value of lime oil. e Value of clove oil. a Value of spearmint (both plants are botanically related). b Value of birch tar oil (both smell smoky and are obtained by destructive distillation of wood). c Value of costus oil (SGS of ambergris is not available). d Value of lime oil. e Value of clove oil. In Thiboud’s database, certain material was described with a given attribute as main or ancillary descriptor if that odor character was clearly recognizable or just noticeable, respectively. Sensors 2013, 13 0.180 galbanum 113 7 0 3 animal −0.142 castoreum 0 0 83 100 tart (dry) 0.180 dry 19 63 0 38 smoky −0.144 leathery 0 1 104 100 aldehyde 0.177 aldehydic 140 0 0 0 amber.+costus e −0.161 ambery 0 0 137 100 bay oil 0.165 bay 5 11 0 34 erogenic −0.161 sensual 0 0 70 100 lavender oil 0.128 lavender 204 26 0 6 eugenol −0.182 clove f 0 26 0 50 clary sage oil 0.126 clary sage 83 62 0 21 spicy −0.182 spicy 163 244 1 30 vegetable 0.126 herbaceous 165 7 2 3 patchouli oil −0.197 patchouli 0 136 253 83 fruity 0.085 fruity 151 24 3 8 vanillin −0.221 vanilla 0 0 301 100 fruity 0.085 peach b 149 7 0 2 olibanum res. −0.249 olibanum 0 3 117 99 fir needle oil 0.061 fir c 0 24 41 82 balsamic −0.249 balsamic 0 0 76 100 coniferous 0.061 pine c 1 87 0 49 sweet −0.280 sweet 0 50 157 88 peppermint oil 0.045 peppermint d 29 2 0 3 sweet −0.280 tonka g 0 1 287 100 oakmoss res. −0.008 oakmoss 0 0 139 100 musk+coumarin h −0.323 musk 0 0 698 100 earthy −0.008 mossy 0 0 252 100 powdery −0.323 powdery 0 0 376 100 jasmine absolute −0.010 jasmine 0 671 0 50 powdery −0.323 warm 0 0 137 100 472 Sensors 2013, 13 Sensors 2013, 13 Because of this, odor descriptors can be coded numerically as 0 if that term is not applied, 1 if it is applied as ancillary descriptor, and 2 for the main attributes. These coded descriptions are shown in Table 3 for 6 relevant attributes: (i) fresh and citrus, which account for the fresh odor character, (ii) balsamic and oriental, which are dissimilar to fresh, and (iii) floral and agrestic, with an intermediate refreshing tone (Figure 1). By adding the values in columns fresh, citrus, floral and agrestic, and subtracting the columns balsamic and oriental, it results a numeric variable from −3 to 6 that can be interpreted as sensory scores on a fresh scale (Sfresh in Table 3). Interestingly, Sfresh is significantly correlated with SI (r = −0.85, p < 10−4), CS (r = −0.77, p < 10−4) and SGS (r = −0.61, p < 10−4). The tight correlation once again evidences that fresh odor character reflects odorant tenacity. 473 Sensors 2013, 13 Sensors 2013, 13 Figure 5. Relationship between fresh odor character and substantivity of reference materials used by Boelens and Haring [12]: scatterplot of p1 (loadings in the formation of PC1) vs. antierogenic index (AI) and substantivity parameters: SI, SGS and EI (values in Table 2). AI, SGS and EI were conveniently transformed as indicated in the legend to range approximately on a 0–100 scale. The fitted regression line (r = −0.81) was obtained after discarding 8 outliers (filled points, highlighted in bold in Table 2). Dashed lines: prediction limits with a confidence level of 95%. Substantivity and AI values of additional materials not included in Table 3 are shown in Table 4. If both tables are merged, it turns out that SI is strongly correlated with CS (r = 0.88, p < 10−4) and SGS (r = 0.83, p < 10−4), but SGS yields a weaker correlation with EI0.5 (r = −0.61, p = 0.0001) probably because many EI values are missing for the least volatile materials. h l ifi d d ( ) iddl ( ) d b 0 10 20 30 40 50 60 70 80 90 100 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 100-3.5·EI0.5 SI SGS/4 (100-AI)/2 p1 (Boelens & Haring, 1981) 0 10 20 30 40 50 60 70 80 90 100 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 100-3.5·EI0.5 SI SGS/4 (100-AI)/2 p1 (Boelens & Haring, 1981) Substantivity and AI values of additional materials not included in Table 3 are shown in Table 4. If both tables are merged, it turns out that SI is strongly correlated with CS (r = 0.88, p < 10−4) and SGS (r = 0.83, p < 10−4), but SGS yields a weaker correlation with EI0.5 (r = −0.61, p = 0.0001) probably because many EI values are missing for the least volatile materials. Substantivity and AI values of additional materials not included in Table 3 are shown in Table 4. If both tables are merged, it turns out that SI is strongly correlated with CS (r = 0.88, p < 10−4) and SGS (r = 0.83, p < 10−4), but SGS yields a weaker correlation with EI0.5 (r = −0.61, p = 0.0001) probably because many EI values are missing for the least volatile materials. Substantivity and AI values of additional materials not included in Table 3 are shown in Table 4. Sensors 2013, 13 If both tables are merged, it turns out that SI is strongly correlated with CS (r = 0.88, p < 10−4) and SGS (r = 0.83, p < 10−4), but SGS yields a weaker correlation with EI0.5 (r = −0.61, p = 0.0001) probably because many EI values are missing for the least volatile materials. Poucher [18] classified odorants as top notes (CS < 15), middle notes (15 ≤ CS ≤ 60) and base notes (CS >60). Interestingly, most materials listed in Table 3 up to elemi oil are basically encountered in top notes (SI << 50) and their CS is below 15. Moreover, materials from vetiver oil to tolu can be regarded as base notes (SI ≈ 100) and they exhibit a high substantivity (CS >> 60). Thus, Poucher’s classification based on CS values seems adequate. Some materials with a low CS (top notes) like mimosa absolute, narcissus absolute or copaiba present a high substantivity according to The Good Scents Company (SGS = 400), which suggests that such particular values might not be reliable. Actually, the website does not indicate how this parameter was obtained. Moreover, 5 out of the 8 outliers highlighted in Figure 5 correspond to SGS values. Sensors 2013, 13 Sensors 2013, 13 Sensors 2013, 13 474 Table 3. Substantivity parameters and fresh odor character of the 44 natural materials contained in the semantic odor profile database compiled by Thiboud [1]. Table 3. Substantivity parameters and fresh odor character of the 44 natural materials contained in the semantic odor profile database compiled by Thiboud [1]. contained in the semantic odor profile database compiled by Thiboud [1]. Sensors 2013, 13 Substantivity Odor Character Descriptors f Freshness Material a CS b SI c SGS d EI e Ifresh Icitrus Iagrest Ifloral Ibalsam Ioriental Sfresh g AI h Petitgrain bergamot oil 3 4 28 170 2 2 1 1 0 0 6 100 Bergamot oil 6 0 450 2 2 1 0 0 0 5 23 Jonquil absolute 24 2 0 1 2 0 0 5 −100 Lime oil 2 0 20 800 2 2 0 0 0 0 4 80 Mandarin oil 2 4 8 2 2 0 0 0 0 4 −27 Coriander oil 3 13 8 200 2 0 2 0 0 0 4 −5 Grapefruit oil 6 264 2 2 0 0 0 0 4 Thyme oil 7 39 172 220 2 0 2 0 0 0 4 100 Lemon oil 8 0 4 2 2 0 0 0 0 4 83 Orange Florida oil 11 1 140 970 2 2 0 0 0 0 4 10 Mimosa absolute 14 400 0 0 2 2 0 0 4 −30 Mugwort oil 9 4 16 1 0 2 0 0 0 3 Chamomile oil, Roman 10 112 530 1 0 2 0 0 0 3 −100 Wormwood oil (absinthe) 10 212 500 0 0 2 1 0 0 3 Violet leaf absolute 18 23 400 20 1 0 0 2 0 0 3 0 Cumin oil 4 7 0 0 2 0 0 0 2 −40 Myrtle oil 4 400 0 0 2 2 2 0 2 Rose oil (Bulgarian) 8 50 168 10 1 0 0 1 0 0 2 −80 Galbanum oil 11 3 72 540 1 0 1 0 0 0 2 100 Carrot seed oil 11 96 80 0 0 2 0 0 0 2 −60 Elemi oil 13 20 720 2 1 1 0 2 0 2 100 Clary sage oil 20 21 36 180 0 0 2 0 0 0 2 7 Geranium Bourbon oil 29 50 28 120 1 0 0 1 0 0 2 −70 Tuberose absolute 43 50 304 0 0 0 2 0 0 2 −100 Cardamom oil 30 16 320 0 0 2 0 0 1 1 75 Rose absolute (French) 43 50 168 0 0 0 1 0 0 1 −90 Cedarwood oil Virginia 8 85 388 30 0 0 1 0 0 0 1 10 Narcissus absolute 11 49 400 0 0 0 2 1 0 1 Nutmeg oil 11 52 550 0 0 1 0 0 1 0 −40 Ginger 7 292 150 0 0 0 0 0 0 0 0 Copaiba 6 400 0 0 0 2 2 0 0 Clove bud oil 22 50 188 20 0 0 0 1 0 1 0 −100 Cinnamon leaf oil 22 56 304 30 0 0 1 0 0 1 0 Vetiver oil Bourbon 100 86 400 0 0 0 0 0 0 0 100 Patchouli oil 100 83 400 4 0 0 1 0 0 1 0 −100 Cistus oil (labdanum) 100 100 400 0 0 2 0 2 0 0 −80 Styrax 100 98 400 0 0 0 2 2 0 0 −100 Pepper (black) oil 100 48 400 0 0 0 0 0 1 −1 Opoponax 90 100 400 20 0 1 0 0 2 1 −2 10 Allspice (pimento berry) 100 400 50 0 0 0 0 2 0 −2 −40 Sandalwood oil 100 88 400 0 0 0 0 2 1 −3 −63 Peru 100 400 0 0 0 0 2 1 −3 −100 Benjoin 100 100 400 0 0 0 0 2 1 −3 −100 Tolu 100 100 400 0 0 0 0 2 1 −3 −100 a Sorted by decreasing value of Sfresh. Sensors 2013, 13 b Coefficient of substantivity [18]. c Substantivity index deduced from the H&R Fragrance Guide according to Equation (1). d Substantivity (hours) according to www.thegoodscentscompany.com. e Evaporation index [7]. f Indicator variables corresponding to 6 relevant descriptors. The value 2 indicates a main descriptor in the semantic odor profile, and the value 1 indicates an ancillary descriptor. g Scores of freshness calculated as: Ifresh + Icitrus + Iagrestic + Ifloral − Ibalsamic − Ioriental. h Antierogenic index [4]. a Sorted by decreasing value of Sfresh. b Coefficient of substantivity [18]. c Substantivity index deduced from the H&R Fragrance Guide according to Equation (1). d Substantivity (hours) according to www.thegoodscentscompany.com. e Evaporation index [7]. f Indicator variables corresponding to 6 relevant descriptors. The value 2 indicates a main descriptor in the semantic odor profile, and the value 1 indicates an ancillary descriptor. g Scores of freshness calculated as: Ifresh + Icitrus + Iagrestic + Ifloral − Ibalsamic − Ioriental. h Antierogenic index [4]. Sensors 2013, 13 Sensors 2013, 13 475 Table 4. Substantivity parameters and antierogenic index (AI) of materials not included in Table 3 (sorted approximately by increasing order of substantivity). Substantivity a Substantivity a Material CS SI SGS EI AI Material CS SI SGS EI AI Lavender oil 4 6 12 410 50 Cascarilla oil 29 248 200 0 Neroli oil 5 4 116 210 17 Orange flower absolute 31 15 400 −50 Laurel leaf oil 9 750 Cinnamon bark oil 24 56 372 120 −100 Peppermint oil 8 3 16 300 100 Lily of the valley; orchid 50 Rosewood oil 2 16 12 250 −8 Carnation absolute 50 −30 Hyacinth absolute 11 16 40 Ylang-ylang oil 32 50 140 90 −80 Basil oil 14 6 225 −50 Ambrette seed oil 30 121 20 −100 Palmarosa oil 14 60 50 0 Orris resinoid 40 b 50 −60 Juniper berry oil 18 700 Jasmine absolute 43 50 280 50 −100 Gardenia 14 Heliotrope 70 Rosemary oil 21 5 4 820 100 Civet absolute 79 100 400 −100 Marjoram oil 18 12 600 Tonka resinoid 100 100 400 −55 Verbena resinoid 19 0 Castoreum absolute 100 100 400 −100 a CS: coefficient of subst.; SI: subst. index; SGS: subst. from www.thegoodscentscompany.com; EI: evaporation index. b Average CS of orris concrete, orris absolute and orris oleo-resin. Sensors 2013, 13 Most materials in Table 2 with p1 > 0 smell antierogenic (AI > 0) and, as a result, the correlation between p1 and AI is statistically significant (r = 0.75, p < 10−4). In Table 3, AI is positively correlated with Sfresh (r = 0.42, p = 0.01). By merging Tables 3 and 4, it turns out that AI is also correlated with SI (r = −0.60, p < 10−4), SGS (r = −0.50, p = 0.0004) and CS (r = −0.45, p = 0.0006). Given the relationship between substantivity and freshness, the observed correlations support Jellinek’s interpretation of antierogenous and refreshing as equivalent concepts in odor description. Nonetheless, most floral odors were regarded by Jellinek [4] as erogenic (AI ≤ −50), which is somewhat unexpected because floral scents present an intermediate refreshing tone (Figure 1) and substantivity (SI ≈ 50 in Table 4). The reason might be that the three perfumers who obtained AI values were probably men. Floral scents, which are perceived as feminine, may be regarded by men as erogenic (i.e., arousing sexual desire for the opposite sex). Thus, a larger panel should have been used with the same number of male and female individuals. Actually, Jellinek [4] recognized that his results were preliminary because of the difficulties in reaching an agreement among observers. 4.2. Proposed Hypothesis to Explain the High Substantivity of Erogenic Odors According to Jellinek [4], erogenous scents are those arousing sexual desire. They are basically reminiscent of the human body smell (i.e., odors released by our skin) and somewhat resemble the smell of materials obtained from animals (e.g., civet, castoreum, ambergris or Tonkin musk). This similarity between erogenic and animal is apparent in the B-H database because both descriptors are correlated (r = 0.46) and determine an independent dimension of odor character (see Figure 3 of [2]). Body odorants are obviously easily retained by the skin and, hence, they present a high substantivity. The same applies to animalic materials. This reasoning is consistent with the observed negative correlation between antierogenic odor character and substantivity parameters (Tables 2–4). Taking into account that substantivity depends on physicochemical properties, the tight correlation of substantivity with freshness implies that, in the case of aroma chemicals, their refreshing odor character could be estimated by means of electronic noses [32] and also based on physical properties. In the B-H database, the projections of odorants over PC1 can be interpreted as estimated scores of freshness on a numeric scale. Work in progress suggests that these scores can be predicted from a set of 20 physicochemical parameters and molecular descriptors using multiple linear regression, with a coefficient of determination R2 ≈ 0.52. This preliminary result further suggests that the correlation of fresh odor character with substantivity is primarily innate and evolutionarily hardwired. Nonetheless, olfactory perception of fragrance raw materials can drastically change when applied in a mixture (e.g., a perfume), in a paper blotter or on the skin, thus possibly changing its substantivity. Further research will be necessary to study this issue. The development of standard psychophysical procedures to obtain accurate values of substantivity and scores of freshness for perfumery materials is strongly encouraged. Sensors 2013, 13 476 Sensors 2013, 13 some studies that have discussed several odor maps of perfumery scents [13,28]. Edwards classifies 12% of fragrances in the citrus, green, watery or fruity categories, which are grouped in the fresh family [2]. But fresh is not considered by most fragrance companies as a main olfactory family of perfumes (see Table 1 of [29]). This category is also missing in the classification of fragrances proposed by the French Society of Perfumers [30], Fragrantica, or Osmoz by Firmenich, among many others. The reason could be the lack of consensus in the interpretation of fresh in odor description, as discussed below. Many psychophysical studies have reported that pleasantness is the most salient dimension when a wide range of odors are assessed (for review, see [31]). This dimension did not clearly show up in a previous study of the B-H database by checking the correlation between loadings and hedonic tones from the literature [11]. Although the same methodology could have been applied here, there is enough evidence to support that freshness is the basic construct underlying Figures 2–4. Actually, animal, mossy or earthy are often described as disagreeable odors, but these descriptors appear close to sweet or spicy that are regarded as pleasant. 4.1. Freshness as a Salient Dimension of Odor Perception Smell is a complex multidimensional perception difficult to measure and describe [26]. The underlying constructs of this multivariate space are still poorly understood. A relatively easy procedure is proposed here to study if freshness is a salient dimension of odor maps, by assigning a refreshing tone to odor character descriptors. This issue is of interest for developing standard sensory maps of cosmetic odors. Different works have reported that freshness is a latent dimension in the perceptual space of fragrances (for review, see [2]). However, this idea is not well established yet in olfactory research. Actually, neither fresh nor refreshing are included in the comprehensive Dravnieks’ list of 146 terms most commonly used in odor description [27]. Moreover, the fresh dimension was not considered by 4.4. Interpretation of ‘Fresh’ vs. ‘Refreshing’ in Odor Description The Collins Dictionary & Thesaurus [44] considers the following terms as synonyms of refreshing: new, original, novel, unusual, stimulating, innovative, cooling, invigorating, etc.. Probably based on the semantic associations, different studies have found that refreshing and invigorating are similar odor descriptors [19]. Actually, a fresh fragrance is usually considered invigorating, nature inspired, reminiscent of early morning air or sea breeze and it is typified by green, citrus notes [1]. Chrea et al. [45] found that refreshed and revitalized were interpreted in the same way by French speakers, and both terms were classified in an emotional factor of odor perception called energizing/refreshing. The same association was found in a further study carried out in Liverpool (UK) and Singapore, and the underlying factor was named energy [42]. Refreshing and stimulating are regarded as orthogonal dimensions of the Odor Effects Diagram [4], but this issue is arguable. In the odor map proposed by Tisserand [46], vivacity and stimulant appear next to erogenic, which is also debatable based on the reported empirical evidence. Given the multiple meanings of refreshing, this term might be interpreted differently according to culture, language, context, age, and experience. This issue may explain why Jellinek [4] regarded refreshing and fresh as different odor descriptors. The former was associated with antierogenous and the latter with green–herbaceous odors, which were supposed to produce antierogenous and stimulating effects. However, this criterion is arguable given the semantic association between refreshing and stimulating. Moreover, odors considered by Jellinek [4] as stimulating are better described as masculine [2]. Although fresh and refreshing may have different connotations in odor description according to language, their different meaning is not clearly established in perfumery and, hence, in my opinion both terms can be used as synonyms for clarity purposes. Sensors 2013, 13 477 Sensors 2013, 13 The human fetus starts the learning process of smelling in the prenatal environment [38]. Flavors from the mother’s diet during pregnancy are transmitted to amniotic fluid and swallowed by the fetus [39], which may provide the foundation for cultural and ethnic differences in odor preference. The human fetus starts the learning process of smelling in the prenatal environment [38]. Flavors from the mother’s diet during pregnancy are transmitted to amniotic fluid and swallowed by the fetus [39], which may provide the foundation for cultural and ethnic differences in odor preference. Despite the general belief that odor perception is basically shaped by learned associations, some studies reveal that certain aspects of olfactory perception might be innate and hardwired. For example, rats show an unconditioned defensive response to the odor of predators [40]. Moreover, recent works have revealed that the hedonic odor character is partly determined by molecular structure [31]. Interestingly, freshness is the most salient perceptual dimension of cosmetic scents and it is also somewhat encoded by molecular structure given the correlation with substantivity. Although many efforts have been carried out to relate molecular structure to the perceived smell [41], odor-structure relationships focused on the fresh olfactory quality have not been reported yet as far as I know. Ferdenzi et al. [42] found that the refreshing/energizing dimension of smell was common to three tested cultures, but other constructs were different. The neurological basis of innate and learned responses to odors has been investigated in mice [43]. 4.4. Interpretation of ‘Fresh’ vs. ‘Refreshing’ in Odor Description 4.3. Conditioned and Unconditioned Factors Influencing Odor Quality Perception It is well established that odor quality perception is strongly influenced by memory [33], learning [34] and culture [35,36]. Social effects affecting the perception of fragrances have also been discussed [37]. Sensors 2013, 13 478 Sensors 2013, 13 Sensors 2013, 13 dimension underlies consumers’ perceptions as to whether a fragrance seems to be more suited for formal evening wear or for informal daytime wear. People feel more comfortable wearing during the day fragrances that do not transmit an erotic message (i.e., smelling antierogenic), which seems quite obvious. Thus, refreshing scents are preferred for daytime wear because they are not perceived as erotic, and daytime is associated with stimulating activities, energy, vigorous actions, etc. Although fresh was long ago proposed as an independent category of odors [47], this particular odor quality is rather subjective and it can be interpreted differently. According to Müller [24], fresh is generally associated in European regions with lemon, lavender, green notes and light floral components. Different studies support this interpretation [2]. For example, a so-called light dimension determined by the descriptors fresh and lemon was found by analyzing numeric odor profiles of 40 compounds rated by French individuals [48]. However, Müller states that sweet and powdery perfumes are also considered as fresh in North America [24]. Taking into account that sweet and powdery are perceived as dissimilar to fresh [2], it is of interest to discuss this controversy. Fresh can be defined in English as (i) newly made or obtained, novel, original, additional, (ii) not stale or deteriorated, (iii) not canned, frozen or preserved, etc. [49]. Consistent with this definition, Schiffman et al. [50] regarded fresh and rotten as two polarities of the same semantic differential scale. By contrast, the first meaning of fresh in French (frais) and Spanish (fresco) is ‘moderately cool’ [51,52], which is antonym of warm. The semantic association between fresh and cool also exists in English, but only when applied to weather, wind or breeze. Thus, fresh air means clean and cool, found outside buildings rather than in a room [49]. In French, the similarity between refreshed, clean and cooling in odor perception has also been reported [45]. I believe that fresh was used originally in the sense of cool, probably long ago by French perfumers. Unfortunately, fresh and cool cannot be regarded nowadays as synonyms because cool has acquired many connotations in perfumery [1]. Cool is often applied to the perception of freshness associated with a trigeminal effect, as it is the case of minty scents. Actually, minty yields the highest correlation with ‘cool, cooling’ (r = 0.82) in the database of Dravnieks [27]. Sensors 2013, 13 Moreover, Harper [53] selected two odorants sharing a minty–fresh odor character (menthol and camphor) as references for ‘cool, cooling’. In order to avoid confusion, I would recommend using fresh, refreshing and antierogenic as synonyms, referred to scents perceived as cooling and not associated with a trigeminal effect. 4.5. Controversies in the Interpretation of ‘Fresh’ in Odor Description The association between refreshing and energizing might correspond to the activation dimension of emotion (arousal) [45], but another interpretation is possible. According to Jellinek [29], the refreshing Sensors 2013, 13 Sensors 2013, 13 Sfresh (Table 3). In the B-H database, the negative correlation between fresh and erogenic (r = −0.24, p < 10−4) partly supports Jellineks’ interpretation, but fresh yields the most negative correlation with powdery (r = −0.58). The reference material for this descriptor smells warm [2], which suggests that the perceptual dimension of freshness is better interpreted as a fresh vs. warm polarity rather than fresh vs. erogenous. This interpretation is supported by further evidence from the literature. Warm perfumes have a high proportion of animalic ingredients [24] and are often described as rich and tenacious [1]. Harper [53] selected costus and amber as standards for warm. Both materials were regarded by Boelens & Haring [12] as references for erogenic, which implies that warm, erogenic and animal are similar descriptors in perfumery. Actually, in a two-dimensional odor map recently reported, warm and sexy appeared close to each other [45]. The origin of this similarity from an evolutionary perspective is not well understood yet, but some hypotheses have been proposed [54]. In a reported analysis of the language of French perfume advertising, it was found that the dimension warm vs. fresh was the most important axis of the semantic field of fragrances [55]. Chrea et al. [45] obtained a two-dimensional odor map from numeric ratings of 24 odorant samples according to 73 affective terms in French, and it was found that refreshed and warm appeared in opposite positions within the pleasant cluster of terms. Jellinek [29] developed a two-dimensional map of fragrances and one of the dimensions was interpreted as warm vs. cool. In a previous study, 140 commercial perfumes were also classified according to a cool vs. warm dimension [56]. Given that cool and warm are terms referring to temperature, these studies support the interpretation of fresh and warm as opposite polarities of a perceptual odor dimension somehow associated with temperature. Interestingly, colors can also be described on a warm vs. cool scale [57]. 4.6. Fresh and Warm as Opposite Polarities of a Perceptual Odor Dimension Based on the semantic meaning, French and Spanish people would easily agree that fresh and warm are two opposite poles of the same perceptual odor dimension, because they will readily associate fresh with cool. The same link probably exists in Japanese, because a fresh dimension that discriminated cold and sour with respect to warm, sweet and sexy was found by Japanese researchers who analyzed with PCA numeric odor profiles of 37 aroma chemicals according to 55 descriptors [32]. Conversely, in English, not everybody would understand the dissimilarity fresh vs. warm unless it is indicated that fresh is used as a synonym of cool. The lack of consensus in the semantic interpretation of freshness might explain why this perception is not easy to define in perfumery [1,24]. Refreshing and erogenous were regarded by Jellinek [4] as opposite polarities of the same dimension. Consistent with this criterion, AI values are positively correlated with p1 (Table 2) and 479 Sensors 2013, 13 480 The olfactory map shown in Figure 1 might reflect an underlying structure of odor perception that discriminates those scents most typically encountered in the different seasons in temperate climates: fresh (winter), floral (spring), warm (summer), and earthy (autumn). The position of summer next to fruity in Figure 1 might suggest that summer scents are perceived as fresh, which is misleading. The correct interpretation is that fresh scents are preferred in summer, probably because they evoke cool temperatures. Conversely, warm and animalic fragrances are preferred in winter [29]. The work of Tisserand [46] leads to the hypothesis that human olfactory perception was shaped throughout evolution in temperate climates to recognize the characteristic odors of each season. This theory is debatable because our sense of smell did not evolve under a constant climate. Moreover, it is uncertain if the fresh odor character is perceived differently by human races that developed in climates with slight weather variations along the year, or by people living in such climates. The proposed theory seems very simplistic given the complexity of factors affecting olfactory perception, but it deserves to be further investigated because it might provide the fundamental basis to better understand certain psychological aspects of olfactory perception. For example, it is still poorly understood to what extent the perception of freshness depends on culture, language, context, age, etc. It would also be of interest to study why sweet fragrances are preferred by women, or why cosmetic odors are perceived as masculine vs. feminine. 5. Conclusions Odor freshness is an underlying dimension in the perceptual space of perfumery scents, as further studied here. Fresh odors are typical in top notes, while those most dissimilar to fresh are encountered in base notes. This relationship between freshness and substantivity is well known by perfumers, but it has not received much attention yet by the scientific community. The present work reports that the correlation between perceptual freshness and odorant substantivity is quite strong (p1 vs. SI in Table 1: r = −0.82; Sfresh vs. SI in Table 3: r = −0.85). This result is important because substantivity depends on physicochemical properties, which provides the fundamental basis to predict odor freshness based on molecular structure. The interpretation of fresh in perfumery as synonym of cool and antonym of warm suggests that this psychological dimension might be associated with temperature and may reflect a structure of odor perception shaped throughout evolution in temperate climates. Thus, fresh would correspond to scents typically encountered in the cool season, while warm would be evoked by odors found in nature during summer. 3. Edwards, M. Fragrances of the World: Parfums du Monde 2010, 26th ed.; Fragrances of the World: Sidney, BC, Canada, 2010; pp. 6–8. 4.7. Proposed Hypotheses to Explain the Fresh/Warm Dimension of Odor Character Taking into account the alternation of seasons in temperate climates, with cool temperatures in winter and warmer conditions in summer, the ability of human olfaction to perceive scents as fresh vs. warm might be explained from an evolutionary perspective. In summer, as a result of the hot weather, only those odorants with higher substantivity would be found in the natural environment, while light scents could be encountered at cooler temperatures. This hypothesis is consistent with the fact that fresh scents are characterized by a lower substantivity, while the opposite applies to warm odors. The idea that olfactory perceptual space in humans might reflect chronobiological annual rhythms was put forward by Tisserand [46]. Starting from the four elements (air, fire, earth and water) long ago proposed by Greek philosophers like Hippocrates, he discussed their associations with the four seasons and with moods evoked by essential oils. It was found that the resulting structure had certain resemblance to the Odor Effects Diagram [4]. This diagram has been recently discussed in detail [2]. Tisserand suggested that erogenic scents evoked odors typically encountered in summer, while those associated with winter such as watery were regarded as antierogenic. Autumn was associated with earthy, which is intuitively appealing because this descriptor has a neutral refreshing tone (p1 ≈ 0). Moreover, earthy is the attribute most dissimilar to floral (Figure 1), and the latter would correspond to the typical smells of spring. 2. Zarzo, M.; Stanton, D.T. Understanding the underlying dimensions in perfumers’ odor perception space as a basis for developing meaningful odor maps. Atten. Percept. 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Ou, L.C.; Luo, M.R.; Sun, P.L.; Hu, N.C.; Chen, H.S.; Guan, S.S.; Woodcock, A.; Caivano, J.L.; Huertas, R.; Treméau, A.; Billger, M.; Izadan, H.; Richter, K. A cross-cultural comparison of colour emotion for two-colour combinations. Color Res. Appl. 2012, 37, 23–43. Sensors 2013, 13 © 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
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Modelling of tropospheric NO2 using WRF-Chem with optimized temporal NOx emission profiles derived from in-situ observations - Comparisons to in-situ, satellite, and MAX-DOAS observations over central Europe For stations within Germany, biases of −5.9 % to +50.3 % were obtained when comparing average noontime NO2 concentrations at different altitudes. Outside of Germany, where lower resolution emission data was used, biases of up to +78.6 % were observed. Overall, the study demonstrates that temporal modulation of emission data is crucial for modelling 15 tropospheric NO2 realistically. Modelling of tropospheric NO2 using WRF-Chem with optimized temporal NOx emission profiles derived from in-situ observations - Comparisons to in-situ, satellite, and MAX-DOAS observations over central Europe Leon Kuhn1, 2, Steffen Beirle2, Vinod Kumar2, Sergey Osipov2, 4, Andrea Pozzer2, Tim Bösch5, Rajesh Kumar3, and Thomas Wagner1, 2 Rajesh Kumar3, and Thomas Wagner1, 2 1Institute for Environmental Physics, University of Heidelberg, Germany 2Max Planck Institute for Chemistry, Mainz, Germany 3National Center for Atmospheric Research, Boulder, United States of America 4Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia 5Institute for Environmental Physics, University of Bremen, Germany 1Institute for Environmental Physics, University of Heidelberg, Germany 2Max Planck Institute for Chemistry, Mainz, Germany 3National Center for Atmospheric Research, Boulder, United States of America 4Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia 5Institute for Environmental Physics, University of Bremen, Germany 1Institute for Environmental Physics, University of Heidelberg, Germany 2Max Planck Institute for Chemistry, Mainz, Germany 3National Center for Atmospheric Research, Boulder, United States of America 4Red Sea Research Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia 5Institute for Environmental Physics, University of Bremen, Germany Correspondence: Leon Kuhn (l.kuhn@mpic.de) Abstract. We present a WRF-Chem simulation over central Europe with a high spatial resolution of 3 km×3 km and a focus on nitrogen dioxide (NO2). A regional emission inventory, issued by the German Environmental Agency, with a spatial resolution of 1 km×1 km is used. We demonstrate, that by precise temporal modulation of the emission data (use of "temporal profiles"), significant improvement in model accuracy over existing simulations is achieved. Simulated NO2 surface concentrations are compared to measurements from a total of 275 in-situ measurement stations in Germany, where the model was able to reproduce 5 average noontime NO2 concentrations with a bias of +0.9 % and R = 0.76. A comparison between modelled NO2 vertical column densities (VCDs) and satellite observations from TROPOMI (TROPOspheric Monitoring Instrument) is conducted, where crucial aspects of the observation process, such as altitude-dependent NO2 sensitivity as well as the influence of clouds and a priori assumptions of the retrieval, are taken into account. Simulations and satellite observations are shown to agree with a 5 10 model bias of −6.6 % and R = 0.84 for monthly means. Lastly, simulated NO2 concentration profiles are compared to profiles 10 obtained from Multiaxis Differential Optical Absorption Spectroscopy (MAX-DOAS) measurements of five European ground stations using the profile retrieval algorithms from the Mexican MAX-DOAS fit (MMF) and the Mainz Profile Algorithm (MAPA). https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Modelling of regional atmospheric chemistry and transport (RCT) is an important discipline in the field of air quality research. While observational data is often only available at coarse spatial or temporal resolutions, model data can be generated on regular 1 Introduction Modelling of regional atmospheric chemistry and transport (RCT) is an important discipline in the field of air quality research. While observational data is often only available at coarse spatial or temporal resolutions, model data can be generated on regular 1 1 (2015); Chowdhury et al. 30 (2021)). Monitoring and predicting realistic distributions of NO2 in the troposphere is therefore of ongoing political and scientific interest. Past modelling efforts with focus on tropospheric NO2 have typically resulted in very similar general outcomes: while researchers found good agreement between modelled and observed meteorological data (such as wind speeds and air temper- been widely recognized among the scientific community (see e.g. Faustini et al. (2014); Mills et al. (2015); Chowdhury et al. 30 (2021)). Monitoring and predicting realistic distributions of NO2 in the troposphere is therefore of ongoing political and scientific interest. Past modelling efforts with focus on tropospheric NO2 have typically resulted in very similar general outcomes: while researchers found good agreement between modelled and observed meteorological data (such as wind speeds and air temper- been widely recognized among the scientific community (see e.g. Faustini et al. (2014); Mills et al. (2015); Chowdhury et al. 30 (2021)). Monitoring and predicting realistic distributions of NO2 in the troposphere is therefore of ongoing political and scientific interest. Past modelling efforts with focus on tropospheric NO2 have typically resulted in very similar general outcomes: while researchers found good agreement between modelled and observed meteorological data (such as wind speeds and air temper- ature), systematic disagreements between modelled surface NO2 concentrations and in-situ observations from ground based 35 measuring stations were found. Visser et al. (2019) report on the results of a WRF-Chem simulation over central Europe in which noontime NO2 surface concentrations and VCDs are underestimated by 38.5 % and approximately 15 %, respectively. In a comparison of monthly mean NO2 VCDs, R-values between 0.82 and 0.92 were obtained. The authors identify an un- derestimation of soil emissions in their emission inventory (TNO-MACC-III, short for "Monitoring Atmospheric Composition ature), systematic disagreements between modelled surface NO2 concentrations and in-situ observations from ground based 35 measuring stations were found. Visser et al. (2019) report on the results of a WRF-Chem simulation over central Europe in which noontime NO2 surface concentrations and VCDs are underestimated by 38.5 % and approximately 15 %, respectively. In a comparison of monthly mean NO2 VCDs, R-values between 0.82 and 0.92 were obtained. The authors identify an un- derestimation of soil emissions in their emission inventory (TNO-MACC-III, short for "Monitoring Atmospheric Composition and Climate" by the Netherlands Organisation for Applied Scientific Research) as a possible explanation. Kuik et al. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. grids of much higher spatio-temporal resolution. Modern RCT models can therefore be used to systematically investigate the 20 processes of transport and (photo)-chemical conversion that trace gases are subject to upon emission into the atmosphere. Most importantly, however, they allow for predictions of trace gas concentrations when observational data is not available and they are also used for operational air quality forecasting. Thus, they give valuable insight into the dynamics of air quality in polluted regions of the earth. RCT models can be "fully coupled", meaning that they include interactions between chemistry grids of much higher spatio-temporal resolution. Modern RCT models can therefore be used to systematically investigate the 20 processes of transport and (photo)-chemical conversion that trace gases are subject to upon emission into the atmosphere. Most importantly, however, they allow for predictions of trace gas concentrations when observational data is not available and they are also used for operational air quality forecasting. Thus, they give valuable insight into the dynamics of air quality in polluted regions of the earth. RCT models can be "fully coupled", meaning that they include interactions between chemistry and meteorology, or "offline", meaning that they simulate chemistry and meteorology separately. Examples for state of the art 25 RCT models are WRF-Chem (Grell et al., 2005), COSMO/MESSy (Kerkweg and Jöckel, 2012), Lotos-Euros (Manders et al., 2017), CAM-chem (Emmons et al., 2020), and CHIMERE (Menut et al., 2021). Nitrogen dioxide (NO2) is one of the most relevant chemical species for air quality in polluted regions. It is toxic to humans and acts as a precursor for ozone (O3), a key pollutant of urban smog. The hazardous impact of NO2 on human health has and meteorology, or "offline", meaning that they simulate chemistry and meteorology separately. Examples for state of the art 25 RCT models are WRF-Chem (Grell et al., 2005), COSMO/MESSy (Kerkweg and Jöckel, 2012), Lotos-Euros (Manders et al., 2017), CAM-chem (Emmons et al., 2020), and CHIMERE (Menut et al., 2021). Nitrogen dioxide (NO2) is one of the most relevant chemical species for air quality in polluted regions. It is toxic to humans and acts as a precursor for ozone (O3), a key pollutant of urban smog. The hazardous impact of NO2 on human health has been widely recognized among the scientific community (see e.g. Faustini et al. (2014); Mills et al. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. the clear consensus that RCT simulations tend to underestimate tropospheric NO2 and that the emission inventories and their 55 preprocessing play a key role in resolving that problem. A number of publications have theorized that an apparent model bias can be explained because groundbased in-situ measure- ments systematically overestimate NO2 concentrations. Conventional in-situ methods often utilize molybdenum converters, which were found to be cross sensitive to other reactive nitrogen species, such as NH3 or PAN. This issue was discussed e.g. the clear consensus that RCT simulations tend to underestimate tropospheric NO2 and that the emission inventories and their 55 preprocessing play a key role in resolving that problem. the clear consensus that RCT simulations tend to underestimate tropospheric NO2 and that the emission inventories and their 55 preprocessing play a key role in resolving that problem. A number of publications have theorized that an apparent model bias can be explained because groundbased in-situ measure- ments systematically overestimate NO2 concentrations. Conventional in-situ methods often utilize molybdenum converters, which were found to be cross sensitive to other reactive nitrogen species, such as NH3 or PAN. This issue was discussed e.g. the clear consensus that RCT simulations tend to underestimate tropospheric NO2 and that the emission inventories and their 55 preprocessing play a key role in resolving that problem. A number of publications have theorized that an apparent model bias can be explained because groundbased in-situ measure- ments systematically overestimate NO2 concentrations. Conventional in-situ methods often utilize molybdenum converters, which were found to be cross sensitive to other reactive nitrogen species, such as NH3 or PAN. This issue was discussed e.g. by Dunlea et al. (2007), Steinbacher et al. (2007), Boersma et al. (2009), and Visser et al. (2019). However, a systematic in- 60 vestigation to prove a general bias of the NO2 in-situ measurements in Europe is still missing. Furthermore, the corresponding European authority claims that their instruments follow strict regulations (defined by the European Norm EN 14221) and that the overestimation of NO2 was found to be smaller than 7 % in test measurements (German Environmental Agency (a)). In this paper, we show the results of a WRF-Chem simulation over central Europe for the month of May 2019 with a spatial resolution of 3 km × 3 km. (2016) 40 run a WRF-Chem simulation with the same emission inventory as Visser et al. (2019) over the region of Berlin, Germany, and observe even stronger underestimations of surface NO2 by more than 50 % during daytime and a strong overestimation at night-time in a similar comparison. The study reveals that increasing the spatial resolution (including downscaling of the used emission data) of the model from 15 km × 15 km to 1 km × 1 km slightly improves agreement, but not to a satisfying and Climate" by the Netherlands Organisation for Applied Scientific Research) as a possible explanation. Kuik et al. (2016) 40 run a WRF-Chem simulation with the same emission inventory as Visser et al. (2019) over the region of Berlin, Germany, and observe even stronger underestimations of surface NO2 by more than 50 % during daytime and a strong overestimation at night-time in a similar comparison. The study reveals that increasing the spatial resolution (including downscaling of the used emission data) of the model from 15 km × 15 km to 1 km × 1 km slightly improves agreement, but not to a satisfying degree. In a subsequent publication, the authors attribute the disagreements to underestimations in their emission data (see Kuik 45 et al., 2018). Mar et al. (2016) report that the choice of the chemical mechanism used by WRF-Chem barely impacts the NOx underestimation by comparing simulations using the RADM2 and MOZART-4 chemical mechanisms. Kumar et al. (2021) demonstrate in a simulation with the MECO(n) model system over Germany using TNO-MACC-III and regional emission data from the German Environmental Agency (UBA), that agreement between modelled NO2 concentrations and in-situ observa- tions improves greatly when diurnal and seasonal variability is added to the yearly resolved emission data using reasonably 50 chosen hourly and monthly weighting factors ("temporal profiles"). This temporal upsampling has become common practice among the air quality modelling community and standard values for such temporal profiles have established (see Crippa et al. (2020b); Kumar et al. (2021) and the references within). Kumar et al. (2021) used improved temporal profiles derived from empirical proxies, e.g. car counts on highways at different hours of the day. Altogether the contemporary literature comes to 2 CC BY 4.0 License. 30° W 12° E 54° E 62° N 50° N 38° N 26° N D1 D2 ‚ Figure 1. Geographical coverage of the WRF-Chem simulation. The spatial resolutions of the outer domain D1 and the inner domain D2 are 15 km × 15 km, and 3 km × 3 km, respectively. ‚ Figure 1. Geographical coverage of the WRF-Chem simulation. The spatial resolutions of the outer domain D1 and the inner domain D2 are 15 km × 15 km, and 3 km × 3 km, respectively. 2 WRF-Chem simulation setup 85 WRF-Chem (Weather Research and Forecasting model with Chemistry, see Grell et al., 2005) is a mesoscale RCT model. We run WRF-Chem on a twofold nested domain over central Europe for the month of May, 2019, see Fig. 1. The spatial resolutions of the outer and inner domain (called D1, and D2 from hereon) are 15 km×15 km, and 3 km×3 km (with 320×245 pixels and 500 × 430 pixels, respectively). The temporal resolution of the simulation is 60 seconds on D1 and 12 seconds on D2. Output files are written daily for D1 and hourly for D2. The simulation uses the Thompson microphysics scheme (see Thompson et al., 90 2008), the RRTMG (Rapid Radiative Transfer Model for General Circulation Models long- and shortwave radiation scheme, see Iacono et al., 2008), the Monin-Obukhov similarity scheme for surface layer modelling (see Monin and Obukhov, 1954), the NOAH Land-Surface Model (see Niu et al., 2011), the YSU boundary layer scheme (see Hong, 2010), and the Grell- Devenyi ensemble scheme for cumulus modelling (see Grell and Dévényi, 2002). For modelling of chemistry, the MOZART chemical mechanism (see Emmons et al., 2010) is coupled to the GOCART aerosol mechanism (see Chin et al., 2000) along 95 with the TUV full photolysis scheme (Madronich (1987); Tie et al. (2003)), which deploys climatological O3 and O2 columns. Dry deposition is calculated according to Wesely (1989). Spectral nudging (see e.g. Omrani et al., 2012) to ERA5 reanalysis data (see Hersbach and Dee, 2017) is used. The full list of WRF-Chem simulation options (the "namelist") can be found in the supplement of this article. The simulation uses a total of 43 vertical levels in terrain-following coordinates (see Table A1 chemical mechanism (see Emmons et al., 2010) is coupled to the GOCART aerosol mechanism (see Chin et al., 2000) along 95 with the TUV full photolysis scheme (Madronich (1987); Tie et al. (2003)), which deploys climatological O3 and O2 columns. Dry deposition is calculated according to Wesely (1989). Spectral nudging (see e.g. Omrani et al., 2012) to ERA5 reanalysis data (see Hersbach and Dee, 2017) is used. The full list of WRF-Chem simulation options (the "namelist") can be found in the supplement of this article. The simulation uses a total of 43 vertical levels in terrain-following coordinates (see Table A1 for the layer heights up to 6 km). The goal is to quantify the level of agreement between simulated NO2 concentrations and VCDs 65 and corresponding results from different observational datasets. In particular, we investigate whether the NO2 biases of the simulation can be resolved by optimization of the temporal profiles. For this purpose our simulation results are compared to three reference datasets: 1. Surface NO2 concentrations measured by a network of in-situ instruments over Germany, operated by the German Environmental Agency (UBA, see German Environmental Agency (b)) 2. Tropospheric NO2 vertical column densities (VCDs) measured by TROPOMI (TROPOspheric Monitoring Instrument) on the Sentinel 5 precursor satellite (specifically the NO2 PAL data product as described in Eskes et al., 2019). This includes recomputing the air mass factors (AMFs) of the retrieval based on our simulation results. 3. NO2 concentration profiles obtained from five MAX-DOAS instruments that partake in the FRM4DOAS project (see Fayt et al., 2021) 3. NO2 concentration profiles obtained from five MAX-DOAS instruments that partake in the FRM4DOAS project (see Fayt et al., 2021) 75 75 We show that when temporal profiles are chosen such that the simulation shows good agreement with in-situ measurements, the simulation also agrees well with satellite and MAX-DOAS observations. This demonstrates that the total NOx (= NO2 + NO) budget assumed in the used emission inventories is realistic. As a direct consequence of this finding, improved simulation strategies like ours could be used for more precise air quality modelling with the focus on NO2, NOx or other species in the future. 80 The paper is structured as follows: Sect. 2 describes the setup of our WRF-Chem simulation and the preprocessing of emis- sion data in detail. Sect. 3 shows the results of our simulation and compares them to a "classical" simulation with empirically derived temporal profiles. Furthermore, the simulated data is compared to the above mentioned observational datasets. In sect. 4 the results are discussed and conclusions are drawn. future. 80 The paper is structured as follows: Sect. 2 describes the setup of our WRF-Chem simulation and the preprocessing of emis- sion data in detail. Sect. 3 shows the results of our simulation and compares them to a "classical" simulation with empirically derived temporal profiles. Furthermore, the simulated data is compared to the above mentioned observational datasets. In sect. 4 the results are discussed and conclusions are drawn. 3 3 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. et al., 2020). Additionally, CAM-chem yields the boundary conditions for D1. D2 receives boundary conditions online from the WRF-Chem simulation running on D1. et al., 2020). Additionally, CAM-chem yields the boundary conditions for D1. D2 receives boundary conditions online from the WRF-Chem simulation running on D1. et al., 2020). Additionally, CAM-chem yields the boundary conditions for D1. D2 receives boundary conditions online from the WRF-Chem simulation running on D1. 2.1 Emission preprocessing Outside of Germany the EDGARv5 emission inventory with a moderate spatial resolu- tion (0.1◦× 0.1◦or ∼11 km × 7 km) is used (see Crippa et al., 2020a). Since UBA-E does not include organic anthropogenic emissions, EDGARv5 is used on the entirety of D1 and D2 for organic species. Non-methane volatile organic compounds 115 (NMVOCs) are provided as lumped species and are speciated according to Huang et al. (2017). An alternative to EDGARv5 would have been the TNO-MAC-III emission inventory (Kuenen et al., 2014) which comes at a higher spatial resolution of 0.0625◦× 0.125◦but was only available for the year 2011 at the time the simulation was run. Since EDGARv5 was available for 2015 and NOx emissions have steadily decreased over the past years (see e.g. Anenberg et al., 2022), EDGARv5 was considered a more reasonable choice. 120 EDGARv5 (in 2015) and UBA-E have time resolutions of 1 month and 1 year, respectively. However, many emissions follow strong diurnal and seasonal patterns. For example, emissions from car traffic are expected to be lower at nighttime and higher at daytime and agricultural emissions typically occur in specific months of the year. EDGARv5 can still resolve emission variations on a monthly time-resolution but is incapable of resolving diurnal patterns. UBA-E provides only annual emissions, considered a more reasonable choice. 120 EDGARv5 (in 2015) and UBA-E have time resolutions of 1 month and 1 year, respectively. However, many emissions follow strong diurnal and seasonal patterns. For example, emissions from car traffic are expected to be lower at nighttime and higher at daytime and agricultural emissions typically occur in specific months of the year. EDGARv5 can still resolve emission variations on a monthly time-resolution but is incapable of resolving diurnal patterns. UBA-E provides only annual emissions, therefore without any temporal pattern. The solution to this problem is to scale the coarsely resolved emission data to an hourly 125 resolution using presumed hourly, daily, and monthly emission scaling factors, called temporal profiles. The emission rate EX,k(m,d,h,lat, lon) of a species X from sector k at month m, day d, and hour h at fixed latitude lat and longitude lon is given as EX,k(m,d,h,lat, lon) = ˆEX,k(lat, lon) · pmonthly,k(m) · pdaily,k(d) · phourly,k(h) (1) therefore without any temporal pattern. 2 WRF-Chem simulation setup 85 Both domains D1 and D2 receive initial conditions from the CAM-chem model (Emmons 100 4 2.1 Emission preprocessing The WRF-Chem simulation is driven by different emission inventories for different emission sectors (see also Table 1). Emis- sions from biomass burning are taken from the Fire Inventory from NCAR (FINN, see Wiedinmyer et al., 2011) with a spatio- 105 temporal resolution of 1 km × 1 km and 24 hours. Biomass burning emissions are assumed to have a diurnal variation with a peak in the early afternoon (1 pm local time) and are distributed vertically in the model following the plumerise parametrization of Freitas et al. (2007). Biogenic emissions are computed online using an implementation of the MEGAN model (see Guenther et al., 2006). For non-organic anthropogenic emissions a combination of two emission inventories is used: Over Germany, an inventory of high spatial resolution (1 km × 1 km, resampled to 0.01◦× 0.01◦or ∼1.1 km × 0.7 km) is provided by the 110 German Environmental Agency (Umweltbundesamt, UBA, see Strogies et al., 2020). From hereon, this emission inventory will be referenced as UBA-E. Using the UBA-E emission data over Germany enables modelling of NO2 distributions at an unprecedented high spatial resolution. Outside of Germany the EDGARv5 emission inventory with a moderate spatial resolu- tion (0.1◦× 0.1◦or ∼11 km × 7 km) is used (see Crippa et al., 2020a). Since UBA-E does not include organic anthropogenic an inventory of high spatial resolution (1 km × 1 km, resampled to 0.01◦× 0.01◦or ∼1.1 km × 0.7 km) is provided by the 110 German Environmental Agency (Umweltbundesamt, UBA, see Strogies et al., 2020). From hereon, this emission inventory will be referenced as UBA-E. Using the UBA-E emission data over Germany enables modelling of NO2 distributions at an unprecedented high spatial resolution. Outside of Germany the EDGARv5 emission inventory with a moderate spatial resolu- tion (0.1◦× 0.1◦or ∼11 km × 7 km) is used (see Crippa et al., 2020a). Since UBA-E does not include organic anthropogenic an inventory of high spatial resolution (1 km × 1 km, resampled to 0.01◦× 0.01◦or ∼1.1 km × 0.7 km) is provided by the 110 German Environmental Agency (Umweltbundesamt, UBA, see Strogies et al., 2020). From hereon, this emission inventory will be referenced as UBA-E. Using the UBA-E emission data over Germany enables modelling of NO2 distributions at an unprecedented high spatial resolution. 2.1 Emission preprocessing The solution to this problem is to scale the coarsely resolved emission data to an hourly 125 resolution using presumed hourly, daily, and monthly emission scaling factors, called temporal profiles. The emission rate EX,k(m,d,h,lat, lon) of a species X from sector k at month m, day d, and hour h at fixed latitude lat and longitude lon is given as E ( d h l l ) ˆE (l l ) ( ) (d) (h) (1) EX,k(m,d,h,lat, lon) = ˆEX,k(lat, lon) · pmonthly,k(m) · pdaily,k(d) · phourly,k(h) (1) X,k(lat, lon) · pmonthly,k(m) · pdaily,k(d) · phourly,k(h) (1) X,k(m,d,h,lat, lon) = ˆEX,k(lat, lon) · pmonthly,k(m) · pdaily,k(d) · phourly,k(h) (1) where ˆEX,k denotes the total emission budget of species X from sector k in the emission inventory and pmonthly, pdaily, and 130 phourly the monthly, daily, and hourly temporal profiles. The individual profiles are normalized to 12 (annual cycle), 7 (weekly cycle), and 24 (diurnal cycle), respectively. Because different emission sectors follow vastly different temporal patterns, the temporal profiles are defined for each sector individually. An overview of the emission sectors is given in Table 1. The total where ˆEX,k denotes the total emission budget of species X from sector k in the emission inventory and pmonthly, pdaily, and 130 phourly the monthly, daily, and hourly temporal profiles. The individual profiles are normalized to 12 (annual cycle), 7 (weekly cycle), and 24 (diurnal cycle), respectively. Because different emission sectors follow vastly different temporal patterns, the temporal profiles are defined for each sector individually. An overview of the emission sectors is given in Table 1. The total where ˆEX,k denotes the total emission budget of species X from sector k in the emission inventory and pmonthly, pdaily, and 130 phourly the monthly, daily, and hourly temporal profiles. The individual profiles are normalized to 12 (annual cycle), 7 (weekly cycle), and 24 (diurnal cycle), respectively. Because different emission sectors follow vastly different temporal patterns, the temporal profiles are defined for each sector individually. An overview of the emission sectors is given in Table 1. The total 5 5 N/A = "not available" N/A = not available † Relative contribution of this sector to the overall emission budget (yearly for UBA-E, and for the month of May for EDGARv5) of NOx https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. where Xlump is the lump of species that contains species X (i.e. Xlump = NOx when X = NO2). 140 In principle, WRF-Chem also supports vertical distribution of trace gas emissions. This is reasonable to consider, given that many strong emissions, like those from combustion stacks, take place at elevated altitudes. In analogy to the mentioned temporal and speciation profiles, this can be modelled using an additional vertical emission profile. Suggestions for vertical emission profiles are given in Bieser et al. (2011) and Pozzer et al. (2009). In our study we omit the use of vertical profiles and inject all emissions into the lowest model layer (0 - 8 m). This simplification was made seeing that vertical profiles would add 145 another dimension of complexity to our main goal, which is to optimize the temporal profiles. Furthermore, precise information on the emission height is often not available. Lastly, assuming a well-mixed troposphere, vertical profiles can be expected to be relevant only in the close vicinity of strong elevated emission sources. inject all emissions into the lowest model layer (0 - 8 m). This simplification was made seeing that vertical profiles would add 145 another dimension of complexity to our main goal, which is to optimize the temporal profiles. Furthermore, precise information on the emission height is often not available. Lastly, assuming a well-mixed troposphere, vertical profiles can be expected to be relevant only in the close vicinity of strong elevated emission sources. inject all emissions into the lowest model layer (0 - 8 m). This simplification was made seeing that vertical profiles would add 145 another dimension of complexity to our main goal, which is to optimize the temporal profiles. Furthermore, precise information on the emission height is often not available. Lastly, assuming a well-mixed troposphere, vertical profiles can be expected to be relevant only in the close vicinity of strong elevated emission sources. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 1. Emission sectors in the UBA-E and EDGARv5 emission inventories Name Contribution† (UBA-E) [%] Contribution† (EDGARv5) [%] traffic (no resuspension) 43.8 38.7 power industry 18.5 15.0 agricultural soils 10.1 4.5 energy for buildings 7.3 6.6 manufacturing industry 7.2 15.8 non-metallic minerals production 2.5 0.0 production of chemicals 2.4 0.3 shipping 2.1 2.1 iron and steel production 1.8 < 0.1 oil refineries and transformation industry 1.7 1.8 aviation landing and take-off 1.2 1.8 railways, pipelines, and off-road transport 0.9 2.3 production of food, pulp, and paper 0.3 0.3 manure management 0.1 1.1 fuel exploitation 0.1 0.0 solid waste incineration 0.1 0.1 non-ferrous metal production 0 < 0.1 non-energy use of fuels < 0.1 0.0 agricultural waste burning N/A 0.3 fossil fuel fires N/A < 0.1 aviation climbing and descent N/A 5.9 aviation cruise N/A 3.4 Table 1. Emission sectors in the UBA-E and EDGARv5 emission inventories Name Contribution† (UBA-E) [%] Contribution† (EDGARv5) [%] N/A = not available † Relative contribution of this sector to the overall emission budget (yearly for UBA-E, and for the month of May for EDGARv5) of NOx N/A = not available † Relative contribution of this sector to the overall emission budget (yearly for UBA-E, and for the month of May for EDGARv5) of NOx emission rate of species X is obtained by summation over all emission sectors k emission rate of species X is obtained by summation over all emission sectors k EX(m,d,h,lat, lon) = X k ˆEX,k(lat, lon) · pmonthly,k(m) · pdaily,k(d) · phourly,k(h) (2) 35 EX(m,d,h,lat, lon) = X k ˆEX,k(lat, lon) · pmonthly,k(m) · pdaily,k(d) · phourly,k(h) (2) 135 (2) Some species are only implicitly contained in the emission inventories. For example, EDGARv5 and UBA-E specify NOx emissions, but the partitioning into NO and NO2 must be chosen by the user via a speciation profile pspec(k). Equation (2) then generalizes to Some species are only implicitly contained in the emission inventories. For example, EDGARv5 and UBA-E specify NOx emissions, but the partitioning into NO and NO2 must be chosen by the user via a speciation profile pspec(k). Equation (2) then generalizes to (3) 6 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3. Gradient-based optimization methods depend on hyperparameters, such as the step size. If these are not picked correctly from the start, the optimization may converge slowly or diverge entirely. This becomes unfeasible, when a single forward run takes days to compute. 3. Gradient-based optimization methods depend on hyperparameters, such as the step size. If these are not picked correctly from the start, the optimization may converge slowly or diverge entirely. This becomes unfeasible, when a single forward run takes days to compute. 3. Gradient-based optimization methods depend on hyperparameters, such as the step size. If these are not picked correctly from the start, the optimization may converge slowly or diverge entirely. This becomes unfeasible, when a single forward run takes days to compute. Using a conventional optimization routine, where the gradient of a loss function determines the outcome of a single optimiza- 175 tion step, would require to encode aspects 1-3 in the form of mathematical constraints. This makes a rigorous treatment of the problem extremely complex. The manual optimization approach, however, does not require a mathematical formulation and can thus take the discussed aspects into consideration more easily. We run the optimization under the following conditions: 1. Only the most relevant emission sectors are modified during the process. According to Table 1 these are: traffic (no resuspension), power industry, agricultural soils, energy for buildings, and manufacturing industry. 1. Only the most relevant emission sectors are modified during the process. According to Table 1 these are: traffic (no resuspension), power industry, agricultural soils, energy for buildings, and manufacturing industry. 2. Speciation of lumped species follows the recommendations of Huang et al. (2017) and is not further optimized. For NOx, the partitioning is assumed to be 87.5 % NO and 12.5 % NO2. This choice reflects the fact that NOx from combustion processes is mostly emitted as NO, which mostly oxidizes to NO2 over time. Literature values for typical NO2/NOx ratios in anthropogenic emissions range from lower values (e.g. 5.3 %, as reported by Wild et al. (2017) and 7.8 %, as reported by Jimenez et al. (2000)) to much higher values (e.g. 39 %, as reported by Richmond-Bryant et al. (2017) and 36 %, as reported by Costantini et al. (2016)). 3. In order to improve generalization of the temporal profiles, the optimization is performed using simulated and observed data from May 2018. Additionally, we accelerate the optimization by only using two weeks of simulation time for each optimization step. The final temporal profiles are evaluated in a full-month simulation for the year 2019, as described in sect. 2. 190 Figure 2 gives an overview of the optimization process in a total of 3 steps. Figure 2h reveals that the overall NOx budget assumed for the simulated month is realistic, meaning that the corresponding monthly weighting factor is reasonable. Similarly, the daily profiles from Kumar et al. (2021) were taken over without changes. What remains are the hourly profiles of each sector. Prior to the optimization, simulated NOx was too low at daytime and too high at night. Therefore, the hourly profiles were Figure 2 gives an overview of the optimization process in a total of 3 steps. Figure 2h reveals that the overall NOx budget assumed for the simulated month is realistic, meaning that the corresponding monthly weighting factor is reasonable. Similarly, the daily profiles from Kumar et al. (2021) were taken over without changes. What remains are the hourly profiles of each sector. Prior to the optimization, simulated NOx was too low at daytime and too high at night. Therefore, the hourly profiles were adjusted accordingly. For example, in step 1 (red line), only a single change was made to the traffic sector profile (subfigure a) 195 in order to boost simulated NOx values in the morning (∼6 AM). The principle was extended to the remaining hourly profiles in the next two steps. The full list of emission profiles used in our simulation is attached to the supplement of this article. 2.2 Optimization of temporal profiles The key difference between previously published RCT simulations and ours is the optimization of the temporal emission 150 profiles. The goal is to find temporal profiles that minimize the model’s mean NOx bias over the course of the day. The mean relative bias is computed as bias = 1 N N X i=1 xsim,i −xobs,i xobs,i (4) bias = 1 N N X i=1 xsim,i −xobs,i xobs,i (4) measurements of background NOx surface concentrations as reference. A detailed explanation of the dataset is given in sect. 155 3.1. Equation (3) hints towards the complexity of the optimization problem: Because the emission inventories include dozens of emission sectors, each with their own emission profiles, this poses an optimization problem with many degrees of freedom. In addition, a single WRF-Chem simulation of just one month takes days to finish even on modern supercomputers. This circumstance makes it nearly impossible to optimize the emission profiles using standard methods like gradient descent. For our simulation we have therefore optimized the emission profiles manually. By "manual optimization" we mean the iterative 160 process of running WRF-Chem, evaluating the simulation results against in-situ observations, and slightly nudging the temporal profiles in a direction in which better agreement between simulation and observations can be expected. Due to the short lifetime of NOx the observed concentrations closely follow the temporal profiles. As an initial starting point, the temporal profiles from Kumar et al. (2021) were used. Although the proposed optimization method is rather unconventional, it has a few important 1. The hourly profiles of many sectors have characteristic shapes, e.g. a peak in the hourly profile of the traffic sector during the morning rush hour. These should be at least coarsely preserved during the optimization process in order to maintain realistic emission behaviour. 1. The hourly profiles of many sectors have characteristic shapes, e.g. a peak in the hourly profile of the traffic sector during the morning rush hour. These should be at least coarsely preserved during the optimization process in order to maintain realistic emission behaviour. 2. Because the optimization problem is ill-posed, it is often unclear, of which sector the profiles should be tuned further in order to improve the simulation. Sometimes, the spatial distribution of a specific emission sector matches that of the 170 model error, indicating that this sector should be prioritized. 7 7 2.3 Chemical boundary conditions Overview of the optimization process of the hourly emission profiles based on the simulation results and in-situ measurements of May 2018. The profiles used by Kumar et al. (2021) are drawn in blue and our final optimized profiles in orange. Therefore, in addition to the tuning of temporal emission profiles, we further conduct a tuning Therefore, in addition to the tuning of temporal emission profiles, we further conduct a tuning of O3 BCs in the same fashion. As a result, a O3 BC scaling factor of ∼0.85 was determined. The results discussed in sect. 3 verify that scaling of the O3 BCs 205 yields an improvement to the resulting O3 concentrations. As a result, a O3 BC scaling factor of ∼0.85 was determined. The results discussed in sect. 3 verify that scaling of the O3 BCs 205 yields an improvement to the resulting O3 concentrations. 2.3 Chemical boundary conditions As described, our simulation uses CAM-chem model results as chemical boundary conditions (BCs) on the outer domain D1 and initial conditions (ICs) on both domains. In practice, it shows that simulated NO2 and NO concentrations are mostly 200 insensitive to BCs and ICs. O3 concentrations, however, are highly sensitive to the O3 BCs. At the same time, O3 is known to be a crucial reaction partner of NOx chemistry, mostly for oxidizing NO in the reaction As described, our simulation uses CAM-chem model results as chemical boundary conditions (BCs) on the outer domain D1 and initial conditions (ICs) on both domains. In practice, it shows that simulated NO2 and NO concentrations are mostly 200 insensitive to BCs and ICs. O3 concentrations, however, are highly sensitive to the O3 BCs. At the same time, O3 is known to be a crucial reaction partner of NOx chemistry, mostly for oxidizing NO in the reaction NO + O3 →NO2 + O2 (R1) (R1) NO + O3 →NO2 + O2 8 AMT paper
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 8.3 cm * 5.1297 cm (single col. Fig., golden ratio) 17.5 cm * 10.8156 cm (double col. Fig., golden ratio) https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. AMT paper
 6.96986 in * 9. 8.3 cm * 5.129 17 5 cm * 10 8 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. (a) (a) (d) (g) (b) (e) (h) (c) (f) (i) [µg m-3] Initial configuration Step 1 Step 2 Step 3 In-situ measurements (background stations) Figure 2. Overview of the optimization process of the hourly emission profiles based on the simulation results and in-situ measurements of May 2018. The profiles used by Kumar et al. (2021) are drawn in blue and our final optimized profiles in orange. (a) (a) (b) (c) (b) (c) (e) (f) (d) (f) (e) (f) (i) (g) (h) (i) [µg m-3] Initial configuration Step 1 Step 2 Step 3 In-situ measurements (background station Figure 2. Overview of the optimization process of the hourly emission profiles based on the simulation results and in-situ measuremen (g) (h) (i) [µg m-3] I iti l fi ti St 1 St 2 St 3 I it (i) (h) (h) [µg m-3] In-situ measurements (background stations) Figure 2. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.1 Comparison of surface concentrations Given that the used emission inventories treat NO and NO2 as lumped NOx, this validates our choice of temporal Comparing S-old and S-new shows the vast difference in simulation accuracy achieved by using the optimized temporal 230 profiles. S-old consistently overestimates the NOx concentration at night, but underestimates it at daytime. Particularly around noontime the simulated NOx concentrations are low-biased by approximately −20 %. S-new achieves much better agreement with the in-situ measurements: The mean NOx concentration is reproduced with minimal deviations (< 2 µg m−3), regardless of daytime. Given that the used emission inventories treat NO and NO2 as lumped NOx, this validates our choice of temporal profiles. 235 The individual time series of NO2 and NO also show good agreement with the in-situ observations, with minimal to no deviations at noontime. However, discrepancies during nighttime between simulation and observations remain (< 4.7 µg m−3 for NO2 and < 4.1 µg m−3 for NO). S-old shows a consistent overestimation of O3 of approximately 20 µg m−3 regardless of daytime, which is reduced to profiles. 235 The individual time series of NO2 and NO also show good agreement with the in-situ observations, with minimal to no deviations at noontime. However, discrepancies during nighttime between simulation and observations remain (< 4.7 µg m−3 for NO2 and < 4.1 µg m−3 for NO). S-old shows a consistent overestimation of O3 of approximately 20 µg m−3 regardless of daytime, which is reduced to profiles. 235 The individual time series of NO2 and NO also show good agreement with the in-situ observations, with minimal to no deviations at noontime. However, discrepancies during nighttime between simulation and observations remain (< 4.7 µg m−3 for NO2 and < 4.1 µg m−3 for NO). S-old shows a consistent overestimation of O3 of approximately 20 µg m−3 regardless of daytime, which is reduced to approximately 10 µg m−3 in S-new. The improvements are mostly attributed to the adjustments to the O3 boundary conditions 240 described in sect. 2.3. A version of Fig. 3 showing traffic stations instead of background stations can be found in appendix A (Fig. A1). The NO2 concentration timeline of the traffic observations has a fundamentally different shape compared to the background observations, with practically no noontime low and no evening peak. As expected, the modelled and observed concentration timelines do not agree well for the traffic stations. 3 Results We compare the results of two WRF-Chem simulations for May 2019 (one with the temporal profiles used by Kumar et al. (2021) called "S-old", and one with optimized profiles called "S-new") to the dataset of in-situ surface concentration mea- surements and show that S-new outperforms S-old in every regard. We then continue to compare S-new to the remaining 210 observational datasets of satellite and MAX-DOAS measurements. 9 9 3.1 Comparison of surface concentrations The left panel (subfigures a-d) is restricted to the first ten days of the simulated time period (01 May 2019 - 10 May 2019) for easier readability. The right panel (subfigures e-h) shows the average diurnal concentrations obtained from averaging over all days of the simulation period. ∆xt = max(|xt −xt−1|,|xt −xt+1|) (5) 225 i.e. the maximal hour-to-hour variation in concentration. Figure 3 shows the average surface concentrations of NO2, NO, NOx, and O3 of both simulations S-old and S-new as well as the corresponding in-situ measurements. The left panel (subfigures a-d) is restricted to the first ten days of the simulated time period (01 May 2019 - 10 May 2019) for easier readability. The right panel (subfigures e-h) shows the average diurnal concentrations obtained from averaging over all days of the simulation period. ∆xt = max(|xt −xt−1|,|xt −xt+1|) (5) 225 i.e. the maximal hour-to-hour variation in concentration. Figure 3 shows the average surface concentrations of NO2, NO, NOx, and O3 of both simulations S-old and S-new as well as the corresponding in-situ measurements. The left panel (subfigures a-d) is restricted to the first ten days of the simulated time period (01 May 2019 - 10 May 2019) for easier readability. The right panel (subfigures e-h) shows the average diurnal concentrations obtained from averaging over all days of the simulation period. (5) Comparing S-old and S-new shows the vast difference in simulation accuracy achieved by using the optimized temporal 230 profiles. S-old consistently overestimates the NOx concentration at night, but underestimates it at daytime. Particularly around noontime the simulated NOx concentrations are low-biased by approximately −20 %. S-new achieves much better agreement with the in-situ measurements: The mean NOx concentration is reproduced with minimal deviations (< 2 µg m−3), regardless of daytime. Given that the used emission inventories treat NO and NO2 as lumped NOx, this validates our choice of temporal Comparing S-old and S-new shows the vast difference in simulation accuracy achieved by using the optimized temporal 230 profiles. S-old consistently overestimates the NOx concentration at night, but underestimates it at daytime. Particularly around noontime the simulated NOx concentrations are low-biased by approximately −20 %. S-new achieves much better agreement with the in-situ measurements: The mean NOx concentration is reproduced with minimal deviations (< 2 µg m−3), regardless of daytime. 3.1 Comparison of surface concentrations In-situ measurements of trace gas surface concentrations in Germany are conducted by the UBA. They are available for NO2, NO and O3 as hourly mean values. A total of 434 UBA measuring stations are distributed over Germany. 92 % of the sta- tions use a chemiluminescence based measuring method. The remaining 8 % use other methods (cavity enhanced phase shift 215 spectroscopy, diffuse sampling, or photolysis conversion). Of all UBA stations, 63 % are classified as "background", 30 % as "traffic", and 7 % as "industry". In this study we only take "background" stations into consideration. This choice was made seeing that NO2 concentration can vary strongly near traffic over distances of only 10 - 100 m (see e.g. Beckwith et al., 2019) and timescales of a few minutes. Given that our simulation runs at a spatial resolution of 3 km × 3 km, it is expected to show poor agreement with the traffic stations. Many of the publications referenced in sect. 1 have also restricted their reference data 220 to "background" in-situ measurements. In order to compare the simulated surface concentrations to the in-situ measurements, they are interpolated from the WRF- Chem simulation grid to the geolocations of the UBA stations. The uncertainty ∆xt of the simulated surface concentrations and the in-situ reference values at time step t is computed via poor agreement with the traffic stations. Many of the publications referenced in sect. 1 have also restricted their reference data 220 to "background" in-situ measurements. In order to compare the simulated surface concentrations to the in-situ measurements, they are interpolated from the WRF- Chem simulation grid to the geolocations of the UBA stations. The uncertainty ∆xt of the simulated surface concentrations and the in-situ reference values at time step t is computed via In order to compare the simulated surface concentrations to the in-situ measurements, they are interpolated from the WRF- Chem simulation grid to the geolocations of the UBA stations. The uncertainty ∆xt of the simulated surface concentrations and the in-situ reference values at time step t is computed via ∆xt = max(|xt −xt−1|,|xt −xt+1|) (5) 225 i.e. the maximal hour-to-hour variation in concentration. Figure 3 shows the average surface concentrations of NO2, NO, NOx, and O3 of both simulations S-old and S-new as well as the corresponding in-situ measurements. 3.1 Comparison of surface concentrations Interestingly, although this was not considered during the optimization process, the temporal profiles we have obtained for the traffic sector (see Fig. 2a) have a shape similar to the NOx 245 10 0 10 20 30 NO2 conc. [µg m 3] (a) (a) 0 10 20 30 (e) (e) Monthly average 0.0 2.5 5.0 7.5 10.0 12.5 NO conc. [µg m 3] (b) (b) 0.0 2.5 5.0 7.5 10.0 12.5 (f) (f) 0 10 20 30 40 NOx conc. [µg m 3] (c) (c) 0 10 20 30 40 (g) (g) Wed, 05-01 00:00 Thu, 05-02 00:00 Fri, 05-03 00:00 Sat, 05-04 00:00 Sun, 05-05 00:00 Mon, 05-06 00:00 Tue, 05-07 00:00 Wed, 05-08 00:00 Thu, 05-09 00:00 Fri, 05-10 00:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 Date and time 25 50 75 100 125 O3 conc. [µg m 3] (d) 00:00 04:00 08:00 12:00 16:00 20:00 00:00 Time of day 25 50 75 100 125 (h) UBA measurements S-new S-old Figure 3. Time series of NO2, NO, NOx, and O3 surface concentrations. The red lines show reference values obtained from the UBA https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0 10 20 30 NO2 conc. [µg m 3] (a) (a) 0 10 20 30 (e) (e) Monthly average 0.0 2.5 5.0 7.5 10.0 12.5 NO conc. [µg m 3] (b) (b) 0.0 2.5 5.0 7.5 10.0 12.5 (f) (f) 0 10 20 30 40 NOx conc. [µg m 3] (c) (c) 0 10 20 30 40 (g) (g) Wed, 05-01 00:00 Thu, 05-02 00:00 Fri, 05-03 00:00 Sat, 05-04 00:00 Sun, 05-05 00:00 Mon, 05-06 00:00 Tue, 05-07 00:00 Wed, 05-08 00:00 Thu, 05-09 00:00 Fri, 05-10 00:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 Date and time 25 50 75 100 125 O3 conc. [µg m 3] (d) 00:00 04:00 08:00 12:00 16:00 20:00 00:00 Time of day 25 50 75 100 125 (h) UBA measurements S-new S-old Figure 3. Time series of NO2, NO, NOx, and O3 surface concentrations. The red lines show reference values obtained from the UBA background in-situ stations. The green and blue lines show the simulation results of S-old and S-new, respectively. 3.1 Comparison of surface concentrations (a) - (d) display a time series spanning multiple days (01 May 2019 - 10 May 2019) and (e) - (f) display the corresponding average concentration values over the entire month of May 2019. Monthly average Figure 3. Time series of NO2, NO, NOx, and O3 surface concentrations. The red lines show reference values obtained from the UBA background in-situ stations. The green and blue lines show the simulation results of S-old and S-new, respectively. (a) - (d) display a time series spanning multiple days (01 May 2019 - 10 May 2019) and (e) - (f) display the corresponding average concentration values over the entire month of May 2019. Figure 3. Time series of NO2, NO, NOx, and O3 surface concentrations. The red lines show reference values obtained from the UBA background in-situ stations. The green and blue lines show the simulation results of S-old and S-new, respectively. (a) - (d) display a time series spanning multiple days (01 May 2019 - 10 May 2019) and (e) - (f) display the corresponding average concentration values over the entire month of May 2019. 11 Table 2. Statistical summary for the surface concentration comparison (all hours of the day, "background" stations only) Table 2. Statistical summary for the surface concentration comparison (all hours of the day, "background" stations only) Table 2. Statistical summary for the surface concentration comparison (all hours of the day, "background" sta al summary for the surface concentration comparison (all hours of the day, "background" stations only) y p y g y Mean [µg m−3] RMSE [µg m−3] Bias R Trace gas UBA S-new S-old S-new S-old S-new S-old S-new S-old NO2 11.9 13.6 15.9 3.7 6.4 +14.3 % +34.0 % 0.77 0.72 NO 2.5 1.7 1.8 1.9 1.8 -30.0 % -27.8 % 0.63 0.65 NOx 14.3 15.3 17.7 4.2 6.6 +6.7 % +23.4 % 0.76 0.67 O3 66.2 73.4 78.5 12.2 16.8 +10.9 % +18.7 % 0.85 0.82 Table 3. Statistical summary for the surface concentration comparison (at noon, "background" stations only) Mean [µg m−3] RMSE [µg m−3] Bias R Trace gas UBA S-new S-old S-new S-old S-new S-old S-new S-old NO2 8.1 8.1 6.8 1.7 2.0 +0.9 % -15.7 % 0.76 0.75 NO 2.1 2.1 1.5 0.5 0.8 -1.5 % -31.8 % 0.72 0.66 NOx 10.2 10.2 8.3 2.0 2.7 +0.4 % -19.1 % 0.77 0.75 O3 84.9 91.4 100.2 11.1 19.0 +7.6 % +18.0 % 0.72 0.58 Table 3. Statistical summary for the surface concentration comparison (at noon, "background" stations only) time series of the traffic stations (see Fig. A1g), whereas the corresponding initial profile deviates quite strongly in shape. This can be seen as further validation of our results. time series of the traffic stations (see Fig. A1g), whereas the corresponding initial profile deviates quite strongly in shape. This can be seen as further validation of our results. Figure 4 emphasizes the improvement in modelling accuracy via scatter plots for NO2, NO, NOx, and O3 surface concen- trations at noontime. The results of S-new are consistently closer to the 1:1 line and linear regressions through the point clouds of each plot yield slopes much closer to 1 with similar (small) intercepts compared to S-old. For example, comparing Fig. 4a 250 and Fig. 4b shows slopes of 0.84 (S-new) and 0.73 (S-old) for NO2. A version of Fig. 4 showing values for all hours of the day can be found in appendix A (see Fig. A2). A summary of corresponding statistical diagnostics is given in Tables 2 and 3 for all hours of the day and noontime only, respectively. The Root Mean Square Error (RMSE) is computed according to 250 of each plot yield slopes much closer to 1 with similar (small) intercepts compared to S-old. For example, comparing Fig. 4a 250 and Fig. 4b shows slopes of 0.84 (S-new) and 0.73 (S-old) for NO2. A version of Fig. 4 showing values for all hours of the day can be found in appendix A (see Fig. A2). A summary of corresponding statistical diagnostics is given in Tables 2 and 3 for all hours of the day and noontime only, respectively. The Root Mean Square Error (RMSE) is computed according to RMSE = sPN i=1 (xsim,i −xobs,i)2 N (6) N where xobs,i denotes the i-th observation and xsim,i the corresponding simulated value. Here the RMSE is computed from the 255 averages over all stations used to generate Fig. 3 and 4. where xobs,i denotes the i-th observation and xsim,i the corresponding simulated value. Here the RMSE is computed from the 255 averages over all stations used to generate Fig. 3 and 4. where xobs,i denotes the i-th observation and xsim,i the corresponding simulated value. Here the RMSE is computed from the 255 averages over all stations used to generate Fig. 3 and 4. Comparing the entries of Tables 2 and 3 shows that S-new consistently outperforms S-old by showing lower RMSE, lower bias, and higher R-values. Most importantly, the biases of total and noontime NOx are reduced from +23.4 % to +6.7 % and from −19.1 % to +0.4 %, respectively. Similarly, the biases of total and noontime NO2 are reduced from +34.0 % to +14.3 % and from −15.7 % to +0.9 %. There appears to be only one minor exception to the overall improvement, namely total NO (see 260 Comparing the entries of Tables 2 and 3 shows that S-new consistently outperforms S-old by showing lower RMSE, lower bias, and higher R-values. Most importantly, the biases of total and noontime NOx are reduced from +23.4 % to +6.7 % and from −19.1 % to +0.4 %, respectively. Similarly, the biases of total and noontime NO2 are reduced from +34.0 % to +14.3 % Comparing the entries of Tables 2 and 3 shows that S-new consistently outperforms S-old by showing lower RMSE, lower bias, and higher R-values. Most importantly, the biases of total and noontime NOx are reduced from +23.4 % to +6.7 % and from −19.1 % to +0.4 %, respectively. Similarly, the biases of total and noontime NO2 are reduced from +34.0 % to +14.3 % and from −15.7 % to +0.9 %. There appears to be only one minor exception to the overall improvement, namely total NO (see 260 12 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0 5 10 15 20 Simulated mass concentration [µg m 3] (a) (a) (a) (a) (a) (a) (a) (a) NO2 (S-new) f(x) = 0.84x + 1.39 0 2 4 6 8 10 12 14 0 5 10 15 (b) (b) (b) (b) (b) (b) (b) (b) NO2 (S-old) f(x) = 0.73x + 0.87 0 1 2 3 4 0 2 4 Simulated mass concentration [µg m 3] (c) (c) (c) (c) (c) (c) (c) (c) NO (S-new) f(x) = 0.89x + 0.20 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 2 3 4 (d) (d) (d) (d) (d) (d) (d) (d) NO (S-old) f(x) = 0.51x + 0.37 0 5 10 15 20 0 10 20 Simulated mass concentration [µg m 3] (e) (e) (e) (e) (e) (e) (e) (e) NOx (S-new) f(x) = 0.88x + 1.25 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 0 5 10 15 20 (f) (f) (f) (f) (f) (f) (f) (f) NOx (S-old) f(x) = 0.72x + 0.95 100 150 ed mass on [µg m 3] (g) (g) (g) (g) (g) (g) (g) (g) O3 (S-new) f(x) = 0.60x + 40.52 100 150 (h) (h) (h) (h) (h) (h) (h) (h) O3 (S-old) f(x) = 0.52x + 55.92 c⃝Author(s) 2023. CC BY 4.0 License. 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0 5 10 15 20 Simulated mass concentration [µg m 3] (a) (a) (a) (a) (a) (a) (a) (a) NO2 (S-new) f(x) = 0.84x + 1.39 0 2 4 6 8 10 12 14 0 5 10 15 (b) (b) (b) (b) (b) (b) (b) (b) NO2 (S-old) f(x) = 0.73x + 0.87 0 1 2 3 4 0 2 4 Simulated mass concentration [µg m 3] (c) (c) (c) (c) (c) (c) (c) (c) NO (S-new) f(x) = 0.89x + 0.20 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 2 3 4 (d) (d) (d) (d) (d) (d) (d) (d) NO (S-old) f(x) = 0.51x + 0.37 0 5 10 15 20 0 10 20 Simulated mass concentration [µg m 3] (e) (e) (e) (e) (e) (e) (e) (e) NOx (S-new) f(x) = 0.88x + 1.25 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 0 5 10 15 20 (f) (f) (f) (f) (f) (f) (f) (f) NOx (S-old) f(x) = 0.72x + 0.95 0 20 40 60 80 100 120 Observed mass concentration [µg m 3] 0 50 100 150 Simulated mass concentration [µg m 3] (g) (g) (g) (g) (g) (g) (g) (g) O3 (S-new) f(x) = 0.60x + 40.52 0 20 40 60 80 100 120 Observed mass concentration [µg m 3] 0 50 100 150 (h) (h) (h) (h) (h) (h) (h) (h) O3 (S-old) f(x) = 0.52x + 55.92 Figure 4. Scatter plots of simulated vs. observed surface concentrations of NO2, NO, NOx and O3. One scatter point resembles the mean over all "background" stations at noontime of one day. The left panel (a) - (g) shows the results of the new simulation (S-new) and the right panel (b) - (h) shows the results of the simulation with the original temporal profiles (S-old). The dashed line in each subplot marks the 1:1 line. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. second line of Table 2). However, the difference in RMSE between S-new and S-old is on the scale of ∼0.1 µg m−3 for NO, which is minuscule second line of Table 2). However, the difference in RMSE between S-new and S-old is on the scale of ∼0.1 µg m−3 for NO, which is minuscule. 0 2 4 6 8 10 12 14 0 5 10 15 (b) (b) (b) (b) (b) (b) (b) (b) NO2 (S-old) f(x) = 0.73x + 0.87 (d) (d) (d) (d) (d) (d) (d) (d) NO (S-old) 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0 5 10 15 20 Simulated mass concentration [µg m 3] (a) (a) (a) (a) (a) (a) (a) (a) NO2 (S-new) f(x) = 0.84x + 1.39 0 0 5 10 15 ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (b) (b) (b) (b) (b) (b) (b) (b) (d) (d) (d) (d) (d) (d) (d) (d) (f) (f) (f) (f) (f) (f) (f) (f) Simulated mass (h) (h) (h) (h) (h) (h) (h) (h) 0 20 40 60 80 100 120 Observed mass concentration [µg m 3] 0 50 100 150 f(x) = 0.52x + 55.92 Figure 4. Scatter plots of simulated vs. observed surface concentrations of NO2, NO, NOx and O3. One scatter point resembles the mean over all "background" stations at noontime of one day. The left panel (a) - (g) shows the results of the new simulation (S-new) and the right panel (b) - (h) shows the results of the simulation with the original temporal profiles (S-old). The dashed line in each subplot marks the 1:1 line. 13 3.2 Comparison of NO2 vertical column densities As a second diagnostic, we compare simulated NO2 vertical column densities (VCDs) to observations of the TROPOMI (TROPOspheric Monitoring Instrument, see Veefkind et al., 2012) satellite instrument. The satellite overpass occurs daily, 265 typically at 13:30 local time in central Europe, with a pixel size of 3.5 km × 5.5 km in NADIR viewing geometry. TROPOMI measures spectra of backscattered sunlight from earth’s surface, from which tropospheric slant column densities (SCDs) are computed using Differential Optical Absorption Spectroscopy (DOAS, see Platt and Stutz, 2008). Tropospheric VCDs are obtained from tropospheric SCDs via VCDtrop = SCDtrop AMFtrop (7) 270 where AMFtrop stands for the tropospheric air mass factor. The tropospheric AMF depends on a number of atmospheric and surface conditions and assumptions, e.g. cloud properties, the stratospheric column, surface albedo, and the relative a priori NO2 profile. In the TROPOMI retrieval, the NO2 a priori profiles are taken from the TM5 global CT model (see Krol et al. (2005); Williams et al. (2017)), with a spatial resolution of ∼1◦× 1◦≈110 km × 70 km where our simulation domain D2 is (7) located. A detailed description of the retrieval algorithm can be found in van Geffen et al. (2022) and Eskes et al. (2021). From 275 hereon we exclusively refer to tropospheric VCDs and SCDs, respectively. Simulated VCDs are obtained by vertical integration of box VCDs (= partial columns) from WRF-Chem. The WRF-Chem output is interpolated to the vertical grid of the TM5 model. The box VCDs are computed by multiplying the NO2 concentration within each model grid cell with its vertical extent. The partial columns are then summed up: located. A detailed description of the retrieval algorithm can be found in van Geffen et al. (2022) and Eskes et al. (2021). From 275 hereon we exclusively refer to tropospheric VCDs and SCDs, respectively. Si l t d VCD bt i d b ti l i t ti f b VCD ( ti l l ) f WRF Ch Th WRF Ch Simulated VCDs are obtained by vertical integration of box VCDs (= partial columns) from WRF-Chem. The WRF-Chem output is interpolated to the vertical grid of the TM5 model. The box VCDs are computed by multiplying the NO2 concentration within each model grid cell with its vertical extent. TM5 model. It has been demonstrated that replacing TM5 a priori profiles with profiles from high-resolution RCT models improves the VCD retrieval significantly (see e.g. Ialongo et al. (2020); Tack et al. (2021); Liu et al. (2021)). TM5 model. It has been demonstrated that replacing TM5 a priori profiles with profiles from high-resolution RCT models improves the VCD retrieval significantly (see e.g. Ialongo et al. (2020); Tack et al. (2021); Liu et al. (2021)). TM5 model. It has been demonstrated that replacing TM5 a priori profiles with profiles from high-resolution RCT models improves the VCD retrieval significantly (see e.g. Ialongo et al. (2020); Tack et al. (2021); Liu et al. (2021)). We incorporate the AKs and our simulated high-resolution NO2 a priori profiles by computing corrected observed VCDs following Eskes et al. (2019): We incorporate the AKs and our simulated high-resolution NO2 a priori profiles by computing corrected observed VCDs following Eskes et al. (2019): We incorporate the AKs and our simulated high-resolution NO2 a priori profiles by computing corrected observed VCDs following Eskes et al. (2019): obs · AMFtrop AMF · P l<13 cl · ∆hl P l<13 cl · ∆hl · Al (9) (9) where VCDobs denotes the observed NO2 VCD, AMFtrop the tropospheric AMF, and A the averaging kernel vector. where VCDobs denotes the observed NO2 VCD, AMFtrop the tropospheric AMF, and A the averaging kernel vector. 2. Each observed VCD has an associated q-value that describes the retrieval quality on a range from 0 (bad) to 1 (good) (see van Geffen et al., 2022). q-values of ≥0.75 also require the cloud radiance fraction (crf) to be smaller than 0.5, which effectively acts as a cloud filter. The effect of clouds on the measurement process depends on the height of the cloud layer: High clouds can shield off lower layers of the atmosphere, decreasing the NO2 sensitivity to almost zero below the cloud layer. Vice versa, cloud layers directly below NO2 layers can enhance the observed NO2 absorption due to the increased cloud albedo. If cloud layers and NO2 layers are at the same altitude, NO2 absorption can be increased by an enhanced light path due to multiple scattering. The influence of clouds on satellite measurements of NO2 has been discussed e.g. in Martin et al. (2002), Kokhanovsky and Rozanov (2008), and Liu et al. (2021). 3.2 Comparison of NO2 vertical column densities The partial columns are then summed up: (8) sim X l<13 l l ( ) where VCDsim denotes the simulated VCD, l the TM5 model layer index, cl the NO2 concentration in layer l, and ∆hl the vertical extent of layer l. Usually the summation would be conducted up until layer ltp, the tropopause layer of the TM5 model. Typically, ltp ≈16. WRF-Chem, however, is designed to be a tropospheric model, meaning that model results within the tropopause region should be used with caution. We observe an increase in modelled NO2 concentrations above 10 km altitude (which equates approximately to layer 13 in the TM5 model, see Fig. A3), which is likely due to stratosphere-troposphere 285 exchange. We therefore limit the summation of box VCDs to the lowest 13 layers. Then, pairs of simulated and observed VCDs are obtained by interpolating the WRF-Chem data to the horizontal TROPOMI grid in space (linear) and time (nearest neighbour). In order to make a representative comparison between simulated and observed VCDs, the following further aspects are taken into consideration: 290 In order to make a representative comparison between simulated and observed VCDs, the following further aspects are taken into consideration: 290 1. TROPOMI is not equally sensitive to all layers of the troposphere. This circumstance is described by the averaging kernels (AKs, see van Geffen et al., 2022). Thus the AMF depends on relative NO2 a priori profiles provided by the 1. TROPOMI is not equally sensitive to all layers of the troposphere. This circumstance is described by the averaging kernels (AKs, see van Geffen et al., 2022). Thus the AMF depends on relative NO2 a priori profiles provided by the 14 We apply a q-filter that removes all observations with q < 0.75, as recommended by Eskes et al. (2019). Figure 5 shows the result of our comparison in the form of monthly mean values (i.e. the total average over May 2019). Subfigures a-e show the comparison between simulated NO2 VCDs and TROPOMI observations using the TM5 NO2 a priori Subfigures a-e show the comparison between simulated NO2 VCDs and TROPOMI observations using the TM5 NO2 a priori profiles. Strong enhancements occur in west Germany (Ruhr region) and along the Rhine river. Moderate enhancements occur 310 over logistical hotpots (e.g. the port of Hamburg with a nearby coal power plant), and larger cities (e.g. Berlin and Munich; see Fig. 8 for an overview of the geographical regions mentioned here). Subfigure c shows the difference between simulated and observed VCDs and reveals the model’s tendency to overestimate the observed VCDs. The overestimation is close to zero in rural regions but reaches up to 1016 molec. cm−2 over hotspot regions. The simulated and observed VCDs correlate with profiles. Strong enhancements occur in west Germany (Ruhr region) and along the Rhine river. Moderate enhancements occur 310 over logistical hotpots (e.g. the port of Hamburg with a nearby coal power plant), and larger cities (e.g. Berlin and Munich; see Fig. 8 for an overview of the geographical regions mentioned here). Subfigure c shows the difference between simulated and observed VCDs and reveals the model’s tendency to overestimate the observed VCDs. The overestimation is close to zero in rural regions but reaches up to 1016 molec. cm−2 over hotspot regions. The simulated and observed VCDs correlate with an R-value of 0.83, an RMSE of 1.23 · 1015 molec. cm−2, and a bias of +17.6 %. Subfigures f-j show the same comparison, 315 but with recomputed airmass factors according to eq. (9). The comparison improves significantly with an R-value of 0.84, an RMSE of 1.11 · 1015 molec. cm−2, and a bias of only −6.6 %. Subfigures k-o show the spatial distribution of simulated and observed surface NO2 concentrations. Model overestimations of NO2 VCD and surface concentration occur in similar geographic regions, particularly in the Ruhr region, along the Rhine an R-value of 0.83, an RMSE of 1.23 · 1015 molec. cm−2, and a bias of +17.6 %. Subfigures f-j show the same comparison, 315 but with recomputed airmass factors according to eq. (9). The comparison improves significantly with an R-value of 0.84, an RMSE of 1.11 · 1015 molec. cm−2, and a bias of only −6.6 %. Subfigures k-o show the spatial distribution of simulated and observed surface NO2 concentrations. Model overestimations of NO2 VCD and surface concentration occur in similar geographic regions, particularly in the Ruhr region, along the Rhine Subfigures k-o show the spatial distribution of simulated and observed surface NO2 concentrations. Model overestimations of NO2 VCD and surface concentration occur in similar geographic regions, particularly in the Ruhr region, along the Rhine river and the port of Hamburg This hints towards a possible overestimation of NO emissions in these regions 320 Subfigures k-o show the spatial distribution of simulated and observed surface NO2 concentrations. Model overestimations of NO2 VCD and surface concentration occur in similar geographic regions, particularly in the Ruhr region, along the Rhine river, and the port of Hamburg. This hints towards a possible overestimation of NOx emissions in these regions. 320 river, and the port of Hamburg. This hints towards a possible overestimation of NOx emissions in these regions. 320 Figure 6 presents further comparisons of simulated and observed NO2 VCDs in the style of Fig. 5. Here, we only show comparisons using reprocessed observations according to eq. (9). Subfigures a-e show the comparison restricted to Germany, where the high-resolution UBA-E emission inventory was used. Compared to the full domain, an increase in RMSE (1.46 · 1015 molec. cm−2) and decrease in bias (+3.3%) is observed. The increase in RMSE can be attributed to the Ruhr region in , p g p x g Figure 6 presents further comparisons of simulated and observed NO2 VCDs in the style of Fig. 5. Here, we only show comparisons using reprocessed observations according to eq. (9). Subfigures a-e show the comparison restricted to Germany, where the high-resolution UBA-E emission inventory was used. Compared to the full domain, an increase in RMSE (1.46 · 1015 molec. cm−2) and decrease in bias (+3.3%) is observed. The increase in RMSE can be attributed to the Ruhr region in west Germany which is one of the most polluted regions within our simulation domain Subfigures f-j show the simulation 325 west Germany, which is one of the most polluted regions within our simulation domain. Subfigures f-j show the simulation 325 15 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 53°N 50°N 47°N 17°E 11°E 5°E WRF-Chem (d) (a) (b) (c) (d) (e) (f) (g) (h) Figure 7. Comparison of simulated and observed NO2 VCDs for two exemplary individual days. (a)-(d) shows 23 May 2019, (e)-(h) shows 15 May 2019. Gray regions are filtered due to the strict masking of clouded pixels. (d) 53°N 50°N 47°N 17°E 11°E 5°E WRF-Chem (d) (a) (b) (c) (d) (d) (h) (e) (f) (g) (h) (e) (f) (g) (g) Figure 7. Comparison of simulated and observed NO2 VCDs for two exemplary individual days. (a)-(d) shows 23 May 2019, (e)-(h) shows 15 May 2019. Gray regions are filtered due to the strict masking of clouded pixels. domain with Germany excluded. Although there are still some strongly polluted regions where WRF-Chem underestimates the NO2 VCD (e.g. the Netherlands) there are also large regions with model underestimation (e.g. Paris, France, Poland, Austria, and Hungary). This results in an overall mean bias of −9.6% on the displayed domain. Fi 6k h h i h f ll i l i d i i h i i l l fil b K l (2021) domain with Germany excluded. Although there are still some strongly polluted regions where WRF-Chem underestimates the NO2 VCD (e.g. the Netherlands) there are also large regions with model underestimation (e.g. Paris, France, Poland, Austria, and Hungary). This results in an overall mean bias of −9.6% on the displayed domain. Figure 6k-o shows the comparison on the full simulation domain using the original temporal profiles by Kumar et al. (2021), Figure 6k-o shows the comparison on the full simulation domain using the original temporal profiles by Kumar et al. (2021), referred to as S-old previously. Although the linear fit in subfigure o is remarkably good with a slope of 1.00, the overall mean 330 bias is −10.2%. This is in agreement with sect. 3.1, where it was shown that S-old suffers from a low bias of −15.7 % for NO2 surface concentrations around noon time. However, in stark contrast to the surface concentrations, which were 16.6 % lower in S-old, the simulated NO2 VCDs are reduced by only 3.6 % in comparison to S-new. This shows that surface NO2 concentrations are highly sensitive to the hourly profiles (i.e. the diurnal distribution of emissions), while the integrated NO2 column mostly depends on the total emission budget. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 53°N 50°N 47°N 17°E 11°E 5°E WRF-Chem ure 5. Comparison of simulated and observed NO2 VCDs (a)-(j) and surface NO2 concentrations (k)-(o). The first row (a)-(e) shows comparison to the original observations using low-resolution TM5 a priori NO2 profiles. The second row (f)-(j) shows the comparison reprocessed observations using high-resolution WRF-Chem a priori profiles and averaging kernels. All satellite observations were icted to cases with q ≥0.75. The third row (k)-(o) shows a comparison between simulated and observed surface NO2 concentrations m "background" stations. 53°N 50°N 47°N 17°E 11°E 5°E WRF-Chem Figure 5. Comparison of simulated and observed NO2 VCDs (a)-(j) and surface NO2 concentrations (k)-(o). The first row (a)-(e) shows the comparison to the original observations using low-resolution TM5 a priori NO2 profiles. The second row (f)-(j) shows the comparison with reprocessed observations using high-resolution WRF-Chem a priori profiles and averaging kernels. All satellite observations were restricted to cases with q ≥0.75. The third row (k)-(o) shows a comparison between simulated and observed surface NO2 concentrations from "background" stations. 16 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 53°N 50°N 47°N 17°E 11°E 5°E WRF-Chem (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (n) (o) ure 6. Further comparisons of simulated and observed NO2 VCDs. The first row (a)-(e) shows the comparison only for Germany. The nd row (f)-(j) shows the comparison for the entire simulation domain except for Germany. The third row (k)-(o) shows a comparison re instead of the newly optimized temporal profiles, the original ones by Kumar et al. (2021) were used. (e) WRF-Chem 53°N 50°N 47°N 17°E 11°E 5°E WRF-Chem (a) (b) (c) (d) (e) (e) 47 N (f) (g) (h) (i) (j) (j) (i) (f) (g) (h) (i) (j) (f) (g) (h) (i) (j) (h) (n) (o) (k) (l) (m) (n) (o) (m) Figure 6. Further comparisons of simulated and observed NO2 VCDs. The first row (a)-(e) shows the comparison only for Germany. The second row (f)-(j) shows the comparison for the entire simulation domain except for Germany. The third row (k)-(o) shows a comparison where instead of the newly optimized temporal profiles, the original ones by Kumar et al. (2021) were used. 17 17 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1473 Preprint. The two displayed days were chosen because they cover the most pol- luted regions of the simulation domain well. In comparison to monthly means, the modelled NO2 VCDs are smeared out. This is most likely an artefact of the simulated wind directions and speeds. In a monthly mean, such effects are expected to cancel out 340 statistically. Furthermore, a significant decrease in R-value (0.66 and 0.80) and an increase in RMSE (2.32·1015 molec cm−2 and 1.92·1015 molec cm−2) is observed in comparison to monthly means (R = 0.84, RMSE = 1.11·1015 molec cm−2). This is expected, seeing that averaging reduces the statistical noise of both datasets. Noise sources that contribute statistically in- clude the DOAS fit error (from the SCD retrieval), uncertainties of the AMF (e.g. due to clouds) and meteorological model variables. Table 4 summarizes the results of this section. 345 340 335 referred to as S-old previously. Although the linear fit in subfigure o is remarkably good with a slope of 1.00, the overall mean 330 bias is −10.2%. This is in agreement with sect. 3.1, where it was shown that S-old suffers from a low bias of −15.7 % for NO2 surface concentrations around noon time. However, in stark contrast to the surface concentrations, which were 16.6 % lower in S-old, the simulated NO2 VCDs are reduced by only 3.6 % in comparison to S-new. This shows that surface NO2 concentrations are highly sensitive to the hourly profiles (i.e. the diurnal distribution of emissions), while the integrated NO2 column mostly depends on the total emission budget. 335 So far only monthly means were considered in the evaluation. Figure 7 shows the comparison for two exemplary individual days: 23 May and 15 May 2019. When looking at single days, large regions of the maps are filtered due to the strict masking of So far only monthly means were considered in the evaluation. Figure 7 shows the comparison for two exemplary individual days: 23 May and 15 May 2019. When looking at single days, large regions of the maps are filtered due to the strict masking of 18 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 4. Statistical summary of the results shown in Fig. 5, 6, and 7 Referring to NO2 a priori profiles Mean (sim.) Mean (obs.) Slope Intercept RMSE Bias R Fig. 5a-e TM5 2.52 2.14 1.39 -0.04 1.23 +17.6 % 0.83 Fig. 5f-j WRF-Chem 2.52 2.70 1.15 -0.06 1.11 -6.6 % 0.84 Germany only, Fig. 6a-e WRF-Chem 3.48 3.37 1.38 -0.12 1.46 +3.3 % 0.83 without Germany, Fig. 6f-j WRF-Chem 2.30 2.54 1.08 -0.04 1.01 -9.6 % 0.83 old temporal profiles, Fig. 6k-o WRF-Chem 2.41 2.68 1.00 -0.03 0.97 -10.2 % 0.83 single day (23 May 2019, Fig. 7a-d) WRF-Chem 2.78 3.15 0.80 0.03 2.32 -11.8 % 0.66 single day (15 May 2019, Fig. 7e-h) WRF-Chem 2.89 2.89 1.13 -0.04 1.92 -0.3 % 0.80 Means and RMSE are given in units of [1015 molec. cm−2] clouded pixels. This is the case for all days of May 2019. The two displayed days were chosen because they cover the most pol- luted regions of the simulation domain well. In comparison to monthly means, the modelled NO2 VCDs are smeared out. This is most likely an artefact of the simulated wind directions and speeds. In a monthly mean, such effects are expected to cancel out 40 statistically. Furthermore, a significant decrease in R-value (0.66 and 0.80) and an increase in RMSE (2.32·1015 molec cm−2 and 1.92·1015 molec cm−2) is observed in comparison to monthly means (R = 0.84, RMSE = 1.11·1015 molec cm−2). This is expected, seeing that averaging reduces the statistical noise of both datasets. Noise sources that contribute statistically in- clude the DOAS fit error (from the SCD retrieval), uncertainties of the AMF (e.g. due to clouds) and meteorological model variables. Table 4 summarizes the results of this section. 45 clouded pixels. This is the case for all days of May 2019. The two displayed days were chosen because they cover the most pol- luted regions of the simulation domain well. In comparison to monthly means, the modelled NO2 VCDs are smeared out. This is most likely an artefact of the simulated wind directions and speeds. In a monthly mean, such effects are expected to cancel out 40 15 2 clouded pixels. This is the case for all days of May 2019. Means and RMSE are given in units of [1015 molec. cm−2] 3.3 Comparison of NO2 concentration profiles As a third diagnostic we compare modelled NO2 concentration profiles to profiles obtained from MAX-DOAS (Multi-Axis Differential Optical Absorption Spectroscopy) measurements. In contrast to the data shown in sect. 3.1 and 3.2, profile com- parison allows for assessment of the model’s capability to capture vertical distributions of NO2. MAX-DOAS measurements As a third diagnostic we compare modelled NO2 concentration profiles to profiles obtained from MAX DOAS (Multi Axis Differential Optical Absorption Spectroscopy) measurements. In contrast to the data shown in sect. 3.1 and 3.2, profile com- parison allows for assessment of the model’s capability to capture vertical distributions of NO2. MAX-DOAS measurements use the DOAS principle (see Platt and Stutz, 2008) to obtain trace gas differential slant column densities (dSCDs) at different 350 elevation angles. By application of an inversion algorithm a discretized concentration vector c is obtained, whose entries de- note the target gas concentration in different atmospheric layers. An overview of different inversion algorithms can be found in Frieß et al. (2019). We use data from the FRM4DOAS network (see Fayt et al., 2021) which applies the Mexican MAX-DOAS fit (MMF, see Friedrich et al., 2019) and the Mainz Profile Algorithm (MAPA, see Beirle et al., 2019). While MMF is based on use the DOAS principle (see Platt and Stutz, 2008) to obtain trace gas differential slant column densities (dSCDs) at different 350 elevation angles. By application of an inversion algorithm a discretized concentration vector c is obtained, whose entries de- note the target gas concentration in different atmospheric layers. An overview of different inversion algorithms can be found in Frieß et al. (2019). We use data from the FRM4DOAS network (see Fayt et al., 2021) which applies the Mexican MAX-DOAS fit (MMF, see Friedrich et al., 2019) and the Mainz Profile Algorithm (MAPA, see Beirle et al., 2019). While MMF is based on optimal estimation (see Rodgers, 2000), MAPA uses Monte Carlo simulation in order to determine profile shape parameters 355 which combine (possibly lifted) box profiles and exponential profiles. Purely exponential concentration profiles, however, can not be obtained from MAPA. 19 Germany Belgium Netherlands Hamburg Berlin Munich Rhine river Ruhr region Heidelberg Mainz De Bilt Uccle Bremen Paris France Czechia Austria Figure 8. Part of central Europe covering Germany, Belgium and Netherlands with important geographical regions marked in red. The blue stars show the locations of the MAX-DOAS measurements reported in sect. 3.3. Figure 8. 3.3 Comparison of NO2 concentration profiles Part of central Europe covering Germany, Belgium and Netherlands with important geographical regions marked in red. The blue stars show the locations of the MAX-DOAS measurements reported in sect. 3.3. Five MAX-DOAS instruments are operated within our simulation domain D2: Mainz (Germany), Bremen (Germany), Hei- delberg (Germany), De Bilt (Netherlands), and Uccle (Belgium). Figure 8 shows the locations of these stations. A single station Five MAX-DOAS instruments are operated within our simulation domain D2: Mainz (Germany), Bremen (Germany), Hei- delberg (Germany), De Bilt (Netherlands), and Uccle (Belgium). Figure 8 shows the locations of these stations. A single station typically yields 2-4 NO2 profile measurements per hour. In order to compare simulated and observed profiles, the WRF-Chem 360 dataset is interpolated to the geolocations and measurement times of the MAX-DOAS instruments. We use nearest-neighbour interpolation in space and time. The uncertainty of the simulated NO2 profiles is obtained as the standard deviation of the surrounding eight WRF-Chem grid cells. The MMF inversion algorithm provides averaging kernels (AKs), represented by a (h × The MMF inversion algorithm provides averaging kernels (AKs), represented by a (h × h) matrix, where h is the number of atmospheric layers considered. Here, h = 20, comprising retrieval altitudes up to 4 km. Using the AKs, the MAX-DOAS 365 measuring sensitivity to different altitudes can be applied to the simulated profiles. For this purpose the simulated NO2 profiles are vertically interpolated to the layers of the MAX-DOAS retrieval grid (linear interpolation). Then, the AKs are applied via csim, corr = Acsim + (1l −A)cap (10) of atmospheric layers considered. Here, h = 20, comprising retrieval altitudes up to 4 km. Using the AKs, the MAX-DOAS 365 measuring sensitivity to different altitudes can be applied to the simulated profiles. For this purpose the simulated NO2 profiles are vertically interpolated to the layers of the MAX-DOAS retrieval grid (linear interpolation). Then, the AKs are applied via csim, corr = Acsim + (1l −A)cap (10) (10) csim, corr = Acsim + (1l −A)cap where csim, corr is the corrected simulated profile, A the averaging kernel matrix, csim the orig where csim, corr is the corrected simulated profile, A the averaging kernel matrix, csim the original simulated profile, 1l the unity matrix, and cap the a priori profile see (see Rodgers and Connor, 2003). MAPA neither provides AKs, nor depends on an 370 a priori profile. 3.3 Comparison of NO2 concentration profiles All profiles flagged as "erroneous" by MAPA were dismissed from the evaluation. Figure 9 shows averaged NO2 profiles from the MAX-DOAS station Mainz, Germany, in the time window from 11 AM to 02 PM for a selection of individual days. The aim is to give an overview of the variety of observed and modelled profile shapes. 20 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 NO2 concentration [1011 molec. cm 3] 0 1 2 3 4 Altitude [km] (a) 2019-05-03 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (b) 2019-05-17 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 NO2 concentration [1011 molec. cm 3] 0 1 2 3 4 Altitude [km] (c) 2019-05-01 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (d) 2019-05-27 NO2 profiles in Mainz (11 AM - 02 PM) https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 NO2 concentration [1011 molec. cm 3] 0 1 2 3 4 Altitude [km] (a) 2019-05-03 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (b) 2019-05-17 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 NO2 concentration [1011 molec. cm 3] 0 1 2 3 4 Altitude [km] (c) 2019-05-01 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (d) 2019-05-27 NO2 profiles in Mainz (11 AM - 02 PM) MAX-DOAS (MAPA) MAX-DOAS (MMF) A priori profile WRF-Chem (with AK) WRF-Chem (without AK) DERP007 (UBA, Background) DERP009 (UBA, Background) DERP010 (UBA, Traffic) DERP011 (UBA, Traffic) Figure 9. Comparison of exemplary profiles for Mainz around noontime (11 AM - 02 PM) for four different days: (a) 03 May 2019, (b) 17 May 2019, (c) 01 May 2019, (d) 27 May 2019. Additionally, average NO2 surface concentrations measured by UBA measuring stations within a radius of 5 km of the MAX-DOAS instrument are drawn as colored scatter markers at 0 m altitude. NO2 profiles in Mainz (11 AM - 02 PM) NO2 profiles in Mainz (11 AM - 02 PM) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 NO2 concentration [1011 molec. cm 3] 0 1 2 3 4 Altitude [km] (a) 2019-05-03 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (b) 2019-05-17 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 NO2 concentration [1011 molec. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 9a shows a typical scenario: On this day (03 May 2019) observation and simulation show good general agreement. All three datasets (MMF, MAPA, and WRF-Chem) yield profiles of similar shape. Above an altitude of ∼2 km the profiles quickly approach zero, which is characteristic for the transitioning regime between the planetary boundary layer and the free troposphere. At low altitudes of 0-150 m the simulated profile has a strong exponential tail, typical for the surface layer into which most NOx emissions are injected (the lowest ∼30 m). Furthermore, the simulated profiles show good agreement with 380 the measurements of the nearby UBA in-situ stations. High vertical resolution turns out to be crucial for the comparison to the observed surface concentrations: In the FRM4DOAS dataset, MAPA and MMF were operated with a vertical resolution of 200 m. In a direct comparison, the concentrations obtained from MAPA or MMF in the lowest layer (0-200 m) underestimate the in-situ observations at the surface because of the limited vertical resolution of the MAX-DOAS measurements and the rather coarse retrieval grid used by FRM4DOAS. Meanwhile, the lowest layer of our WRF-Chem simulation only spans 0 - 8 m, 385 which allows for a much more representative comparison. Subfigure 9b shows a day on which an elevated NO2 layer was detected by MMF. This occurs regularly at all MAX-DOAS stations in this study and is characterized by strongly enhanced NO2 concentrations at higher altitudes (here at ∼500 m). Ele- vated layers are typically caused by elevated emissions, e.g. from a power plant stack at a few hundred meters height. Addition- coarse retrieval grid used by FRM4DOAS. Meanwhile, the lowest layer of our WRF-Chem simulation only spans 0 - 8 m, 385 which allows for a much more representative comparison. Subfigure 9b shows a day on which an elevated NO2 layer was detected by MMF. This occurs regularly at all MAX-DOAS stations in this study and is characterized by strongly enhanced NO2 concentrations at higher altitudes (here at ∼500 m). Ele- vated layers are typically caused by elevated emissions, e.g. from a power plant stack at a few hundred meters height. Addition- ally, transport events which advect NO2 from the surface layer could be the cause. However, no corresponding enhancements 390 can be seen in the simulated NO2 profile. As described in sect. 3.3 Comparison of NO2 concentration profiles cm 3] 0 1 2 3 4 Altitude [km] (c) 2019-05-01 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (d) 2019-05-27 NO2 profiles in Mainz (11 AM - 02 PM) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 NO2 concentration [1011 molec. cm 3] 0 1 2 3 4 Altitude [km] (a) 2019-05-03 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (b) 2019-05-17 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 NO2 concentration [1011 molec. cm 3] 0 1 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 NO2 concentration [1011 molec. cm 3] 0 1 2 3 4 Altitude [km] (c) 2019-05-01 0 1 2 3 4 5 NO2 concentration [1011 molec. cm 3] (d) 2019-05-27 (c) Altitude [km] NO2 concentration [1011 molec. cm 3] Figure 9. Comparison of exemplary profiles for Mainz around noontime (11 AM - 02 PM) for four different days: (a) 03 May 2019, (b) 17 May 2019, (c) 01 May 2019, (d) 27 May 2019. Additionally, average NO2 surface concentrations measured by UBA measuring stations within a radius of 5 km of the MAX-DOAS instrument are drawn as colored scatter markers at 0 m altitude. Additionally, the average NO2 surface concentrations measured by UBA stations within a radius of 5 km of the MAX-DOAS instrument are drawn as colored scatter markers at 0 m altitude. 375 Additionally, the average NO2 surface concentrations measured by UBA stations within a radius of 5 km of the MAX-DOAS instrument are drawn as colored scatter markers at 0 m altitude. 375 375 21 2.1, we do not use vertical emission profiles in the WRF-Chem simulation. Furthermore, it is possible that the overall spatial resolution of our simulation limits its ability to model compa- rable elevated trace gas abundances. MAX-DOAS instruments typically have a field of view of ∼1◦, which is small enough to resolve e.g. plumes from power plant stacks. Our WRF-Chem simulation has a spatial resolution of 3 km × 3 km × 100 m ally, transport events which advect NO2 from the surface layer could be the cause. However, no corresponding enhancements 390 can be seen in the simulated NO2 profile. As described in sect. 2.1, we do not use vertical emission profiles in the WRF-Chem simulation. Furthermore, it is possible that the overall spatial resolution of our simulation limits its ability to model compa- rable elevated trace gas abundances. MAX-DOAS instruments typically have a field of view of ∼1◦, which is small enough to resolve e.g. plumes from power plant stacks. Our WRF-Chem simulation has a spatial resolution of 3 km × 3 km × 100 m at ∼500 m altitude and therefore dilutes trace gas concentrations into comparably large grid cell volumes. Lastly, horizontal 395 concentration gradients can be expected near strong emission sources. These can lead to apparently elevated profiles, because the MAX-DOAS profile inversion makes the simplifying assumption of horizontally homogeneous distributions. Comparison of simulated and observed elevated urban emissions is therefore an advanced problem and not further adressed here. Subfigure 9c shows a day on which the agreement between model and observations was poor, with deviations of > 100 %. at ∼500 m altitude and therefore dilutes trace gas concentrations into comparably large grid cell volumes. Lastly, horizontal 395 concentration gradients can be expected near strong emission sources. These can lead to apparently elevated profiles, because the MAX-DOAS profile inversion makes the simplifying assumption of horizontally homogeneous distributions. Comparison of simulated and observed elevated urban emissions is therefore an advanced problem and not further adressed here. Subfigure 9c shows a day on which the agreement between model and observations was poor, with deviations of > 100 %. g y g p , Such days are outliers and do not represent the overall quality of the model or the measurements, however, they should not 400 remain unmentioned. Possible reasons for such deviations include e.g. falsely modelled wind directions (affecting trace gas transport) or cloud cover (affecting photolysis). The scatter plots (subfigures b, d, f) show a strong correlation between simulation and measurements Figure 10 shows the results of the three German MAX-DOAS locations Mainz, Bremen, and Heidelberg. Overall, the three stations show qualitatively similar profiles. At Bremen (see subfigures c-d), a consistent elevated layer at ∼1000 m altitude was detected, which could be explained by multiple power plants in the near vicinity of the instrument. This has been discussed in detail by Bösch (2018). The scatter plots (subfigures b, d, f) show a strong correlation between simulation and measurements and yield linear fits with slopes of 1.32 for Mainz, 1.15 for Bremen, and 0.97 for Heidelberg. The results shown in Fig. 11 420 show similar agreement in De Bilt but larger deviations in Uccle. As a result, the slope of 1.58 for the linear fit in Fig. 11d is significantly larger than for the other stations. Table 5 gives a statistical summary of the results shown in Fig. 10 and Fig. 11. As an additional diagnostic, a correlation analysis between observed (MMF) and simulated (with AKs applied) NO2 con- centrations in the lowest 0-200 m above the surface is shown in Appendix A (see Fig. A4). Subfigure 9d shows a day on which the modelled profile has an extremely steep exponential gradient in combination with a relatively thin boundary layer. At the transition from the boundary layer to the surface layer (∼30 - 50 m above ground), Such days are outliers and do not represent the overall quality of the model or the measurements, however, they should not 400 remain unmentioned. Possible reasons for such deviations include e.g. falsely modelled wind directions (affecting trace gas transport) or cloud cover (affecting photolysis). Subfigure 9d shows a day on which the modelled profile has an extremely steep exponential gradient in combination with a relatively thin boundary layer. At the transition from the boundary layer to the surface layer (∼30 - 50 m above ground), a relatively thin boundary layer. At the transition from the boundary layer to the surface layer ( 30 50 m above ground), the simulated profiles shows an increase of almost 400 %. Interestingly, on this day (27 May 2019), one of the background 405 stations (DERP009) measured more than twice the surface concentration compared to a nearby traffic station (DERP011). This phenomenon was observed on only one other day (10 May 2019) of the simulated month, on which very similar profile shapes were observed. Furthermore, no MAPA profile is available on this day due to the filtering by error flags. Also, there is good agreement between modelled and observed profiles above the surface layer. It is therefore plausible to assume that the steep the simulated profiles shows an increase of almost 400 %. Interestingly, on this day (27 May 2019), one of the background 405 stations (DERP009) measured more than twice the surface concentration compared to a nearby traffic station (DERP011). This phenomenon was observed on only one other day (10 May 2019) of the simulated month, on which very similar profile shapes were observed. Furthermore, no MAPA profile is available on this day due to the filtering by error flags. Also, there is good agreement between modelled and observed profiles above the surface layer. It is therefore plausible to assume that the steep 22 22 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 5. Statistical summary of the results shown in Fig. 10 and 11 Location Mean (sim.) Mean (obs.) Slope Intercept RMSE Bias Mainz 3.05 2.03 1.32 0.38 1.35 +50.3 % Bremen 1.83 1.94 1.15 -0.41 1.42 -5.9 % Heidelberg 2.57 1.98 0.97 0.66 0.93 +30.1 % De Bilt 2.40 2.05 1.16 0.02 0.54 +16.9 % Uccle 4.11 2.30 1.58 0.48 2.44 +78.6 % Means, intercept, and RMSE are given in units of [1010 molec. cm−3]. NO2 gradient towards the surface is not due to a faulty model, but, on the contrary, indicates the model’s ability to capture 410 scenarios in which e.g. vertical mixing is mostly suppressed and large portions of the emitted NO2 remain in the surface layer. In order to condense the remaining evaluation, we will focus on monthly noontime averages from hereon, i.e. plots of the same structure as shown in Fig. 9, but averaged over the entire simulation period of May 2019 (Fig. 10 and 11). In both figures the right-side panel shows scatter plots of averaged NO2 concentrations (WRF-Chem vs. MMF, due to availability of AKs) at diff t ltit d 415 NO2 gradient towards the surface is not due to a faulty model, but, on the contrary, indicates the model’s ability to capture 410 scenarios in which e.g. vertical mixing is mostly suppressed and large portions of the emitted NO2 remain in the surface layer. In order to condense the remaining evaluation, we will focus on monthly noontime averages from hereon, i.e. plots of the same structure as shown in Fig. 9, but averaged over the entire simulation period of May 2019 (Fig. 10 and 11). In both figures the right-side panel shows scatter plots of averaged NO2 concentrations (WRF-Chem vs. MMF, due to availability of AKs) at different altitudes. 415 Figure 10 shows the results of the three German MAX-DOAS locations Mainz, Bremen, and Heidelberg. Overall, the three stations show qualitatively similar profiles. At Bremen (see subfigures c-d), a consistent elevated layer at ∼1000 m altitude was detected, which could be explained by multiple power plants in the near vicinity of the instrument. This has been discussed in detail by Bösch (2018). 4 Discussion and conclusion 425 We have presented new WRF-Chem simulation results with specific focus on NO2. The simulation was run on a domain over central Europe with a spatial resolution of 3 km×3 km for the month of May 2019. Over Germany, a new emission inventory from the German Environmental Agency with a resolution of 1 km × 1 km was used. Outside of Germany, the EDGARv5 emission inventory with a resolution of 11 km×7 km was used. A new set of temporal emission profiles for the most important emission sectors (traffic, power industry, agricultural soils, energy for buildings, and manufacturing industry) was determined 430 by comparing simulated NOx surface concentrations to corresponding in-situ measurements in Germany. Once good agreement was found, the model results were also compared to satellite measurements of the NO2 VCD and NO2 concentration profiles emission sectors (traffic, power industry, agricultural soils, energy for buildings, and manufacturing industry) was determined 430 by comparing simulated NOx surface concentrations to corresponding in-situ measurements in Germany. Once good agreement was found, the model results were also compared to satellite measurements of the NO2 VCD and NO2 concentration profiles 23 0 1 2 3 4 Altitude [km] (MMF) 17.5 cm * 10.8156 cm (double col. Fig., golden ratio) 0 1 2 3 4 Altitude [km] MAX-DOAS (MAPA) MAX-DOAS (MMF) WRF-Chem (with AK) WRF-Chem (without AK) Simulated NO2 concentration (with AK) UBA stations Figure 10. Comparison of averaged NO2 profiles for Mainz, Bremen, and Heidelberg around noontime (11 AM - 02 PM). The left column (a), (c), (e) shows the NO2 profiles as obtained by MMF, MAPA, and WRF-Chem. The right column (b), (d), (f) shows the corresponding scatter plots of averaged NO2 concentrations at different altitudes (here: WRF-Chem vs. MMF). imulated NO2 concentration (with AK) Figure 10. Comparison of averaged NO2 profiles for Mainz, Bremen, and Heidelberg around noontime (11 AM - 02 PM). The left column (a), (c), (e) shows the NO2 profiles as obtained by MMF, MAPA, and WRF-Chem. The right column (b), (d), (f) shows the corresponding scatter plots of averaged NO2 concentrations at different altitudes (here: WRF-Chem vs. MMF). 24 0 1 2 3 4 Altitude [km] 0 1 2 3 4 Altitude [km] MAX-DOAS (MAPA) MAX-DOAS (MMF) WRF-Chem (with AK) WRF-Chem (without AK) Simulated NO2 concentration (with AK) (MMF) Figure 11. Like Fig. 10, but for the stations De Bilt and Uccle. 4 Discussion and conclusion 425 Simulated NO2 concentration (with AK) (MMF) mulated NO2 concentration (with AK) (MMF) Figure 11. Like Fig. 10, but for the stations De Bilt and Uccle. obtained from MAX-DOAS measurements. The main focus of the study was to investigate, whether RCT simulations can be improved by fine-tuning of the temporal emission profiles and whether good agreement with all three observational datasets can be achieved. 5 obtained from MAX-DOAS measurements. The main focus of the study was to investigate, whether RCT simulations can be improved by fine-tuning of the temporal emission profiles and whether good agreement with all three observational datasets obtained from MAX-DOAS measurements. The main focus of the study was to investigate, whether RCT simulations can be improved by fine-tuning of the temporal emission profiles and whether good agreement with all three observational datasets can be achieved. 435 In sect. 3.1 modelled surface concentrations of NO2, NO, NOx and O3 were compared to in-situ measurements. Here, we used simulation results of two WRF-Chem runs: Firstly, a run with conventional temporal profiles used in previously pub- lished literature, and secondly, a run with our newly optimized temporal profiles. The optimized simulation showed significant improvements over the conventional simulation. In particular, the diurnal variation of trace gas concentration was much more accurately reproduced using the new temporal profiles. NOx biases were reduced from +23.4 % to +6.7 % (all hours of the day 440 considered) and from -19.1 % to +0.4 % (at noontime). NO2 biases were reduced from +34.0 % to +14.3 % (all hours of the day considered) and from -15.7 % to +0.9 % (at noontime). The corresponding correlation coefficients also slightly improved. This way, the noon-time underestimations of NOx and NO2 could be resolved without any changes to the total emission budget. 25 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Underestimations like these were identified in recent literature, but have remained an open issue so far. Despite the significant improvements in NOx and NO2 modelling accuracy, some discrepancies remain: While NOx concentrations appear to be ac- 445 curate throughout the day, the NO2/NO ratio is too large before and after noontime. It was found that this problem can not be resolved by changing the speciation ratio of NOx, which can be explained by the rapid conversion of NO to NO2 directly after emission. It is therefore more likely that this issue relates to chemistry: Both O3 and VOCs oxidize NO to NO2, and a slight model overestimation of O3 (+10.9 %) was found. Additionally, O3 concentrations are influenced by VOCs. However, the reference data includes no measurements of VOCs, which means no direct validation of the modelled VOC concentrations is 450 possible. If such measurements become available in the future, the methods described in this article can be extended to further improve the simulation results. In sect. 3.2 modelled tropospheric NO2 VCDs were compared to TROPOMI measurements. In order to make a representative comparison the air mass factors of the TROPOMI retrieval were recomputed using the high-resolution NO2 profiles from our possible. If such measurements become available in the future, the methods described in this article can be extended to further improve the simulation results. In sect. 3.2 modelled tropospheric NO2 VCDs were compared to TROPOMI measurements. In order to make a representative comparison, the air mass factors of the TROPOMI retrieval were recomputed using the high-resolution NO2 profiles from our simulation. The distribution of modelled and observed NO2 VCDs was found to be similar and agrees with previously reported 455 simulation results. Monthly means of simulated and observed VCDs were shown to correlate strongly (R = 0.84) with a model bias of −6.6%. Recomputing the air mass factors using the NO2 profiles from the regional model was found to be crucial for good agreement: With the original air mass factors the model bias was much larger (+17.6 %). Comparison between the spatial distribution of NO2 VCDs and surface concentration revealed that in regions where the simulation overestimated the simulation. The distribution of modelled and observed NO2 VCDs was found to be similar and agrees with previously reported 455 simulation results. Monthly means of simulated and observed VCDs were shown to correlate strongly (R = 0.84) with a model bias of −6.6%. Recomputing the air mass factors using the NO2 profiles from the regional model was found to be crucial for good agreement: With the original air mass factors the model bias was much larger (+17.6 %). Comparison between the spatial distribution of NO2 VCDs and surface concentration revealed that in regions where the simulation overestimated the VCDs, it also tended to overestimate the surface concentrations. This was observed in the strongly polluted regions of west 460 Germany and the Netherlands. On the other hand, a general tendency for model underestimation in less polluted regions was found. This observation hints towards faulty NOx emissions in the corresponding regions. While surface concentrations were shown to be highly sensitive to the choice of temporal profiles, only a weak dependence was found for the NO2 VCDs. In a comparison to the simulation run with conventional temporal profiles, NO2 VCDs were only +3.6 % larger on average. The VCDs, it also tended to overestimate the surface concentrations. This was observed in the strongly polluted regions of west 460 Germany and the Netherlands. On the other hand, a general tendency for model underestimation in less polluted regions was found. This observation hints towards faulty NOx emissions in the corresponding regions. While surface concentrations were shown to be highly sensitive to the choice of temporal profiles, only a weak dependence was found for the NO2 VCDs. In a comparison to the simulation run with conventional temporal profiles, NO2 VCDs were only +3.6 % larger on average. The surface concentrations, on the other hand, were 16.6 % larger. 465 In sect. 3.3 modelled noontime NO2 concentration profiles were compared to profiles from MAX-DOAS measurements at 5 locations in central Europe from the FRM4DOAS network. The inversion algorithms MMF and MAPA were used. General agreement in the overall shape and magnitude of the profiles was found, with per-station concentration biases of +50.3 % (Mainz), −5.9 % (Bremen), +30.1 % (Heidelberg), +16.9 % (De Bilt) and +78.6 % (Uccle). Two qualitative differences surface concentrations, on the other hand, were 16.6 % larger. 465 In sect. 3.3 modelled noontime NO2 concentration profiles were compared to profiles from MAX-DOAS measurements at 5 locations in central Europe from the FRM4DOAS network. The inversion algorithms MMF and MAPA were used. General agreement in the overall shape and magnitude of the profiles was found, with per-station concentration biases of +50.3 % (Mainz), −5.9 % (Bremen), +30.1 % (Heidelberg), +16.9 % (De Bilt) and +78.6 % (Uccle). Two qualitative differences between observed and simulated profiles (as produced by WRF-Chem, i.e. without application of averaging kernels) were 470 identified: Firstly, the simulated profiles show steep NO2 gradients close to the surface and, furthermore, agree well with the collocated surface concentration measurements. The observed profiles do not resolve the surface layer, most likely due to the coarse retrieval grid with 200 m layer height. As a consequence, the observed profiles tend to underestimate the surface concentration measurements. Secondly, elevated NO2 layers were identified in some of the observed profiles, but not in the between observed and simulated profiles (as produced by WRF-Chem, i.e. without application of averaging kernels) were 470 identified: Firstly, the simulated profiles show steep NO2 gradients close to the surface and, furthermore, agree well with the collocated surface concentration measurements. The observed profiles do not resolve the surface layer, most likely due to the coarse retrieval grid with 200 m layer height. As a consequence, the observed profiles tend to underestimate the surface concentration measurements. Secondly, elevated NO2 layers were identified in some of the observed profiles, but not in the corresponding simulated profiles. Here, the lack of vertical emission profiles, the overall spatial resolution of our WRF-Chem 475 simulation, and the influence of horizontal concentration gradients on the MAX-DOAS inversion procedure were identified as possible explanations. corresponding simulated profiles. Here, the lack of vertical emission profiles, the overall spatial resolution of our WRF-Chem 475 simulation, and the influence of horizontal concentration gradients on the MAX-DOAS inversion procedure were identified as possible explanations. 26 profiles can be applied wherever suitable observational data is available, making the method viable for large parts of the world 490 and other seasons. In the future, our method of deriving temporal profiles could be made more rigorous by experimenting with modern optimization algorithms. This will take time and require the synergy of both, modelling and inversion expertise, but has the potential to make our method applicable to larger domains and simulation periods with less effort. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. We draw two main conclusions: Firstly, our study shows that the model accuracy for NOx and NO2 in RCT simulations can be strongly improved by optimizing the corresponding hourly emission profiles. In particular, the typical underestimations of noontime NOx and NO2 can be resolved. We have described a flexible manual approach for temporal profile tuning, which 480 only requires an iterative chain of model forward runs and comparison to corresponding in-situ observations. Secondly, the intercomparison of the observational datasets used for model validation (here: in-situ measurements, satellite observations, and MAX-DOAS measurements) are overall consistent and agree with our simulation results. It is important to emphazise the scope of our study. We have shown exemplary temporal profiles and simulation results which only represent central Europe in mid-summer. Influences such as regulations for industrial processes, changes in the 485 prevailing energy sources, or reduction of cars as a primary means of personal transport may impact the hourly profiles in the future. Furthermore, it is likely that the hourly profiles inhibit a seasonal dependence. Therefore, the temporal profiles we have presented here should not be seen as generally valid, but rather as a specific result obtained in the framework of our case study for the purpose of proving how sensitive simulation results are to them. However, our general procedure of finding temporal profiles can be applied wherever suitable observational data is available, making the method viable for large parts of the world 490 and other seasons. In the future, our method of deriving temporal profiles could be made more rigorous by experimenting with modern optimization algorithms. This will take time and require the synergy of both, modelling and inversion expertise, but has the potential to make our method applicable to larger domains and simulation periods with less effort. profiles can be applied wherever suitable observational data is available, making the method viable for large parts of the world 490 and other seasons. In the future, our method of deriving temporal profiles could be made more rigorous by experimenting with modern optimization algorithms. This will take time and require the synergy of both, modelling and inversion expertise, but has the potential to make our method applicable to larger domains and simulation periods with less effort. Data availability. All data are available from the authors upon request. Data availability. All data are available from the authors upon request. Author contributions. LK, TW, SB, and VK developed the question of research. LK and SO carried out the optimization process for the 495 temporal emission profiles. VK, LK, SO, and RK conducted the WRF-Chem simulations. LK, VK, TW, SO, SB, AP, and TB made the comparison between simulation and observational datasets. LK wrote the paper, with all authors contributing by revising it interactively. Author contributions. LK, TW, SB, and VK developed the question of research. LK and SO carried out the optimization process for the 495 temporal emission profiles. VK, LK, SO, and RK conducted the WRF-Chem simulations. LK, VK, TW, SO, SB, AP, and TB made the comparison between simulation and observational datasets. LK wrote the paper, with all authors contributing by revising it interactively. Competing interests. The authors declare that they have no conflict of interest. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1473 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. References Anenberg, S., Mohegh, A., Goldberg, D., Kerr, G., Brauer, M., Burkart, K., Hystad, P., Larkin, A., Wozniak, S., and Lamsal, L.: Long-term 505 trends in urban NO2 concentrations and associated paediatric asthma incidence: estimates from global datasets, The Lancet Planetary Health, 6, https://doi.org/10.1016/s2542-5196(21)00255-2, 2022. 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Data analysis and visualization were performed in Python 3.9 including standard libraries such as numpy, scipy, pandas, netCDF4, matplotlib, and basemap. For processing of emission data the HERMES software was used. One of the co-authors (R. Kumar) is from the National Center for Atmospheric Research, which is sponsored by the National Science Foundation of the United States. Acknowledgements. We acknowledge the Umweltbundesamt and Deutscher Wetterdienst for providing in situ measurement data and the UBA emission inventory. Furthermore, we acknowledge Udo Frieß and François Hendrick for providing access to and guidance through 500 the FRM4DOAS datasets. Data analysis and visualization were performed in Python 3.9 including standard libraries such as numpy, scipy, pandas, netCDF4, matplotlib, and basemap. For processing of emission data the HERMES software was used. One of the co-authors (R. 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K., Al-Saadi, J. A., Orlando, J. J., and Soja, A. J.: The Fire INventory from NCAR (FINN): a high resolution global model to estimate the emissions from open burning, Geoscientific Model Development, 4, 625– 641, https://doi.org/10.5194/gmd-4-625-2011, 2011. 670 670 641, https://doi.org/10.5194/gmd-4-625-2011, 2011. 670 Wild, R. J., Dubé, W. P., Aikin, K. C., Eilerman, S. J., Neuman, J. A., Peischl, J., Ryerson, T. B., and Brown, S. S.: On- road measurements of vehicle NO2/NOx emission ratios in Denver, Colorado, USA, Atmospheric Environment, 148, 182–189, https://doi.org/10.1016/j.atmosenv.2016.10.039, 2017. Williams, J. E., Boersma, K. F., Le Sager, P., and Verstraeten, W. W.: The high-resolution version of TM5-MP for optimized satellite i l d i i d lid i G i ifiM d l D l 10 721 750 h //d i /10 5194/ d 10 721 2017 2017 675 Wild, R. J., Dubé, W. P., Aikin, K. C., Eilerman, S. J., Neuman, J. A., Peischl, J., Ryerson, T. Exact layer bottoms and tops depend on location and time. The values given here are averages. B., and Brown, S. S.: On- road measurements of vehicle NO2/NOx emission ratios in Denver, Colorado, USA, Atmospheric Environment, 148, 182–189, https://doi.org/10.1016/j.atmosenv.2016.10.039, 2017. Wild, R. J., Dubé, W. P., Aikin, K. C., Eilerman, S. J., Neuman, J. A., Peischl, J., Ryerson, T. B., and Brown, S. S.: On- road measurements of vehicle NO2/NOx emission ratios in Denver, Colorado, USA, Atmospheric Environment, 148, 182–189, https://doi.org/10.1016/j.atmosenv.2016.10.039, 2017. Williams, J. E., Boersma, K. F., Le Sager, P., and Verstraeten, W. W.: The high-resolution version of TM5-MP for optimized satellite retrievals: description and validation, Geoscientific Model Development, 10, 721–750, https://doi.org/10.5194/gmd-10-721-2017, 2017. 675 Williams, J. E., Boersma, K. F., Le Sager, P., and Verstraeten, W. W.: The high-resolution version of TM5-MP for optimized satellite retrievals: description and validation, Geoscientific Model Development, 10, 721–750, https://doi.org/10.5194/gmd-10-721-2017, 2017. 675 675 retrievals: description and validation, Geoscientific Model Development, 10, 721–750, https://doi.org/10.5194/gmd-10-721-2017, 2017. 675 32 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Appendix A: Additional figures and tables 33 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table A1. Layer extent of the lowest 24 layers in our WRF-Chem simulation Layer number Layer bottom [m] Layer top [m] Layer number Layer bottom [m] Layer top [m] 1 0 8 13 1370 1546 2 8 33 14 1546 1697 3 33 66 15 1697 1841 4 66 125 16 1841 1937 5 125 209 17 1937 2035 6 209 310 18 2035 2183 7 310 429 19 2183 2374 8 429 575 20 2374 2661 9 575 741 21 2661 3142 10 741 935 22 3142 3907 11 935 1178 23 3907 4762 12 1178 1370 24 4762 5643 Exact layer bottoms and tops depend on location and time The values given here are averages r number Layer bottom [m] Layer top [m] Layer number Layer bottom [m] Layer top [m] Table A2. Statistical summary of the results shown in Fig. A4 Table A2. Statistical summary of the results shown in Fig. A4 Location Mean (sim.) Mean (obs.) Slope Intercept RMSE Bias R Mainz 11.69 7.18 0.07 11.15 10.42 +62.8 % 0.13 Bremen 9.09 4.68 0.64 6.12 8.63 +94.3 % 0.29 Heidelberg 8.21 9.60 0.15 6.78 17.06 -14.5 % 0.38 De Bilt 8.62 6.71 0.69 3.97 6.31 +28.5 % 0.56 Uccle 17.69 11.24 0.57 11.32 13.35 +57.3 % 0.45 Means, intercept, and RMSE are given in units of [1010 molec. cm−3]. 34 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0 10 20 30 40 50 60 NO2 conc. [µg m 3] (a) (a) 0 10 20 30 40 50 60 (e) (e) Monthly average 0 20 40 60 80 100 NO conc. [µg m 3] (b) (b) 0 20 40 60 80 100 (f) (f) 120 140 3] (c) (c) 120 140 (g) (g) UBA measurements S-new S-old 0 10 20 30 40 50 60 NO2 conc. [µg m 3] (a) (a) 0 10 20 30 40 50 60 (e) (e) Monthly average 0 20 40 60 80 100 NO conc. [µg m 3] (b) (b) 0 20 40 60 80 100 (f) (f) 0 20 40 60 80 100 120 140 NOx conc. [µg m 3] (c) (c) 0 20 40 60 80 100 120 140 (g) (g) Wed, 05-01 00:00 Thu, 05-02 00:00 Fri, 05-03 00:00 Sat, 05-04 00:00 Sun, 05-05 00:00 Mon, 05-06 00:00 Tue, 05-07 00:00 Wed, 05-08 00:00 Thu, 05-09 00:00 Fri, 05-10 00:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 Date and time 25 50 75 100 125 O3 conc. [µg m 3] (d) 00:00 04:00 08:00 12:00 16:00 20:00 00:00 Time of day 25 50 75 100 125 (h) UBA measurements S-new S-old 0 10 20 30 40 50 60 NO2 conc. [µg m 3] (a) (a) 0 10 20 30 40 50 60 (e) (e) Monthly average 0 20 40 60 80 100 NO conc. [µg m 3] (b) (b) 0 20 40 60 80 100 (f) (f) 0 20 40 60 80 100 120 140 NOx conc. Table A2. Statistical summary of the results shown in Fig. A4 [µg m 3] (c) (c) 0 20 40 60 80 100 120 140 (g) (g) Wed, 05-01 00:00 Thu, 05-02 00:00 Fri, 05-03 00:00 Sat, 05-04 00:00 Sun, 05-05 00:00 Mon, 05-06 00:00 Tue, 05-07 00:00 Wed, 05-08 00:00 Thu, 05-09 00:00 Fri, 05-10 00:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 12:00 Date and time 25 50 75 100 125 O3 conc. [µg m 3] (d) 00:00 04:00 08:00 12:00 16:00 20:00 00:00 Time of day 25 50 75 100 125 (h) UBA measurements S-new S-old Figure A1. Like Fig. 3, but for traffic stations instead of background stations. Figure A1. Like Fig. 3, but for traffic stations instead of background stations. 35 0 5 10 15 20 25 30 0 10 20 30 Simulated mass concentration [µg m 3] (a) (a) (a) (a) (a) (a) (a) (a) NO2 (S-new) f(x) = 0.88x + 3.18 0 5 10 15 20 25 30 0 10 20 30 (b) (b) (b) (b) (b) (b) (b) (b) NO2 (S-old) f(x) = 1.15x + 2.26 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 0 5 10 15 20 Simulated mass concentration [µg m 3] (c) (c) (c) (c) (c) (c) (c) (c) NO (S-new) f(x) = 0.46x + 0.58 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 0 5 10 15 20 (d) (d) (d) (d) (d) (d) (d) (d) NO (S-old) f(x) = 0.41x + 0.76 0 10 20 30 40 0 20 40 Simulated mass concentration [µg m 3] (e) (e) (e) (e) (e) (e) (e) (e) NOx (S-new) f(x) = 0.71x + 5.17 0 10 20 30 40 0 20 40 (f) (f) (f) (f) (f) (f) (f) (f) NOx (S-old) f(x) = 0.82x + 5.92 0 20 40 60 80 100 120 Observed mass concentration [µg m 3] 0 50 100 150 Simulated mass concentration [µg m 3] (g) (g) (g) (g) (g) (g) (g) (g) O3 (S-new) f(x) = 0.74x + 24.35 0 20 40 60 80 100 120 Observed mass concentration [µg m 3] 0 50 100 150 (h) (h) (h) (h) (h) (h) (h) (h) O3 (S-old) f(x) = 0.82x + 24.28 Figure A2. Like Fig. 4, but for all hours of the day instead for noontime only. Error bars were omitted and a color coding (without physical meaning) applied for easier readability. Table A2. Statistical summary of the results shown in Fig. A4 Simulated mass 3 (h) (h) (h) (h) (h) (h) (h) (h) Simulated mass 3 Figure A2. Like Fig. 4, but for all hours of the day instead for noontime only. Error bars were omitted and a color coding (without physical meaning) applied for easier readability. 36 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. [1010 molec cm-3] Figure A3. Average simulated NO2 profile, vertically interpolated to the TM5 grid. Figure A3. Average simulated NO2 profile, vertically interpolated to the TM5 grid. 37 37 https://doi.org/10.5194/egusphere-2022-1473 Preprint. Discussion started: 6 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0 1 2 3 4 5 (a) Mainz (b) Bremen 0 1 2 3 4 5 NO2 concentration (WRF-Chem with AK, 0-200 m) [1011 molec. cm 3] (c) Heidelberg 0 1 2 3 4 5 NO2 concentration (MMF, 0-200 m) [1011 molec. cm 3] (d) De Bilt 0 1 2 3 4 5 NO2 concentration (MMF, 0-200 m) [1011 molec. cm 3] 0 1 2 3 4 5 (e) Uccle Figure A4. Correlation analysis between observed (MMF) and simulated (averaging kernels applied) NO2 concentrations in the lowes 0-200 m above the surface. In each subplot, the dashed line marks the 1:1 line and the solid line is a linear fit through the point cloud. The R-values and further statistical diagnostics of each plot are found in Table A2. Bremen De Bilt NO2 concentration (MMF, 0-200 m) [1011 molec. cm 3] NO2 concentration (MMF, 0-200 m) [1011 molec. cm 3] NO2 concentration (MMF, 0-200 m) [1011 molec. cm 3] Figure A4. Correlation analysis between observed (MMF) and simulated (averaging kernels applied) NO2 concentrations in the lowest 0-200 m above the surface. In each subplot, the dashed line marks the 1:1 line and the solid line is a linear fit through the point cloud. The R-values and further statistical diagnostics of each plot are found in Table A2. Figure A4. Correlation analysis between observed (MMF) and simulated (averaging kernels applied) NO2 concentrations in the lowest 0-200 m above the surface. In each subplot, the dashed line marks the 1:1 line and the solid line is a linear fit through the point cloud. The R-values and further statistical diagnostics of each plot are found in Table A2. 38
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Using Natural Language Processing to Explore Mental Health Insights From UK Tweets During the COVID-19 Pandemic: Infodemiology Study (Preprint)
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Corresponding Author: Email: chris.marshall@io.nihr.ac.uk (JMIR Infodemiology 2022;2(1):e32449) doi: 10.2196/32449 (JMIR Infodemiology 2022;2(1):e32449) doi: 10.2196/32449 Using Natural Language Processing to Explore Mental Health Insights From UK Tweets During the COVID-19 Pandemic: Infodemiology Study Christopher Marshall, BSc, PhD; Kate Lanyi, BSc, MRes; Rhiannon Green, BSc, MRes; Georgina C Wilkins, BSc, PhD; Fiona Pearson, BSc, MRes, PhD; Dawn Craig, BSc, MSc National Institute for Health Research Innovation Observatory, Newcastle University, Newcastle, United Kingdom National Institute for Health Research Innovation Observatory, Newcastle University, Newcas Corresponding Author: Christopher Marshall, BSc, PhD National Institute for Health Research Innovation Observatory Newcastle University The Catalyst Newcastle, NE4 5TG United Kingdom Phone: 44 0191 2082259 Email: chris.marshall@io.nihr.ac.uk Abstract Background: There is need to consider the value of soft intelligence, leveraged using accessible natural language processing (NLP) tools, as a source of analyzed evidence to support public health research outputs and decision-making. Objective: The aim of this study was to explore the value of soft intelligence analyzed using NLP. As a case study, we selected and used a commercially available NLP platform to identify, collect, and interrogate a large collection of UK tweets relating to mental health during the COVID-19 pandemic. Methods: A search strategy comprised of a list of terms related to mental health, COVID-19, and lockdown restrictions was developed to prospectively collate relevant tweets via Twitter’s advanced search application programming interface over a 24-week period. We deployed a readily and commercially available NLP platform to explore tweet frequency and sentiment across the United Kingdom and identify key topics of discussion. A series of keyword filters were used to clean the initial data retrieved and also set up to track specific mental health problems. All collated tweets were anonymized. Results: We identified and analyzed 286,902 tweets posted from UK user accounts from July 23, 2020 to January 6, 2021. The average sentiment score was 50%, suggesting overall neutral sentiment across all tweets over the study period. Major fluctuations in volume (between 12,622 and 51,340) and sentiment (between 25% and 49%) appeared to coincide with key changes to any local and/or national social distancing measures. Tweets around mental health were polarizing, discussed with both positive and negative sentiment. Key topics of consistent discussion over the study period included the impact of the pandemic on people’s mental health (both positively and negatively), fear and anxiety over lockdowns, and anger and mistrust toward the government. Conclusions: Using an NLP platform, we were able to rapidly mine and analyze emerging health-related insights from UK tweets into how the pandemic may be impacting people’s mental health and well-being. This type of real-time analyzed evidence could act as a useful intelligence source that agencies, local leaders, and health care decision makers can potentially draw from, particularly during a health crisis. (JMIR Infodemiology 2022;2(1):e32449) doi: 10.2196/32449 Marshall et al Marshall et al JMIR INFODEMIOLOGY JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 1 (page number not for citation purposes) KEYWORDS Twitter; mental health; COVID-19; sentiment; lockdown; soft intelligence; artificial intelligence; machine learning; natural language processing https://infodemiology.jmir.org/2022/1/e32449 https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX JMIR INFODEMIOLOGY Marshall et al applying NLP can aid health researchers in gaining insights from large, unstructured data sets, such as Twitter. However, the true value of this type of work, including the data set itself, analysis methods, and how it might be integrated into more formal public health research outputs, is still uncertain. For example, a lot of previous work so far has focused on the use of internally developed, bespoke tools or packages, which tend to require a certain level of technical expertise around machine learning (ML) in order operate effectively. However, as methods continue to mature, we are seeing a growing number of “off-the-shelf” solutions become available, which appear to be more accessible and require less technical understanding of the underlying ML concepts. AND mental health OR anxiety OR depression OR anxious OR depressed OR depressing OR trauma OR traumatic OR “obsessive compulsive disorder” OR OCD OR vulnerable OR loneliness OR lonely OR isolated OR isolation OR sleep OR stress OR stressful OR self-harm OR self-harming OR suicide OR suicidal OR well-being mental health OR anxiety OR depression OR anxious OR depressed OR depressing OR trauma OR traumatic OR “obsessive compulsive disorder” OR OCD OR vulnerable OR loneliness OR lonely OR isolated OR isolation OR sleep OR stress OR stressful OR self-harm OR self-harming OR suicide OR suicidal OR well-being Search terms were identified through discussion within the research team and scanning recent literature around mental health. Once the strategy had been agreed upon, it was reviewed by a topic expert and information specialist. We then began prospectively searching for and scraping relevant tweets using Twitter’s advanced search application interface [15]. A geolocation filter was applied to the search strategy to limit the collection of tweets to those posted in the United Kingdom only. Search terms were identified through discussion within the research team and scanning recent literature around mental health. Once the strategy had been agreed upon, it was reviewed by a topic expert and information specialist. We then began prospectively searching for and scraping relevant tweets using Twitter’s advanced search application interface [15]. A geolocation filter was applied to the search strategy to limit the collection of tweets to those posted in the United Kingdom only. In this article, we report the findings from our analysis of relevant tweets in the United Kingdom collected over a 24-week period, from July 23, 2020 to January 6, 2021. Introduction COVID-19 was identified as a new type of coronavirus in early January 2020 [1]. Since then, the disease has rapidly spread to and affected almost all parts of the world. In the United Kingdom, the first outbreak was reported on January 31, 2020, with a national lockdown following on March 26, 2020. Shortly before this, COVID-19 was declared a global pandemic by the World Health Organization (WHO) on March 11, 2020 [2,3]. The COVID-19 pandemic continues to have a profound effect on mental health [4]. In a key position paper published in June 2020, the authors explored the current and future potential psychological, social, and neuroscientific effects of COVID-19 and set out a series of priorities and longer-term strategies for mental health research [4]. One of the immediate research priorities presented in the paper was “surveillance.” In particular, the authors suggested that finding useful ways to monitor and analyze data on the mental health effects of the COVID-19 pandemic across the whole population, as well as vulnerable subgroups, was essential [4]. The aim of this study was to further explore the value of soft intelligence as a meaningful source of evidence, which, when analyzed using an accessible NLP platform, can support public health research activity. In this article, we report the findings from a case study that examined a large collection of tweets relating to mental health posted from the United Kingdom during the COVID-19 pandemic. With over 300 million active monthly users, Twitter is one of the most popular social media platforms available. Twitter is a free microblogging service that enables its users to post, read, and respond to each other’s “tweets” (ie, short messages limited to 280 characters). Social media data are being increasingly used as a data source to inform health-related research, with the potential for offering a more efficient means of data collection over traditional, time-consuming, and costly survey-based methods [5]. In particular, Twitter has been used to monitor, track trends, and disseminate health information during past viral pandemics [6-9]. Further, previous studies have successfully leveraged Twitter data for the assessment of public sentiments, attitudes, and opinions concerning health-related issues [10,11]. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 2 (page number not for citation purposes) Data Collection An advanced AI-based, text analytics platform using NLP was used to initially analyze the tweets. The analytics platform, “Wordnerds,” is described by its developers as a “text analysis and insights platform using machine learning techniques” [13]. In particular, this off-the-shelf platform supports analysis of metadata, topic, and sentiment to understand the context of a tweet and to group tweets together into topic clusters that contain tweets relating to each other or discussing similar issues. This facilitates a more accurate and sophisticated insight into the vaccine conversation on Twitter compared with methodologies that rely solely on a qualitative count of single words, phrases, or hashtags [14]. Channels of soft intelligence like Twitter, leveraged using novel artificial intelligence (AI) techniques (including natural language processing [NLP]), offer an opportunity for real-time analysis of public attitudes, sentiments, and key topics of discussion [12]. As aforementioned, previous case studies have shown that We developed a search strategy comprising a list of terms related to COVID-19, the lockdown, and mental health to search (or “scrape”) for relevant tweets (see Textbox 1). Textbox 1. Search strategy for relevant tweets. Corona OR covid OR lockdown AND mental health OR anxiety OR depression OR anxious OR depressed OR depressing OR trauma OR traumatic OR “obsessive compulsive disorder” OR OCD OR vulnerable OR loneliness OR lonely OR isolated OR isolation OR sleep OR stress OR stressful OR self-harm OR self-harming OR suicide OR suicidal OR well-being Corona OR covid OR lockdown https://infodemiology.jmir.org/2022/1/e32449 Data Analysis We used Wordnerds to interrogate the tweets. The developers state that their platform uses a range of different technologies in order to deliver its various analyses, including contextual word embeddings and collocation methods [13]. To date, we have found no other published studies coordinated by an academic research group that have used this specific tool. The NLP platform was then used to explore the sentiment (ie, positive, neutral, or negative) of the whole corpus of tweets. Sentiment was determined using contextual word embedding techniques, including classification of grammar to understand how words interact [16]. Using the platform, we were able to track and determine the weekly frequency of tweets relevant to our initial search strategy Textbox 3. Keyword filters used to identify specific mental health problems. Textbox 3. Keyword filters used to identify specific mental health problems. Textbox 3. Keyword filters used to identify specific mental health problems. Anxiety: “anxious,” “anxiety” Depression: “depression,” “depressed,” “depressing” Stress: “stress,” “stressful,” “stressed” Loneliness: “loneliness,” “lonely,” “alone” Following sentiment analysis, the platform’s topic analysis feature was used to identify and cluster key emerging topics of discussion, both with positive and negative underlying sentiment. For this analysis, the platform automatically clustered key topics of positive and negative discussion using topic collocation methods. This is a probabilistic method of identifying interesting sentence fragments and words that occur frequently together within a data set. The results of the platform’s topic analysis were examined, and its findings were summarized by 2 of the authors (KL and RG). These summaries were checked by 2 further authors (CM and GCW). Following sentiment analysis, the platform’s topic analysis feature was used to identify and cluster key emerging topics of discussion, both with positive and negative underlying sentiment. For this analysis, the platform automatically clustered key topics of positive and negative discussion using topic collocation methods. This is a probabilistic method of identifying interesting sentence fragments and words that occur frequently together within a data set. The results of the platform’s topic analysis were examined, and its findings were summarized by 2 of the authors (KL and RG). These summaries were checked by 2 further authors (CM and GCW). notable events or issues that occurred throughout the study period are summarized in Table 1 (weeks 1 to 12) and Table 2 (weeks 13 to 24). Textbox 2. Keyword filter used to clean the data set. Textbox 2. Keyword filter used to clean the data set. mood OR “mental health” OR depression OR anxiety OR anxious OR depressed OR depressing OR trauma OR traumatic OR OCD OR compulsive OR vulnerable OR loneliness OR lonely OR isolated OR sleep OR stress OR stressful OR self-harm OR self-harming OR suicide OR suicidal OR anorexia OR anorexic OR bulimia OR bulimic OR eating disorder OR binge eating OR OFSED (other specified feeding or eating disorder) from the United Kingdom between July 23, 2020 and January 6, 2021. We also tracked the frequency of subsets of these tweets that incorporated terms for specific mental health problems, as listed in Textbox 3. Data Analysis As shown in Figure 1, the highest volume of tweets occurred week commencing (w/c) October 29, 2020, with 51,340 tweets. The lowest volume was observed w/c December 3, 2020, with 12,622 tweets. The data show a fairly consistent baseline trend over the study period. Spikes in the volume of tweets occurred in September, October, and December, typically during periods leading up to (or during) a major change in social distancing and lockdown measures across the United Kingdom. Due to the high volume of tweets collected, the topic analysis was split between 2 equal time periods. The first covered summer 2020 to autumn 2020, when lockdown restrictions were relaxed. The second covered the autumn to winter period in 2020, when regional and then further national lockdown restrictions were introduced. The first peak was observed w/c September 17, 2020, the week after the introduction of the “rule of 6,” whereby a mix of 6 people from any household could meet indoors or outdoors. A similar peak was observed w/c October 8, 2020. This was the week leading up to the government’s introduction of a new tiered system, whereby regions across the United Kingdom were allocated to 1 of 3 tiers (and later a fourth tier) based on prevalence of COVID-19. Higher tiers corresponded with tighter restrictions, including closing nonessential businesses and limits placed on social gatherings. Ethical Considerations Institutional review board approval was not sought as this study used only publicly available data. All posts were de-identified, and there was no direct interaction with Twitter users. The largest peak was observed w/c October 29, 2020, the week before the second national lockdown began. The final peak occurred w/c December 31, 2020, the week leading up to the start of a third national lockdown. For all 3 national lockdowns, all nonessential businesses were closed, and UK residents were restricted from meeting anyone outside of their “social bubble” (ie, their household or, for people living alone, themselves plus one other household). Preparing and Cleaning the Data All collated tweets were anonymized. Before analyzing the data, the retrieved results were run through a final keyword filter. This filter was comprised of a series of terms and keywords associated with mental health problems to help ensure a more relevant, cleaner, and less noisy data set for analysis. For XSL•FO RenderX XSL•FO RenderX Marshall et al JMIR INFODEMIOLOGY were added alongside the original terms. example, general terms such as “isolation” and “well-being” were filtered out. Further, terms associated with eating disorders JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 3 (page number not for citation purposes) https://infodemiology.jmir.org/2022/1/e32449 JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 4 (page number not for citation purposes) Tweet Volume We captured and collated 286,902 tweets posted by users in the United Kingdom from July 23, 2020 to January 6, 2021. The volume of tweets by week, together with key events taking place during this study period, is visualized in Figure 1. Further JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 3 (page number not for citation purposes) XSL•FO RenderX JMIR INFODEMIOLOGY JMIR INFODEMIOLOGY Marshall et al Marshall et al Figure 1. Volume of tweets from July 23, 2020 to January 6, 2021. Key events during the study period included the (1) rule of 6 (up to 6 people from any number of households could meet indoors or outdoors), (2) tier system (regions across England were assigned a tier from 1 to 3 based on epidemiological indicators, and these tiers dictated the restrictions in that area, such as which businesses could open and how many individuals could meet in a group during national lockdown—nonessential businesses were closed and people were prohibited from meeting outside of their support bubble). Table 1. Notable events that occurred from July 23, 2020 to October 14, 2020. Events Weeks Weeks 1-2 • “Health Protection Regulations 2020” comes into force (ie, mandatory wearing of face masks in most indoor establishments • Gyms, swimming pools, and other indoor sports facilities reopen. • England reports highest number of excess deaths in Europe for February to June. • Major incident is declared in Greater Manchester after rise in cases. Weeks 3-4 • Stricter measures are reintroduced in Preston, Lancashire. • England’s revamped contact-tracing app begins public trials. • General Certificate of Secondary Education (GCSE) results are published with grades based on teachers’ assessments. • The Education Secretary confirms free appeals for A-Level and GCSE. Weeks 5-6 • Ban on property evictions is extended until September 20. • Greater Manchester Police report breaking up 126 illegal gatherings. • Thousands of lockdown protesters gather in Trafalgar Square. Weeks 7-8 • Health experts express doubt about mass testing plan: “Operation Moonshot.” • World Suicide Prevention Day is on September 10. • “Rule of 6” for indoor and outdoor gatherings is announced. Weeks 9-10 • Local lockdown measures are announced for Newcastle. • Restrictions are relaxed for childcare purposes between households. • Couples in established relationships can meet without social distancing. • Second version of National Health Service (NHS) contact-tracing app becomes publicly available. • £10,000 (US $13,035) fine for failing to self-isolate is announced. Weeks 11-12 • Lockdown restrictions are tightened in the Northeast. • Tier system comes into force, replacing local lockdowns. • World Mental Health Day occurs on October 10. Table 1. Notable events that occurred from July 23, 2020 to October 14, 2020. https://infodemiology.jmir.org/2022/1/e32449 Marshall et al JMIR INFODEMIOLOGY Table 2. Marshall et al Notable events that occurred from October 15, 2020 to January 6, 2021 Events Weeks Weeks 13-14 • Shielding ends for clinically vulnerable • Additional financial support to businesses in Tier 3 is announced. Weeks 15-16 • Month-long national lockdown in England from November 5, 2020 is announced. • Clinically vulnerable are asked to shield again. • Furlough scheme is extended until March 2021. • Pfizer/BioNTech press release announces vaccine is 90% effective. • Travel window for university students to return for Christmas is announced. Weeks 17-18 • Government publishes their “staying mentally well this winter” guidance. • Plans to ease restrictions for 5 days over Christmas are announced. Weeks 19-20 • Indoor care home visits can resume subject to lateral flow test. • Medicines and Healthcare products Regulatory Agency (MHRA) approves Pfizer/BioNTech vaccine for rollout in the United Kingdom. • Joint Committee on Vaccination and Immunisation (JCVI) publishes vaccine priority groups. • National lockdown ends, and Tier system resumes. Most regions in England are placed in Tier 2 or 3. • Shielding ends for clinically vulnerable. • First COVID-19 vaccine is administered. Weeks 21-22 • Self-isolation period is reduced from 14 days to 10 days. • New variant of the virus is identified. • Further regions of the United Kingdom enter highest Tier. • Furlough scheme is extended to April 2021. • Large parts of Southeast England move into the new, stricter Tier 4. • Christmas relaxation period is reduced from 5 days to 1 day. • Travel restrictions for South Africa are enforced. • Most of England will enter Tier 4 from December 26, 2020 is announced. Weeks 23-24 • MHRA approves AstraZeneca/Oxford vaccine. • JCVI publishes updated guidance. • UK Prime Minister announces third national lockdown from January 4, 2021, with schools remaining closed. December 3, 2020. The “Anxiety” filter returned the highest total number of tweets, and the “Loneliness” filter returned the lowest (see Table 3). The trend in tweet frequency for each filter mirrored the trend reported for the overall data set over the study period. December 3, 2020. The “Anxiety” filter returned the highest total number of tweets, and the “Loneliness” filter returned the lowest (see Table 3). The trend in tweet frequency for each filter mirrored the trend reported for the overall data set over the study period. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 5 (page number not for citation purposes) Textbox 4. Examples of positive and negative tweets classified by the NLP platform. Example tweets classified with positive sentiment ...Since lockdown my anxiety has dramatically gotten so much better. I used to get stress spots, panic attacks before presentations, etc. Na bruh, working from home has been a life saver... If anyone is struggling with lockdown (or wants to reduce stress/anxiety). I would really recommend trying gratitude journaling. I’ve shared some tips below – I hope they’ll be helpful …Never been so happy to set foot in a gym today. I feel so much happier getting back into a routine and pleased to see I haven’t lost much strength through pregnancy and lockdown. It’s made me realise how important it is for my own mental health... Example tweets classified with negative sentiment ...I’m finding this lockdown harder than the last one. For me it's not just the weather, it’s having no end in sight which is a struggle when you suffer with anxiety and depression... …genuinely think a second lockdown would completely destroy my mental health. I’m nervous about the next few weeks/months... Feeling absolute rubbish today, feel like my mental health is debilitating can’t bring myself to get out of bed, my only escape is drinking with to forget these awful times Sentiment Analysis Of the 286,902 tweets, 34,347 (11.97%) were identified as having positive sentiment, 217,728 (75.89%) as having neutral sentiment, and 34,827 (12.14%) as having negative sentiment, with an overall sentiment score of 50% assigned. A score below 50% suggests negative sentiment, and a score greater than 50% Here, the overall score of 50% indicates neutral sentiment across all tweets over the study period. Figure 3 visualizes the weekly change in sentiment over the study period from July 23, 2020 to January 6, 2021. Textbox 4. Examples of positive and negative tweets classified by the NLP platform. Mental Health Problems Figure 2. Volume of tweets from July 23, 2020 to January 6, 2021. suggests positive sentiment. Textbox 4 presents a selection of example tweets that the NLP platform classified as both positive and negative. Sentiment Analysis Mental Health Problems Table 3 summarizes the volume of tweets that utilized at least one of the terms related to anxiety, depression, stress, or loneliness. In total, 113,312 (39.50%) of the 286,902 tweets scraped through the initial search strategy related to anxiety, depression, stress, or loneliness. During the first half of the analysis period (w/c July 23, 2020 to w/c October 22, 2020), tweet volume between the 4 mental health problem filters varied. In particular, tweets related to the “Anxiety” filter were consistently posted most often, and tweets relating to the “Loneliness” filter were consistently posted least. There was a spike in volume across all 4 filters w/c October 29, 2020, the start of the second national lockdown. Following the spike, the volume of tweets across all of the filters was broadly similar for the remainder of the analysis period. Figure 2 presents the volume of tweets utilizing terms related to anxiety, depression, stress, or loneliness around each keyword filter over the study period. Across all of the mental health problems that were focused on here, the highest volume of tweets was observed w/c October 29, 2020, and the lowest volume of tweets occurred w/c Table 3. Keyword filters and resultant volume of tweets for specific mental health problems. Tweets (N=113,312) Keyword terms Filter 37,213 (32.84%) “anxious,” “anxiety” Anxiety 29,523 (26.05%) “depression,” “depressed,” “depressing” Depression 26,725 (23.59%) “stress,” “stressful,” “stressed” Stress 19,851 (17.52%) “loneliness,” “lonely,” “alone” Loneliness https://infodemiology.jmir.org/2022/1/e32449 https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX JMIR INFODEMIOLOGY Marshall et al JMIR INFODEMIOLOGY Figure 2. Volume of tweets from July 23, 2020 to January 6, 2021. Sentiment Analysis Of the 286,902 tweets, 34,347 (11.97%) were identified as having positive sentiment, 217,728 (75.89%) as having neutral sentiment, and 34,827 (12.14%) as having negative sentiment, with an overall sentiment score of 50% assigned. A score below 50% suggests negative sentiment, and a score greater than 50% suggests positive sentiment. Textbox 4 presents a selection of example tweets that the NLP platform classified as both positive and negative. Here, the overall score of 50% indicates neutral sentiment across all tweets over the study period. Figure 3 visualizes the weekly change in sentiment over the study period from July 23, 2020 to January 6, 2021. Textbox 4. Examples of positive and negative tweets classified by the NLP platform. Example tweets classified with positive sentiment Marshall et al JMIR INFODEMIOLOGY Figure 2. Volume of tweets from July 23, 2020 to January 6, 2021. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 6 (page number not for citation purposes) Example tweets classified with positive sentiment ...Since lockdown my anxiety has dramatically gotten so much better. I used to get stress spots, panic attacks before presentations, etc. Na bruh, working from home has been a life saver... If anyone is struggling with lockdown (or wants to reduce stress/anxiety). I would really recommend trying journaling. I’ve shared some tips below – I hope they’ll be helpful If anyone is struggling with lockdown (or wants to reduce stress/anxiety). I would really recommend trying gratitude journaling. I’ve shared some tips below – I hope they’ll be helpful …Never been so happy to set foot in a gym today. I feel so much happier getting back into a routine and pleased to see I haven’t lost much strength through pregnancy and lockdown. It’s made me realise how important it is for my own mental health... Example tweets classified with negative sentiment Example tweets classified with negative sentiment ...I’m finding this lockdown harder than the last one. For me it's not just the weather, it’s having no end in sight which is a struggle when you suffer with anxiety and depression... …genuinely think a second lockdown would completely destroy my mental health. I’m nervous about the next few weeks/months... Feeling absolute rubbish today, feel like my mental health is debilitating can’t bring myself to get out of bed, my only escape is drinking with to forget these awful times Feeling absolute rubbish today, feel like my mental health is debilitating can’t bring myself to get out of bed, my only escape is drinking with to forget these awful times JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 6 (page number not for citation purposes) JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 6 (page number not for citation purposes) https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX JMIR INFODEMIOLOGY Marshall et al Figure 3. Sentiment of tweets from July 23, 2020 to January 6, 2021. Figure 3. Sentiment of tweets from July 23, 2020 to January 6, 2021. Figure 3. Sentiment of tweets from July 23, 2020 to January 6, 2021. Figure 3. Sentiment of tweets from July 23, 2020 to January 6, 2021. Figure 3. Sentiment of tweets from July 23, 2020 to January 6, 2021. Sentiment remained neutral or positive throughout most of the study period. The highest assigned sentiment score, 52%, occurred in weeks 1, 4 to 6, 16, and 18 to 20. The lowest assigned sentiment score, 49%, occurred in week 9 (w/c September 17, 2020). Sentiment remained neutral or positive throughout most of the study period. The highest assigned sentiment score, 52%, occurred in weeks 1, 4 to 6, 16, and 18 to 20. The lowest assigned sentiment score, 49%, occurred in week 9 (w/c September 17, 2020). health throughout the “coronavirus pandemic,” as a critical health issue, was shared widely by people on Twitter. During this period, people openly discussed their mental health and how they had been coping. People also shared praise for specific local and national mental health services, as well as key public figures (eg, Marcus Rashford). Overall, the data show a relatively consistent trend in sentiment over the study period. Example tweets classified with negative sentiment When sentiment fluctuation did occur, it was similar to the trend observed with tweet frequency and coincided with major changes to lockdown or social distancing rules. There was considerable discussion based around “World Mental Health Day” and “World Suicide Prevention Day.” People were sharing helpful strategies (eg, videos, charities, help lines, exercise regimes, healthy eating advice) others could use to protect and maintain their mental health. There were also calls from people to be particularly vigilant and make sure they are checking in with any “vulnerable people” in their life. Topic Analysis In this section, results are reported for 2 time periods, as follows: (1) July 23, 2020 to October 14, 2020 and (2) October 15, 2020 to January 6, 2021. Positive discussion was observed around “working from home.” Various users were sharing helpful resources to support working from home effectively, including strategies that people had found useful during the previous national lockdown. Some people reported that mandatory working from home had helped them to achieve a better work-life balance and reduced their anxiety. Results of the Topic Analysis for Weeks 1 to 12 This section presents the results of a topic analysis based on 115,700 scraped tweets posted from July 23, 2020 to October 14, 2020. ...Never been so happy to set foot in a gym today. I feel so much happier getting back into a routine and pleased to see I haven’t lost much strength through pregnancy and lockdown. It’s made me realise how important it is for my own mental health... ...I eventually took time out from March as it just got too much. My anxiety was through the roof and frankly I was glad of lockdown - I didn’t want to leave the house anyway. I’m in a much better place now thankfully... ...This coming Thursday is world suicide prevention day and we need to be talking about this in the context of covid, lockdown, the economy and more. Most of all we need to talk and respond with compassion and support... xtbox 5. Sample of clustered tweets from weeks 1 to 12 with positive sentiment. ...Never been so happy to set foot in a gym today. I feel so much happier getting back into a routine and pleased to see I haven’t lost much strength through pregnancy and lockdown. It’s made me realise how important it is for my own mental health... ...I eventually took time out from March as it just got too much. My anxiety was through the roof and frankly I was glad of lockdown - I didn’t want to leave the house anyway. I’m in a much better place now thankfully... ...This coming Thursday is world suicide prevention day and we need to be talking about this in the context of covid, lockdown, the economy and more. Most of all we need to talk and respond with compassion and support... https://infodemiology.jmir.org/2022/1/e32449 Summary of Clustered Topics With Negative Sentiment Tables 4 and 5 present the top 10 most discussed topics th occurred throughout the study period. ...went into the year hopeful and ready to turn my life around...then covid happened and the world became a giant prison. Depression slowly came back, anxiety eating away and all emotion and feeling slowly dry up. Mind became a wasteland pretty much. Lifeless and desert... ...I have to get a COVID test done before going back to work at the end of the month and the anxiety I am having from the thought of them sticking that thing up my nose... ...I’m finding this lockdown harder than the last one. For me it's not just the weather, it’s having no end in sight which is a struggle when you suffer with anxiety and depression... ...genuinely think a second lockdown would completely destroy my mental health. I’m nervous about the next few weeks/months... ...I'm all for people following the rules - wear your masks and keep distance when required, but this is very true and will make people more depressed by lack of communication with real people. Depression will be a huge and sad factor if this new lockdown does go ahead. Stay safe... ...This bizarre competition between suicide and Covid deaths needs to end. I understand the mental health issues that can arise from lack of social interaction and other factors from Covid but this weird death competition between the 2 needs to stop... bl 2 i l i f bl h Table 2 summarizes a selection of notable events that occurred during this time period. Summary of Clustered Topics With Negative Sentiment y f p g “Second lockdown” emerged as a key topic of discussion underpinned with negative sentiment. People were sharing their fears, concerns, and anxieties over the prospect of a second national lockdown and the impact this would have on theirs (and other’s) mental health. People recalled and spoke openly about how their mental health had suffered during the previous lockdown, referencing specific problems such as “anxiety,” “depression,” and posttraumatic stress disorder. Some people shared that they had been diagnosed with depression for the first time due to the previous national lockdown. Many people were angry that not enough had been done by the government to protect “vulnerable people” during the previous national lockdown. Suicide was also discussed. People were claiming that suicide rates had increased during lockdown, particularly among younger people. There was widespread sharing of warnings from key educational figures that the pandemic would have long-lasting negative effects on Tweets contained within some of the other topic clusters generated by the platform, such as “care homes” and “covid deaths,” did not appear to be related to mental health. Specific examples of mental health tweets underpinned with negative sentiment from weeks 1 to 12 are presented in Textbox 6. Textbox 6. Sample of clustered tweets from weeks 1 to 12 with negative sentiment" to be consistent with the other textbox p g ...went into the year hopeful and ready to turn my life around...then covid happened and the world became a giant prison. Depression slowly came back, anxiety eating away and all emotion and feeling slowly dry up. Mind became a wasteland pretty much. Lifeless and desert... ...I have to get a COVID test done before going back to work at the end of the month and the anxiety I am having from the thought of them sticking that thing up my nose... ...I’m finding this lockdown harder than the last one. For me it's not just the weather, it’s having no end in sight which is a struggle when you suffer with anxiety and depression... ...genuinely think a second lockdown would completely destroy my mental health. I’m nervous about the next few weeks/months... ...I'm all for people following the rules - wear your masks and keep distance when required, but this is very true and will make people more depressed by lack of communication with real people. Summary of Clustered Topics With Negative Sentiment Depression will be a huge and sad factor if this new lockdown does go ahead. Stay safe... ...This bizarre competition between suicide and Covid deaths needs to end. I understand the mental health issues that can arise from lack of social interaction and other factors from Covid but this weird death competition between the 2 needs to stop... esults of the Topic Analysis for Weeks 13 to 24 his section presents the results of a topic analysis of 171,202 raped tweets posted from October 15, 2020 to January 6, 2021. Table 2 summarizes a selection of notable events that occurred during this time period. Tables 4 and 5 present the top 10 most discussed topics that occurred throughout the study period. ...went into the year hopeful and ready to turn my life around...then covid happened and the world became a giant prison. Depression slowly came back, anxiety eating away and all emotion and feeling slowly dry up. Mind became a wasteland pretty much. Lifeless and desert... ...I have to get a COVID test done before going back to work at the end of the month and the anxiety I am having from the thought of them sticking that thing up my nose... ...I’m finding this lockdown harder than the last one. For me it's not just the weather, it’s having no end in sight which is a struggle when you suffer with anxiety and depression... ...genuinely think a second lockdown would completely destroy my mental health. I’m nervous about the next few weeks/months... ...I'm all for people following the rules - wear your masks and keep distance when required, but this is very true and will make people more depressed by lack of communication with real people. Depression will be a huge and sad factor if this new lockdown does go ahead. Stay safe... ...This bizarre competition between suicide and Covid deaths needs to end. I understand the mental health issues that can arise from lack of social interaction and other factors from Covid but this weird death competition between the 2 needs to stop... Results of the Topic Analysis for Weeks 13 to 24 This section presents the results of a topic analysis of 171,202 scraped tweets posted from October 15, 2020 to January 6, 2021. Table 2 summarizes a selection of notable events that occurr during this time period. Summary of Clustered Topics With Positive Sentiment Specific examples of mental health tweets underpinned with positive sentiment from weeks 1 to 12 are presented in Textbox Specific examples of mental health tweets underpinned with positive sentiment from weeks 1 to 12 are presented in Textbox 5. “Mental health” emerged as a key topic of discussion underpinned with positive sentiment. The importance of mental Textbox 5. Sample of clustered tweets from weeks 1 to 12 with positive sentiment. ...Never been so happy to set foot in a gym today. I feel so much happier getting back into a routine and pleased to see I haven’t lost much strength through pregnancy and lockdown. It’s made me realise how important it is for my own mental health... ...This coming Thursday is world suicide prevention day and we need to be talking about this in the context of covid, lockdown, the economy and more. Most of all we need to talk and respond with compassion and support... JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 7 (page number not for citation purposes) https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX XSL•FO RenderX JMIR INFODEMIOLOGY Marshall et al “children.” Further, some argued that people were using “mental health” as an excuse to avoid further lockdown restrictions. Many people were concerned that a second lockdown would be much worse for people’s mental health than the first (the lockdown coinciding with winter and students returning to university were both seen as contributing negative factors). Those who refused to wear masks were a further source of anxiety for some people, with a high proportion of tweets calling on others to “wear a mask.” People also discussed COVID-19 tests during this period. In particular, some people shared how stressful and anxiety-inducing taking the test, and also waiting for the results, can be. ...went into the year hopeful and ready to turn my life around...then covid happened and the world became a giant prison. Depression slowly came back, anxiety eating away and all emotion and feeling slowly dry up. Mind became a wasteland pretty much. Lifeless and desert... ...went into the year hopeful and ready to turn my life around...then covid happened and the world became a giant prison. Depression slowly came back, anxiety eating away and all emotion and feeling slowly dry up. Mind became a wasteland pretty much. Lifeless and desert... ...I have to get a COVID test done before going back to work at the end of the month and the anxiety I am having from the thought of them sticking that thing up my nose... ...I’m finding this lockdown harder than the last one. For me it's not just the weather, it’s having no end in sight which is a struggle when you suffer with anxiety and depression... ...genuinely think a second lockdown would completely destroy my mental health. I’m nervous about the next few weeks/months... ...I'm all for people following the rules - wear your masks and keep distance when required, but this is very true and will make people more depressed by lack of communication with real people. Depression will be a huge and sad factor if this new lockdown does go ahead. Stay safe... ...This bizarre competition between suicide and Covid deaths needs to end. I understand the mental health issues that can arise from lack of social interaction and other factors from Covid but this weird death competition between the 2 needs to stop... Results of the Topic Analysis for Weeks 13 to 24 Table 2 summarizes a selection of notable events that occur during this time period. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 9 (page number not for citation purposes) Suicide figures are up 200% since lockdown. Could two followers please copy and re-post this tweet? We’re trying to demonstrate that someone is always listening. Call 116 123 (Samaritans UK). Just two. Any two. Copy, not RT. #MentalHealth #SuicidePrevention #SuicideAwareness important to remember with all the 200% increase in suicide tweets. Raising awareness is fantastic, but make sure it’s factual. #SuicidePrevention. https://infodemiology.jmir.org/2022/1/e32449 Results of the Topic Analysis for Weeks 13 to 24 This section presents the results of a topic analysis of 171,202 scraped tweets posted from October 15, 2020 to January 6, 2021. Tables 4 and 5 present the top 10 most discussed topics that occurred throughout the study period. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 8 (page number not for citation purposes) https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX XSL•FO RenderX Marshall et al JMIR INFODEMIOLOGY Table 4. Top 10 most discussed positive and negative topics from July 23, 2020 to October 14, 2020. Number of tweets Clustered topic Ranking Topics with positive sentiment 5499 “mental health” 1 1513 “vulnerable people” 2 491 “lockdown anxiety” 3 348 “communities... support” 4 327 “health... important” 5 266 “coronavirus pandemic” 6 257 “work from home” 7 246 “support... provide” 8 234 “improved... health” 9 233 “world day” 10 Topics with negative sentiment 5192 “mental health” 1 1421 “people... vulnerable” 2 1037 “children... coronavirus” 3 743 “covid... deaths” 4 566 “wear a mask” 5 419 “test for covid” 6 411 “lockdown... gone” 7 405 “care homes” 8 377 “second lockdown” 9 365 “anxiety... depression” 10 Table 4. Top 10 most discussed positive and negative topics from July 23, 2020 to October 14, 2020. Clustered topic Ranking https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX XSL•FO RenderX Marshall et al JMIR INFODEMIOLOGY Table 5. Top 10 most discussed positive and negative topics from October 15, 2020 to January 6, 2020.. Number of tweets Clustered topic Ranking Topics with positive sentiment 10,096 “suicide lockdown” 1 10,031 “mental health” 2 1860 “people…vulnerable” 3 526 “friends or family” 4 451 “need…support” 5 409 “ones you love” 6 306 “community…helping” 7 295 “stress & anxiety 8 275 “night’s sleep” 9 259 “difficult times” 10 Topics with negative sentiment 10,299 “mental health“ 1 1767 “vulnerable people” 2 1025 “going…lockdown” 3 979 “deaths…covid” 4 490 “suicide rates” 5 479 “sleep at night” 6 474 “lockdown…depressing” 7 460 “committed suicide” 8 441 “coronavirus…children” 9 441 “wearing masks” 10 children, young people, disabled people, those with learning difficulties, and those with any pre-existing mental health problems. Some people were calling on the government to provide further support for these groups as lockdown restrictions tightened. Many people were encouraging those that that were struggling to stay connected with others and reach out to “ones you love” and “friends and family.” Summary of Clustered Topics With Positive Sentiment Summary of Clustered Topics With Positive Sentiment During this period, a tweet suggesting suicide rates had risen by 200% since lockdown was shared widely. The tweet contained contact details for a registered UK charity, Samaritans, urging people to reach out for support if needed. This viral tweet resulted in our analysis platform recognizing “suicide lockdown” as a key topic of discussion. The information being reported by this tweet was not accurate [17,18]. “Sleep” emerged as a key topic of discussion with positive sentiment. Many people discussed how important getting a good “night’s sleep” was for their mental health, particularly during these “difficult times.” People shared relaxation techniques they had used, which had helped them to fall asleep, and which may help others too. As with the previous 12 weeks, “mental health” remained a key topic of discussion underpinned with positive sentiment during this period. People discussed how the lockdown had, in some ways, had a positive impact on their mental health. Various people and organizations continued to share practical tips on how to support one’s mental health, particularly around strategies to reduce “stress and anxiety.” Specific examples of mental health tweets underpinned with positive sentiment from weeks 13 to 24 are presented in Textbox Many people continued to express concern for the mental health of perceived “vulnerable people” during lockdown, including Textbox 7. Sample of clustered tweets from weeks 13 to 24 with positive sentiment. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 10 (page number not for citation purposes) XSL•FO RenderX JMIR INFODEMIOLOGY Marshall et al My mental health is sooo much better now it’s a real lockdown again. However I know this isn’t a universal experience. For those who find lockdown harder for whatever reason, I’m a) sending virtual hugs if wanted but b) reminding you it’s STRONG to reach out to a helpline! As we start lockdown 3, a reminder that your situation does not have to be the worst for it to suck and for you to get help! Reach out to your loved ones & professional mental health support if you need it. Stay safe. Such a tough time at the moment for everyone, another lockdown especially in winter can be devastating for mental health. My dm’s are always open for anyone who needs a chat – be kind and check on your loved ones. Summary of Clustered Topics With Negative Sentiment Summary of Clustered Topics With Negative Sentiment “Mental health” continued to be widely discussed during this final 12-week period. Many people reflected on the negative impact that lockdowns had had on their mental health. There were concerns from some that any progress they had made with their mental health would be lost with another lockdown. There was continued anger toward the UK government about a perceived lack of support for those struggling with their mental health. During this period, people shared their thoughts about the vaccine rollout. In particular, people were concerned about the length of time between jabs and the number of canceled vaccination appointments being reported by the media. “Sleep” continued to be a key topic of discussion with many people sharing how they had not been sleeping well. Some people shared how they had been increasing their alcohol intake in an effort to help them sleep. There was a lot of discussion around the looming national lockdown announced for January 4, 2021. Many people expressed concern about how long this lockdown would last and their hope that this would be the final lockdown. Some shared that they would be defying restrictions in order to prioritize their mental health. Others continued to argue that Specific examples of mental health tweets underpinned with negative sentiment from weeks 13 to 24 are presented in Textbox 8. Textbox 8. Sample of clustered tweets from weeks 13 to 24 with negative sentiment. Textbox 8. Sample of clustered tweets from weeks 13 to 24 with negative sentiment. Well lockdown 3.0 has barely started and I can already feel all the hard work I put balancing my mental health slip away Tbh I just hope my mental health doesn’t become as bad as first lockdown Feeling absolute rubbish today, feel like my mental health is debilitating can’t bring myself to get out of bed, my only escape is drinking with to forget these awful times There needs to be more mental health support @BorisJohnson as that will be one of the highest collateral costs of this. People like me are struggling badly in isolation and with mental health issues and there isn’t enough support. https://infodemiology.jmir.org/2022/1/e32449 Summary of Clustered Topics With Positive Sentiment Thanks to local organisations, community groups and faith institutions that have provided vital services, human support and companionship, in person and online, to many vulnerable people during #Covid-19. We will keep working with you all to build stronger and united communities. If anyone is struggling with lockdown (or wants to reduce stress/anxiety). I would really recommend trying gratitude journaling. I’ve shared some tips below – I hope they’ll be helpful #COVID19 wellbeing tip: make sure you get a good night’s sleep! A good rest is so important for your mental and physical health, managing stress and much more. If you’re struggling with sleep, try these tips and check out our Sleep self-help guide. people were using their mental health as an excuse for not following the rules. There was continued worry about how the lockdown would affect perceived “vulnerable people.” Summary of Clustered Topics With Negative Sentiment #Uklockdown #Covid My mental health is already rock bottom due to not seeing my family/friends, being overworked and not sleeping – god help what another lockdown is gonna do to me … everyone is talking about lockdown 3 but carehomes haven’t left lockdown since the first lockdown started. Everyone is ignoring how vulnerable people feel, disables people or careworkers and carers. ...I’ve never had ocd but do have A LOT of anxiety, + have noticed the longer lockdown goes on the more my anxious behaviours start to look like obsessive ones eg. I check my cooker before bed/before leaving my house when I never did that pre lockdown, also started counting.! It’s only our second day of lockdown 3.0 going solo & I’m already finding harder than the others. It could be due to the distinct lack of sleep last night. What are your favourite self care activities to do? I need motivation to get off the sofa today #lockdown #lockdownblues ...everyone is rotting in their own houses and getting depression I’m not saying they’re less important but clearly this going in and out of lockdown isn’t helping anyone is it There needs to be more mental health support @BorisJohnson as that will be one of the highest collateral costs of this. People like me are struggling badly in isolation and with mental health issues and there isn’t enough support. #Uklockdown #Covid My mental health is already rock bottom due to not seeing my family/friends, being overworked and not sleeping – god help what another lockdown is gonna do to me My mental health is already rock bottom due to not seeing my family/friends, being overworked and not sleeping – god help what another lockdown is gonna do to me … everyone is talking about lockdown 3 but carehomes haven’t left lockdown since the first lockdown started. Everyone is ignoring how vulnerable people feel, disables people or careworkers and carers. ...I’ve never had ocd but do have A LOT of anxiety, + have noticed the longer lockdown goes on the more my anxious behaviours start to look like obsessive ones eg. I check my cooker before bed/before leaving my house when I never did that pre lockdown, also started counting.! It’s only our second day of lockdown 3.0 going solo & I’m already finding harder than the others. It could be due to the distinct lack of sleep last night. Limitations In this study, we identified and analyzed 286,902 geolocated tweets posted from users in the United Kingdom from July 23, 2020 to January 6, 2021 using a commercially available NLP platform. The findings showed that there was a fairly consistent trend in the volume of tweets over the study period, with spikes typically occurring during (or leading up to) a major change in social distancing measures in the United Kingdom. The NLP platform calculated an overall sentiment score of 50% indicating neutral sentiment across all tweets over the study period. Similar to volume, major fluctuations in sentiment appeared to coincide with major changes to lockdown rules. Several limitations can be attributed to this study. First, there are still considerable limitations concerning the reliability, accuracy, and transparency of the technologies in play. As an example, on examining the results of the NLP platform’s topic analysis, some of the tweets collated were not relevant to mental health (despite being identified as such). For example, tweets contained within clustered topics like “care homes” and “covid deaths” were expressing anger at the government, rather than negatively discussing mental health problems. Some of the tweets included by the platform in its analysis were posted by businesses or charitable organizations, rather than members of the public. Such tweets, which often advertised local or national mental health services or shared self-improvement strategies, were typically classified by the platform as having positive sentiment. This created a large amount of background noise and skewed the overall sentiment toward positive. Further, tweets were not deduplicated by the platform, nor was there any formal analysis accounting for potential bot traffic. These factors will also have impacted the results. Key topics of discussion that emerged consistently throughout the study period included (1) the impact that the pandemic and resulting lockdowns had been having on people’s mental health, both positive and negative; (2) fear and anxiety around the prospect of prolonged and subsequent lockdowns and how this might (or continue to) affect people’s mental health; and (3) anger and mistrust toward the government concerning a perceived lack of support for people struggling with their mental health. Later in (and less consistently discussed throughout) the study period, other topics linked with mental health emerged, including sleep difficulties, increased alcohol intake, and anxieties concerning testing and the vaccine rollout. Summary of Clustered Topics With Negative Sentiment What are your favourite self care activities to do? I need motivation to get off the sofa today #lockdown #lockdownblues It’s only our second day of lockdown 3.0 going solo & I’m already finding harder than the others. It could be due to the distinct lack of sleep last night. What are your favourite self care activities to do? I need motivation to get off the sofa today #lockdown #lockdownblues ...everyone is rotting in their own houses and getting depression I’m not saying they’re less important but clearly this going in and out of lockdown isn’t helping anyone is it JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 11 (page number not for citation purposes) https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX XSL•FO RenderX Marshall et al JMIR INFODEMIOLOGY this lockdown is starting to get to me – went off my food, crying more and just generally depressed loool examining the value of this type of analyzed evidence and how it might support, link to, and (where appropriate) replace more traditional survey-based methods and data [15,21-24]. Limitations In addition, despite the popularity of Twitter as a social networking tool, its users are not an accurate representation of the overall demographic of a population. Therefore, if we are to consider using Twitter (and similar resources) as a potential intelligence source, we must be mindful of bias concerning the key demographic information among its users (such as age, gender, and socioeconomic status). Before the study, we anticipated that topics of discussion relating to mental health would be mostly underpinned with negative sentiment. It was therefore surprising that the findings of the topic analysis revealed higher levels of positive sentiment across posts associated with mental health. Consistently over the study period, people took to Twitter to share practical tips, strategies, and resources that could be used to support one’s mental health, and the platform was effective in clustering these types of posts with positive sentiment. There was also a number of limitations specific to the NLP platform that we selected. We had originally planned to run the topic analysis using the platform across all of the tweets as a single corpus. However, the platform was not able to process and analyze such a large volume of tweets in one go. Therefore, we had to split and run the topic analysis over 2 time periods (weeks 1 to 12 and weeks 13 to 24). Further, it was not possible to retrospectively search for and collect historic tweets, thus restricting possible options for analysis. Finally, although the broader methodologies that power the platform are touched on by its developers, the finer technical detail is not shared publicly due to commercial reasons. The viral spread of misinformation and “fake news” has represented a critical issue generating mass confusion, fear, and insecurity surrounding COVID-19 [19]. The WHO has repeatedly used the term “infodemic” to describe the sheer overabundance of misinformation being shared throughout the pandemic [20]. Our findings provide further example and insight into the rapid spread of health-related misinformation, particularly via channels of soft intelligence like social media. Specifically, in the case of this study, a copy-and-paste tweet campaign falsely claiming that suicide rates had increased by 200% since the first lockdown was shared widely by users. This particular cluster of tweets ranked as the topmost discussion topic during weeks 13 to 24 over the study period. https://infodemiology.jmir.org/2022/1/e32449 JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 12 (page number not for citation purposes) Acknowledgments This project is funded by the National Institute for Health Research (NIHR; HSRIC-2016-1009/Innovation Observatory). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. Authors' Contributions CM was responsible for project supervision; the conception, design, and conduct of the study; leading the development of the initial manuscript; and managing its revision. KL, RG, and GCW were responsible for data analysis, data interpretation, and critical review and revision of the manuscript. FP and DC provided critical review of the manuscript. Conclusions In this work, we analyzed a large collection of UK tweets relating to mental health during the COVID-19 pandemic to further explore the value of soft intelligence leveraged using NLP. Using a specialist, off-the-shelf, NLP platform, we collated a large corpus of tweets over a 24-week period and carried out various analyses to explore the volume, sentiment, and key trends and topics of discussion. Overall, the results of this study demonstrate that using NLP to mine and analyze sources of soft intelligence (like Twitter) can yield useful health-related insights, which agencies, local leaders, and health care decision makers can potentially draw from. These findings contribute to a growing body of literature Our findings provide further evidence that this type of research is potentially a highly useful and efficient means to gain a rapid JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 12 (page number not for citation purposes) JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 12 (page number not for citation purposes) XSL•FO RenderX JMIR INFODEMIOLOGY Marshall et al real-time analysis and intelligence may be particularly useful in helping shape rapid and reactive public health engagement and communication strategies during a health crises like COVID-19. real-time analysis and intelligence may be particularly useful in helping shape rapid and reactive public health engagement and communication strategies during a health crises like COVID-19. understanding of the key messages, concerns, and issues people are facing at scale. In the case of this reported study, we were able to draw insights into how the pandemic may be impacting people’s mental health and well-being by examining both the topic and sentiment specific to the UK population. This type of understanding of the key messages, concerns, and issues people are facing at scale. In the case of this reported study, we were able to draw insights into how the pandemic may be impacting people’s mental health and well-being by examining both the topic and sentiment specific to the UK population. This type of Conflicts of Interest The National Institute for Health Research (NIHR) Innovation Observatory purchased a license to use and evaluate the natural language processing (NLP) platform, Wordnerds. However, as independent users, the authors have no vested interest in the tool. References The National Institute for Health Research (NIHR) Innovation Observatory purchased a license to use and evaluate the natural language processing (NLP) platform, Wordnerds. 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COVID-19 vaccine-related discussion on Twitter: topic modeling and sentiment analysis. J Med Internet Res 2021 Jun 29;23(6):e24435 [FREE Full text] [doi: 10.2196/24435] [Medline: 34115608] 24. Valdez D, Ten Thij M, Bathina K, Rutter LA, Bollen J. Social media insights into US mental health during the COVID-19 pandemic: longitudinal analysis of Twitter data. J Med Internet Res 2020 Dec 14;22(12):e21418 [FREE Full text] [doi: 10.2196/21418] [Medline: 33284783] References URL: https://www.wordnerds.ai/ uk-rail-twitter-sentiment-study-2019 [accessed 2022-03-13] JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 13 (page number not for citation purposes) JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 13 (page number not for citation purposes) JMIR INFODEMIOLOGY Marshall et al 17. Appleby L, Richards N, Ibrahim S, Turnbull P, Rodway C, Kapur N. Suicide in England in the COVID-19 pandemic: Early observational data from real time surveillance. Lancet Reg Health Eur 2021 May;4:100110 [FREE Full text] [doi: 10.1016/j.lanepe.2021.100110] [Medline: 34557817] 18. Krishna R. There is no evidence that suicides have increased 200% under lockdown. Full Fact. URL: https://fullfact.org/ online/suicide-200-percent/ [accessed 2022-03-13] 19. Freiling I, Krause NM, Scheufele DA, Brossard D. Believing and sharing misinformation, fact-checks, and accurate information on social media: The role of anxiety during COVID-19. New Media & Society 2021 Apr 22:146144482110114. [doi: 10.1177/14614448211011451] 20. Eysenbach G. How to fight an infodemic: the four pillars of infodemic management. J Med Internet Res 2020 Jun 29;22(6):e21820 [FREE Full text] [doi: 10.2196/21820] [Medline: 32589589] 21. Al Baghal T, Sloan L, Jessop C, Williams ML, Burnap P. Linking Twitter and survey data: the impact of survey mode and demographics on consent rates across three UK studies. Social Science Computer Review 2019 Feb 26;38(5):517-532. [doi: 10.1177/0894439319828011] 22. Hung M, Lauren E, Hon ES, Birmingham WC, Xu J, Su S, et al. Social network analysis of COVID-19 sentiments: application of artificial intelligence. J Med Internet Res 2020 Aug 18;22(8):e22590 [FREE Full text] [doi: 10.2196/22590] [Medline: 32750001] 23. Lyu JC, Han EL, Luli GK. COVID-19 vaccine-related discussion on Twitter: topic modeling and sentiment analysis. J Med Internet Res 2021 Jun 29;23(6):e24435 [FREE Full text] [doi: 10.2196/24435] [Medline: 34115608] 24. Valdez D, Ten Thij M, Bathina K, Rutter LA, Bollen J. Social media insights into US mental health during the COVID-19 pandemic: longitudinal analysis of Twitter data. J Med Internet Res 2020 Dec 14;22(12):e21418 [FREE Full text] [doi: 10.2196/21418] [Medline: 33284783] Abb i i 17. Appleby L, Richards N, Ibrahim S, Turnbull P, Rodway C, Kapur N. Suicide in England in the COVID-19 pandemic: Early observational data from real time surveillance. Lancet Reg Health Eur 2021 May;4:100110 [FREE Full text] [doi: 10.1016/j.lanepe.2021.100110] [Medline: 34557817] j 18. Krishna R. There is no evidence that suicides have increased 200% under lockdown. Full Fact. URL: https://fullfact.org/ online/suicide-200-percent/ [accessed 2022-03-13] 19. Freiling I, Krause NM, Scheufele DA, Brossard D. Believing and sharing misinformation, fact-checks, and accurate information on social media: The role of anxiety during COVID-19. New Media & Society 2021 Apr 22:146144482110114. [doi: 10.1177/14614448211011451] 19. Freiling I, Krause NM, Scheufele DA, Brossard D. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 14 (page number not for citation purposes) Abbreviations AI: artificial intelligence ML: machine learning NIHR: National Institute for Health Research NLP: natural language processing w/c: week commencing WHO: World Health Organization Edited by T Mackey; submitted 28.07.21; peer-reviewed by H Lee, R Castilla-Puentes, W Zhang; comments to author 04.09.21; revised version received 15.10.21; accepted 12.01.22; published 31.03.22 Please cite as: Marshall C, Lanyi K, Green R, Wilkins GC, Pearson F, Craig D Using Natural Language Processing to Explore Mental Health Insights From UK Tweets During the COVID-19 Pandemic: Infodemiology Study JMIR Infodemiology 2022;2(1):e32449 URL: https://infodemiology.jmir.org/2022/1/e32449 doi: 10.2196/32449 PMID: 36406146 ©Christopher Marshall, Kate Lanyi, Rhiannon Green, Georgina C Wilkins, Fiona Pearson, Dawn Craig. Originally published in JMIR Infodemiology (https://infodemiology.jmir.org), 31.03.2022. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Infodemiology, is properly cited. The complete bibliographic information, a link to the original publication on https://infodemiology.jmir.org/, as well as this copyright and license information must be included. JMIR Infodemiology 2022 | vol. 2 | iss. 1 | e32449 | p. 14 (page number not for citation purposes) https://infodemiology.jmir.org/2022/1/e32449 XSL•FO RenderX XSL•FO RenderX
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Interspecific variation in responses to microclimate by terrestrial isopods: implications in relation to climate change
ZooKeys
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http://zoobank.org/0C528E3A-EBE5-4BA4-891A-54CAF94182B4 Citation: Hassall M, Moss A, Dixie B, Gilroy JJ (2018) Interspecific variation in responses to microclimate by terrestrial isopods: implications in relation to climate change. In: Hornung E, Taiti S, Szlavecz K (Eds) Isopods in a Changing World. ZooKeys 801: 5–24. https://doi.org/10.3897/zookeys.801.24934 Copyright Mark Hassall et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Interspecific ZooKeys 801: 5–24 (2018) doi: 10.3897/zookeys.801.24934 http://zookeys.pensoft.net Interspecific ZooKeys 801: 5–24 (2018) doi: 10.3897/zookeys.801.24934 http://zookeys.pensoft.net Interspecific ZooKeys 801: 5–24 (2018) doi: 10.3897/zookeys.801.24934 http://zookeys.pensoft.net responses to microclima RESEARCH ARTICLE Interspecific variation in responses to microclimate by terrestrial isopods: implications in relation to climate change Mark Hassall1, Anna Moss1,2, Bernice Dixie1, James J. Gilroy1 1 School of Environmental Sciences, University of East Anglia, Norwich, NR4 7TJ, UK 2 School of Social Sciences, University of Dundee, Dundee, DD1 4HN, UK Corresponding author: Mark Hassall (mhassall49@googlemail.com) Academic editor: Katalin Szlavecz  |  Received 10 March 2018  |  Accepted 28 October 2018  |  Published 3 December 2018 Introduction In this review we draw attention to an important contribution that soil biologists, in particular those who study the biology of terrestrial isopods, can potentially make to the current debate as to how global climate change may influence components of the global carbon cycle. Currently the greatest uncertainty in modelling the global carbon cycle is not the fluxes across the ocean atmosphere interface or fluxes relating to net primary production but in modelling carbon fluxes within the soil. Globally ten times more carbon dioxide is emitted from soils than from all anthropogenic sources com­ bined (Adger and Brown 1995, IPCC 2013). Anthropogenic induced climate change has caused shifts in both temperature and rainfall patterns across a range of geographic scales (Rosenzweig et al. 2008). Climate models predict global surface temperatures will rise by 0.3–4.8 °C by 2100 (IPCC 2014). A major challenge of current ecological research is to determine how ecosystem processes will respond to future environmental conditions (Santotja et al. 2017). It is widely acknowledged that food webs play pivotal roles in carbon cycles, including emissions of carbon dioxide from soils under climate change but this is rarely considered in modelling carbon fluxes (Pelini et al. 2015), while understanding of organismic physiological responses under climate change re­ mains very rudimentary (Schmitz 2013). One question that is currently being debated is the extent to which soil animals can be modelled grouped together in broad trophic categories or whether they need to be considered at a finer level of functional trait or taxonomic resolution, depending upon their individual responses to changes in micro- climate (Jiguet et al. 2011, Dixie et al. 2015). Terrestrial isopods potentially form a useful model system with which to address this question because their behaviour, physiology, and life histories have been so extensively studied. Carbon dioxide emissions from soils are mediated predominately by microbial me­ tabolism (Fig. 1), which, when not constrained by moisture availability, is a function of temperature (Schlesinger 1977). There is thus the potential for increased soil temperatures to accelerate emissions of carbon dioxide from soils. The further accumulation of CO2 in the atmosphere could then lead to further increases in temperature, leading to a posi­ tive feedback (Fig. 1) (Crowther et al. 2015, Thakur et al. 2018). This process is of most concern in regions with the highest pools of soil organic matter, such as tundra regions and temperate grasslands (Melillo et al. Keywords Aggregation, CO2 emissions from soils, feeding behaviour, future rainfall patterns, life history traits, norms of reaction, response curves, soil animals Abstracth The importance of considering species-specific biotic interactions when predicting feedbacks between the effects of climate change and ecosystem functions is becoming widely recognised. The responses of soil an­ imals to predicted changes in global climate could potentially have far-reaching consequences for fluxes of soil carbon, including climatic feedbacks resulting from increased emissions of carbon dioxide from soils. The responses of soil animals to different microclimates can be summarised as norms of reaction, in order to compare phenotypic differences in traits along environmental gradients. Thermal and moisture reaction norms for physiological, behavioural and life history traits of species of terrestrial isopods differing in their morphological adaptations for reducing water loss are presented. Gradients of moisture reaction norms for respiratory rates and thermal reaction norms for water loss, for a species from the littoral zone were steeper than those for species from mesic environments. Those for mesic species were steeper than for those from xeric habitats. Within mesic species, gradients of thermal reaction norms for aggregation were steeper for Oniscus asellus than for Porcellio scaber or Armadillium vulgare, and moisture reaction norms for sheltering and feeding behaviours were steeper for Philoscia muscorum than for either P. scaber or A. vulgare. These differences reflect differences in body shape, permeability of the cuticle, and development of pleopodal lungs. The implications of differences between different species of soil animals in response to microclimate on the possible influence of the soil fauna on soil carbon dynamics under future climates are discussed. In conclusion a modelling approach to bridging the inter-disciplinary gap between carbon cycling and the biology of soil animals is recommended. Mark Hassall et al. / ZooKeys 801: 5–24 (2018) Mark Hassall et al. / ZooKeys 801: 5–24 (2018) 6 Introduction 2017). However, recent evidence indicates cur­ rent carbon stores in other soils may also be changing from sinks to sources (Carrera et al. 2011) due to greater increases in soil respiration than in rates of carbon input to the soils. The possible consequences of this positive feedback cycle could potentially be reduced if soil moisture were to decrease as a result of changes in rainfall patterns (Fig. 1). Climate models predict that globally rainfall will increase but it is predicted to vary strongly both spatially and temporally. Spatially, greater increases are projected Interspecific variation in responses to microclimate by terrestrial isopods... 7 7 Figure 1. A conceptual diagram illustrating some of the pathways by which changes in global climate could potentially impact on rates of carbon dioxide emissions from soils. Both changes in temperature and in the levels and patterns of rainfall have strong direct effects on the metabolism of bacteria and fungi but their ecology and metabolism are also regulated by the extent to which they are stimulated by soil animals. Both functional (e.g., behavioural and physiological) responses and numerical (both life history and popu­ lation) responses of soil animals are affected by their microclimate. This is in turn affected by larger scale changes in temperature and rainfall. Therefore, as well as their direct effect on microbial metabolism, these climatic variables have a strong indirect effect by influencing the behavioural, physiological, life history, and population processes of soil animals such as isopods. Figure 1. A conceptual diagram illustrating some of the pathways by which changes in global climate could potentially impact on rates of carbon dioxide emissions from soils. Both changes in temperature and in the levels and patterns of rainfall have strong direct effects on the metabolism of bacteria and fungi but their ecology and metabolism are also regulated by the extent to which they are stimulated by soil animals. Both functional (e.g., behavioural and physiological) responses and numerical (both life history and popu­ lation) responses of soil animals are affected by their microclimate. This is in turn affected by larger scale changes in temperature and rainfall. Therefore, as well as their direct effect on microbial metabolism, these climatic variables have a strong indirect effect by influencing the behavioural, physiological, life history, and population processes of soil animals such as isopods. Introduction for high latitudes and mid-latitude wet regions but decreases in subtropical dry regions and many mid-latitude regions (IPCC 2013). Temporally, summer rainfall in temper­ ate regions, such as west and south Europe, is predicted to occur in fewer, but more intense episodes. These will result in longer periods of summer drought which could potentially constrain temperature-induced increases in soil carbon fluxes, including by reducing the stimulation of microbial metabolism by moisture dependent soil animals. g The microbially mediated emissions of carbon dioxide from soils are strongly influ­ enced by soil animals acting as key system regulators (Fig. 1) (Anderson 1991, Lavelle Mark Hassall et al. / ZooKeys 801: 5–24 (2018) 8 and Spain 2001, Krab et al. 2010, Coleman et al. 2017). The significance of direct and indirect consequences of global climate change on ecosystem processes mediated by invertebrates, including their indirect effects on heterotroph respiration through interactions with microorganisms, are widely recognised (Pelini et al. 2015). However, although they are likely to have a major impact on ecosystems in the future, they are as yet understudied (Blankinship et al. 2011, Carrera et al. 2011, Del Toro et al. 2015).if It is becoming increasingly apparent that the importance of interspecific differences in key traits not only leads to different species having different functional roles (Dias et al. 2013, Zimmer et al. 2002) but also causes different species to be affected differently by climate change (Veldhuis et al. 2017). Because ecosystem science has yet to embrace how principles of evolutionary ecology explain the ways in which organisms mediate ecosys­ tem carbon dynamics (Schmitz 2013), there is an urgent need to increase our understand­ ing of how differential impacts of climate change on basic biological traits of individual species can lead to changes in community structure and function (Jiguet et al. 2011). Traits of many soil animals are sensitive to relative humidity, making the soil fauna vulnerable to their activities being curtailed by changes in soil moisture (Krab et al. 2010, Crowther et al. 2015). Isopods are the most successful group of crustaceans to have made the transition to the terrestrial environment in terms of their abundance, distribution, and diversity, and have colonised a wide range of terrestrial habitats from the littoral zone to deserts (Edney 1954, Warburg 1987). They have become promi­ nent members of the arthropod macro-decomposer guild in many ecosystems (Sutton 1980). Introduction 2014) and their eco-physiology (Spencer and Edney 1954, Edney 1968, Warburg 1987, 1989, Hornung 2011). We used the selected species to explore possible implications of inter-specific differences in responses to micro- climate for the role of soil animals in soil processes, including regulation of microbi­ ally mediated soil carbon dynamics (Heemsbergen et al. 2004), under predicted climate change scenarios. Figure 2. A schematic representation of a typical thermal response curve for enzymes (simplified from Huey and Kingsolver 1989). The temperature optimum is the temperature at which performance reaches its maximal level or peak performance. The performance breadth defines how steeply peaked (stenother­ mal) or broadly plateaued (eurythermal) the response curve is. Any part or the whole of such a curve can be considered to be a reaction norm of a genotype representing a range of phenotypes expressed across an environmental gradient, in this example, of temperature. Schmidt and Wägele 2001, Broly et al. 2014) and their eco-physiology (Spencer and Edney 1954, Edney 1968, Warburg 1987, 1989, Hornung 2011). We used the selected species to explore possible implications of inter-specific differences in responses to micro- climate for the role of soil animals in soil processes, including regulation of microbi­ ally mediated soil carbon dynamics (Heemsbergen et al. 2004), under predicted climate change scenarios. Schmidt and Wägele 2001, Broly et al. 2014) and their eco-physiology (Spencer and Edney 1954, Edney 1968, Warburg 1987, 1989, Hornung 2011). We used the selected species to explore possible implications of inter-specific differences in responses to micro- climate for the role of soil animals in soil processes, including regulation of microbi­ ally mediated soil carbon dynamics (Heemsbergen et al. 2004), under predicted climate change scenarios. Introduction Their morphology, physiology, behaviour, life histories, and ecology are known to be strongly influenced by micro-climates. Isopods therefore form good model soil animals for testing hypotheses concerning how inter-specific differences in their physi­ ological, behavioural and life history traits will lead to interspecific differences in re­ sponses to predicted future changes in climate.f In this review we examine the extent to which traits of different species of terres­ trial isopods known to have different strategies for reducing water loss in the terrestrial environment, respond to temperature and relative humidity. Phenotypic responses to a gradient in the environment can be summarised as norms of reaction. A reaction norm is the set of phenotypes produced in a range of environments (Schmalhausen 1949, Stearns 1992) and represents either the whole or part of a response curve of an organ­ ism to any environmental gradient as for example illustrated by the thermal response curve for enzymes shown in Fig. 2. Differences in the gradients of performance response curves, such as that in Fig. 2, denote how quickly an organism changes its performance for a given change in an environmental gradient. A steep gradient indicates a strong response and a high level of phenotypic plasticity over that region of the environmental gradient. Reaction norms can be very effective in comparing responses not just between different species as has been demonstrated very clearly by Dias et al. (2013) but also to different environmental gradients such as soil moisture and temperature.f We investigated differences in reaction norms of physiological, behavioural, and life history traits in response to differences in temperature and relative humidity for a range of isopod species chosen to reflect differences in their eco-morphology (Schmalfuss 1984, Interspecific variation in responses to microclimate by terrestrial isopods... 9 9 Figure 2. A schematic representation of a typical thermal response curve for enzymes (simplified from Huey and Kingsolver 1989). The temperature optimum is the temperature at which performance reaches its maximal level or peak performance. The performance breadth defines how steeply peaked (stenother­ mal) or broadly plateaued (eurythermal) the response curve is. Any part or the whole of such a curve can be considered to be a reaction norm of a genotype representing a range of phenotypes expressed across an environmental gradient, in this example, of temperature. Schmidt and Wägele 2001, Broly et al. Behavioural responses to microclimate: thermal norms of reaction for aggregating behaviour Case study 1. Here we compare differences in aggregating behaviour of three species of isopod as described by Dixie et al. (2015) who give full details of the methodology including those of the statistical analyses used.f Differences in the degree of aggregation, at 90% relative humidity, between three species of isopods differing in their adaptations to reduce water loss in the terrestrial environment, are shown in Fig. 3. Oniscus asellus (Linnaeus, 1758) was most suscep­ tible to water loss, the Armadillidium vulgare (Latreille, 1804) is morphologically the best adapted to withstand desiccation while Porcellio scaber (Latreille, 1804) has an intermediate level of morphological adaptation but also uses aggregation as a key be­ havioural tactic in its overall desiccation avoidance strategy. Mark Hassall et al. / ZooKeys 801: 5–24 (2018) 10 Figure 3. Aggregation of isopod species differing in desiccation resistance at different temperatures. Mean ± 1 SE aggregation indices (variance:mean ratio) at 90% relative humidity. a P. scaber (F 4,249 = 3.76, p < 0.01 b A. vulgare (F 4,249 = 1.97, P < 0.01) c O. asellus (F 4, 249 = 12.22, P < 0.001) d thermal reaction norms fo aggregation expressed as quadratic response curves for: P. scaber (dashed line): y = -11.519 + 1.526× - 0.04×2 A. vulgare (solid line): y = -3.534 + 0.574× 0.016×2; O. asellus (dotted line): y = -5.890 + 0.814× – 0.018×2 Figure 3. Aggregation of isopod species differing in desiccation resistance at different temperatures. Mean ± 1 SE aggregation indices (variance:mean ratio) at 90% relative humidity. a P. scaber (F 4,249 = 3.76, p < 0.01) b A. vulgare (F 4,249 = 1.97, P < 0.01) c O. asellus (F 4, 249 = 12.22, P < 0.001) d thermal reaction norms for aggregation expressed as quadratic response curves for: P. scaber (dashed line): y = -11.519 + 1.526× - 0.04×2; A. vulgare (solid line): y = -3.534 + 0.574× 0.016×2; O. asellus (dotted line): y = -5.890 + 0.814× – 0.018×2. The temperature response curve of P. scaber (Fig. 3a, d) resembles the theoretical one in Fig. Behavioural responses to microclimate: thermal norms of reaction for aggregating behaviour 2 in being an upwardly convex curve, with a peak in the aggregation index of 2.93 at 19 °C, a performance breadth at 90% of the peak of 4.7 °C and a gradient of the reaction norm from 14 °C to 17 °C of + 0.8 increase in aggregation index for this 3 °C rise in temperature. At temperatures higher than 19 °C the curve of the ag­ gregation index decreases due to more of the animals leaving the clumps and moving around the arena sometimes climbing up the walls in an attempt to escape to find more favourable lower temperatures (Dixie et al. 2015). Armadillidium vulgare shows a broadly similar pattern of response (Fig. 3b, d), but with a lower peak of 1.9 in the aggregation index, again at 19 °C. The response curve is shallower than for P. scaber with a performance breadth of 6 °C at 90% of the peak value and a gradient for the reaction norm for the aggregation index between 14 -17 °C of +0.3. As for P. scaber, aggregation decreases at temperatures higher that the peak temperature. Interspecific variation in responses to microclimate by terrestrial isopods... 11 The temperature response curve for aggregation of O. asellus (Fig. 3c, d) has a higher peak aggregation index of 3.35 at a higher temperature of 21.3 °C, a wide per­ formance breadth of 6.2 °C at 90% of the peak value and a gradient for its thermal norm of reaction between 14 -17 °C of 1.5 in the aggregation index over this 3 °C range. This was approximately twice as steep as for P. scaber and three times as steep as for A. vulgare. The decline following the peak for O. asellus was less pronounced than for either of the other two species. Overall these results show that these three species have substantially different pat­ terns of thermal response curves for aggregating behaviours, reflecting differences in their morphological adaptations to terrestrial life. Behavioural responses to microclimate: moisture norms of reaction for sheltering and feeding behaviours Many terrestrial isopods aggregate in shelter sites, particularly during the day, often un­ der stones or pieces of wood where moisture from the soil maintains a more favourable relative humidity than in more open sites. They then emerge to forage at night when temperatures are lower and relative humidity is higher. Sheltering behaviour is thus of central importance in reducing mortality due to desiccation while also reducing the risk of being eaten by diurnal predators, such as insectivorous birds.f Case study 2. Effects of substrate moisture content and relative humidity on sheltering and feeding behaviour were investigated by Moss (2007) under controlled temperature conditions in the laboratory. Three species were studied: A. vulgare, Philoscia muscorum (Scopoli, 1763) and P. scaber differing in morphological and physiological adaptations to the terrestrial environment (Dias et al. 2012). The ex­ perimental mesocosms (220mm × 150mm × 80mm deep) were lined with plaster of Paris covered by 50mm sand with a feeding tray at one end and a shelter at the other, as described in further detail by Moss (2007). Two simulated rainfall re­ gimes were used: high simulated rainfall, representing current mean summer daily rainfall intensity of 1.65 mm day-1 calculated from British Atmospheric Data Cen­ tre records for Lacock, Wiltshire, England (51°43'N, 2°11'W) during 1996–2006 when rainfall events occurred on average once every 72h. The low simulated rainfall regime was half the intensity of the high rainfall regime, 0.83 mm day-1, represent­ ing a scenario with 810 ppm concentration of atmospheric CO2 by 2080 which is predicted to result in a 50% reduction in summer rainfall in south west England (Hulme et al. 2002). For both regimes the simulated rainfall was administered at the start of each 72h period using a seedling watering can with 0.6 mm diameter holes in the rose with the shelter and food trays temporarily covered. Replicate arenas but without animals were used to monitor percentage moisture content of the substrate by taking samples of sand each morning and evening. Differences in the moisture conditions in the mesocosms were caused by both differences in intensity of simu­ Mark Hassall et al. / ZooKeys 801: 5–24 (2018) 12 12 lated rainfall and the progressive drying out of mesocosms which occurred with time between rainfall events. Starting moisture level following high rainfall level ≈ 20% and for low rainfall level ≈ 10%. Behavioural responses to microclimate: moisture norms of reaction for sheltering and feeding behaviours Eight replicate mesocosms were used for each rainfall treatment, with six identical control boxes for monitoring substrate moisture content. Each mesocosm contained twenty individuals of each species. The behaviours of all animals over a 72-hour period were classified according to their location in the mesocosm and activity categories, including sheltering and presence in the feeding area. It was not possible to observe movements of mouthparts so time spent in the feeding area was assumed to be propor­ tional to time spent feeding (Dias et al. 2012). Regression analyses of behavioural traits on substrate moisture were conducted. The results in Fig. 4 and Table 1 show that Ph. muscorum spent significantly less time sheltering than either of the two other species under the highest moisture condi­ tions. It also had the steepest gradient for its moisture reaction norm of -2.16, com­ pared with values of -1.05 for A. vulgare and -0.19 for P. scaber (Table 1). Porcellio scaber showed the least change in the proportion of time it spent sheltering because it sheltered for more than 95% of the time under all substrate moisture conditions. In contrast A. vulgare and Ph. muscorum sheltered for 74% and 51% respectively of the time under the moistest conditions (Table 1). Ph. muscorum also had the steepest gradient for its moisture reaction norm for feeding of 0.55 (Fig. 4b), which was significantly higher than those for either P. scaber (0.05) or A. vulgare (0.03) (Table 1). Time spent feeding decreased from 11.5% when moisture content of the sand was 20%, down to zero under the driest conditions when none of the three species spent a significant amount of time feeding.i Overall Ph. muscorum showed significantly steeper moisture reaction norms than did either of the other species just as O. asellus, which similarly lacks pleopodal lungs, had a steeper gradient for its thermal reaction norm for aggregation than did either P. scaber or A. vulgare. Life history responses to microclimate: thermal and moisture norms of reaction for growth and survivorship Case study 3. Both thermal and moisture reaction norms for the key life history traits of growth and mortality were compared for O. asellus and Porcellio dilatatus Brandt, 1833 by Dixie et al. (2015) who give full details of the methodology and statistical analyses used.h The 2×2 factorial experimental design for investigating both relative growth rates and mortality rates permits comparison of thermal and moisture reaction norms simultaneous­ ly. Growth rates of O. asellus increased significantly at 5 °C higher temperatures under the drier, 70% relative humidity conditions but did not grow significantly faster at the higher temperatures in the moister, 90% humidity (Table 2). Growth of P. dilatatus increased with temperature to a similar extent under both experimental relative humidity conditions. All four of the moisture reaction norms for a 20% decrease in moisture are steeper than any Interspecific variation in responses to microclimate by terrestrial isopods... 13 13 Interspecific variation in responses to microclimate by terrestrial isopods... 13 Figure 4. Moisture reaction norms for a) sheltering and b) feeding behaviours with changing sand moisture content (time spent in behaviour as percentages of total observed behaviours). Lines represent linear regression models: A. vulgare (solid line) (sheltering: y = 95.24 – 1.05×; feeding: y = 0.32 + 0.03), P. scaber (dashed line) (sheltering: y = 96.72 – 0.19×; feeding: y= 0.22 + 0.05×), Ph. muscorum (dotted line) (sheltering: y = 93.14 – 2.16×; feeding: y = 0.36 + 0.55x). Further regression statistics and number of observations (N), are given in Table 1. Figure 4. Moisture reaction norms for a) sheltering and b) feeding behaviours with changing sand moisture content (time spent in behaviour as percentages of total observed behaviours). Lines represent linear regression models: A. vulgare (solid line) (sheltering: y = 95.24 – 1.05×; feeding: y = 0.32 + 0.03), P. scaber (dashed line) (sheltering: y = 96.72 – 0.19×; feeding: y= 0.22 + 0.05×), Ph. muscorum (dotted line) (sheltering: y = 93.14 – 2.16×; feeding: y = 0.36 + 0.55x). Further regression statistics and number of observations (N), are given in Table 1. of the thermal reaction norms for a 5 °C temperature difference for either species. This indicates a greater sensitivity to a 20% change in relative humidity than to a rise of 5 °C in temperature, suggesting that perhaps these species might respond more to predicted future changes in rainfall than to predicted increases in temperature. Life history responses to microclimate: thermal and moisture norms of reaction for growth and survivorship The difference in gradients of reaction norms between species (Table 2) reflects the higher rates of growth for P. dilatatus under all four combinations of temperature and relative humidity conditions. Mortality rates (Table 3) were very low at 90% relative humidity; none of the O. asellus and very few of the P. dilatatus died at this relative humidity. The gradients of thermal reaction norms at 90% relative humidity were therefore not significantly Mark Hassall et al. / ZooKeys 801: 5–24 (2018) 14 Table 1. Regression statistics for moisture reaction norms for sheltering and feeding behaviours of three species of isopods, Armadillidium vulgare, Porcellio scaber, and Philoscia muscorum, in laboratory arenas. Abbreviations: N: number of observations; a and b: parameters of regression equation; †: significant dif­ ferences at P < 0.001(t test). Regression statistics Sheltering Feeding N a b t a b t A. vulgare 1648 95.24 -1.05 -21.84† 0.32 0.03 3.77† P. muscorum 824 93.14 -2.16 -25.07† 0.36 0.55 15.32† P. scaber 1648 96.72 -0.19 -8.39† 0.22 0.05 6.84† Comparison of gradients Sheltering Feeding A. vulgare vs. P. muscorum 18.99† -30.19† A. vulgare vs. P. scaber -25.08† -3.96† P. muscorum vs. P. scaber -44.43† 28.36† Table 2. Relative growth rates (mg g-1 day-1) of Oniscus asellus and Porcellio dilatatus. Three way ANOVA: temperature F1,72 = 5.15, P = 0.026; humidity F1,72 = 88.62, P < 0.001; species F1,72 = 41.53, P < 0.001. Reac­ tion norms are derived from the differences in response to the two temperature and two humidity conditions. Table 2. Relative growth rates (mg g-1 day-1) of Oniscus asellus and Porcellio dilatatus. Three way ANOVA: temperature F1,72 = 5.15, P = 0.026; humidity F1,72 = 88.62, P < 0.001; species F1,72 = 41.53, P < 0.001. Reac­ tion norms are derived from the differences in response to the two temperature and two humidity conditions. Temperature °C Relative humidity % Moisture reaction norms 90% 70% O. asellus 13.5 °C 34 ± 6 2 ± 0.1 -32 18.5 °C 32 ± 2.5 17 ± 9 -15 Thermal reaction norms -2 15 P. dilatatus 13.5 °C 66 ± 0.5 15 ± 4 -51 18.5 °C 77 ± 8 28 ± 4 -49 Thermal reaction norms 11 13 Table 3. Mortality rates (numbers dying container -1 7days-1) of O. asellus and P. dilatatus. Mann Whit­ ney U test for O. Life history responses to microclimate: thermal and moisture norms of reaction for growth and survivorship asellus: temperature NS, humidity U = 1851 P < 0.001; for P. dilatatus: temperature U = 2754, P = 0.016, humidity U = 2277, P < 0.001. Reaction norms are derived from the differences in response to the two temperature and two humidity conditions. Temperature °C Relative humidity % Moisture reaction norms 90% 70% O. asellus 13.5 °C 0 0.73 ± 0.18 0.73 18.5 °C 0 0.58 ± 0.12 0.58 Thermal reaction norms 0 0.46 P. dilatatus 13.5 °C 0.09 ± 0.04 0.78 ± 0.16 0.69 18.5 °C 0.03 ± 0.03 0.18 ± 0.08 0.15 Thermal reaction norms 0.06 0.60 different to zero. Under drier conditions, at 70% relative humidity, more individuals of both species died at 13.5 °C than at 18.5 °C, leading to steeper thermal reaction norms at 70% relative humidity than at 90% relative humidity. different to zero. Under drier conditions, at 70% relative humidity, more individuals of both species died at 13.5 °C than at 18.5 °C, leading to steeper thermal reaction norms at 70% relative humidity than at 90% relative humidity. Overall, 20% lower relative humidity resulted in steeper moisture reaction norms for mortality rates than for thermal reaction norms resulting from a 5 °C rise in tem­ perature, with O. asellus being more susceptible to drier conditions than P. dilatatus. Interspecific variation in responses to microclimate by terrestrial isopods... 15 Physiological responses to microclimate: temperature and moisture norms of reaction for respiratory rate and water loss One of the drivers of differences between species of isopods in their behavioural and life history responses to differences in temperature and moisture is their different physiological adaptations to the terrestrial environment. In this section we compare responses of terrestrial isopod species, occurring in a wide range of biomes representing a gradient of moisture conditions. Moisture reaction norms for differences in respiratory rate for O. asellus, P. scaber, and A. vulgare are shown in Fig. 4 together with that for Ligia oceanica (Linnaeus, 1767). The pattern of differences between the physiological reaction norms closely parallels that of behavioural reaction norms (Figs. 3, 4) with the gradient for O. asellus over a range of a 50% difference in relative humidity being substantially higher than for P. scaber and A. vulgare. However the gradient for L. oceanica, a littoral species, is more than double that for O. asellus reflecting the higher stress that would result from a reduction in relative humidity for a species that lives in a habitat with abundant avail­ ability of crevices with saturated micro-climates. y This interspecific comparison is extended for species from a wider range of habitats for thermal reaction norms for the physiological process of water loss in Fig. 6. The pattern for L. oceanica, O. asellus and P. scaber is similar to that for the moisture reac­ tion norms for respiratory rate. Ph. muscorum has a higher rate than O. asellus, which, together with P. scaber, has a slightly steeper moisture reaction norm than either A. vul­ gare or Armadillidium nasatum (Budde-Lund, 1885). All five of these species are found in mesic habitats. The three species of Buddelundia from semi-arid habitats in South Australia all have lower gradients for their water loss thermal reaction norms than any of the European species from mesic habitats. Least affected by differences in tempera­ ture are species from xeric habitats including those found in the Negev and North Afri­ can deserts such as Hemilepistus reaumurii (Milne-Edwards, 1840). The two Armadillo species and Porcellio olivieri (Audouin, 1826) also occur in desert environments but not so exclusively, as they are also found in other dry Mediterranean habitats. These three species have gradients for their thermal reaction norms that are intermediate be­ tween those of the desert specialists (including Venezillo arizonicus Mulaik & Mulaik, 1942) and the mesic species. Discussion The range of morphological adaptations in body shape, respiratory surfaces of the pleopods and cuticle of isopods have been comprehensively described (Edney 1968, Warburg 1992, Schmalfuss 1984, Hornung 2011, Csonka et al. 2013, Broly et al. 2014) and reflect a moisture gradient in the habitats in which they are found, from littoral, through mesic to xeric, including desert. It can therefore be predicted that there will be a parallel gradient in the magnitude of physiological responses to changes in microclimate reflected in the gradients of thermal and moisture reaction norms. Mark Hassall et al. / ZooKeys 801: 5–24 (2018) 16 Figure 5. Gradients of moisture reaction norms for respiration of isopods differing in their resistance to desiccation. Reaction norms over the range 50–100% relative humidity for respiratory rates measured as rates of oxygen uptake (mm3 mm-2 body surface h-1) (Edney 1968). Key to species: Lo Ligia oceanica, Oa Oniscus asellus, Ps Porcellio scaber, Av Armadillidium vulgare. Key to habitats: littoral (blue), mesic (green). Figure 5. Gradients of moisture reaction norms for respiration of isopods differing in their resistance to desiccation. Reaction norms over the range 50–100% relative humidity for respiratory rates measured as rates of oxygen uptake (mm3 mm-2 body surface h-1) (Edney 1968). Key to species: Lo Ligia oceanica, Oa Oniscus asellus, Ps Porcellio scaber, Av Armadillidium vulgare. Key to habitats: littoral (blue), mesic (green). This hypothesis was supported for respiratory rate moisture reaction norms (Fig. 5), as L. oceanica, the species with the most primitive morphological adaptations to the terrestrial environment, had the steepest reaction norm, while O. asellus, which does not have pleopodal lungs, had a steeper moisture reaction norm than either P. scaber or A. vulgare, both of which have well developed pleopodal lungs. Similarly, gradients of thermal reaction norms for water loss were substantially higher for L. oceanica than for the more fully terrestrial species O. asellus and Ph. muscorum, both of which had steeper gradients for their thermal reaction norms than members of either of the Porcellio species or A. vulgare (Fig. 6). There was a clear trend of thermal reaction norms for species from semi-arid environments in South Australia and xeric habitats in Europe, including specialist desert species, all having shallower thermal reaction norms for rates of water loss. Discussion Thus it can be predicted that those spe­ cies from mesic environments will be more sensitive to changes in rainfall patterns, including longer periods of summer drought, than those from xeric habitats. Given that these patterns of inter-specific differences in physiological traits for both thermal and moisture reaction norms reflect differences in morphology (Schmal­ fuss 1984, Broly et al. 2015), it can be predicted that there would also be a parallel pattern in thermal and moisture reaction norms for behavioural and life history traits. Aggregation in isopods is a behavioural tactic that reduces respiratory rates and water loss (Allee 1926), further elaborated by Friedlander (1965), Cloudsey-Thompson and Constaninou (1987), Caubet et al. (2008), and Broly et al. (2014). This morphologi­ cally related hypothesis is supported by both the peak of the thermal response curve and the gradient of the thermal reaction norm for aggregation being higher for O. asel­ lus than for either P. scaber or A. vulgare. Interspecific variation in responses to microclimate by terrestrial isopods... 17 17 Dif­ ferences in the gradients of reaction norms of life history traits could thus result in differences in the way abundances of these species might change under future climates. f An important conclusion of this overview is that, due to inter-specific differences in morphological, physiological, behavioural and life history traits, different species of isopods are likely to respond very differently to predicted changes in global climate. In­ terspecific differences in response to changes in temperature (Hickling et al. 2006) are now well documented for other taxa such as birds (Pearce-Higgins et al. 2015), Lepi­ doptera (McDermott Long et al. 2017), Orthoptera (Buckley et al. 2015) and Odo­ nata (Stoks et al. 2014). In contrast, much less is known about ecological responses to predicted changes in rainfall patterns (Urban et al. 2016). Such responses are likely to be particularly pronounced for soil animals due to their activity and ecology being so strongly influenced by soil moisture, as shown by Blankinship et al. (2011) who found that precipitation limited all taxa and trophic groups in forest floors. l If the abundance of some species declines, it is not yet known whether other spe­ cies, less sensitive to changes in microclimate, will respond by expanding their realised niches. It is known that interspecific competition for high quality foods between dif­ ferent species of isopods does occur in the field (Hassall and Dangerfield 1989) so it is possible that total guild densities may change less than species composition of guilds for macro-decomposers. It is known that differential impacts on basic biological pa­ rameters of individual species can modify fundamental characteristics of community structure and function (Jiguet et al. 2011). For isopods it is possible that the func­ tional role of the whole isopod guild in stimulating microbial activity may alter less as a result of future climate change than might be expected from effects on particularly sensitive individual species. If other species in the guild, less sensitive to changes in micro-climate, expanded their realised niches in response to the decline of a more sensitive species, this could buffer consequences of climate change for overall decom­ position rates. It is yet to be tested whether net community level responses, resulting from differential impacts of climate change on individual species, will impact on the extent to which future changes in rainfall patterns might mitigate potential increased temperature/ induced carbon dioxide emission feedbacks. Figure 6. Thermal reaction norm gradients for evaporation rate (water loss) for isopods from biomes differ­ ing in availability of moisture. Evaporation rate (g g-1 h-1× 10-2) standardised to a temperature range of 3.5 °C (from Edney 1951; Warburg 1965, 1987, 1989). Key to species: Lo Ligia oceanica, Ph Philoscia muscorum, Oa Oniscus asellus, Ps Porcellio scaber, Av Armadillidium vulgare, Ao Armadillo officinalis, Aa Armadillo albomargi­ natus, Po Porcellio olivieri, An Armadillium nasatum, Ba Buddelundia albinogrisescens, Bf Buddelundia frontosa, Va Veneziillo arizonicus, Bsp Buddelundia spp. probably lateralis, Hr Hemilepistus reaumurii, Sf Schizidium fes­ tai. Key to habitats: littoral (blue), mesic (green), xeric (yellow), semi-arid (orange) habitats in South Australia. Sheltering behaviour is another tactic evolved in isopods to help reduce water loss by taking refuge in more humid shelter sites. Again, Ph. muscorum had a significantly higher gradient for its sheltering moisture reaction norm than either P. scaber or A. vul­ gare. Similarly, feeding behaviour moisture reaction norms of Ph. muscorum decreased more steeply than for either of the other two species. Thus within three species, all typical in mesic environments, there was a consistent trend in all of these behavioural traits, with species without pleopodal lungs being more sensitive to changes in micro­ climate than species better adapted to resist desiccation. This again indicates that there are significant inter-specific differences which suggest those species least adapted to the terrestrial environment might be more susceptible to potential changes in micro- climate resulting from changes in future patterns of rainfall. It would be logical to predict that such a trend might also apply to growth rates but that was not supported by the data because thermal reaction norms were very similar for O. asellus and P. dilatatus while the gradient for moisture reaction norms at both temperatures were higher for P. dilatatus. P. dilatatus had higher growth rates under all four combinations of temperature and relative humidity, possibly because the microcli­ mate actually experienced by P. dilatatus was moister than that experienced by O. asel­ lus due to P. dilatatus behavioural trait of burrowing into the sand substrates thereby being subject to a higher relative humidity in its immediate microclimate. 18 Mark Hassall et al. / ZooKeys 801: 5–24 (2018) These life history traits of growth and survivorship are important correlates of fitness and have a very strong influence on the population dynamics of isopods. It is widely agreed however, more focus is required on biotic interactions to clarify the potential feedbacks between climate change and soil carbon dynamics (Carrera et al. 2011). The importance of species-specific responses to climate change has been highlighted for Collembola by Krab et al. (2010) and Makkonen et al. (2011), who found that in­ terspecific faunal trait variation provided a valuable tool in predicting animal responses to climate change. However, it is noteworthy that Crowther et al. (2015) conclude that the regulatory effects of interspecific interactions are rarely considered in climate feed­ back studies. Pelini et al. (2015) extend this conclusion further by arguing that without isolating and including the significant impact of invertebrates, climate models will be incomplete, hindering well-informed policy decisions. Because the adaptive value of fundamental biological traits in isopods have been so comprehensively studied and Interspecific variation in responses to microclimate by terrestrial isopods... 19 because isopods form such a prominent component of so many soil macro-arthropod communities, we propose that terrestrial isopods represent excellent model systems for further investigation of species specific responses to predicted climate change and their consequences for soil carbon dynamics. What are the most pressing needs for future research in this field? Our understanding of the global carbon cycle is predominantly encapsulated in mod­ els. Our understanding of terrestrial isopod biology, in contrast, is mostly based on results of empirical studies. There is a very important need to bridge the gap between these two contrasting approaches and methodologies in these completely different dis­ ciplines. Considering the whole series of symposium volumes on the Biology of Ter­ restrial Isopods from 1984 until 2018, models may qualify as an endangered species. We know that carbon dynamics in the soil is the least well understood part of the global carbon cycle. Whilst, it is extremely complicated, models by definition are sim­ plifications of reality. Models can never represent the full complexity of the real world, that is not their function, but what they have are both holistic and heuristic properties. Holistic in that it should be easier to appreciate emergent properties of a system as a whole from a model of it, rather than from detailed studies of individual components. Heuristic in that models should generate testable predictions that cannot be made on the basis of studying individual components in isolation. Terrestrial isopods are soil animals about which we have a wealth of knowledge based on empirical studies on their anatomy, behaviour, physiology, life history, and ecology. What we do not have is integrative models of different aspects of their biology that can then be interfaced with those of other soil animals and ultimately with those of micro-or­ ganisms. This integrative approach would significantly contribute to our understanding of how global climate change will affect the soil component of the global carbon cycle. Where can we start? Models do not necessarily have to be numerical, at least initially. A topographic map is a model of a landscape. At a glance, that piece of paper only tens of cm wide, shows us where hills, mountains, rivers and bridges are in landscapes at much larger scales. By us­ ing a map we can predict how best to travel from A to B. What is very urgently needed in soil biology is some comparably simplified maps of the interactions involving the soil animals under our feet. A starting place could be for the context of many future studies to be introduced using conceptual box and arrow models of the system under study (Fig. 1). Our skills as scientists lie in how effectively we can simplify the real world we are studying by being brave enough to make a whole array of untested assumptions.h g g g The next stage could then be to parameterise these models using the extensive empirical data available in the literature, making assumptions we know to be simplistic Mark Hassall et al. / ZooKeys 801: 5–24 (2018) 20 but which enable us to make further quantitative predictions. Progressively testing these predictions experimentally to validate the models further could then increase our understanding of the system. g A problem will always be integrating models representing different levels of or­ ganisation which, from previous studies, range from molecules to communities. These may initially appear to be measured in different currencies but ultimately, while we are working within the paradigm of neo-Darwinian evolutionary theory, the answer to this problem may be in using fitness, or at least fitness correlates, to equate different traits at the individual level. Moving from the individual level to the ecosystem function level could then at least be based on a sound theoretical foundation.h The “functional traits” approach (Moretti et al. 2017) may be one way forward. Splitting whole trophic groups, treated as homogeneous entities in some large scale models, into functional groups has been an important step in the right direction. Tak­ ing account of interspecific differences in traits within functional groups and incor­ porating the diversity of responses into systems models could be a further important step. Where can we start? It would appear that the new generation of isopod biologists is now uniquely well placed to do this, thus giving them the potential to fill an extremely important gap in 21st Century science in a way that is a meaningful contribution to our understanding of the effects mankind is having by interfering with the global carbon cycle and thus global climate systems. We wish younger colleagues all success with such a challenging venture at such an exciting time and best wishes with carrying the science of terrestrial isopod biology to new horizons. Acknowledgements MH is very grateful to Stephen Sutton for initiating, stimulating, and encouraging his interest in the biology of terrestrial isopods. We also thank Martin Zimmer for suggest­ ing an earlier version of this paper, NERC for a studentship for AM, and to the guest editors of this volume for inviting us to contribute to these symposium proceedings. 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https://openalex.org/W3134838293
https://journal.ksiop.or.kr/index.php/KJIOP/article/download/371/378
Korean
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Validation for Korean Version of Kaleidoscope Career Attitude Measurement
Han'gug simlihag hoeji. san'eob mich jo'jig/Korean journal of industrial and organizational psychology
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* 이 논문은 2019년 대한민국 교육부와 한국연구재단의 인문사회분야 신진연구자지원사업의 지원을 받아 수행된 연구임(NRF-2019S1A5A8038225). †교신저자 : 정보영, 현대자동차 인재개발팀 책임매니저, viautumn@gmail.com 한국심리학회지: 산업및조직 Korean Journal of Industrial and Organizational Psychology 2021. Vol. 34, No. 1, 51-79 https://doi.org/10.24230/kjiop.v34i1.51-79 한국심리학회지: 산업및조직 Korean Journal of Industrial and Organizational Psychology 2021. Vol. 34, No. 1, 51-79 https://doi.org/10.24230/kjiop.v34i1.51-79 한국심리학회지: 산업및조직 Korean Journal of Industrial and Organizational Psychology 2021. Vol. 34, No. 1, 51-79 https://doi.org/10.24230/kjiop.v34i1.51-79 한국판 칼레이도스코프 경력태도 측정도구 타당화 연구* 정 보 영† 현대자동차 정 보 영† 현대자동차 이 재 은 상지대학교 이 재 은 상지대학교 이 연구의 목적은 Sullivan과 그의 동료들(2009)이 개발한 칼레이도스코프 경력태도 척도를 우 리나라의 기업근로자 대상으로 타당화하는 데 있다. 이를 위해 수정델파이법을 통해 내용타 당도를 검증하고, 남녀 기업근로자를 대상으로 2개의 표본자료를 수집하여(N=347, N=631) 구인타당도, 변별타당도, 준거타당도를 검증하였다. 연구결과는 다음과 같다. 첫째, 수정델파 이법을 통해 번안된 칼레이도스코프 경력태도 측정문항의 내용타당도를 확인하고 국내 기업 근로자에게 적용할 수 있는 3문항을 개발하였다. 둘째, 칼레이도스코프 경력태도 측정문항은 원척도와 같이 3개 요인으로 구성되었으며, 최종 13개 문항으로 구성된 측정도구의 적합도 가 적절하게 도출되어 이를 채택하였다. 셋째, 교차타당도 분석 결과 새로운 표본(N=631)에 서 13개 문항의 측정모형 적합도가 양호하였으며 남녀 성별 집단에서도 동일한 척도로 활용 될 수 있음을 확인하였다. 한편, 칼레이도스코프 경력태도 중 균형과 도전 인식에서 성별 집 단의 잠재평균 차이가 유의한 것으로 나타났다. 연구결과를 종합하여 한국판 칼레이도스코 프 경력태도가 타당하고 신뢰할 수 있는 척도임을 밝혔으며, 이를 토대로 연구의 의의와 한 계점, 향후 연구를 위한 제언 등이 논의되었다. 주요어: 칼레이도스코프경력, 만화경경력, 측정도구, 타당화, 잠재평균분석 * 이 논문은 2019년 대한민국 교육부와 한국연구재단의 인문사회분야 신진연구자지원사업의 지원을 받아 수행된 연구임(NRF-2019S1A5A8038225). †교신저자 : 정보영, 현대자동차 인재개발팀 책임매니저, viautumn@gmail.com - 51 - 한국심리학회지: 산업및조직 고 있다(McMahon, 2014; Savickas, 2012). 그러나 국내 뿐 아니라 국외에서도 칼레이도스코프 경력에 대한 연구는 아직까지 크게 확산되지 못하고 있는 실정이다. 이는 칼레이도스코프 경력이 특정 시점에서의 근로자 경력태도를 확인하고 이와 관련된 다양한 변인이 무엇인 지 밝히는 것에 초점을 두고 있기 보다는 근 로자의 생애에 걸친 경력패턴의 변화를 설명 하는 것을 목적으로 하고 있기 때문이라 짐작 된다. 최근 많은 근로자들이 경험하고 있는 급격 한 환경변화는 개인의 경력에 대한 태도와 경 력개발의 기준을 모두 바꾸어 놓고 있다. 조 직 중심의 경력개발에서 개인 주도의 경력개 발로 구심점이 이동한 경력 패러다임의 변화 는 더 이상 새로운 관점이 아닌 것으로 여겨 지고 있다. 조직 구성원들의 경력에 관한 태 도와 경력개발 의사결정 등과 관련하여 새로 운 개념 제시와 관점 채택이 필요한 시점이다. 이러한 경력 패러다임의 변화와 함께 국내외 경력 연구자들은 새로운 경력태도에 관심을 갖고 연구해 왔다. 새로운 경력태도와 관련하 여 프로티언 경력(protean career), 무경계 경력 (boundaryless career)과 함께 자주 거론되는 모 형이 Mainiero와 Sullivan(2005)이 제안한 칼레이 도스코프 경력모형(Kaleidoscope Career Model: KCM)으로(Baruch, 2007; Sullivan & Baruch, 2009), 국내에서는 만화경 경력모형, 또는 만 화경 경력태도로 번안되기도 하였다. Mainiero 와 Sullivan(2005)은 환경변화에 따라 기존의 경력 개념에 변화가 필요하다고 설명하면서, 만화경이 반사에 의해 무한한 패턴을 만드는 것과 같이 개인은 경력과 관련하여 3개의 주 요한 경력 파라미터(career parameter: 진정성 (authenticity), 균형(balance), 도전(challenge))를 회 전시키며 다양한 경력 패턴을 만들어 간다고 보았다. 한국판 칼레이도스코프 경력태도 측정도구 타당화 연구* 이를 통해 향후 칼레이도스코프 경 력태도 관련 연구의 방향을 제시하고자 한다. 를 확인할 수 있을 것이다. 이 연구는 우리나라 기업근로자를 대상으로 하여 칼레이도스코프 경력태도 측정도구를 타당화하는 것을 목적으로 한다. 이를 위해 Sullivan, Forret, Carraher와 Mainiero(2009)가 개발 한 칼레이도스코프 경력태도 측정도구를 번안 하여 내용타당도를 확인하고, 탐색적 요인분 석과 확인적 요인분석을 통해 구성개념 타당 도를 검증하고자 한다. 또한 칼레이도스코프 경력태도의 구성개념과 관련된 일과 삶의 균 형, 프로티언 경력태도, 도전적 직무행동, 경 력만족, 고용가능성, 경력몰입 등의 변인과의 관계를 분석하여 준거관련 타당도를 확인하고 자 한다. 이를 통해 향후 칼레이도스코프 경 력태도 관련 연구의 방향을 제시하고자 한다. 이론적 배경 이론적 배경 한국판 칼레이도스코프 경력태도 측정도구 타당화 연구* 즉 개인은 자신의 생애 경력발달 단 계에서 나타날 수 있는 정체성 갈등을 해결해 나가면서 경력 발전의 중요한 축이 되는 경력 파라미터를 개인의 정체성에 맞게 조정하며 경력과 관련된 의사결정을 수행해간다는 것이 다(Kirk, 2016). 칼레이도스코프 경력모형은 삶 의 전체적인 맥락 안에서 구성주의적 관점을 갖고 개인의 경력개발에 초점을 두어야 한다 는 최근 경력 관련 연구자들의 주장을 수용하 선행연구에서 칼레이도스코프 경력태도는 주로 특정 집단의 경력에 대한 태도를 이해하 거나 경력 관련 행동에 차이가 있는 두 집단 의 특성을 비교할 목적으로 활용되어 왔다. 예를 들어, 여성(August, 2011; Cabrera, 2007; Shaw & Leberman, 2015), 기업가(Sullivan, Forret, Mainiero, & Terjesen, 2007), 교원(Carraher, Crocitto & Sullivan, 2014)의 칼레이도스코프 경 력태도를 구명하고자 한 연구가 수행되었으며, 성차(Mainiero & Gibson, 2018)나 세대차(Sullivan, Forret, Carraher, & Mainiero, 2009)를 밝히고자 한 연구들이 있었다. Sullivan과 Baruch(2009)는 개인의 경력 관련 의사결정에 있어 진정성, 균형, 도전의 속성이 점점 더 중요해지고 있 다고 설명하였으나, 서구 문화권에서 다양한 집단을 대상으로 칼레이도스코프 경력모형이 적용되어 온 것과 달리 국내 근로자를 대상으 로 칼레이도스코프 경력태도를 측정하거나 칼레이도스코프 경력모형을 적용한 연구는 매우 부족한 실정이다. 이에 우리나라 근로 자에게도 칼레이도스코프 경력을 구성하는 세 개의 경력 파라미터가 유효한지 검증할 필요가 있다. 경력 패러다임의 변화와 함께 나타난 근로 자의 새로운 경력태도를 대표하는 프로티언 - 52 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 경력태도는 주도적으로 자신의 가치에 기초하 여 경력을 개발하는 개인의 태도(Hall, 2004)로 자기주도적 경력관리와 개인가치 지향이라는 두 가지 하위요인으로 구성된다. 많은 연구에 서 프로티언 경력태도는 고용가능성, 주관적 경력성공, 경력만족, 직무만족, 조직몰입 등 개인의 긍정적인 경력, 조직 태도와 정적으로 관련되어 있어(Lee, Park, & Kim, 2019), 프로티 언 경력이 새로운 경력 패러다임 시대에 적응 적인 경력태도임을 밝힌 바 있다. 이 연구의 주요 관심인 칼레이도스코프 경력태도는 프로 티언 경력태도에서 강조하고 있는 ‘개인 가치 지향’의 실체를 보다 상세하게 설명해 준다는 측면에서 의의가 있다. 칼레이도스코프 경력 에서 강조하고 있는 세 가지 경력 파라미터가 개인이 지향하는 구체적인 가치가 될 수 있기 때문이다. 칼레이도스코프 경력은 ‘현대사회에 서 경력과 관련하여 개인이 추구하는 가치는 무엇인가?’, ‘이러한 가치는 전체 경력개발의 단계에서 어떻게 달라지는가?’, ‘개인의 경력 과 관련하여 변화된 가치는 경력관련 태도와 행동에 어떠한 영향을 미치는가?’ 등의 질문에 답변을 가능하게 한다. 를 확인할 수 있을 것이다. 이 연구는 우리나라 기업근로자를 대상으로 하여 칼레이도스코프 경력태도 측정도구를 타당화하는 것을 목적으로 한다. 이를 위해 Sullivan, Forret, Carraher와 Mainiero(2009)가 개발 한 칼레이도스코프 경력태도 측정도구를 번안 하여 내용타당도를 확인하고, 탐색적 요인분 석과 확인적 요인분석을 통해 구성개념 타당 도를 검증하고자 한다. 또한 칼레이도스코프 경력태도의 구성개념과 관련된 일과 삶의 균 형, 프로티언 경력태도, 도전적 직무행동, 경 력만족, 고용가능성, 경력몰입 등의 변인과의 관계를 분석하여 준거관련 타당도를 확인하고 자 한다. 칼레이도스코프경력모형 세 개의 파라미터는 진정성(authenticity), 균형(balance), 도전(challenge) 으로 구성되며, 진정성은 진정한 자기 자신이 되는 것으로, 균형은 일(work)과 일 외적인 요 구 사이의 균형으로, 도전은 개인을 자극하는 일과 경력에서의 발전으로 정의된다. 이러한 세 개의 파라미터는 생애주기 동안 모두 활성 화되어 있으나, 특정 시기에 어떤 파라미터가 강하게 나타나는가에 따라 개인의 경력 패턴 이 결정되게 된다. 보다 구체적으로 개인은 경력 파라미터에 기반하여 직무요구, 제한, 기 회와 개인이 얻게 될 가치, 이익, 관계 등을 비교하며 최적의 상태(best fit)를 추구한다는 것이다. 즉 개인의 생애에 걸친 경력 의사결 정과 그 결과로 나타나는 경력패턴을 경력 파 라미터를 중심으로 이해할 수 있다는 데 모형 의 의의가 있다. 칼레이도스코프 경력태도는 Sullivan과 그의 동료들(2009)이 개발한 측정도구를 통해 측정 할 수 있다. 칼레이도스코프 경력 측정도구는 세 개의 하위요인과 15문항으로 구성되어 있 으며, (1) 나를 전혀 표현하지 못하고 있다 부 터 (5) 나를 매우 잘 표현하고 있다 까지 자기 보고식 5점 LIkert 척도로 측정하게 된다. Sullivan과 그의 동료들(2009)의 연구는 칼레이 도스코프 경력태도의 세대 간 차이를 확인하 기 위한 목적으로 실시되었는데, 칼레이도스 코프 경력태도 측정도구의 구인타당도는 보고 되지 않았다. 다만 각 경력파라미터의 내적 일치도는 진정성 .76, 균형 .81, 도전 .84로 보 고되었다. 또한 경력 파라미터 간 상관관계의 범위는 .48-.56으로 중간 정도의 상관을 보였 다. 이 연구에서는 세대차 외에도 통제변인으 로 성별, 자녀 수, 혼인상태, 교육수준, 소득과 칼레이도스코프 경력의 상관관계도 분석하였 는데 남성에 비해 여성이 진정성(.14)과 균형 (.08)에서 유의한 정적 상관관계를 보였고, 자 녀 수와 진정성(.08), 균형(.27), 도전(.07) 간 상 관관계가 유의하였으며, 기혼일 때 균형(.18)과 의 상관관계가 유의하였다. 또한, 교육수준과 칼레이도스코프경력모형 칼레이도스코프 경력모형은 처음 제안된 당 시 여성의 경력경로 발달의 특징을 깊이 조명 하고자 하였다. 특히 기존의 전통적인 경력 경로가 아닌 새로운 경력 패러다임을 강조하 면서 경력과 관련된 남녀의 차이를 주요하게 다루었다. Mainiero와 Sullivan(2006)은 남성의 경 우 하나 혹은 두 개의 조직에서 일하는 경우 뿐만 아니라 다양한 조직을 거치더라도 상향 이동을 지향하는 전통적인 경력패턴을 갖는 반면, 대다수 여성들의 경력이동은 비선형적 이며 노동시장을 떠났다가 재진입하는 등 이 동을 반복하는 경향을 갖고 있다고 강조하면 서 칼레이도스코프 경력모형을 통해 여성의 경력 의사결정을 설명할 수 있다고 하였다. 칼레이도스코프 경력태도에서 강조하고 있 는 도전, 진정성, 일과 삶의 균형 등은 최근 일터에서의 경험, 리더십, 일-가정 균형 등 구 성원들의 일과 관련된 태도에서 중요한 가치 로 다루어지고 있다. 따라서 칼레이도스코프 경력태도가 높은 근로자일수록 보다 효과적인 경력 관련 태도 및 행동을 보일 가능성이 높 을 것으로 판단된다(Kim & Lee, 2020). 이 연구 에서는 칼레이도스코프 경력태도의 응답값이 경력 관련 긍정적인 태도 및 행동과 유의한 관계를 갖는지 검증하고자 한다. 이를 통해 칼레이도스코프 경력태도의 크기가 갖는 의미 - 53 - 한국심리학회지: 산업및조직 칼레이도스코프 경력모형(KCM)은 세 개의 평면거울이 반사에 의해 무한한 패턴을 만드 는 것과 같이 개인이 경력 관련 의사결정을 내릴 때, 세 개의 주요 경력 파라미터(career parameter)를 회전시키며 다양한 경력 패턴을 만들어간다고 설명한다. 세 개의 파라미터는 진정성(authenticity), 균형(balance), 도전(challenge) 으로 구성되며, 진정성은 진정한 자기 자신이 되는 것으로, 균형은 일(work)과 일 외적인 요 구 사이의 균형으로, 도전은 개인을 자극하는 일과 경력에서의 발전으로 정의된다. 이러한 세 개의 파라미터는 생애주기 동안 모두 활성 화되어 있으나, 특정 시기에 어떤 파라미터가 강하게 나타나는가에 따라 개인의 경력 패턴 이 결정되게 된다. 보다 구체적으로 개인은 경력 파라미터에 기반하여 직무요구, 제한, 기 회와 개인이 얻게 될 가치, 이익, 관계 등을 비교하며 최적의 상태(best fit)를 추구한다는 것이다. 즉 개인의 생애에 걸친 경력 의사결 정과 그 결과로 나타나는 경력패턴을 경력 파 라미터를 중심으로 이해할 수 있다는 데 모형 의 의의가 있다. 환경, 맥락적 상황에 따라 변화한다. 경력태도 는 개인의 경력과 관련된 특정한 행동을 예측 하는 변인으로 다루어져 왔으며, 주요 연구도 경력태도의 결과변인에 초점을 맞추어 이루 어져왔다. 선행연구에서는 주로 전통적 경력 태도(traditional career attitude), 프로티언 경력 태도(protean career attitude), 무경계 경력태도 (boundaryless career attitude) 등 경력과 관련된 특정한 관점을 강조해 왔다. 이 연구에서는 칼레이도스코프 경력모형에서 제시된 칼레이 도스코프 경력태도에 초점을 맞추고 경력태도 를 설명하고자 한다. 칼레이도스코프 경력모형(KCM)은 세 개의 평면거울이 반사에 의해 무한한 패턴을 만드 는 것과 같이 개인이 경력 관련 의사결정을 내릴 때, 세 개의 주요 경력 파라미터(career parameter)를 회전시키며 다양한 경력 패턴을 만들어간다고 설명한다. 칼레이도스코프경력태도의측정 경력태도(career attitude)는 경력에 대한 신념, 지식, 사고 등 인지적 요소와 선호되는 경력 인지 아닌지에 대한 평가적 요소 또는 정서적 요소, 특정 방향으로 행동하려는 행동 의도 및 경향에 해당하는 행동적 요소를 포괄하는 개념이다. 태도는 다양한 환경에 의해 학습되 고, 특정한 행동을 유발하며, 특정한 사물, 사 건에 대해 일관된 선호 또는 비선호를 갖게 하는 특징이 있다(Fishbein & Ajzen, 1975). 또한, 태도는 가변적 속성을 갖고 있어 개인의 경험, - 54 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 진정성(.07), 도전(.10) 간 상관관계가 유의하였 으며, 소득수준은 균형(.07)과 도전(.11) 간 상 관관계가 유의한 것으로 보고되었다. Shaw와 Leberman(2015)은 뉴질랜드의 여성 CEO 7명을 대상으로 한 심층 인터뷰를 통해 경력 파라미터의 하위 주제를 확인하여 칼레이도 스코프 경력모형을 확장하였는데, 구체적으로 진정성에는 열정과 관계구축이, 균형에는 자 기인식과 조직 내 영향이, 도전에는 기회수용 과 남성중심 환경에서의 업무수행이 도출되 었다. 한편, 최근 수행된 Kim과 Lee(2020)의 연구 에서 칼레이도스코프 경력태도를 측정하고 구 인타당도를 확인하였다. 이 연구는 Mainiero와 Gibson(2017)의 16문항으로 구성된 칼레이도스 코프 측정도구를 활용하였는데, 탐색적 요인 분석 과정에서 도전과 균형의 측정 문항이 부 적으로 할당되어 선행연구에서 제시하고 있는 칼레이도스코프 경력태도의 요인 구조를 확인 하는 데 한계가 있었다. 정량적 접근만 활용 했을 때 나타나는 이러한 한계점을 극복하기 위해, 이 연구는 델파이 조사를 실시하여 칼 레이도스코프 측정도구 번안 내용을 검토하고 국내 근로자들의 경력개발 환경에 적용할 수 있는 문항을 새롭게 추가하고자 하였다. 한편 칼레이도스코프 경력태도의 세 가지 경력 파라미터에 대한 태도를 각각 비교하여 성차나 세대차와 같이 집단 간 차이가 있음을 밝히는 데 초점을 둔 연구들도 수행되었다. 먼저 성차와 관련된 연구를 살펴보면, Sullivan 과 Mainiero(2007)는 응답자 중 대부분(84%)의 남성들은 초기-중기-말기의 경력단계에서 도전 -진정성-균형에 각각 순서대로 초점을 두는 알 파 칼레이도스코프 경력 패턴을 갖고 있는 반 면, 대부분의 여성(83%)들은 초기에는 도전, 중기에는 균형, 말기에는 진정성에 초점을 두 는 베타 칼레이도스코프 경력 패턴을 보였다 고 설명하였다. Cabrera(2009)도 25명의 여성을 심층 인터뷰한 질적 연구를 통해 베타 경력 패턴이 여성에게 있어서 가장 많이 나타나 는 패턴임을 밝혔다. 최근 수행된 Mainiero와 Gibson(2017)의 연구에서는 경력중기의 남녀 실업자를 대상으로 칼레이도스코프 경력태도 의 차이를 밝혔는데, 여성은 남성에 비해 균 형을 훨씬 중요하게 생각하고 있었으며, 경력 후기로 갈수록 남녀 모두 진정성을 더 강조 하는 것으로 나타났다. Elley-Brown, Pringle과 Harris(2018)가 교육분야 뉴질랜드 여성 직업인 을 대상으로 한 연구에서는 경력 중기 여성에 게서 알파 경력에 대한 선호가 관찰되었으며, 진정성은 여성에게 여전히 중요한 파라미터임 을밝혔다 칼레이도스코프경력태도관련선행연구 칼레이도스코프 경력태도를 활용하여 차이를 밝힌 연구는 제한적이나 직업가 경력성공에 대한 준거에 대한 연구를 통 레이도스코프 경력태도의 성차를 유추 있다. 예를 들어, Parker와 Chusmir(1992) 력성공의 준거를 성별에 따라 비교하였 여성은 남성에 비해 가족관계, 개인적 직업안정성 등을 중요하게 생각하고 에 대한 중요성은 낮게 평가하였다. D Murphy(2006)의 연구에서는 여성은 남성 해 균형과 관계를 중요한 직업적 성공 여기고 있었다. 국내 연구에서는 Kim과 (2014)가 성별에 따라 경력성공 준거에 인식에 차이가 있음을 밝혔는데, 여성은 칼레이도스코프 경력모형을 적용한 세대차 연구도 수행되었다. Sullivan과 그의 동료들 (2009)은 X세대의 경우 베이비부머 세대에 비 해 진정성과 균형에 대한 높은 요구를 갖고 있는 것으로 나타났으나 도전에 대한 세대 차는 발견되지 않았다고 설명하였다. August (2011)는 경력후기 여성을 대상으로 질적 연구 를 수행한 결과 여성은 상대적으로 젊을 때 도전을 더 강조하는 것으로 나타났으며, 경력 후기로 갈수록 진정성과 균형을 강조하고 있 다고 설명하였다. 프 경력태도의 성별, 세대별 집단 간 차이를 다룬 연구에서도 칼레이도스코프 경력태도의 각 경력 파라미터의 평균 차이를 분석하고 있 다. 이는 각 집단에서의 동일한 측정을 가정 한다. 그러나 실제로 이러한 집단 간에(예. 남 성 vs. 여성) 칼레이도스코프 경력에 대한 측 정동일성이 검증되지 않았다는 점에서 한계가 있다. 칼레이도스코프 경력태도와 성차 칼레이도스코프 경력모형은 남성과 다른 여 성의 경력태도를 제안하기 위한 목적으로 처 음 발표되었으므로 칼레이도스코프 경력태도 의 성차는 선행연구에서 중요하게 다루어져 왔다. 그러나 칼레이도스코프 경력태도의 차 이를 실증적으로 검증한 연구는 제한적으로 수행되었다. 예를 들어 Mainiero와 Gibson(2017) 은 미국의 남녀 경력 중기 실업자(N=744)를 대상으로 칼레이도스코프 경력태도의 성차를 검증하였는데, 경력중기 여성은 남성보다 균 형을 더 강조하는 것을 밝혔다. 칼레이도스코프 경력태도와 관련된 선행연 구를 종합해보면, 칼레이도스코프 경력모형이 제안된 이후 새로운 기업 환경에서의 경력을 설명하기에 칼레이도스코프 경력 모형이 적절 한 지에 대한 연구와 경력과 관련된 태도에서 의 성차, 세대차를 칼레이도스코프 경력모형 으로 설명할 수 있는지에 초점을 맞추고 연구 들이 수행되어 온 것을 알 수 있다. 특히 여 성의 생애사건과 경력단계에 따른 경력 파라 미터의 변화를 분석한 질적 연구들이 많이 수 행되었다. 그러나 다음과 같은 한계점이 관찰 된다. 첫째, 칼레이도스코프 경력태도의 측정 도구가 여러 문화에 걸쳐 타당한지 측정도구 를 타당화 한 연구는 찾아보기 어렵다. 둘째, 칼레이도스코프 경력모형이 현대인의 경력 태 도를 설명하는데 타당한 지 확인하기 위해 특 정 집단을 대상으로 한 질적 연구 방법에 초 점을 두고 있어 다양한 대상을 포괄한 양적 연구의 수행이 요구된다. 셋째, 칼레이도스코 프 경력태도의 성차와 관련하여 여성이 알파 경력패턴을 갖는다는 결과와 베타 경력패턴을 갖는다는 상반된 연구결과가 존재하므로 보다 다양한 맥락적 변인을 종합적으로 고려한 연 구설계가 필요하다. 마지막으로 칼레이도스코 칼레이도스코프 경력태도를 활용하여 성별 차이를 밝힌 연구는 제한적이나 직업가치나 경력성공에 대한 준거에 대한 연구를 통해 칼 레이도스코프 경력태도의 성차를 유추할 수 있다. 칼레이도스코프경력태도관련선행연구 Mainiero와 Sullivan(2005)이 처음으로 칼레이 도스코프 경력모형을 제안한 이후 이를 기반 으로 한 양적, 질적 연구와 이론 연구들이 다 수 수행되어 왔다. 특히 후속연구들은 세 개 의 경력 파라미터가 실제로 존재하는 지에 대 한 근거를 제시하였는데, Sullivan과 그의 동료 들(2009)은 928명을 대상으로 칼레이도스코프 경력태도가 타당하게 구분될 수 있음을 밝혔 으며, Cabrera(2007)는 497명의 전문직 여성을 대상으로 한 연구에서 62%의 응답자가 자신 의 경력에 대한 초점이 시간에 따라 변화되었 다고 기술하였음을 설명하였다. 또한 Baruch와 그의 동료들(2014)은 칼레이도스코프 경력모형 과 계획행동이론에 따라 교원의 연구년에 대 한 의사결정을 설명하는 모델을 제안하였다. - 55 - 한국심리학회지: 산업및조직 칼레이도스코프 경력모형을 적용한 세대차 연구도 수행되었다. Sullivan과 그의 동료들 (2009)은 X세대의 경우 베이비부머 세대에 비 해 진정성과 균형에 대한 높은 요구를 갖고 있는 것으로 나타났으나 도전에 대한 세대 차는 발견되지 않았다고 설명하였다. August (2011)는 경력후기 여성을 대상으로 질적 연구 를 수행한 결과 여성은 상대적으로 젊을 때 도전을 더 강조하는 것으로 나타났으며, 경력 후기로 갈수록 진정성과 균형을 강조하고 있 다고 설명하였다. 칼레이도스코프 경력태도와 관련된 선행연 구를 종합해보면, 칼레이도스코프 경력모형이 제안된 이후 새로운 기업 환경에서의 경력을 설명하기에 칼레이도스코프 경력 모형이 적절 한 지에 대한 연구와 경력과 관련된 태도에서 의 성차, 세대차를 칼레이도스코프 경력모형 으로 설명할 수 있는지에 초점을 맞추고 연구 들이 수행되어 온 것을 알 수 있다. 특히 여 성의 생애사건과 경력단계에 따른 경력 파라 미터의 변화를 분석한 질적 연구들이 많이 수 행되었다. 그러나 다음과 같은 한계점이 관찰 된다. 첫째, 칼레이도스코프 경력태도의 측정 도구가 여러 문화에 걸쳐 타당한지 측정도구 를 타당화 한 연구는 찾아보기 어렵다. 둘째, 칼레이도스코프 경력모형이 현대인의 경력 태 도를 설명하는데 타당한 지 확인하기 위해 특 정 집단을 대상으로 한 질적 연구 방법에 초 점을 두고 있어 다양한 대상을 포괄한 양적 연구의 수행이 요구된다. 셋째, 칼레이도스코 프 경력태도의 성차와 관련하여 여성이 알파 경력패턴을 갖는다는 결과와 베타 경력패턴을 갖는다는 상반된 연구결과가 존재하므로 보다 다양한 맥락적 변인을 종합적으로 고려한 연 구설계가 필요하다. 마지막으로 칼레이도스코 프 경력태도의 성별, 세대별 집단 간 차 다룬 연구에서도 칼레이도스코프 경력태 각 경력 파라미터의 평균 차이를 분석하 다. 이는 각 집단에서의 동일한 측정을 한다. 그러나 실제로 이러한 집단 간에( 성 vs. 여성) 칼레이도스코프 경력에 대 정동일성이 검증되지 않았다는 점에서 한 있다. 칼레이도스코프 경력태도와 성차 칼레이도스코프 경력모형은 남성과 다 성의 경력태도를 제안하기 위한 목적으 음 발표되었으므로 칼레이도스코프 경력 의 성차는 선행연구에서 중요하게 다루 왔다. 그러나 칼레이도스코프 경력태도 이를 실증적으로 검증한 연구는 제한적 수행되었다. 예를 들어 Mainiero와 Gibson 은 미국의 남녀 경력 중기 실업자(N= 대상으로 칼레이도스코프 경력태도의 성 검증하였는데, 경력중기 여성은 남성보 형을 더 강조하는 것을 밝혔다. 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 에 비해 경제적 안정, 일과 삶의 균형, 평생 직장, 행복/즐거움 등을 더 중요하게 인식하 는 것으로 나타났다. 일반적으로 여성의 경 우 출산과 육아 뿐 아니라 생애단계에 따라 주어지는 돌봄 노동에 대한 기대로 균형에 대한 가치를 중요하게 생각할 것으로 가정할 수 있다. 에 비해 경제적 안정, 일과 삶의 균형, 평생 직장, 행복/즐거움 등을 더 중요하게 인식하 는 것으로 나타났다. 일반적으로 여성의 경 우 출산과 육아 뿐 아니라 생애단계에 따라 주어지는 돌봄 노동에 대한 기대로 균형에 대한 가치를 중요하게 생각할 것으로 가정할 수 있다. (true to oneself)’으로 일터에서의 삶, 또는 자기 자신을 진정한 나로 받아들일 수 있는지와 관 련된다(Mainiero & Sullivan, 2005). 이는 개인의 내적 가치와 연관되어 있을 것이라 가정할 수 있으며, 실제로 여성을 대상으로 한 질적 연 구에서 진정성을 유지하기 위해서는 개인의 가치와 조직의 가치를 정렬하는 것이 중요하 다는 것을 발견하였다(Shaw & Leberman, 2015). 따라서 칼레이도스코프 경력태도의 진정성 파 라미터는 프로티언 경력태도와 유의한 상관관 계를 가질 것이라 가정하였다. 가설 1. 여성은 남성에 비해 칼레이도스코 프 경력태도 중 균형에 대한 인식이 유의미하 게 높을 것이다. 가설 2. 칼레이도스코프 경력태도는 프로티 언 경력태도와 유의한 정적 상관관계를 가질 것이다. 칼레이도스코프 경력태도와 프로티언 경력 태도, 일-삶의 균형, 도전적 직무경험 프로티언 경력태도는 경력에 대한 새로운 사고방식으로 ‘개인적인 가치를 토대로 경력 을 선택하는 자기주도적이고 자유의지를 반영 한 경력 관련 태도’를 말한다(Briscoe & Hall, 2006, p.6). 즉 프로티언 경력태도는 환경의 변 화 뿐 아니라 개인이 갖고 있는 가치에 따라 자신의 경력을 주도적으로 개발하고 관리하는 것이다(Hall, 2004). 프로티언 경력태도는 크게 두 가지 하위요인으로 구성되는데, 자기주도 적 경력관리 태도(self-directed career management attitude)와 가치기반 경력태도(value-driven career attitude)로 나눌 수 있다(Briscoe, Hall, & DeMuth, 2006). 자기주도적 경력관리 태도는 스스로 경력관리의 주체가 되어 주도적인 역 할을 수행하는 것을 의미하며, 가치기반 경력 태도는 외적 보상보다는 개인의 고유한 가치 에 기반하여 심리적 성공을 추구하는 정도를 의미한다(Briscoe & Hall, 2006). 프로티언 경력 태도의 두 가지 하위요인은 칼레이도스코프 경력태도의 하위요인 중 진정성 파라미터와 유사하다. 진정성은 ‘나 자신에게 진실한 것 일-삶의 균형(work-life balance: WLB)은 근로 자가 일 영역과 삶의 영역 간 균형을 이루기 위해 개인적 자원을 필요한 역할 영역에 적 절하게 배분하는 것을 의미한다(Lee & Choi, 2019). Kim과 Park(2008)은 개인적 자원에는 시 간, 심리적 에너지, 신체적 에너지 등이 있을 수 있으며, 적절한 분배를 통해 삶에 대한 통 제감을 갖고 삶에 만족한 상태를 의미한다고 설명하였다. 칼레이도스코프 경력태도의 하위 요인 중 균형 파라미터는 개인의 일과 삶에 있어서 균형을 유지하는 것이 얼마나 중요한 지에 대한 개인의 인식을 반영한다(Mainiero & Sullivan, 2005). 칼레이도스코프경력태도관련선행연구 예를 들어, Parker와 Chusmir(1992)는 경 력성공의 준거를 성별에 따라 비교하였는데, 여성은 남성에 비해 가족관계, 개인적 성취, 직업안정성 등을 중요하게 생각하고 지위 에 대한 중요성은 낮게 평가하였다. Dyke와 Murphy(2006)의 연구에서는 여성은 남성에 비 해 균형과 관계를 중요한 직업적 성공으로 여기고 있었다. 국내 연구에서는 Kim과 Cha (2014)가 성별에 따라 경력성공 준거에 대한 인식에 차이가 있음을 밝혔는데, 여성은 남성 - 56 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 실제로 선행연구에서 혼인여부 와 자녀의 수는 칼레이도스코프 경력태도 중 균형과 유의한 상관관계를 갖는 것으로 나타 났으며, 이러한 경력 파라미터를 중요하게 생 각하는 근로자는 실제 일-삶의 균형을 높게 유지할 것이라 가정할 수 있다. - 57 - 한국심리학회지: 산업및조직 한국심리학회지: 산업및조직 가설 3. 칼레이도스코프 경력태도는 일-삶의 균형과 유의한 정적 상관관계를 가질 것이다. 심리적 과정 또는 결과로 설명되기도 한다 (Aryee & Tan, 1992). 경력몰입은 몰입 대상에 대한 심리적 애착이라는 관점에서 직무몰입 및 조직몰입과 유사하지만, 몰입의 대상이 조 직이 아니라 개인의 내재적인 경력 목표라는 관점에서, 그리고 특정 시점이 아닌 장기적인 속성을 갖는다는 점에서 차이가 있다(Cho & Lee, 2017). 개인의 경력몰입은 현재의 경력을 지속적으로 유지하고자 하는 동기를 나타내므 로 많은 연구에서 경력 적응의 지표로 활용되 어 왔다. 도전적 직무경험은 현재 일상적으로 사용 하고 있는 업무수행 전략이나 방식이 불충분 하여 과업을 수행하는 새로운 방법이 요구되 는 업무활동을 의미한다(Lee & Cho, 2019). 도 전적 직무경험은 개인으로 하여금 새로운 학 습의 기회나 동기를 제공해주는 직무 특성을 포함하고 있고(McCauley, Ruderman, Ohlott, & Morrow, 1994), 이를 통해 개인은 지식, 기술, 능력, 통찰력, 가치 등을 개발하여 성장할 수 있게 된다. 칼레이도스코프의 도전 파라미터 는 개인이 지속적으로 학습할 수 있는 긍정적 이고 도전적인 환경을 구축하는 것과 관련된 다(Shaw & Leberman, 2015). 도전은 개인이 업 무로부터 오는 책임을 두려워하지 않고, 새로 운 일에서 성장의 기회를 발견하는 것을 의미 한다(Sullivan et al., 2009). 따라서 칼레이도스코 프 경력태도 중 도전 파라미터는 도전적 직무 경험과 높은 상관관계를 가질 것이라 가정할 수 있다. 주관적 경력성공은 개인의 경력과 관련된 성취나 만족감이라고 정의되며, 현재 직무를 포함한 개인의 모든 경력에서의 성공에 대 한 주관적 평가로 정의된다(Seibert, Crant, & Kraimer, 1999). 주관적 경력성공을 연구한 많 은 선행연구들은 경력만족을 주관적 경력성공 의 지표로 측정해 왔으며(Gattiker & Larwood, 1986; Judge, Cable, Boudreau, & Bretz, 1995), 최 근에는 경력에 대한 주관적 만족감 외에 개인 의 지속적인 고용가능성에 대한 인식을 주관 적 경력성공의 하위요인으로 포함해 왔다(Lee & Lee, 2016). 개인이 자신의 경력에 대해 긍정 적으로 평가하고 인식하는 것과 관련된 주관 적 경력성공은 개인의 경력 결과에 대한 주관 적인 판단으로 많은 선행연구에서 경력개발 노력에 대한 결과변인으로 다루어져 왔다(Kim & Moon, 2018). 가설 4. 칼레이도스코프 경력태도는 도전적 직무경험과 유의한 정적 상관관계를 가질 것 이다. 칼레이도스코프 경력태도와 경력몰입, 주관 적 경력성공 경력몰입은 자신의 직업에 대한 일반적 태 도 중 하나로 개인이 선택한 직업에서 지속적 으로 일하고자 하는 동기이다(Blau, 1985). 연구방법 중 즐거움과 균형을 중요하게 생각하는 유형 과 도전과 기여를 중요하게 생각하는 유형이 있었는데, 두 가지 유형은 다른 유형(지위와 도전 중심 유형, 보상과 안정 중심 유형)에 비 해 경력만족이 더 높은 것으로 나타났다. 즐 거움, 균형, 도전과 같은 경력성공의 준거가 칼레이도스코프 경력태도의 진정성, 균형, 도 전과 유사한 개념이라고 가정하면, 칼레이도 스코프 경력태도의 각 하위요인의 값이 높은 것이 보다 적응적인 것을 의미하는 것으로 해 석할 수 있을 것이다. 따라서 이 연구에서는 칼레이도스코프 경력태도의 하위요인의 수준 이 높은 것은 주관적 경력성공 그리고 경력몰 입과 유의미한 정적 상관관계를 가질 것이라 가정하였다. 첫째, 번역된 도구의 심리측정적 등가성 (psychometric equivalence)을 확보하기 위해 번역 -역번역법(back-translation method)을 실시하였다 (Brislin, 1973). 번역-역번역법은 번역문의 정확 도를 교차검증하기 위해 목표어로 번역된 도 구를 다른 사람에 의해 원어(source language)로 다시 번역하여 원래의 도구와 역번역된 도구 를 비교하는 것이다. 이에 연구자는 측정도구 원문을 개발한 주저자 Sullivan의 동의를 얻어, Sullivan과 그의 동료들(2009)이 제시한 칼레이 도스코프 경력태도 세 개 요인 15문항을 번역 -역번역 하였다. 최초 번역은 연구자 2인이 합의를 통해 실시하였다. 역번역 과정에서 미 국에서 10년 이상 거주하고 HRD 석사 학위 를 취득하여 한국어와 영어 모두 능숙한 이중 언어 구사자(bilingual) 1명에게 연구 도구에 대 한 배경설명 없이 도구를 역번역 하도록 하였 다. 대표적으로 진정성 파라미터의 5문항 중 하나로 ‘지금 당장 내 꿈을 따라갈 수 있다면, 나는 그렇게 할 것이다.’는 ‘If I can follow my dream right now, I will’로 번역되었고, 균형 파라 미터 5문항 중 하나인 ‘일과 가정의 균형을 이루 는 것은 내 인생의 신성한 목표다.’는 ‘Achieving work-life balance is the sacred goal of my life.’ 로, 도전 파라미터 5문항 중 하나인 ‘나는 좌절을 극복해야 할 문제가 아니라 해결책 이 필요한 도전으로 본다.’는 ‘I see setbacks as “challenges” that require solutions rather than “problems” to be overcome.’으로 번역되었다. 둘째구인화및예비문항에대해내용타당 가설 5. 칼레이도스코프 경력태도는 경력몰 입과 유의한 정적 상관관계를 가질 것이다. 가설 6. 칼레이도스코프 경력태도는 주관적 경력성공과 유의한 정적 상관관계를 가질 것 이다. 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 경 력몰입은 자신의 직무에 대한 개인의 동일시 나 현재의 경력목표를 추구해 나가는 과정에 서 어려움을 감수할 수 있는 의지로써 정서적, 칼레이도스코프 경력태도와 경력몰입, 주관 적 경력성공 한편, Lee(2018)는 X세대와 Y세대의 경력성 공 준거에 대한 인식을 비교하면서 즐거움, 균형, 도전과 같은 내부 지향의 주관적인 준 거들을 포함시켰으며 Y세대의 경력성공 인식 프로파일을 분석하고 개인의 경력성공 중요도 및 성취도 인식 수준을 비교한 바 있다. 이 연구에서 Y세대의 경력성공 인식의 잠재유형 - 58 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 중 즐거움과 균형을 중요하게 생각하는 유형 과 도전과 기여를 중요하게 생각하는 유형이 있었는데, 두 가지 유형은 다른 유형(지위와 도전 중심 유형, 보상과 안정 중심 유형)에 비 해 경력만족이 더 높은 것으로 나타났다. 즐 거움, 균형, 도전과 같은 경력성공의 준거가 칼레이도스코프 경력태도의 진정성, 균형, 도 전과 유사한 개념이라고 가정하면, 칼레이도 스코프 경력태도의 각 하위요인의 값이 높은 것이 보다 적응적인 것을 의미하는 것으로 해 석할 수 있을 것이다. 따라서 이 연구에서는 칼레이도스코프 경력태도의 하위요인의 수준 이 높은 것은 주관적 경력성공 그리고 경력몰 입과 유의미한 정적 상관관계를 가질 것이라 가정하였다. 연구1. 내용타당도분석 선행연구 검토를 통해 한국형 칼레이도스코 프 경력태도를 측정하는 데 있어 Sullivan과 그 의 동료들(2009)의 도구가 가장 근접한 조사도 구라는 연구자 간 합의를 도출하고, 해당 도 구를 번역-역번역법을 통해 국문화한 뒤, 수 정델파이법을 활용하여 전문가들의 의견을 취 합함으로써 측정도구 초안을 개발하였다. 둘째, 구인화 및 예비문항에 대해 내용타당 도를 확인하기 위해 수정델파이법을 활용하였 다. 일반적으로 개발 완료된 측정도구는 하위 요인별 3~15 문항 정도로 구성할 것을 권고 - 59 - 한국심리학회지: 산업및조직 문으로 각 문항에 대한 수정 의견을 기재하 도록 하였다. 2라운드는 1라운드에서 수정된 문항과 타 전문가의 코멘트, 본인의 이전 응 답 등을 종합적으로 고려하여 타당성 정도를 Likert 5점 척도에 응하게 하고, 개방형 질문으 로 각 문항에 대한 최종 의견을 기재하도록 하였다. 문으로 각 문항에 대한 수정 의견을 기재하 도록 하였다. 2라운드는 1라운드에서 수정된 문항과 타 전문가의 코멘트, 본인의 이전 응 답 등을 종합적으로 고려하여 타당성 정도를 Likert 5점 척도에 응하게 하고, 개방형 질문으 로 각 문항에 대한 최종 의견을 기재하도록 하였다. 하고 있으므로(Walsh & Betz, 1995), 델파이 연 구 결과에 따른 문항 삭제 등 도구 수정을 고 려하여 파라미터별로 7개씩 총 21개로 구성된 칼레이도스코프 경력태도 측정도구 예비문항 을 선정하였다. 앞서 실시한 역번역 결과로 얻은 3개 요인 15문항 외에, 선행연구 분석을 토대로 연구자 2인이 상호 합의하여 요인별 2 개 문항을 추가하였다. 진정성(Authenticity) 파 라미터에 포함한 문항은 ‘나는 내 삶과 내 일 을 통해 진정한 나를 찾을 것이다.’와 ‘내가 나아갈 길에 대한 가치와 신념은 외부의 압박 에도 흔들리지 않는다.’ 등 2문항 이고, 균형 (Balance) 파라미터에는 ‘나에게는 가족이 가장 큰 우선순위이며, 그 다음이 일이다.’, ‘나는 가족으로부터 얻는 행복과 일에서 얻는 성취 를 동시에 추구한다.’ 등 2문항을 포함하였으 며, 도전(Challenge) 파라미터에는 ‘나에게 있어 새로운 변화란 나를 성장시킬 수 있는 기회이 다.’, ‘나는 도전적인 과제를 수행할 때 살아있 음을 느낀다.’ 등 2문항을 새로 개발하여 포함 하였다. * CVR: Content Validity Ratio 결 과 수정델파이 1라운드 결과, 진정성 1번 문항 은 번역이 어색하다는 의견 및 문장 수정 의 견에 따라 ‘나는 나에게 적합한 더 큰 삶의 의미를 찾길 원한다’로 수정하였다. 진정성 6 번 문항 ‘나는 내 삶과 내 일을 통해 진정한 나를 찾을 것이다’는 삶과 일이 개념적으로 ‘과’로 묶이는 동일한 자격이 아니라고 사료된 다는 전문가 의견과 강한 어조를 사용한 경우 점수분포가 상대적으로 낮아질 수 있어 주의 를 요한다는 전문가 의견을 반영하여 ‘나는 내 일을 통해 내 삶 속에서 진정한 나를 찾길 바란다’로 수정하였다. 진정성 7번 문항 ‘내가 나아갈 길에 대한 가치와 신념은 외부의 압박 에도 흔들리지 않는다’는 ‘나아갈 길’ 및 ‘외 부 압박’이라는 개념이 모호하고 범위를 특정 할 수 없다는 지적에 근거하여 ‘내 진로에 대 한 가치와 신념은 어떠한 환경이나 조건에도 흔들리지 않는다’로 수정하였다. 한편, 전문가 가 제안한 ‘나에게는 마음, 정신, 신체 건강이 통합된 만족감이 중요하다’ 는 문항을 새롭게 추가하였다. 수정델파이 전문가 패널의 선정기준은 다음 과 같다. 먼저, 박사학위를 소지하고 있고 칼 레이도스코프 경력태도와 유관한 경력개발 또 는 진로발달 관련 변인 도구개발 경력이 있거 나 HRD 현업에 10년 이상 종사한 전문가 5인 을 선정하였으며, 측정 대상이 되는 근로자 중 박사학위를 소지하고 진정성, 균형, 도전을 경력발달 과정에서 경험했다고 응답한 경력 10년 이상의 여성과학기술인 5인을 포함하여 총 10인을 전문가 패널로 선정하였다. 수정델 파이는 총 2라운드로 구성하였다. 1라운드에 서는 전문가 패널로 하여금 파라미터에 각 문 항이 위치해 있는 것이 적절한지 타당성 정도 를 Likert 5점 척도에 응하게 하고, 개방형 질 균형 2번 문항 ‘나는 항상 가족의 요구에 우선하여 나의 일을 배치한다’는 가족의 요구 에 우선하기보다는 가족 요구와 나의 일을 지 - 60 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 * CVR: Content Validity Ratio 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 포함하였으나, 2라운드 결과 매우 낮은 내용 타당도비율(CVR)값을 보여 최종 삭제하였다. 이에 따라, 2라운드에 걸친 델파이 분석 결과 최종 진정성 파라미터 6문항, 균형 파라미터 6 문항, 도전 파라미터 6문항이 개발되었다. 도전 2번 문항에서 ‘좌절’은 전문가 의견을 바탕으로 ‘어려움’ 으로 순화하였다. 도전 3번 문항 ‘추가된 업무 책임은 나에게 걱정거리가 못 된다’ 는 번역투가 어색하다는 다수 전문 가의 의견에 따라 ‘나는 기존보다 업무가 추 가되어도 크게 걱정하지 않는다’로 수정하였 다. 도전 4번 문항 ‘내 주위 사람들은 나를 매 우 목표 지향적인 사람이라고 설명한다.’는 경 력태도 상의 도전이 지나치게 목표 지향성과 유사한 현상으로 이해될 수 있어 주의가 요구 된다는 전문가 의견에 따라 ‘내 주위 사람들 은 나를 목표 지향적인 사람이라고 얘기한다’ 로 수정하였다, 도전 7번 문항 ‘나는 도전적인 과제를 수행할 때 살아있음을 느낀다’는 도전 적 직무 경험과 같은 외부 변수와 중복이 우 려된다는 전문가 의견에 따라 삭제하였다. 연구방법 조사대상 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 균형을 이루는 것은 내 인생의 신성한 목표 다’ 는 ‘신성한’이라는 단어의 국내 맥락에서 의 쓰임 및 정서를 감안하여야 한다는 의견을 반영하여 ‘일과 가족의 균형을 이루는 것은 속적으로 균형 있게 조정한다는 뜻이라고 판 단한 전문가 2인의 의견을 반영하여 ‘나는 가 족의 요구를 고려하여 나의 커리어를 계획한 다’ 로 수정하였다. 균형 4번 문항 ‘일과 삶의 items First Round Second Round Mean S.D. Median CVR Decision Mean S.D. Median CVR Decision Authenticity 1 4.10 0.88 4 0.40 Modify 4.40 0.70 5 0.80 Accept 2 4.80 0.42 5 1.00 Maintain 4.70 0.48 5 1.00 Accept 3 4.60 0.70 5 0.80 Maintain 4.80 0.42 5 1.00 Accept 4 4.20 0.79 4 0.60 Modify 4.50 0.71 5 0.80 Accept 5 4.70 0.48 5 1.00 Maintain 4.70 0.48 5 1.00 Accept 6 3.90 0.99 4 0.40 Modify 4.40 0.84 5 0.60 Modify/ Accept 7 3.70 1.16 4 0.20 Modify 4.00 0.94 4 0.60 Delete + - - - - Generate 3.10 1.10 3 -0.40 Delete Balance 1 4.20 0.63 4 0.80 Maintain 4.50 0.53 5 1.00 Accept 2 3.60 1.43 4 -0.20 Modify 4.40 0.70 5 0.80 Accept 3 4.10 1.20 4 0.80 Maintain 4.30 0.67 4 0.80 Accept 4 4.50 1.08 5 0.60 Modify 4.60 0.70 5 0.80 Accept 5 3.50 1.45 4 0.40 Modify 4.30 0.82 5 0.60 Accept 6 3.10 1.20 3 -0.20 Delete - - - - - 7 4.00 0.82 4 0.40 Modify 4.60 0.52 5 1.00 Accept Challenge 1 4.60 0.97 5 0.80 Maintain 4.90 0.32 5 1.00 Accept 2 3.90 1.12 5 0.40 Modify 4.70 0.48 5 1.00 Accept 3 4.10 0.99 4 0.60 Modify 4.20 0.63 4 0.80 Accept 4 3.90 1.10 4 0.20 Modify 4.60 0.70 5 0.80 Accept 5 4.50 0.71 5 0.80 Maintain 4.40 0.70 5 0.80 Accept 6 4.40 0.84 5 0.60 Modify 4.90 0.32 5 1.00 Accept 7 3.80 1.03 4 0.20 Delete - - - - - * l d Table 1 Summary of the result from modified delphi analysis Summary of the result from modified delphi analysis - 61 - 한국심리학회지: 산업및조직 포함하였으나, 2라운드 결과 매우 낮은 내용 타당도비율(CVR)값을 보여 최종 삭제하였다. 이에 따라, 2라운드에 걸친 델파이 분석 결과 최종 진정성 파라미터 6문항, 균형 파라미터 6 문항, 도전 파라미터 6문항이 개발되었다. 내 인생의 중요한 목표다’로 수정하였다. 균형 6번 문항은 CVR값이 낮고 균형 1번 문항과 사실상 같은 의미를 내포한다는 전문가 의견 에 따라 삭제하였다. 결 과 칼레이도스코프 경력태도 칼레이도스코프 경력태도는 연구1을 통해 내용타당도를 확보한 18개 문항(진정성, 균형, 도전 각 6개 문항)으로 구성되었다. Sullivan과 그의 동료들(2009)이 개발한 원척도는 5점 Likert로 응답하도록 하였으나 5점 Likert에 비 해 6점 Likert 응답을 활용하는 것이 신뢰도와 변별도를 유의미하게 향상시킨다는 Chomeya (2010)의 연구결과에 따라 6점 Likert 척도(1= 전혀 동의하지 않는다. 2=별로 동의하지 않는 다. 3=동의하지 않는 편이다. 4=동의하는 편 이다. 5=동의한다. 6=매우 동의한다)를 활용 하여 측정하였다. 탐색적요인분석 요인분석에 필요한 기본가정을 표본이 충족 시키는 지 확인하기 위해 KMO(Kaiser-Meyer- Olkin) 검정과 Bartlett이 제안한 구형성 검정을 실시하였다. KMO는 .921로 1에 근접하므로 적합하다고 보았고, Bartlett 검정값은 근사 카 이제곱 3209.433, p<.001 로 나타나 적절하므 로 요인분석의 선행조건을 만족한다고 판단하 였다. 탐색적 요인분석을 위해 고유값은 1보 다 큰 조건으로, 베리맥스 회전을 통해 최대 우도법으로 요인을 추출한 결과 3개의 요인 이 도출되었다(표 2 참조). 스크리도표를 함께 참고한 결과 3요인 모형이 가장 적합한 것으 로 나타났다. 도전 파라미터의 내적 일관성 (cronbach α)은 .882, 균형은 .869, 진정성은 .832 였으며, 전체적으로는 .911 로 나타났다. 탐색 적 요인분석 결과 원 도구와 마찬가지로 3개 의 요인 구조가 도출되었으나, 균형 1번과 진 정성 4번 등 일부 문항의 공통성이 .4 미만으 로 나타났으며, 진정성 3번, 5번, 6번은 도전 요인에도 요인부하량이 .4 이상으로 나타나 확인적 요인분석을 통해 요인구조를 재확인하 고자 하였다. 조사대상 연구 자료 수집을 위해 온라인 조사업체에 의뢰하여 국내의 다양한 기업에 근무하고 있 는 근로자를 대상으로 온라인 설문조사를 실 시하였다. 조사 대상을 선정함에 있어 성별, 연령을 고려하여 비율표집 하였고, 기타 고 려할 수 있는 인구통계학적 요인은 무선화 (randomize)함을 원칙으로 하였다. 전체 2,857명 을 대상으로 설문지가 발송되어 476명이 응답 을 시도하였으며(응답률 16.7%), 재직 근로자 가 아니거나 중간에 설문응답을 포기한 89명 을 제외한 396명의 응답자가 설문응답을 완료 하였다. 396명 중 응답이 불성실한 43명의 데 이터를 제외하고 최종적으로 347명의 응답자 료를 분석하였다. 347명의 인구통계학적 특성 은 다음과 같다. 성별은 남성이 193명(55.6%), 여성이 154명(44.4%)이고, 연령은 20~24세 24 명(6.9%), 25~29세 44명(12.7%), 30~34세 45명 (13.0%), 35~39세 48명(13.8%), 40~44세 43명 (12.4%), 45~49세 47명(13.5%), 50~54세 37명 (10.7%), 55~59세 32명(9.2%), 60세 이상 27명 (7.8%)이었다. 직급은 사원급 103명(29.7%), 대 수정델파이 2라운드 결과, 진정성 6번 문항 ‘나는 내 일을 통해 내 삶 속에서 진정한 나 를 찾길 바란다’는 ‘찾는다’는 동사를 ‘발견한 다’ 는 행위로 변경하면 내적인 자각, 성찰을 의미할 것이라는 의견을 반영하여 최종 ‘나는 일을 통해 내 삶 속에서 진정한 나를 발견하 기를 원한다’로 수정 선택하였다. 진정성 7번 문항 ‘내 진로에 대한 가치와 신념은 어떠한 환경이나 조건에도 흔들리지 않는다’는 칼레 이도스코프 경력태도의 경우 진로에 대한 가 치와 신념이 변한다는 전제에 기반한 개념이 므로, 진정성 문항이라 하더라도 ‘불변’의 논 리를 사용하는 것은 무리라는 전문가 의견을 반영하여 최종 삭제하였다. 진정성 8번 문항 은 1라운드에서 전문가 의견에 따라 문항으로 - 62 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 인하였으며, 탐색적 요인분석을 통해 확인된 요인구조를 바탕으로 요인 및 문항의 구조적 관계를 검증하기 위해 확인적 요인분석을 실 시하였다. 적합도 지수는 TLI, CFI, RMSEA, SRMR을 활용하였다. 리급 74명(21.3%), 과장급 62명(17.9%), 차장급 32명(9.2%), 부장급 43명(12.4%), 임원 이상 33 명(9.5%) 이었다. 학력은 박사 이상 4명(1.2%), 석사 24명(6.9%), 대졸 231명(66.6%), 전문대졸 46명(13.3%), 고졸이하 42명(12.1%) 이었다. 조사도구 분석방법 칼레이도스코프 경력태도의 이론적 구성개 념이 심리측정적으로 일치하는지 확인하기 위 해 통계 프로그램 IBM SPSS AMOS 25.0을 이 용하여 기술통계, 신뢰도 분석, 요인분석을 실 시하였다. 먼저 연구 1을 통해 확인한 18개 문항의 칼레이도스코프 경력태도 측정도구의 요인구조를 확인하기 위해 탐색적 요인분석을 실시하였다. 탐색적 요인분석은 최대우도법, 직교회전으로 추출하였으며 아이겐값을 기준 으로 요인의 수를 검토하였다. 요인부하량과 공통성을 검토하여 삭제할 문항이 있는지 확 - 63 - 한국심리학회지: 산업및조직 한국심리학회지: 산업및조직 Table 2 The result of exploratary factor analysis (N=347) Items Factors communality 1 2 3 Challenge 4 Most people would describe me as being very goal-directed. .763 .157 .167 .635 Challenge 5 I thrive on work challenges and turn work problems into opportunities for change. .714 .266 .246 .642 Challenge 2 I view setbacks not as “problems” to be overcome but as “challenges” that require solutions. .687 .201 .314 .611 Challenge 6 For me, a new change is an opportunity to grow. .661 .227 .389 .640 Challenge 3 Added work responsibilities don’t worry me. .644 .074 .097 .429 Challenge 1 I continually look for new challenges in everything I do. .610 .192 .342 .525 Balance 5 Nothing matters more to me right now than balancing work with my family responsibilities. .096 .796 .176 .674 Balance 3 My work is meaningless if I cannot take the time to be with my family. .102 .776 .111 .625 Balance 4 Achieving balance between work and family is life’s holy grail. .067 .742 .322 .659 Balance 6 I pursue the happiness of my family and the achievement of my work at the same time. .213 .682 .308 .605 Balance 1 If necessary, I would give up my work to settle problematic family issues or concerns. .191 .589 .030 .384 Balance 2 I constantly arrange my work around my family needs. .352 .571 -.011 .450 Authenticity 2 I hunger for greater spiritual growth in my life. .205 .165 .750 .632 Authenticity 1 I hope to find a greater purpose to my life that suits who I am. .115 .148 .709 .538 Authenticity 4 If I could follow my dream right now, I would. .280 .118 .531 .374 Authenticity 5 I want to have an impact and leave my signature on what I accomplish in life. .421 .186 .514 .476 Authenticity 6 I want to find genuine me in my life through work. 분석방법 .458 .086 .513 .480 Authenticity 3 I have discovered that crises in life offer perspectives in ways that daily living does not. .436 .185 .476 .451 Eigen Value 3.728 3.293 2.808 % of Variance Explained: 54.607 20.711 18.296 15.600 Cronbach α: .911 .882 .869 .832 Table 2 The result of exploratary factor analysis (N=347) - 64 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 연구방법 조사대상 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 삭제하였다. 문항 삭제 후 확인적 요인분석을 다시 실시한 결과 χ2 =161.788(df=62, p<.001), CFI=.957, TLI=.945, RMSEA=.068 [하한값: .055, 상한값: .081], SRMR=.049로 모형의 적합 도가 전반적으로 개선된 것으로 나타났다(표 3 참조). 삭제하였다. 문항 삭제 후 확인적 요인분석을 다시 실시한 결과 χ2 =161.788(df=62, p<.001), CFI=.957, TLI=.945, RMSEA=.068 [하한값: .055, 상한값: .081], SRMR=.049로 모형의 적합 도가 전반적으로 개선된 것으로 나타났다(표 3 참조). 확인적요인분석 확인적요인분석 탐색적 요인분석 결과를 통해 확인된 진정 성, 균형, 도전 파라미터를 잠재변인으로 설 정하고, 측정 문항을 측정변인으로 하여 잠 재변인과 측정변인 간 경로를 연결하고 확인 적 요인분석을 실시하였다. 그 결과, 모형적 합도 측면에서 χ2=415.826(p<.001), CFI=.880, TLI=.911, RMSEA=.072[하한값: .064, 상한값: .080], SRMR=.056로 나타나 χ2값과 CFI가 적 합도 기준에 부합하지 않는 것으로 나타났 다. 이에 문항의 요인부하량과 다중상관자승 (Squared Mutilple Correlations: SMC) 값을 검토하 였다. 모든 문항의 요인부하량은 .5 이상으로 적합하였으나, 진정성1(나는 나에게 적합한 더 큰 삶의 의미를 찾기를 원한다), 진정성4(지금 당장 내 꿈을 따라갈 수 있다면, 나는 그렇게 할 것이다), 균형1(필요하다면, 나는 가족과 관 련된 문제를 해결하기 위해 일을 포기할 것이 다), 균형2(나는 가족의 요구를 고려하여 나의 커리어를 계획한다), 도전3(나는 기존에 수행 하던 업무에 추가적인 과업이 주어져도 크게 걱정하지 않는다)의 SMC 값이 .4 미만으로 나 타났다. 이에 문항을 세부적으로 검토하여 연 구자 간 합의를 통해 5개 문항을 삭제하였다. 진정성1번 문항은 진정성 2번 문항과 오차 상 관이 높게 나타나 두 문항 간 측정내용이 유 사하여 이를 삭제하는 것이 적절하다고 판단 하였으며, 진정성 4번, 균형 1번, 균형 2번은 유연성이 낮은 우리나라 노동시장의 맥락적 특성을 고려할 때 국내 근로자의 경력 태도를 측정하기에 적합하지 않은 문항으로 판단하였 다. 마지막으로 도전 3번 문항은 추가적인 과 업에 대한 스트레스 감내성으로 도전에 대한 다른 측정문항들과 성격이 다르다고 판단하여 연구3: 교차타당도, 잠재평균차이, 준거관련타당도분석 조사대상 연구3을 위한 표본은 별도의 자료수집을 통 해 확보되었다. 연구2와 마찬가지로 2020년 7 월 기준 전체 근로자의 연령분포 비율을 확인 하여 연령별 비율 표집을 실시하였다. 온라인 전문조사업체에 의뢰하여 전체 4,456명을 대상 으로 설문지가 발송되었고 846명이 응답을 시 도하였으며(응답률 19.0%), 재직 근로자가 아 니거나 중간에 설문응답을 포기한 212명을 제 외한 634명의 응답자가 설문응답을 완료하였 다. 634명 중 응답이 불성실한 3명의 데이터 를 제외하고 최종적으로 631명의 응답자료를 분석하였다. 631명의 인구통계학적 특성은 다음과 같다. 성별은 남성이 347명(55.0%), 여 성이 284(45.0%)명이고, 연령은 20~24세 29명 (4.6%), 25~29세 75명(11.9%), 30~34세 82명 (13.0%), 35~39세 88명(13.9%), 40~44세 82명 (13.0%), 45~49세 83명(13.2%), 50~54세 73명 (11.6%), 55~59세 61명(9.7%), 60세 이상 58명 (9.2%) 이었다. 직급은 사원급 163명(25.8%), 대 - 65 - 한국심리학회지: 산업및조직 통해 번안된 바 있다. 예시문항은 ‘나는 일(직 장)과 삶 두 영역에 적절히 시간을 배분하고 사용하고 있다’이다. Valcour(2007)의 연구에서 신뢰도(cronbach’s α)는 .93으로 보고되었고, Lee(2012)의 연구에서는 .836으로 나타났다. 이 연구에서 측정한 일-삶의 균형 5문항의 신뢰 도(cronbach’s α)는 .876이었다. 리급 135명(21.4%), 과장급 135명(21.4%), 차장 급 65명(10.3%), 부장급 84명(13.3%), 임원 이 상 49명(7.8%) 이었다. 학력은 박사 이상 14명 (2.2%), 석사 65명(10.3%), 대졸 401명(63.5%), 전문대졸 84명(13.3%), 고졸이하 67명(10.6%) 였다. 혼인 여부는 미혼이 234명(37.1%), 기혼 이 385명(61.0%) 이며, 12명(1.9%)은 기타(별거, 이혼, 사별)에 응답하였다. 리급 135명(21.4%), 과장급 135명(21.4%), 차장 급 65명(10.3%), 부장급 84명(13.3%), 임원 이 상 49명(7.8%) 이었다. 학력은 박사 이상 14명 (2.2%), 석사 65명(10.3%), 대졸 401명(63.5%), 전문대졸 84명(13.3%), 고졸이하 67명(10.6%) 였다. 혼인 여부는 미혼이 234명(37.1%), 기혼 이 385명(61.0%) 이며, 12명(1.9%)은 기타(별거, 이혼, 사별)에 응답하였다. 도전적 직무경험은 De Pater와 그의 동료들 (2009)이 개발한 7문항의 척도를 사용하였다. De Pater와 그의 동료들(2009)의 연구에서는 도 전적 직무경험이 하위요인이 없는 단일요인 구성개념으로 제시되었고 신뢰도는 .74로 양 호하였다. 이 연구에서는 De Pater와 그의 동 료들(2009)이 개발한 척도를 Lee와 Lee(2016)가 번역한 도전적 직무경험 7문항 척도를 활용하 였다. 예시문항은 ‘나는 우리 부서에서 전략적 변화를 시도하는 일을 담당한다’이며 신뢰도 (cronbach’s α)는 .912로 양호하였다. χ2=161.788(df=62, p<.001), CFI=.957, TLI=.945, RMSEA=.068 [하한값: .055, 상한값: .081], SRMR=.049 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 Path B β S.E. t AVE CR 진정성 (Authenticity) → 나는 내 인생에서 더 큰 정신적 성장을 갈망한다. (I hunger for greater spiritual growth in my life.) 1 .657 .515 .809 → 나는 인생의 위기를 통해 새로운 관점을 얻을 수 있다는 것을 알고 있다. (I have discovered that crises in life offer perspectives in ways that daily living does not.) 1.190 .686 .112 10.664*** → 나는 스스로 성취한 것을 통해 영향력을 갖고 내 발자취를 남기고 싶다. (I want to have an impact and leave my signature on what I accomplish in life.) 1.222 .712 .111 10.971*** → 나는 일을 통해 내 삶 속에서 진정한 나를 발견하기를 원한다. (I want to find genuine me in my life through work.) 1.213 .717 .110 11.032*** 균형 (Balance) → 가족과 함께 하는 시간을 낼 수 없다면, 내 일은 무의미하다. (My work is meaningless if I cannot take the time to be with my family.) 1 .745 .606 .860 → 일과 가족의 균형을 이루는 것은 내인생의 중요한 목표다. (Achieving balance between work and family is life’s holy grail.) .959 .826 .065 14.783*** → 지금 당장 나의 일과 가족의 균형을 맞추는 것보다 더 중요한 것은 없다. (Nothing matters more to me right now than balancing work with my family responsibilities.) .951 .820 .065 14.682*** → 나는 가족으로부터 얻는 행복과 일에서 얻는 성취를 동시에 추구한다. (I pursue the happiness of my family and the achievement of my work at the same time.) .850 .771 .061 13.835*** 도전 (Challenge) → 나는 내가 하는 모든 일에서 끊임없이 새로운 도전을 추구한다. (I continually look for new challenges in everything I do.) 1 .720 .624 .892 → 나는 어려움을 극복해야 할 “문제”가 아니라 해결책이 필요한 “도전”으 로 본다. (I view setbacks not as “problems” to be overcome but as “challenges” that require solutions.) 1.058 .790 .076 14.001*** → 내 주위 사람들은 나를 목표지향적인 사람이라고 얘기한다. (Most people would describe me as being very goal-directed.) 1.120 .744 .085 13.205*** → 나는 업무상 어려움을 잘 극복하고, 발생된 문제는 개선의 기회로 활용 한다. (I thrive on work challenges and turn work problems into opportunities for change.) 1.002 .800 .071 14.179*** → 나에게 있어 새로운 변화란 나를 성장시킬 수 있는 기회이다. 조사도구 칼레이도스코프 경력태도는 연구 2를 통해 도출된 13문항을 활용하여 측정하였다. 진정 성 4문항, 균형 4문항, 도전 5문항으로 구성되 며, 문항은 표 3에 기술된 것과 같다. 프로티언 경력태도는 개인의 가치나 관심에 따라 자신의 경력을 자기주도적으로 개발하 고 관리하는 경력을 의미하며(Briscoe & Hall, 2006), 가치지향성과 자기주도성의 2가지 하위 요인으로 구성된다(Briscoe, Hall, & Demuth, 2006). 이 연구에서는 Briscoe, Hall과 DeMuth (2006)의 측정도구를 번안한 Shin과 Jeong(2014) 의 도구를 활용하였다. 예시문항은 ‘내 경력의 성공과 실패에 대한 책임은 나에게 있다.’(자 기주도성), ‘나는 경력을 평가할 때, 임금이나 승진보다는 나의 고유한 가치를 더 추구한 다.’(가치지향성)이다. 이 연구에서 하위요인 의 신뢰도(cronbach’s α)는 자기주도성 .853, 가치지향성 .838였으며, 전체 문항의 신뢰도 (cronbach’s α)는 .890이었다. 주관적 경력성공은 경력만족과 고용가능성 을 각각 측정하여 합산한 값을 활용하였다. Greenhaus와 그의 동료들(1990)이 개발한 도구 를 바탕으로 Shim(2011)이 번안한 도구를 사용 하였다. 경력만족의 측정도구는 경력 전반에 대한 만족도와 목표달성, 기술과 능력, 보수, 직급 측면에서의 만족도를 묻는 5문항으로 구 성되었으며, Shim(2011)의 연구에서 신뢰도 (cronbach’s α)는 .88 이었다. 고용가능성은 Johnson(2001)이 개발한 도구를 바탕으로 Chang 과 Park(2004)이 번안한 도구를 사용하였다. 고 용가능성의 측정도구는 6문항의 5점 LIkert로 측정하도록 되어있으며 Chang과 Park(2004)의 연구에서 신뢰도는 .90였다. 이 연구에서는 경 력만족과 고용가능성의 신뢰도(cronbach’s α)는 각각 .919, .881이었으며, 전체 주관적 경력성 공의 신뢰도(cronbach’s α)는 .917였다. 일-삶의 균형을 측정하기 위해 Valcour(2007) 가 개발한 일-가정 균형에 대한 만족도를 측 정하는 5문항을 활용하였다. 이 측정도구는 일과 가정 균형에 대한 전반적 만족도를 측정 하고 있으며, 국내에서는 Lee(2012)의 연구를 - 66 - 교차타당도분석결과 교차타당도분석결과 교차타당도를 확인하기 위해 연구 2의 표본 1(N=347)을 추정표본으로 하고 연구 3의 표본 2(N=631)를 검증표본으로 하여 모형 적합도를 비교하였다. 교차타당화집단의 모형 적합도를 살펴보면 모수추정집단과 비교했을 때 상대적 으로 양호하지 않은 것을 알 수 있으나 CFI, TLI가 .943, .928로 양호한 수준으로 나타났으 며, RMSEA도 .072로 양호한 범위로 판단할 수 있다. 또한 교차타당성 지수(ECVI: Expected Cross-Validation Index)를 확인한 결과, 모수추정 집단은 .635, 교차타당화집단은 .514로 교차타 당화집단 모형이 더 안정적인 것으로 나타났 다. 즉, 표본이 달라졌음에도 불구하고 칼레이 도스코프의 3요인 측정모형은 타당한 것으로 확인되었다. 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 (For me, a new change is an opportunity to grow.) 1.012 .818 .070 14.488*** χ2=161.788(df=62, p<.001), CFI=.957, TLI=.945, RMSEA=.068 [하한값: .055, 상한값: .081], SRMR=.049 Table 3 The result of confirmative factor analysis (N=347) Table 3 The result of confirmative factor analysis (N=347) → (For me, a new change is an opportunity to grow.) - 67 - 한국심리학회지: 산업및조직 경력몰입은 Blau(1985)가 개발하고 Chang (1997)이 번안한 측정도구를 활용하였다. Chang (1997)가 번안한 척도 중 ‘나는 내가 하고 있 는 직업분야에서 내 경력을 유지하기를 원한 다’ 등 4문항을 활용하였다. Chang(1997)의 연 구에서 보고한 신뢰도(cronbach’s α)는 .76이었 으며, 이 연구의 신뢰도(cronbach’s α)는 .824였 다. 모든 측정변인은 6점 Likert 척도(1=전혀 동의하지 않는다. 2=별로 동의하지 않는다. 3=동의하지 않는 편이다. 4=동의하는 편이다. 5=동의한다. 6=매우 동의한다)를 활용하여 측 정하였다. 분석을 실시하여 준거 변인들과의 관계를 확 인하였다. 연구결과 연구결과 교차타당도분석결과 분석방법 이 연구에서 설정한 구인 모형의 타당도를 다시 한 번 확인하기 위해 표본 2를 대상으로 확인적 요인분석을 실시하였다. 확인적 요인 분석 모형의 적합도와 더불어 기대교차타당성 지수(Expected Cross-Validation Index; ECVI)를 확 인하였다. 또한 다집단 분석을 실시하여 남녀 기업근로자의 측정동일성을 검증하였고, 측정 오차를 통제한 상태에서 칼레이도스코프 경력 태도 평균의 성별 차이를 확인하기 위해 잠재 평균분석을 실시하였다. 마지막으로 한국판 칼레이도스코프와 관련 변인들과의 상관관계 연구 2에서 사용하지 않았던 새로운 표본 2(N=631)을 추정표본으로 하여 확인적 요인 분석을 실시하였다. 연구 2의 결과로 제안된 Table 4 Model χ2 df χ2/df CFI TLI RMSEA ECVI Parameter Estimation Group (N=347) 161.788 62 2.609*** .957 .954 .068 (.055-.081) .635 (.537-.755) Cross-Validity Group (N=631) 266.097 62 4.292*** .943 .928 .072 (.063-.081) .514 (.440-.601) ***p <.001 Table 4 Model comparison for cross-validity analysis Model comparison for cross-validity analysis - 68 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 - 13문항 3요인 모형을 연구모형으로 설정하고 측정모형을 검증하였다. 확인적 요인분석의 적합도 지수는 χ2=266.097(df=62, p<.001), CFI=.943, TLI=.928, RMSEA=.072 [하한값: .063, 상한값: .081], SRMR=.045로 모형의 적 합도는 양호한 것으로 나타났다. 또한 각 요 인별 표준화계수가 모두 .6 이상으로 통계적 으로 유의한 적재량을 보이고 있었으며, 다 중상관자승(SMC) 값도 .4 이상으로 나타났다. 한편, 모든 잠재 변인의 개념 신뢰도(construct reliability)가 .8 이상이었으며, 평균분산추출 (Average Variance Extracted)이 .5 이상으로 나타 나 내적 일관성 및 수렴 타당성을 확보하였다. 칼레이도스코프측정도구의성별에따른잠재 평균차이분석 칼레이도스코프 측정도구의 3요인 모형이 남녀 재직근로자 모두에게 적용할 수 있는 모 형인지 확인하기 위해 다집단분석을 실시하였 다. 먼저 칼레이도스코프 측정도구의 3요인 Model χ2(df) Δχ2(Δdf) Model A 353.941(124) - Model B 372.422(134) 18.481* (10) Model C 393.238(147) 20.816 (13) Model D 414.703(153) 21.466** (6) Note: Model A: unconstrained model; Model B: measurement weights and intercepts; Model D: constrain Table 5 The result of multi-group analysis between 13문항 3요인 모형을 연구모형으로 설정하고 측정모형을 검증하였다. 확인적 요인분석의 적합도 지수는 χ2=266.097(df=62, p<.001), CFI=.943, TLI=.928, RMSEA=.072 [하한값: .063, 상한값: .081], SRMR=.045로 모형의 적 합도는 양호한 것으로 나타났다. 또한 각 요 인별 표준화계수가 모두 .6 이상으로 통계적 으로 유의한 적재량을 보이고 있었으며, 다 중상관자승(SMC) 값도 .4 이상으로 나타났다. 한편, 모든 잠재 변인의 개념 신뢰도(construct reliability)가 .8 이상이었으며, 평균분산추출 (Average Variance Extracted)이 .5 이상으로 나타 나 내적 일관성 및 수렴 타당성을 확보하였다. 모형이 남녀 재직근로자 모두에게 적합한 모 형인지 확인하기 위해 집단 간 형태동일성을 검증하였다. 그 결과 남녀 집단 모두에서 3 요인 모형이 적합한 것으로 나타났다(χ2= 353.941(df=124, p<.001), CFI=.936, TLI=.920, RMSEA=.054 [하한값: .048, 상한값: .061]). 형 태동일성 모형을 기저모형으로 하고 잠재변인 과 측정변인 간 경로를 제약한 측정동일성 모 형을 비교하였다. 분석방법 측정요인 계수의 동일화 제 약을 가한 측정동일성 모형과 기저모형의 χ2 차이검증을 실시한 결과 기저모형(모형 A)과 측정동일성 모형(모형 B)간의 χ2값의 차이는 통계적으로 유의한 것으로 나타났으나(Δχ2(Δ df)=18.481(10), p=.047), 모형A에 비해 적합도 가 근소한 차이로 감소하였으므로 측정동일성 모형을 수용하였다. 다음으로 집단 간 측정오 차의 잔차가 동일하다고 제약한 모형 C와 측 정동일성 모형을 비교하였다. 두 모형 간 χ2 값의 차이는 통계적으로 유의하지 않았다 (Δχ2(Δdf)=20.816(13), p=.077). 따라서 모형 C 를 수용하였다. 마지막으로 집단 간 요인계 칼레이도스코프측정도구의성별에따른잠재 평균차이분석 칼레이도스코프측정도구의성별에따른잠재 평균차이분석 칼레이도스코프측정도구의성별에따른잠재 평균차이분석 칼레이도스코프 측정도구의 3요인 모형이 남녀 재직근로자 모두에게 적용할 수 있는 모 형인지 확인하기 위해 다집단분석을 실시하였 다. 먼저 칼레이도스코프 측정도구의 3요인 Model χ2(df) Δχ2(Δdf) χ2/df CFI TLI RMSEA Model A 353.941(124) - 2.854*** .936 .920 .054 (.048-.061) Model B 372.422(134) 18.481* (10) 2.779*** .934 .923 .053 (.047-.060) Model C 393.238(147) 20.816 (13) 2.675*** .932 .928 .052 (.045-.058) Model D 414.703(153) 21.466** (6) 2.710*** .927 .926 .052 (.046-.058) Note: Model A: unconstrained model; Model B: constrained measurement weights; Model C: constrained measurement weights and intercepts; Model D: constrained measurement weight, intercepts, and covariance The result of multi-group analysis between gender The result of multi-group analysis between gender - 69 - 한국심리학회지: 산업및조직 집단 이상에서 요인의 평균을 동시에 추정할 수 없으므로 남성 근로자를 참조집단으로 하 여 하위 요인의 평균을 0으로 고정한 뒤 여성 근로자의 하위 요인 평균을 추정하였다. 표 6 의 남녀 근로자 집단 간 잠재평균 차이분석을 살펴보면 남성에 비해 여성의 잠재평균이 상 대적으로 낮은 것을 확인할 수 있으며, 진정 성 요인에서의 차이는 유의하지 않았으나, 균 형과 도전에서의 잠재평균차이는 유의한 것으 로 나타났다. Cohen’s d값을 살펴보면 균형의 경우 d값이 -.320, 도전의 경우 -.255로 둘 다 .20을 넘는 것으로 나타나 중간 수준의 효과 크기를 갖는 것(Cohen, 1988)으로 확인되었다. 즉, 남성 재직근로자가 칼레이도스코프 경력 태도 중 균형과 도전을 더 높게 인식하고 있 는 것을 확인할 수 있었다. 따라서 여성이 남 성에 비해 균형에 대한 인식이 더 높을 것이 라는 가설 1은 기각되었다. 수 뿐 아니라 각 잠재변인의 공분산도 동일 하다고 제약한 모형을 모형 C와 비교하였다. χ2차이검증을 실시한 결과 두 모형 간 χ2값 은 통계적으로 유의한 것으로 나타났다(Δχ2 (Δdf)=21.466(6), p=.002). 공분산까지 동일화 제약을 가한 모형 D는 모형 C보다 적합도가 다소 떨어지는 것을 알 수 있으나, χ2 차이검 증의 경우 사례 수에 민감하여 적합한 모형을 기각할 우려가 있으며, CFI가 .01보다 적게 감 소하거나 RMSEA가 적게 증가한 경우에는 적 합도에 차이가 없는 것으로 해석할 수 있다는 주장(Chen, 2007; Cheung & Rensvold, 2002)도 제기된 바 있다. 모형 D가 모형 C와 χ2차이 검증에서 유의한 차이를 보였으나, 모형 D의 적합도가 나쁘다고 해석하기에는 CFI, TLI 지 수가 .9 이상으로 양호하고, CFI의 감소폭이 0.1보다 적고, TLI와 RMSEA는 오히려 증가한 것을 고려할 때 적합도 차이가 없는 것으로 해석할 수도 있다. 따라서 성별에 따라 형태 동일성, 측정동일성, 측정오차의 잔차동일성, 공분산동일성이 모두 인정되므로 집단 간 측 정의 교차타당성이 확보되었다고 볼 수 있다. 집단 간 측정의 동일성 가정이 충족되어 두 집단 간 칼레이도스코프 하위요인의 잠재평균 비교분석을 실시하였다. 잠재평균 비교 시 두 칼레이도스코프 측정도구의 준거관련타당도 분석 A=Authenticity, B=Balance, C=Challenge ***p<.001 A=Authenticity, B=Balance, C=Challenge 칼레이도스코프 측정도구의 준거관련타당도 분석 칼레이도스코프의 구인타당도와 교차타당도 를 확인한 뒤 칼레이도스코프 경력태도의 총 점 및 하위요인과 관련변인 간 상관관계 분석 Table 6 Parameter Male Female t Effect size (Cohen’s d) Authenticity .00 -.06 -1.030 -.015 Balance .00 -.16 -2.734** -.320 Challenge .00 -.16 -2.386* -.255 *p <.05, **p <.01 Table 6 The result of latent mean analysis between gender The result of latent mean analysis between gender - 70 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 을 통해 측정도구의 수렴타당도와 준거관련 타당도를 확인하였다. 먼저, 칼레이도스코프의 총점과 하위요인 간 상관관계 분석을 통해 수 렴타당도를 확인한 결과 칼레이도스코프 경력 태도 총점은 진정성과 .839, 균형과 .785, 도전 과 .866으로 높은 상관을 갖는 것으로 나타났 다. 또한 세 하위요인 간 상관관계를 살펴보 면 .444-.674의 범위를 갖고 있는 것으로 나타 나 칼레이도스코프 경력태도라는 구성개념을 측정하기 위한 내적 타당도를 갖추었다고 판 단할 수 있다. 과 유의미한 관계에 있을 것이라고 가정한 프 로티언 경력태도, 일-삶의 균형, 도전적 직무 경험, 주관적 경력성공, 경력몰입과의 상관분 석을 통해 준거관련 타당도를 확인하였다. 먼 저 자기주도성과 가치지향성의 하위요인으로 구성된 프로티언 경력태도와 칼레이도스코프 경력태도 총점과의 상관관계는 .708로 높은 수준으로 나타났으며, 특히 칼레이도스코프의 하위요인 중에서도 진정성은 자기주도성과 높 은 상관(r=.673, p<.001)을 갖는 것으로 확인 되었다. 칼레이도스코프 경력태도의 총점은 일-삶의 균형(r=.562, p<.001), 도전적 직무경 그 다음으로 칼레이도스코프의 각 하위요인 Table 7 Correlation between kaleidoscope career attitude and related variables (N=631) Variables M SD Korean vesion of Kaleidoscope Career Attitude (KKCA) Result of correlation difference analysis Total KKCA Authenticity Balance Challenge Total KKCA 4.31 0.63 1 Authenticity 4.44 0.71 .839*** 1 Balance 4.35 0.79 .785*** .444*** 1 Challenge 4.15 0.77 .866*** .674*** .482*** 1 Protean Career Attitude 4.22 0.60 .708*** .631*** .446*** .691*** B < A, C Self-directed 4.37 0.62 .732*** .673*** .475*** .683*** B < A, C Value-driven 4.07 0.72 .544*** .467*** .331*** .559*** B < A < C Work-life Balance 4.20 0.76 .562*** .433*** .455*** .507*** A = B = C Challenging job behavior 3.02 0.83 .481*** .353*** .298*** .546*** A = B < C Subjective Career Success 3.69 0.81 .476*** .352*** .290*** .541*** A = B < C Career satisfaction 3.61 0.98 .366*** .236*** .231*** .441*** A = B < C Employability 3.78 0.84 .494*** .407*** .293*** .533*** B < A < C Career commitment 3.62 1.00 .446*** .318*** .280*** .510*** A = B < C Table 7 Correlation between kaleidoscope career attitude and related variables (N=631) Correlation between kaleidoscope career attitude and related variables (N=631) - 71 - 한국심리학회지: 산업및조직 하였다. 칼레이도스코프 측정도구의 준거관련타당도 분석 델파이조사를 통해 수정된 문항을 설 문조사 도구로 개발, 347명의 기업근로자를 대상으로 측정하고 탐색적 및 확인적 요인분 석을 실시하여 구성개념 타당도를 검증하였다. 마지막으로 새롭게 수집된 631명의 기업 근로 자 표본을 대상으로 칼레이도스코프 경력태도 와 관련 변인을 함께 측정하여 준거관련 타당 도를 확인하였다. 연구 결과를 통해 얻은 결 론 및 시사점은 다음과 같다. 험(r=.481, p<.001), 주관적 경력성공(r=.476, p<.001), 경력몰입(r=.446, p<.001)과도 유의미 한 상관관계를 갖는 것으로 나타났다. 칼레이 도스코프 하위요인 중 도전은 도전적 직무경 험과의 상관관계가 다른 하위요인에 비해 가 장 큰 것으로 나타났으며(r=.546, p<.001), 균 형은 일-삶의 균형과 r=.455 (p.<.001)로 유의 한 상관관계를 가졌다. 따라서 가설 2, 3, 4, 5, 6은 모두 지지되었다. 한편, Fisher의 Z 변환점수를 통해 칼레이도 스코프 경력 파라미터와 관련 변인 간 상관관 계의 차이가 있는 지 분석하였다. 유의수준은 .05에서 양측검정을 통해 통계적 유의도를 판 단하였다. 일과 삶의 균형과 칼레이도스코프 경력 하위 파라미터 간 상관관계는 유의한 차 이를 보이지 않았으나, 프로티언 경력태도와 의 상관관계에서는 균형(.446)에 비해 진정성 (.631, z=-4.667, p<.000)과 도전(.691, z=-6.559, p<.000)이 유의하게 높은 것으로 나타났으며, 일 삶의 균형, 도전적 직무행동, 주관적 경력 성공, 경력몰입에서는 진정성, 균형에 비해 도 전과의 상관관계가 유의하게 높은 것을 확인 할 수 있었다. 이는 도전 파라미터에 응답값 이 높은 사람들의 경우 자신의 경력 결과를 더 긍정적으로 평가하고 경력에 보다 몰입하 고 있음을 시사한다. 첫째, 문화적 배경에 따라 잠재변인을 측정 하는 구체적인 행동요소들이 달라질 수 있는 데, 이 연구는 델파이, 두 차례의 표집을 통해 반복 검증하여 타당도를 확보한 도구 개발 절 차를 거친 바, 칼레이도스코프의 한국판 측정 도구를 타당화했다는 데 의의가 있다. 최근 Kim과 Lee(2020)는 중소기업 초기 경력자를 대 상으로 칼레이도스코프 경력태도를 측정하여 탐색적 요인분석을 실시하고, 균형과 도전에 서 각각 한 문항씩 삭제된 도구를 활용했는데, 제거된 문항에는 이 연구에서 제거한 균형 1 번 (필요하다면, 나는 가족과 관련된 문제를 해결하기 위해 일을 포기할 것이다) 문항이 포함되어 있었다. 우리나라와 같이 노동시장 이 경직된 사회적 맥락에서는 퇴직 후 유사한 조건의 일을 구하는 것이 상대적으로 어려우 므로 해당 문항은 우리나라 문화에 적용하기 에 부적절한 것으로 판단된다. 이 연구는 정 성적 접근과 정량적 접근을 모두 활용한 통합 적 접근법(mixed method)을 바탕으로 수정델파 이법과 두 개의 표본을 통한 반복 검증을 통 해 원도구의 측정 문항 중 우리나라 기업근로 자에게 적용하기 어려운 문항을 가려냄과 동 시에 우리나라 기업근로자의 칼레이도스코프 경력태도를 측정하는 추가문항(진정성-나는 일 을 통해 내 삶 속에서 진정한 나를 발견하기 논 의 이 연구의 목적은 칼레이도스코프 경력태도 의 한국판 측정도구를 개발하는 데 있다. 칼레이도스코프 측정도구의 준거관련타당도 분석 이 를 위해 문헌고찰, 번역-역번역 방법을 통해 마련한 칼레이도스코프 경력태도 측정도구 초 안을 수정델파이법을 통해 내용타당도를 확인 - 72 - 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 를 원한다; 균형-나는 가족으로부터 얻는 행복 과 일에서 얻는 성취를 동시에 추구한다; 도 전-나에게 있어 새로운 변화란 나를 성장시킬 수 있는 기회이다)을 개발하고 타당도를 검증 했다는 점에서 의의가 있다. 이 연구를 통해 최종적으로 마련된 14문항은 후속연구에서 지속적으로 연구결과를 축적할 필요가 있다. 둘째, 한국판 칼레이도스코프 경력태도를 측정하는 3요인의 14문항은 서로 다른 두 표 본에서도 구인타당도가 확인되었으며, 남녀 성별 집단에서도 동일한 측정도구를 활용할 수 있는 것으로 확인되었다. 칼레이도스코프 경력태도를 구성하는 진정성, 균형, 도전의 3 요인 모형은 서로 다른 표본에서도 안정적으 로 도출되었다. 한편, 칼레이도스코프 경력태 도는 개인이 생애 단계에서 어떤 경력 파라미 터를 중요하게 생각하는지를 핵심으로 하고 있으며, 세 가지 파라미터의 절대 점수보다는 3요인 점수의 프로파일이 중요한 정보를 제공 한다. 선행연구도 이를 고려하여 알파 패턴, 베타 패턴 등으로 명명하면서 칼레이도스코프 경력 파라미터를 중심으로 판단해 왔다. 예를 들어, Sullivan과 Mainiero(2007)는 응답자 중 대 부분(84%)의 남성들은 초기-중기-말기의 경력 단계에서 도전-진정성-균형에 각각 초점을 주 는 알파 칼레이도스코프 경력 패턴을 갖고 있 는 반면, 대부분의 여성(83%)들은 초기에는 도 전, 중기에는 균형, 말기에는 진정성에 초점을 두는 베타 칼레이도스코프 경력 패턴을 보였 다고 보고하였다. 최근 수행된 Mainiero와 Gibson(2017)의 연구에서는 경력중기의 남녀 실업자를 대상으로 칼레이도스코프 경력태도 의 차이를 밝혔는데, 여성이 남성에 비해 균 형을 훨씬 중요하게 생각하고 있었으며, 경력 후기로 갈수록 남녀 모두 진정성을 더 강조하 는 것으로 나타났다. 이 연구에서는 진정성과 도전의 상관관계(r=.674)에 비해 진정성과 균 형(r=.444), 도전과 균형(r=.482)의 상관관계는 상대적으로 낮게 나타나 칼레이도스코프의 경 력 파라미터가 배타적인 양상으로 존재할 가 능성을 시사한다. 따라서 칼레이도스코프 경 력태도를 연구하고 해석하는 데 있어 칼레이 도스코프 경력태도의 프로파일을 분석하고 이 를 토대로 한 연구를 진행할 필요가 있다. 셋째, 이 연구는 칼레이도스코프 경력태도 중 균형 파라미터에서 여성이 남성에 비해 더 높은 점수를 가질 것이라 가정하였으나, 연구 결과 칼레이도스코프의 잠재평균은 남성이 여 성보다 높은 것을 밝혔다. 특히, 균형 및 도전 에서 잠재평균 차이가 유의미했으며, 균형에 서의 차이는 효과크기가 가장 큰 것으로 나타 났다. 이는 여성보다 남성들이 칼레이도스코 프의 전반적인 응답값이 상대적으로 높음을 의미하며, 여성들이 남성보다 균형을 더 강조 한다는 선행연구(Mainiero & Gibson, 2017)와는 상반된 결과이다. Mainiero와 Gibson(2017)의 선 행연구에서 칼레이도스코프 경력태도의 성별 차이를 밝히는 데 있어 경력단계를 함께 고려 했기 때문에 이러한 차이가 나타난 것으로 판 단된다. 단순히 남녀 간 성별의 차이가 아니 라 경력단계 또는 생애발달 단계에서 주어지 는 다양한 과업과 역할 변화에 따라 성차가 존재할 수 있다는 것이다. 한국 사회에서 일 반적으로 여성에 비해 남성의 평균적인 지위, 급여수준이 높고 고용형태가 보다 안정적이라 는 것은 잘 알려진 사실이다. 칼레이도스코프 측정도구의 준거관련타당도 분석 칼레이도스코프 경력태도의 차이가 이러한 사회맥락적 특성 및 보편적 인식에 따른 것인지, 또는 남성이 경력과 관련하여 변화된 가치를 여성보다 용 이하게 받아들인 결과인지 확인하는 연구가 는 것으로 나타났다. 이 연구에서는 진정성과 도전의 상관관계(r=.674)에 비해 진정성과 균 형(r=.444), 도전과 균형(r=.482)의 상관관계는 상대적으로 낮게 나타나 칼레이도스코프의 경 력 파라미터가 배타적인 양상으로 존재할 가 능성을 시사한다. 따라서 칼레이도스코프 경 력태도를 연구하고 해석하는 데 있어 칼레이 도스코프 경력태도의 프로파일을 분석하고 이 를 토대로 한 연구를 진행할 필요가 있다. 를 원한다; 균형-나는 가족으로부터 얻는 행복 과 일에서 얻는 성취를 동시에 추구한다; 도 전-나에게 있어 새로운 변화란 나를 성장시킬 수 있는 기회이다)을 개발하고 타당도를 검증 했다는 점에서 의의가 있다. 이 연구를 통해 최종적으로 마련된 14문항은 후속연구에서 지속적으로 연구결과를 축적할 필요가 있다. 둘째, 한국판 칼레이도스코프 경력태도를 측정하는 3요인의 14문항은 서로 다른 두 표 본에서도 구인타당도가 확인되었으며, 남녀 성별 집단에서도 동일한 측정도구를 활용할 수 있는 것으로 확인되었다. 칼레이도스코프 경력태도를 구성하는 진정성, 균형, 도전의 3 요인 모형은 서로 다른 표본에서도 안정적으 로 도출되었다. 한편, 칼레이도스코프 경력태 도는 개인이 생애 단계에서 어떤 경력 파라미 터를 중요하게 생각하는지를 핵심으로 하고 있으며, 세 가지 파라미터의 절대 점수보다는 3요인 점수의 프로파일이 중요한 정보를 제공 한다. 선행연구도 이를 고려하여 알파 패턴, 베타 패턴 등으로 명명하면서 칼레이도스코프 경력 파라미터를 중심으로 판단해 왔다. 예를 들어, Sullivan과 Mainiero(2007)는 응답자 중 대 부분(84%)의 남성들은 초기-중기-말기의 경력 단계에서 도전-진정성-균형에 각각 초점을 주 는 알파 칼레이도스코프 경력 패턴을 갖고 있 는 반면, 대부분의 여성(83%)들은 초기에는 도 전, 중기에는 균형, 말기에는 진정성에 초점을 두는 베타 칼레이도스코프 경력 패턴을 보였 다고 보고하였다. 최근 수행된 Mainiero와 Gibson(2017)의 연구에서는 경력중기의 남녀 실업자를 대상으로 칼레이도스코프 경력태도 의 차이를 밝혔는데, 여성이 남성에 비해 균 형을 훨씬 중요하게 생각하고 있었으며, 경력 후기로 갈수록 남녀 모두 진정성을 더 강조하 셋째, 이 연구는 칼레이도스코프 경력태도 중 균형 파라미터에서 여성이 남성에 비해 더 높은 점수를 가질 것이라 가정하였으나, 연구 결과 칼레이도스코프의 잠재평균은 남성이 여 성보다 높은 것을 밝혔다. 특히, 균형 및 도전 에서 잠재평균 차이가 유의미했으며, 균형에 서의 차이는 효과크기가 가장 큰 것으로 나타 났다. 이는 여성보다 남성들이 칼레이도스코 프의 전반적인 응답값이 상대적으로 높음을 의미하며, 여성들이 남성보다 균형을 더 강조 한다는 선행연구(Mainiero & Gibson, 2017)와는 상반된 결과이다. Mainiero와 Gibson(2017)의 선 행연구에서 칼레이도스코프 경력태도의 성별 차이를 밝히는 데 있어 경력단계를 함께 고려 했기 때문에 이러한 차이가 나타난 것으로 판 단된다. 단순히 남녀 간 성별의 차이가 아니 라 경력단계 또는 생애발달 단계에서 주어지 는 다양한 과업과 역할 변화에 따라 성차가 존재할 수 있다는 것이다. 한국 사회에서 일 반적으로 여성에 비해 남성의 평균적인 지위, 급여수준이 높고 고용형태가 보다 안정적이라 는 것은 잘 알려진 사실이다. 칼레이도스코프 측정도구의 준거관련타당도 분석 칼레이도스코프 경력태도의 차이가 이러한 사회맥락적 특성 및 보편적 인식에 따른 것인지, 또는 남성이 경력과 관련하여 변화된 가치를 여성보다 용 이하게 받아들인 결과인지 확인하는 연구가 - 73 - 한국심리학회지: 산업및조직 여성 CEO, 실업자, 교사, 은퇴기 여성 등을 대 상으로 연구가 수행된 바 있다. 개인의 주도 성이 강조되는 경력 패러다임 시대의 새로운 경력태도로 지칭되는 칼레이도스코프 경력태 도가 전체 직업군에 적용될 수 있는 개념인지 다양한 표본을 통한 반복 검증 연구를 실시하 여 확인할 필요가 있다. 이러한 연구들은 칼 레이도스코프 경력태도의 개념 뿐 아니라 경 력과 관련된 변인 간 관계와 주요 메커니즘에 대한 이해도 확장시킬 수 있을 것이다. 또한, 기업, 공공기관 등 조직의 인사부서에서 경력 태도 척도를 채택하여 활용할 경우, 보직임면 장면에서 경력태도 진단 및 피드백을 통해 긍 정적인 직원경험(employee experience)을 부여할 수 있을 것이다. 특히 세 가지 경력 파라미터 와 관련하여 경력개발 개입방안을 체계적으로 구조화하여 제공할 필요가 있다. 둘째, 칼레이 도스코프 경력태도의 세대차에 대한 연구도 수행될 필요가 있다. 이 연구에서는 표집의 한계로 인해 세대차이는 다루지 않았으나, Sullivan과 그의 동료들(2009)은 X세대가 베이 비부머 세대에 비해 진정성과 균형에서 유의 하게 높은 것으로 나타났음을 보고하였다. 성 별 뿐 아니라 세대 간 집단에서 진정성, 균형, 도전과 같은 중요한 가치의 차이가 어떠한 지 확인할 필요가 있을 것이다. 마지막으로 경력 단계를 고려한 칼레이도스코프 경력태도에 대 한 연구가 이루어져야 할 것이다. 경력은 조 직 관점에서는 인적자원 차원에서 적시적소에 한정된 역량을 배치 및 활용하기 위한 매개물 로 받아들여지나, 개인 관점에서는 시간의 흐 름에 따른 역량의 발달뿐만 아니라 가족 돌봄 을 포함한 개인의 삶을 영위하는 태도 전반에 관계된 목적물이다. 일의 세계는 계속해서 변 화하고 있으며, 이에 따라 최근에 조직이 학 필요하다. 필요하다. 넷째, 이 연구는 칼레이도스코프 경력태도 의 합산 값을 어떻게 활용할 수 있을지에 대 한 자료를 제공하고 있다는 점에서 의의가 있 다. 선행연구는 주로 칼레이도스코프 경력태 도의 경력파라미터에 따른 패턴을 분석하는데 초점을 두고 있어 전체 칼레이도스코프 경력 태도 값을 어떻게 해석할 수 있을지에 대한 근거를 제시하는 데 한계가 있었다. 이 연구 에서는 칼레이도스코프 경력태도와 관련 변인 간 상관관계를 바탕으로 칼레이도스코프 경력 태도가 보다 적응적인 경력태도와 관련되어 있을 것이라는 가설을 확인하였다. 칼레이도 스코프 경력태도는 프로티언 경력태도, 일-삶 의 균형, 도전적 직무경험과 유의미한 상관관 계를 갖는 것으로 나타났는데, 그 중에서도 전반적으로 도전 파라미터가 준거 변인들과의 상관이 높게 나타났다. 이는 도전이라는 경력 파라미터를 중요하게 생각하는 기업근로자가 자신의 경력과 관련하여 보다 긍정적인 태도 를 갖는 것으로 해석할 수 있다. 최근 경력개 발에서의 개인의 주도성을 강조하는 새로운 경력태도는 고용환경 변화의 가속화와 무관하 지 않으며, 주어진 상황에서 새로운 도전을 추구하고 주도성을 발휘해 나가는 것이 개인 의 경력개발에서도 중요한 역할을 하고 있음 을 시사한다. 이재은․정보영/ 한국판칼레이도스코프경력태도측정도구타당화연구 습하고, 다수의 개인이 채택하는 경력태도는 다양하다. 칼레이도스코프는 세 가지 경력파 라미터의 현재 수준보다는 이러한 경력 파라 미터가 어떻게 달라지는 지에 초점을 두고 있 는 개념이므로 향후 종단 연구나 다양한 경력 단계에서의 칼레이도스코프 경력태도 인식 차 이 연구 등이 이루어져야 할 것이다. 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A study on the work-life balance through multileel analysis: The effect of generational work value and supporting institutions & climate. Unpublished doctorial dissertation, Hanyang University. Lee, Y. G., Park, S. Y., & Kim, J. G. (2019). Meta-analysis of domestic and international researches on protean career attitudes and outcome variables. Journal of Agricultural Education and Human Resource Development, 51(3), 21-41. Lee, J. (2018). Identifying latent profiles based on career success perception among Korean employees: A comparison of generations X and Y. The Korean Journal of Human Resource Development Quarterly, 20(3), 175-203. doi:10.18211/kjhrdq.2018.20.3.007 doi:10.23840/agehrd.2019.51.3.21 Mainiero, L. A., & Gibson, D. E. (2017). The Kaleidoscope Career Model revisited: How midcareer men and women diverge on authenticity, balance, and challenge. Journal of Career Development, 45(4), 361-377. doi:10.1177/0894845317698223 Lee, J. M., & Choi, H. (2019). Influence of organizational culture supporting work-life Balance on well-being and depression mediated by work-life Balance: An application of multi-group analysis across gender and age. Korean Journal of Industrial and Organizational Psychology, 32(1), 1-27 doi:10.24230/kjiop.v32i1.1-27 Mainiero, L. A., & Sullivan, S. E. (2005). Kaleidoscope careers: An alternate explanation for the “opt-out” revolution. Academy of Management Perspectives, 19(1), 106-123. doi:10.5465/ame.2005.15841962 투고일자 : 2020. 10. 19 수정일자 : 2020. 12. 01 확정일자 : 2020. 12. 15 doi:10.24230/kjiop.v32i1.1-27 Lee, J., & Cho, Y. (2019). The Systematic Review and Developing Integrated Model on the Challenging Job Experience. The Korean Journal of Human Resource Development Quarterly, 21(3), 133-163. doi: 10.18211/kjhrdq.2019.21.3.006 Mainiero, L. A., & Sullivan, S. E. (2006). The opt out revolt: Whe people are leaving companies to create kaleidoscope careers. Davies-Black Publishing. doi:10.5860/choice.44-4552 McCauley, C. D., Ruderman, M. N., Ohlott, P. J., & Morrow, J. E. (1994). Assessing the developmental components of managerial jobs. Journal of Applied Psychology, 79(4), 544-560. doi:10.1037/0021-9010.79.4.544 Lee, J., & Lee, C. (2016). A development of integrated evaluation criteria model for measurement of career success of Korean office worker. The Korean Journal of Human Resource Development Quarterly, 18(2), 1-36. Parker, B., & Chusmir, L. H. (1992). A comparison of men and women managers’ and Nonmanagers’ perceptions of success. Human Resource Development Quarterly, 3(1), 73-84. Lee, J., & Lee, C. (2016). The relationship of core self-evaluation and subjective career success of white-collar workers: Mediating effect of challenging job experience and moderated Lee, J., & Lee, C. (2016). The relationship of core self-evaluation and subjective career success of white-collar workers: Mediating effect of challenging job experience and moderated - 77 - 한국심리학회지: 산업및조직 한국심리학회지: 산업및조직 Savickas, M. L. (2012). Life Design: A Paradigm for Career Intervention in the 21st Century. Journal of Counseling & Development, 90(1), 13-19. doi:10.1111/j.1556-6676.2012.00002.x Sullivan, S. E., & Mainiero, L. A. (2007). The changing nature of gender roles, alpha/beta careers and work‐life issues. Career Development International, 12(3), 238-263. doi:10.1108/13620430710745881 Seibert, S. E., Crant, J. M., & Kraimer, M. L. (1999). Proactive personality and career success. Journal of applied psychology, 84(3), 416-427. doi:10.5860/choice.44-4552 Sullivan, S. E., Forret, M. L., Carraher, S. M., & Mainiero, L. A. (2009). Using the kaleidoscope career model to examine generational differences in work attitudes. Career Development International, 14(3), 284-302. doi:10.1108/13620430910966442 Sullivan, S. E., Forret, M. L., Carraher, S. M., & Mainiero, L. A. (2009). Using the kaleidoscope career model to examine generational differences in work attitudes. Career Development International, 14(3), 284-302. doi:10.1108/13620430910966442 Shaw, S., & Leberman, S. (2015). Using the kaleidoscope career model to analyze female CEOs’ experiences in sport organizations. Gender in Management: An International Journal, 30(6), 500-515. doi:10.1108/gm-12-2014-0108 Sullivan, S. E., Forret, M. L., Mainiero, L. A., & Terjesen, S. (2007). What motivates entrepreneurs? An exploratory study of the kaleidoscope career model and entrepreneurship. Journal of Applied Management and Entrepreneurship, 12(4), 4-19. doi:10.1108/13620430910966442 Shim, M. (2011). The protean career orientation and career success: The role of career development. Unpublished doctorial dissertation, Korea University. Shin, S., & Jeong, J. (2014). The Causal Relationships among Subjective Career Success,Job Security, Organizational Career Management Support, Protean Career Attitude, and Protean Career Management Behavior of the Workers in Major Industries, Korea. Journal of Agricultural Education and Human Resource Development, 46(2), 183-213. doi:10.23840/agehrd.2014.46.2.183 Sullivan, S. E., & Baruch, Y. (2009). Advances in Career Theory and Research: A Critical Review and Agenda for Future Exploration. Journal of Management, 35(6), 1542-1571. doi:10.1177/0149206309350082 doi:10.1108/13620430910966442 Shin, S., & Jeong, J. (2014). The Causal Relationships among Subjective Career Success,Job Security, Organizational Career Management Support, Protean Career Attitude, and Protean Career Management Behavior of the Workers in Major Industries, Korea. Journal of Agricultural Education and Human Resource Development, 46(2), 183-213. doi:10.23840/agehrd.2014.46.2.183 Valcour, M. (2007). Work-based resources as moderators of the relationship between work hours and satisfaction with work-family balance. Journal of Applied Psychology, 92(6), 1512-1523. doi:10.1037/0021-9010.92.6.1512 Walsh, W. B., & Betz, N. E. (1995). Tests and assessment. Prentice-Hall, Inc. Sullivan, S. E., & Baruch, Y. (2009). Advances in Career Theory and Research: A Critical Review and Agenda for Future Exploration. Journal of Management, 35(6), 1542-1571. †Corresponding Author: Boyoung Jung, Senior Manager, HRD Team, byjung@hyundai.com * This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea(NRF-2019S1A5A8038225). 한국심리학회지: 산업및조직 doi:10.1177/0149206309350082 Sullivan, S. E., & Baruch, Y. (2009). Advances in Career Theory and Research: A Critical Review and Agenda for Future Exploration. Journal of Management, 35(6), 1542-1571. doi:10.1177/0149206309350082 - 78 - 한국심리학회지: 산업및조직 Korean Journal of Industrial and Organizational Psychology 2021. Vol. 34, No. 1, 51-79 https://doi.org/10.24230/kjiop.v34i1.51-79 한국심리학회지: 산업및조직 Korean Journal of Industrial and Organizational Psychology 2021. Vol. 34, No. 1, 51-79 https://doi.org/10.24230/kjiop.v34i1.51-79 Validation for Korean Version of Kaleidoscope Career Attitude Measurement* Validation for Korean Version of Kaleidoscope Career Attitude Measurement* BoYoung Jung† * This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea(NRF-2019S1A5A8038225). †Corresponding Author: Boyoung Jung, Senior Manager, HRD Team, byjung@hyundai.com JaeEun Lee Sangji University Sangji University Hyundai Motor Company The purpose of this study is to validate the Kaleidoscope career attitude scale developed by Sullivan et al. (2009) to measure the new career attitudes of Korean corporate employees. This study can be summarized as follows: first, the Korean version of the Kaleidoscope career attitude measurement’s psychometric equivalence and contents validity was verified. Second, the Kaleidoscope career attitude measurement items consisted of three factors, equivalent to the original scale. Third, the fit of the measurement model consisting of 13 items was confirmed with the new sample. And it was confirmed that the same scale could be used in the male and female groups. Fourth, the difference in the latent mean was significant in balance and challenge parameters. Comprehensive research results revealed that the Korean version of the Kaleidoscope career attitude is valid and reliable. Based on this result, the significance and limitations of the research and suggestions for future research were discussed. Key words : Kaleidoscope Career Model(KCM), Career Attitude, Measurement, Validation, Latent Mean Analysis - 79 -
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Hypersensitive-like response in Brassica plants is specifically induced by molecules from egg-associated secretions of cabbage white butterflies
Frontiers in ecology and evolution
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Pieris, cabbage, cell death, plant-insect interaction, oviposition-induced response, egg-associated molecular pattern TYPE  Original Research PUBLISHED  01 February 2023 DOI  10.3389/fevo.2022.1070859 TYPE  Original Research PUBLISHED  01 February 2023 DOI  10.3389/fevo.2022.1070859 TYPE  Original Research PUBLISHED  01 February 2023 DOI  10.3389/fevo.2022.1070859 COPYRIGHT © 2023 Caarls, Bassetti, Verbaarschot, Mumm, van Loon, Schranz and Fatouros. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS EDITED BY Maria L. Pappas, Democritus University of Thrace, Greece REVIEWED BY Junji Takabayashi, Kyoto University, Japan Islam S. Sobhy, Cardiff University, United Kingdom Lotte Caarls 1*†, Niccolò Bassetti 1, Patrick Verbaarschot 1, Roland Mumm 2, Joop J. A. van Loon 3, M. Eric Schranz 1 and Nina E. Fatouros 1* 1 Biosystematics Group, Wageningen University & Research, Wageningen, Netherlands, 2 Bioscience, Wageningen University & Research, Wageningen, Netherlands, 3 Laboratory of Entomology, Wageningen University & Research, Wageningen, Netherlands Plants perceive and respond to herbivore insect eggs. Upon egg deposition on leaves, a strong hypersensitive response (HR)-like cell death can be activated leading to egg desiccation and/or dropping. In Brassica spp., including many crops, the HR-like mechanism against eggs of cabbage white butterflies (Pieris spp.) is poorly understood. Using two Brassica species, the crop B. rapa and its wild relative B. nigra, we studied the cellular and molecular plant response to Pieris brassicae eggs and characterized potential insect egg-associated molecular patterns (EAMPs) inducing HR-like cell death. We found that eggs of P. brassicae induced typical hallmarks of early immune responses, such as callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like cell death. However, elevated levels of ethylene production and upregulation of salicylic acid-responsive genes were only detected in a B. nigra accession expressing HR-like cell death. Eggs and egg wash from P. brassicae contains compounds that induced such responses, but the eggs of the generalist moth Mamestra brassicae did not. Furthermore, wash made from hatched Pieris eggs, egg glue, and accessory reproductive glands (ARG) that produce this glue, induced HR-like cell death, whereas washes from unfertilized eggs dissected from the ovaries or removal of the glue from eggs resulted in no or a reduced response. This suggests that there is one or multiple egg associated molecular pattern (EAMP) located in the egg glue a that teresponse in B. nigra is specific to Pieris species. Lastly, our results indicate that the EAMP is neither lipidic nor proteinaceous. Our study expands the knowledge on the mechanism of Brassica-Pieris-egg interaction and is a step closer toward identification of EAMPs in Pieris egg glue and corresponding receptor(s) in Brassica. OPEN ACCESS CITATION Caarls L, Bassetti N, Verbaarschot P, Mumm R, van Loon JJA, Schranz ME and Fatouros NE (2023) Hypersensitive-like response in Brassica plants is specifically induced by molecules from egg-associated secretions of cabbage white butterflies. Front. Ecol. Evol. 10:1070859. doi: 10.3389/fevo.2022.1070859 COPYRIGHT © 2023 Caarls, Bassetti, Verbaarschot, Mumm, van Loon, Schranz and Fatouros. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. frontiersin.org Hypersensitive-like response in Brassica plants is specifically induced by molecules from egg-associated secretions of cabbage white butterflies OPEN ACCESS EDITED BY Maria L. Pappas, Democritus University of Thrace, Greece REVIEWED BY Junji Takabayashi, Kyoto University, Japan Islam S. Sobhy, Cardiff University, United Kingdom *CORRESPONDENCE Lotte Caarls lotte.caarls@wur.nl Nina E. Fatouros nina.fatouros@wur.nl †PRESENT ADDRESS Lotte Caarls, Plant Breeding, Wageningen University and Research, Wageningen, Netherlands SPECIALTY SECTION This article was submitted to Chemical Ecology, a section of the journal Frontiers in Ecology and Evolution RECEIVED 15 October 2022 ACCEPTED 19 December 2022 PUBLISHED 01 February 2023 CITATION Caarls L, Bassetti N, Verbaarschot P, Mumm R van Loon JJA, Schranz ME and Fatouros NE (2023) Hypersensitive-like response in Brassica plants is specifically induced by molecules from egg-associated secretions of cabbage white butterflies. Front. Ecol. Evol. 10:1070859. doi: 10.3389/fevo.2022.1070859 COPYRIGHT © 2023 Caarls, Bassetti, Verbaarschot, Mumm van Loon, Schranz and Fatouros. This is an open-access article distributed under the ter of the Creative Commons Attribution Licens (CC BY). The use, distribution or reproductio in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication 1. Introduction Plants rely on an immune system that regulates the perception of attackers and subsequent activation of inducible defenses (Wilkinson et al., 2019). Perception involves detection of pathogen- derived effector proteins and of molecular patterns, which can derive from different organisms, such as microbes (MAMPs) or herbivores (HAMPs; Gust et al., 2017; Stahl et al., 2018; Van der Burgh and Frontiers in Ecology and Evolution 01 10.3389/fevo.2022.1070859 Caarls et al. prime plants for future larval attack (Fatouros et al., 2008, 2009, 2015; Paniagua Voirol et al., 2020). Anti-aphrodisiacs transferred from the butterfly male to the female during mating were shown to be present in minute amounts in the ARG secretion and were suggested as potential elicitors (Fatouros et al., 2008, 2009). However, glands from unmated females induced HR-like, and therefore another, female-derived elicitor is likely to play a role (Fatouros et al., 2015). In A. thaliana, egg-derived phosphatidylcholines were found to induce production of H2O2, SA and result in cell death as shown by trypan blue staining (Stahl et al., 2020). It is still unclear which compounds from eggs of Pieris spp. and Anthocharis cardamines are detected by Brassica spp. and close relatives that result in HR-like activation, and whether these reside in the eggs themselves or in the secretions surrounding the eggs (Griese et al., 2021). Joosten, 2019). Perception is followed by an early signaling cascade including rapid ion-flux changes and production of reactive oxygen species (ROS; Couto and Zipfel, 2016). Induced defenses typically include reinforcement of extracellular barriers, for example by callose deposition, the production of antimicrobial or insecticidal metabolites and proteins, and a localized, rapid cell death response, the hypersensitive response (Cui et al., 2015; Couto and Zipfel, 2016; Campos et al., 2018; Balint-Kurti, 2019; Dalio et al., 2020). Most in-depth studies on activation of the plant immune system have been performed on interactions with plant pathogens. So far, 119 HAMPs and/or effectors mostly from oral secretions of piercing sucking and chewing herbivores, have been identified, but pattern recognition receptors for HAMPs are just starting to be discovered (Erb and Reymond, 2019; Steinbrenner et al., 2020).h The detection by plants of herbivore eggs deposited on plant tissue is remarkable, as eggs are immobile and seemingly harmless structures. However, insect eggs will turn into feeding larvae and thus pose a future threat to the plant. 2.1. Plant material and rearing of butterflies Black mustard (B. nigra L.) accessions WUR-01 and WUR-02 used, originated from plants that were collected near the river Rhine in Wageningen, Netherlands (N51.96, E05.68). Brassica rapa L. genotypes used in this study (L58, R-o-18, RC-144) were obtained from the Laboratory of Plant Breeding (WUR). Plants were grown in a greenhouse (18 ± 5°C, 50–70% RH, L16, D8) and were used when three to five  weeks old. Besides HR-like cell death, eggs of different insect species induce immune responses similar to pattern-triggered immunity (PTI), including callose deposition, accumulation of ROS and SA, and transcriptome changes of several defense genes, including PATHOGENESIS-RELATED PROTEIN 1 (PR1) in A. thaliana (Little et  al., 2007; Bruessow et  al., 2010; Gouhier-Darimont et  al., 2013; Reymond, 2013; Lortzing et al., 2020). These early signaling defense responses to eggs seem to be conserved across different plant species (Lortzing et al., 2020). Expression of PR1 was also induced in leaves of B. nigra underneath P. brassicae and P. rapae eggs (Fatouros et al., 2014, 2015). Whether brassicaceous species that are natural hosts of Pieris spp., including B. nigra and B. rapa, respond with a general immune response, including ROS, callose and expression of different defense genes, to insect eggs, and whether there is genetic variation for this response, is largely unknown. i Pieris brassicae L. (Lepidoptera: Pieridae) was reared on Brussels sprouts plants (Brassica oleracea var. gemmifera cv. Cyrus) in a climate room (21 ± 1°C, 50%–70% RH, L16: D8) at the Laboratory of Entomology, Wageningen University. Virgin adult females were obtained by isolating female butterflies immediately after eclosion. Otherwise, twenty females and males could mate in a large cage (60 × 60 × 90 cm) and females were used for oviposition in experiments or oviposition on filter paper (grade 3 hw, Sartorius, Germany) for egg wash production (see section below). The cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae) was reared on Brussels sprouts plants in a climate room (21 ± 1°C, 50%–70% RH, L16: D8) at the Laboratory of Entomology, Wageningen University. So far, few studies have identified EAMPs that activate defense against insect eggs (Reymond, 2013; Hilker and Fatouros, 2015; Stahl et al., 2018). In some studies, secretions surrounding eggs were sufficient to elicit defense responses in plants and a few elicitors have been isolated from these secretions (Hilker et al., 2005; Tamiru et al., 2011; Salerno et al., 2013). 2.1. Plant material and rearing of butterflies Most EAMPs were found to be organic compounds of low-molecular weight associated with the eggs, while recently a first proteinaceous compound, diprionin, was found as an elicitor of pine defense against sawfly eggs (Hundacker et al., 2021). In P. brassicae, egg-enveloping secretions are produced by the females’ accessory reproductive gland (ARG) and form a glue-like structure between the eggs and leaves (Beament and Lal, 1957; Fatouros et al., 2012). Treatment of Brassica plants with extracts from ARGs has shown to induce HR-like cell death and plant chemical cues attracting egg parasitoids, and to 1. Introduction Upon detection, plants can mount defenses against eggs that range from plant-mediated desiccation of eggs, egg dropping, egg crushing and the production of ovicidal substances (Hilker and Fatouros, 2015, 2016). Eggs of cabbage white butterflies (Pieris spp.) trigger necrotic lesions in leaves of the black mustard, Brassica nigra that can result in egg-killing by desiccating and/or dropping off singly laid Pieris eggs (Shapiro and DeVay, 1987; Griese et  al., 2017). As the egg-induced plant phenotype resembles an HR, it has been referred to as hypersensitive response-like (“HR-like”; Fatouros et al., 2012). Oviposition by Pieris butterflies has been shown to induce HR-like cell death in several other plants of the Brassicaceae family, although the severity of the response varies between, and within species (Pashalidou et al., 2015; Griese et al., 2020, 2021; Groux et al., 2021; Bassetti et al., 2022).f In this study, we  developed and implemented a method to characterize the compounds from Pieris spp. eggs and egg-enveloping secretions. We specifically addressed: (1) the cellular and molecular response of two Brassica species, (2) the specificity of these responses by comparing eggs and egg washes of Pieris with the generalist moth M. brassicae, (3) the origin of EAMP inducing HR-like cell death, and (4) the chemical nature and stability of the elicitor. Frontiers in Ecology and Evolution 2.5. Dissection of butterfly reproductive tract For all experiments testing the effects of treating plants with egg washes, 10 μL egg wash (i.e., an equivalent of 25 eggs) was pipetted on the abaxial side of the fourth or fifth emerged leaf of 3–4 weeks old B. nigra plants, unless otherwise specified. Symptoms induced by egg wash were scored 4 days after treatment. To quantify severity, a scoring system from 0 to 4 was adapted from Griese et al. (2017). Severity score 0: no visual response; score 1: brown spots underneath eggs or egg wash spot, only visible at abaxial side leaf; score 2: cell death also visible at adaxial side of leaf, spot smaller than 2 mm diameter, score 3: cell death the size of egg wash spot, and score 4: spreading lesion beyond spot of treatment (Supplementary Figure S1). Score 0 and 1 are classified as “non-HR,” score 2–4 are classified as “HR”. In female butterflies, eggs are produced in the ovaries, pass through the common oviduct and are fertilized by sperm released from the bursa copulatrix (BC) into the vagina. Before being expelled through the ovipore, the eggs are covered by secretions released from the accessory reproductive gland (ARG), a paired gland that contains egg-enveloping secretions and glue-like material to attach eggs to leaves (Supplementary Figure S2). For dissection of tissues from the female reproductive tract, namely ARGs and BCs or eggs from the ovarian tubules (hereafter termed “ovarian eggs”), mated P. brassicae females were obtained by pairing a virgin female and virgin male 1 day after eclosion, which were kept together until mating was observed. Virgin females were obtained by keeping them separately shortly after eclosion. Three to four days after eclosion, mated and virgin P. brassicae females were killed by beheading using a scalpel and then dissected. ARGs, BC and ovarian eggs were dissected from females under a stereomicroscope (optical magnification 20×) in 20 mM Mes buffer. Dissected structures were washed overnight (16 h) in 20 mM Mes pH 5.7 using 50 μL per each ARG, 100 μL buffer per each BC or 5 μL buffer per egg. Solution was pipetted off the next morning and frozen until use (modified after Fatouros et al., 2015). For experiments with oviposition on plants, one mated female P. brassicae butterfly was placed in a cage with a B. nigra or B. rapa plant and removed when ~10–25 eggs were laid. 2.4. Histochemical staining tested different buffers and found that using Mes buffer resulted in the most consistent plant response (data not shown). The solution (egg wash) was pipetted into a new tube the next morning. For histochemical staining of plant tissue underneath eggs, P. brassicae egg-laden plants were sampled 24, 48, and 72 h after oviposition by taking a 10 mm diameter leaf disk of the area surrounding the eggs. Similarly this was done with plants treated with egg wash. Eggs were carefully removed with a brush before proceeding with histochemical staining. Pictures of the leaf disks were taken with a Dino-Lite digital microscope (AnMo Electronics Corporation, Taiwan) before staining (with eggs) and after staining (without eggs). As cell death is generally preceded by production of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion (Torres, 2010), we visualized ROS with different histochemical stainings. 3,3′-diaminobenzidine (DAB; Sigma- Aldrich, MO, United States) was used to stain hydrogen peroxide (H2O2). Leaf disks (10 mm Ø) were submersed in 1 mg/mL DAB solution and samples were incubated for 30–60 min in the dark. Nitroblue tetrazolium chloride (NBT; Sigma-Aldrich, MO, United States) was used to stain superoxide radical O2 •−. For this leaf disks (10 mm Ø) were submersed in 0.2% NBT with 50 mM sodium phosphate buffer (pH 7.5) and samples were incubated 30–60 min in the dark. For visualization of cell death, leaves were submersed in 0.08% trypan blue solution (Sigma-Aldrich, MO, United States), overnight (16 h). For all three stainings, destaining of leaves was performed with 96% ethanol. Pictures of leaf disks before staining (with eggs) and after staining (without eggs) were taken with a Dino-Lite digital microscope (AnMo Electronics Corporation, Taiwan). For staining of callose, leaf disks were first destained and then submersed in 0.01% aniline blue (Sigma- Aldrich, MO, United States) with 150 mM K2HPO4 and imaged after at least 2 h of incubation using a DAPI filter on a fluorescence microscope equipped with a DS-5MC camera and NIS elements AR 2.30 software. To obtain egg-enveloping secretions for wash of only egg glue, 1-day old P. brassicae eggs were collected as above, counted, and then carefully removed from the paper using a brush. The spots of secretions underneath eggs were then cut out and washed overnight (16 h) in 1 mL 20 mM Mes buffer (pH 5.7) per 400 eggs. 2.4. Histochemical staining A wash of the pieces of the same filter paper without secretions was used as control. To remove egg-enveloping secretions from eggs, eggs on paper were submersed in 1 mL per 400 eggs of either a solution of 1% bleach and 2% Tween-20 or in 250 mM NaPO4 pH 9.0 for 30 min (two different treatments that were shown to remove egg glue; Jacobs et al., 2013). After treatment, eggs were rinsed once with 1 mL MILLI-Q® water (Sigma-Aldrich, MO, United States), hereafter “MQ water,” and then 1 mL Mes buffer and then washed overnight (16 h) in 20 mM Mes buffer pH 5.7. Eggs that were used to compare where egg-enveloping secretions were not removed, were submersed in Mes for 30 min, rinsed in MQ and Mes, and then washed. To study the induction by eggs of different ages, eggs were collected as above on paper, and then kept at room temperature until they were washed at the end of each day, until they hatched (6–7 days after oviposition). After hatching, young caterpillars were carefully removed using a brush. Empty eggshells and associated secretions (on paper) were then washed overnight (16 h). To collect unfertilized eggs for egg wash, filter paper was pinned underneath a B. nigra leaf of a plant placed in a cage with 10 virgin adult females and then prepared in a similar way as fertilized eggs. Paper sheets with M. brassicae eggs were obtained from a rearing population from the Laboratory of Entomology (WUR). To obtain egg wash, eggs were counted, spots with eggs were cut out and washed in 1 mL 20 mM Mes buffer (pH 5.7) per 400 eggs. The wash was pipetted off the next morning and frozen at −20°C until use. Frontiers in Ecology and Evolution 2.2. Preparation of egg washes The protocol for preparation of egg washes has been newly developed to prepare egg wash free from leaf-surface related chemical compounds which enables us to conduct chemical analysis and identify possible EAMPs. Full procedure is described in Supplementary Data (Supplementary Figure S1). Pieris brassicae eggs were collected on filter paper pinned underneath a B. oleracea leaf in a cage containing 20 mated females. Egg clutches laid on the paper were cut out and submersed in 1 mL 2-(N-morpholino)ethanesulfonic acid (Mes) buffer per 400 eggs, overnight (16 h) without disturbance. Previously, we have 02 frontiersin.org Caarls et al. 10.3389/fevo.2022.1070859 2.7. Phospholipid treatments To compare gene expression between P. brassicae and M. brassicae egg wash, B. nigra plants were treated with 10 μL of either egg wash (400 eggs/mL in Mes buffer) or a negative control (Mes buffer). Six leaf disks (Ø 6 mm) were sampled directly next to the egg wash-treated spot 24 h after treatment. Four plants were used for each treatment as biological replicates. Phosphatidylcholines (PCs) were recently found to be  the main components of P. brassicae egg extracts that show a bioactivity in A. thaliana Col-0 resulting in induction of SA and PR1 marker gene as well as ROS accumulation and cell death formation (Stahl et al., 2020). Thus, we tested whether the same PCs used in Stahl et al. (2020) may be associated with development of HR-like cell death by B. nigra. Phospholipids were ordered from Avanti Polar Lipids (Alabaster, Alabama, United States). Specifically, PC(16:1/16:1), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, catalog no. 850358 and PC(18:1/18:1), 1,2-dioleoyl-sn-glycero-3-phosphocholine, catalog no. 850357 were used for experiments. Phospholipid stock solutions were made in 100% MeOH. For phospholipid application, the MeOH was evaporated under a nitrogen-flux and the phospholipids were dissolved in MQ water with 1% DMSO, 0.5% glycerol, 0.1% Tween 20 by sonication. Phospholipids were applied on B. nigra leaves at different concentrations (1, 5 and 10 μg/μL) that were previously shown to have biological activity in A. thaliana (Stahl et al., 2020). An MQ water solution of 1% DMSO, 0.5% glycerol and 0.1% Tween 20 was used as control. To compare the gene expression of two B. nigra accessions with contrasting ability to develop HR-like cell death, egg wash at higher concentration (~1,000 eggs/mL in demineralised water) was used. Experimental design consisted of two treatments (egg wash, control), two B. nigra accessions (WUR-01 without HR, and WUR-02, developing HR), and three time points after treatment (6, 24, and 48 h after treatment). For each treatment combination, egg wash or control were applied with two droplets of 5 μL on the abaxial side of a single leaf. Leaf disks (Ø 6 mm) were harvested at each time point on the treatment spots and disks from the same treatment on a leaf were pooled. For each treatment combination, a total of 15 plants were used and groups of 3 plants with similar treatments were pooled to compose a total of 5 biological replicates. 2.5. Dissection of butterfly reproductive tract HR-like cell death was scored 4 days after oviposition, using the same scoring system as with egg wash. For experiments with M. brassicae, five mated female moths were placed together with a B. nigra plant in a cage to allow egg deposition overnight (16 h) and removed in the morning. No visible cell death was observed under M. brassicae eggs as was also reported in our previous studies (Fatouros et al., 2012; Griese et al., 2021). 03 frontiersin.org Caarls et al. 10.3389/fevo.2022.1070859 2.6. Treatments of egg wash with heating, proteinase K, freezing, methanol and speedvac extract Netherlands). After sampling leaf disks for the ethylene assay, plants were also treated with P. brassicae egg wash to determine their HR-like cell death phenotype. Netherlands). After sampling leaf disks for the ethylene assay, plants were also treated with P. brassicae egg wash to determine their HR-like cell death phenotype. To characterize the chemical nature of EAMP in the egg wash we exposed it to different treatments. Untreated egg wash was used as a control. To study the stability of the elicitor, the egg wash was heated and frozen. For heating, egg wash was made as described above, collected in microcentifuge tubes and boiled in a water bath for 30 min at 95°C. For freezing treatment, egg wash was frozen at −20°C and then thawed for 1 h at room temperature, then frozen again. Freezing–thawing cycles were repeated up to four times, after which, all washes were used to treat plants. To study if the elicitor was an intact protein, a proteinase treatment was performed, a stock of 20 mg/mL Proteinase K (Qiagen, Cat No./ID: 19133) was diluted 100× in 100 μL egg wash (10% volume, pH 7.2) and incubated with egg wash for 30 min at 37°C. 2.8. Production of ethylene Ethylene production is regularly measured to characterize the induction of early plant immune response by biotic stresses (Fan et al., 2017). Ethylene was measured as previously published (Oome et al., 2014). To measure the plant production of ethylene, in B. nigra, ten plants for each accession (with and without HR-like phenotype) were used. For B. rapa, three plants of each genotype were used. For each plant, leaf disks (3 mm Ø) were sampled from mature leaves of untreated plants and incubated overnight (16 h) in demineralised water. Subsequently, three leaf disks for each biological replicate (a single plant) were randomly chosen and incubated for 5 h in air tight glass vials with in either 400 μL of 20 mM Mes pH 5.7 or 400 μL egg wash (400 eggs/mL in same Mes buffer). After incubation, 1 mL of the headspace of each sample was taken to measure ethylene concentration on a Focus gas chromatograph (Thermo Electron S.p.A., Milan, Italy) equipped with an FID detector and a RT-QPLOT column, 15 m × 0.53 mm ID (Restek, Bellefonte, PA, United States). The system was calibrated with a certified gas of 1.01 μL L−1 (1 ppm) ethylene in synthetic air (Linde Gas Benelux B.V., Schiedam, 2.9. Plant treatments for gene expression experiments To compare gene expression induced by eggs and egg wash, B. nigra plants were treated with either 10 μL of egg wash at lower concentration (400 eggs/mL in Mes buffer) or oviposited with an egg clutch of 10 eggs. Six leaf disks (Ø 6 mm) were sampled with a leaf puncher directly next to the eggs or the egg wash-treated spot at 0, 3, 6, 24, and 48 h, as done in previous research (Fatouros et al., 2014; Griese et al., 2021). For each timepoint, four plants were sampled individually and considered biological replicates. Brassica rapa plants were treated with three single eggs on a single leaf of each plant. Leaf disks (Ø 6 mm) were then harvested next to the eggs at 0, 3, 6, 24, and 96 h after treatment. For each timepoint, three plants were sampled individually and considered biological replicates. 2.7. Phospholipid treatments For each gene expression experiment, samples were snap frozen in liquid nitrogen and stored at −80°C until use. Frontiers in Ecology and Evolution frontiersin.org 2.11. Data analysis All data analysis was carried out in R (R Core Team, 2020). Scoring of HR-like cell death in severity categories were analyzed with a non-parametric method (Kruskal–Wallis test) on HR scores (0, 1, 2, 3, and 4) and different treatments were included as categorical fixed factors. For all other statistical analyses involving comparison of mean values (gene expression, ethylene production), the choice of parametric or non-parametric methods was made after checking the assumptions of normality (Shapiro–Wilk normality test) and homogeneity of variances (Fligner–Killeen test) on the raw data. As parametric methods, Student’s T-test, Welch T-test and ANOVAs followed by Tukey’s honestly significant difference test were used. As non-parametric method, Kruskal–Wallis test followed by pairwise Wilcoxon rank sum test was used. Gene expression data from qRT-PCR were calculated with the ΔΔCt method (Livak and Schmittgen, 2001) and were analyzed on log2-transformed data (parametric test) or on raw data (non-parametric test) specifying time points or treatments as factors. 3. Results 3.1. Pieris eggs triggered cellular responses in Brassica spp. 3. Results 3.1. Pieris eggs triggered cellular responses in Brassica spp. cDNA synthesis kit (Bioline, United Kingdom) according to the manufacturer’s instructions. Real time qRT-PCR reactions were performed using SensiFAST SYBR No-ROX Kit (Bioline, United Kingdom) in 10 μL reaction volumes, containing 3 μL cDNA and 500 nM of primers on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, CA, United  States). The following qRT-PCR program was used: 95°C for 2 min followed by 40 cycles of 95°C for 5 s; primer-specific annealing temperature for 5 s and 72°C for 10 s, with data collection at 72°C, followed by a melt curve analysis. Relative gene expression was calculated with the ΔΔCq method, using GAPDH as reference gene. Primers sequences are available in Supplementary materials (Supplementary Table S1). We first investigated cellular responses against Pieris brassicae oviposition in its natural host, the black mustard Brassica nigra. Reactive oxygen species (ROS) such as superoxide anion (O2 •−) and hydrogen peroxide (H2O2) accumulated underneath eggs at 24 h (Figure 1A). At the same time point, aniline blue staining in B. nigra revealed also callose deposition underneath eggs (Figure 1A). Further, the occurrence of cell death in plant tissue under the eggs was investigated by staining with trypan blue (TB), showing that cell death occurred 72 h after oviposition (Figure 1B). Occasionally, a few TB-stained cells were visible at 48 h (not shown). In B. nigra, we regularly observed a strong, macroscopically visible HR-like cell death that spreads beyond the egg site and stops 96 h after oviposition (Figure 1C). Cellular responses against P. brassicae oviposition were also investigated in B. rapa. ROS accumulation and cell death formation were also detected in B. rapa at 24 h and 72 h after oviposition, respectively (Supplementary Figures S2A,B). Although these early cellular responses appeared similar to what was observed in B. nigra, the macroscopically visible HR-like cell death developed by B. rapa appeared as black necrotic spots that never spread beyond the egg site (Supplementary Figure S2C). 2.10. Expression of genes by real-time qRT-PCR We investigated to which extent the difference in HR-like phenotype between B. nigra accessions WUR-01 and WUR-02 was related to activation of different phytohormones associated with plant immunity. Thus, we quantified expression of genes induced by SA, such as ICS1, PR1, and PR2 (Little et al., 2007), and genes regulated by JA, i.e., MYC2, VSP1, and VSP2 (Reymond et  al., 2004). For experiments on gene expression induced by eggs or egg wash in both B. nigra and B. rapa, RNA extraction was performed according to Oñate-Sánchez and Vicente-Carbajosa (2008). For experiments on gene expression induced by egg wash in two B. nigra accessions, RNA extraction was performed with Direct-zol RNA Miniprep Kit (Zymo Research, CA, United States) following the manufacturer’s protocol. For preparation of cDNA of all experiments, 1 μg of RNA was reverse-transcribed using SensiFAST 04 frontiersin.org Caarls et al. 10.3389/fevo.2022.1070859 3.2. Egg wash induced a plant response similar to the response to eggs Expression at each time point was compared to the 0 h timepoint (Kruskal–Wallis, p < 0.05 for eggs and p < 0.05 for egg wash). (D) Ethylene production in parts per million (ppm) by plants treated with egg wash. (Student’s T-test, p < 0.001). Each treatment consisted of 10 biological replicates. The height of the boxes in C-D represents the first to the third quartile of the range; the horizontal line within the box is the median; the whiskers indicate the data minimum and maximum; and dots represent data points. Asterisk indicates p < 0.05, *** <0.001. A B B A C D D D C FIGURE 2 Plant responses induced by Pieris brassicae eggs and egg wash in Brassica nigra. (A) Picture of responses induced by eggs and egg wash next to each other on a leaf (microscopic image): both eggs and egg wash induce HR-like necrotic spots. (B) Quantification of severity of symptoms induced by egg wash and eggs. Shown is the fraction of plants that were scored in a specific class for each treatment (N = 32 plants for both treatments; Kruskal–Wallis, p > 0.05). ns = no significant difference. (C) BnPR1 expression in plants after egg deposition (red box plots) or treatment with egg wash (blue box plots). Gene expression was measured by qRT-PCR and normalized to the housekeeping gene BnGADPH. Each treatment consisted of 3–4 biological replicates. Expression at each time point was compared to the 0 h timepoint (Kruskal–Wallis, p < 0.05 for eggs and p < 0.05 for egg wash). (D) Ethylene production in parts per million (ppm) by plants treated with egg wash. (Student’s T-test, p < 0.001). Each treatment consisted of 10 biological replicates. The height of the boxes in C-D represents the first to the third quartile of the range; the horizontal line within the box is the median; the whiskers indicate the data minimum and maximum; and dots represent data points. Asterisk indicates p < 0.05, *** <0.001. egg wash compared to incubation with control Mes buffer (Figure 2D; Supplementary Table S4). In addition, there was a significant difference in ethylene produced between plants with contrasting HR-like phenotypes. 3.2. Egg wash induced a plant response similar to the response to eggs To isolate potential EAMPs and to quickly screen plants for HR-like cell death, we developed a method to dissolve egg-enveloping secretions from P. brassicae eggs (Methods section; Supplementary Figure S1). We then compared visually whether the B C A B C FIGURE 1 Plant immunity responses induced in Brassica nigra by Pieris brassicae eggs (Bn). (A) B. nigra leaf 24 h after oviposition with accumulation of O2 •− (NBT staining), H2O2 (DAB staining) and callose deposition (aniline blue staining). (B) B. nigra leaf 72 h after oviposition showing cell death (trypan blue staining). (C) Fully developed HR-like cell death that is macroscopically visible at 96 h after oviposition. A few eggs were removed from the clutch to show the cell death underneath. Stainings were repeated multiple times with similar results. Scale bars = 1 mm. B C B C A A B C C FIGURE 1 Plant immunity responses induced in Brassica nigra by Pieris brassicae eggs (Bn). (A) B. nigra leaf 24 h after oviposition with accumulation of O2 •− (NBT staining), H2O2 (DAB staining) and callose deposition (aniline blue staining). (B) B. nigra leaf 72 h after oviposition showing cell death (trypan blue staining). (C) Fully developed HR-like cell death that is macroscopically visible at 96 h after oviposition. A few eggs were removed from the clutch to show the cell death underneath. Stainings were repeated multiple times with similar results. Scale bars = 1 mm. 05 Frontiers in Ecology and Evolution frontiersin.org 10.3389/fevo.2022.1070859 Caarls et al. A B C D FIGURE 2 Plant responses induced by Pieris brassicae eggs and egg wash in Brassica nigra. (A) Picture of responses induced by eggs and egg wash next to each other on a leaf (microscopic image): both eggs and egg wash induce HR-like necrotic spots. (B) Quantification of severity of symptoms induced by egg wash and eggs. Shown is the fraction of plants that were scored in a specific class for each treatment (N = 32 plants for both treatments; Kruskal–Wallis, p > 0.05). ns = no significant difference. (C) BnPR1 expression in plants after egg deposition (red box plots) or treatment with egg wash (blue box plots). Gene expression was measured by qRT-PCR and normalized to the housekeeping gene BnGADPH. Each treatment consisted of 3–4 biological replicates. 3.2. Egg wash induced a plant response similar to the response to eggs Plants responding with stronger HR-like cell death (score 2 or higher), produced a significantly higher amount of ethylene after incubation with egg wash than plants with no HR-like cell death (Student’s T-test = −4.087, df = 18, p < 0.001; Figure  2D; Supplementary Table S4). Similarly, B. rapa plants developing HR-like cell death, also showed a higher ethylene production upon incubation of leaves with P. brassicae egg wash (Student’s T-test = −3.876, df = 4, p = 0.018; Supplementary Figure S3E). These results suggest that there is an early detection response in plants after contact with egg wash that will ultimately lead to cell death. treatment of leaves with egg wash induced a similar HR-like cell death as the butterfly eggs (Figure 2A). As a control, leaves were treated with only the buffer (no eggs were washed). Symptoms induced by eggs or egg wash were scored after 4 days, and HR-like frequency (proportion of plants showing HR-like) and HR-like severity (mean score of induced symptoms) were compared between the two treatments in B. nigra. HR-like severity after oviposition by eggs or treatment with egg wash did not differ (Kruskal–Wallis: χ2 = 1.33, df = 1, p = 0.24; Figure 2B; Supplementary Table S2). We compared expression of SA-marker gene PR1 in B. nigra oviposited on by P. brassicae butterflies or treated with egg wash. PR1 was significantly upregulated both after oviposition and after treatment with egg wash. In B. nigra, PR1 expression increased at 6 h and was significantly upregulated at 24 h and 48 h after egg deposition (Kruskal– Wallis: χ2 = 12.23, df = 3, p = 0.015; Figure 2C). No significant differences in PR1 expression were found between B. nigra plants treated with eggs or egg wash (Figure 2C; Supplementary Table S3). Similarly, in B. rapa, PR1 expression was significantly upregulated by egg deposition at 24 h and showed further increase at 96 h (Kruskal–Wallis: χ2 = 11.18, df = 4, p = 0.024; Supplementary Figure S2D). Frontiers in Ecology and Evolution 3.5. EAMP derived from female accessory reproductive glands There was no significant difference in eliciting activity of eggs of increasing age, from one-day-old, to five-day-old eggs, although HR-like severity decreased slightly. Egg wash was also prepared from eggshells and secretions that remained on the paper after caterpillars hatched, and this still induced HR-like symptoms (Kruskal–Wallis: χ2 = 3.20, df = 5, p > 0.1; Figure 5A; Supplementary Table S6). 3.3. Variation in HR-like cell death severity is associated with SA-related defenses Asterisks indicate different p-values within the same time point: ** <0.01, *** <0.001, no asterisks indicate no significant difference. followed by Tukey, p < 0.01), PR1 expression induced by M. brassicae egg wash was not different from the control treatment (Figure 4C; Supplementary Table S5). In addition, incubation with M. brassicae egg wash induced lower ethylene production compared to P. brassicae egg wash (Kruskal–Wallis: χ2 = 21.36, df = 2, p < 0.001; Figure 4D), comparable to the ethylene levels previously observed in B. nigra plants lacking a visible cell death (Figure  2D). Overall, eggs of M. brassicae did not induce cell death and only a weak ROS and ethylene response in B. nigra. relative expression of JA-related genes was generally very low and not significantly different between two accessions. SA-marker BnICS1 was significantly expressed at higher levels in plants that showed a visible HR already at 6 h after treatment (Welch’s T-test = −4.901, df = 5.3, p = 0.003). Both, BnPR1 and BnPR2 showed increased expression across the time points, although with a different magnitude between the two accessions. Both genes were already expressed at higher level at 6 h in plants that showed a visible HR (Welch’s T-test = −2.932, df = 5.3, p = 0.03 and Welch’s T-test = −4.850, df = 5.8, p = 0.003 respectively), and the difference with plants not expressing HR increased up to 5-fold (24 h) and 20-fold (48 h) for BnPR1 and 4-fold (24 h) and 5-fold (48 h) for BnPR2. The expression of JA-related genes showed more stable profiles across time points, with a small peak at 24 h in all treatments, but with no significant differences between the two accessions (MYC2, Welch’s T-test = −1.549, df = 5.8 p > 0.05; VSP1, Welch’s T-test = −0.141, df = 3 p > 0.05; VSP2, Welch’s T-test = −0.301, df = 3.5 p > 0.05). 3.3. Variation in HR-like cell death severity is associated with SA-related defenses All SA marker genes were upregulated in B. nigra plants upon egg wash treatment compared to control. However, the magnitude of expression of SA-related genes was significantly different between the two B. nigra accessions at different time points (Figure 3). In contrast, Next, we tested whether egg wash induced ethylene. Brassica nigra leaves responded with higher ethylene production after incubation with 06 frontiersin.org Caarls et al. 10.3389/fevo.2022.1070859 FIGURE 3 Expression of SA- and JA-related defense genes upon P. brassicae egg wash treatment in two different B. nigra accessions. Gene expression was measured with two treatments (egg wash, control), two accessions (no HR and HR) and at three time points (6, 24, and 48 h after treatment). HR phenotype of each plant was scored after sampling for RNA isolation. Gene expression was measured by qPCR-RT and normalized to housekeeping gene BnGADPH. Expression levels were calculated at each time point relative to control treatment (water droplets). Each treatment consisted of 4–5 biological replicates and is reported as boxplots. The height of the boxes in represents the first to the third quartile of the range; the horizontal line within the box is the median; the whiskers indicate the data minimum and maximum; and dots represent data points. At each time point, gene expression between the two accessions was compared (Welch’s T-test). Asterisks indicate different p-values within the same time point: ** <0.01, *** <0.001, no asterisks indicate no significant difference. FIGURE 3 Expression of SA- and JA-related defense genes upon P. brassicae egg wash treatment in two different B. nigra accessions. Gene expression was measured with two treatments (egg wash, control), two accessions (no HR and HR) and at three time points (6, 24, and 48 h after treatment). HR phenotype of each plant was scored after sampling for RNA isolation. Gene expression was measured by qPCR-RT and normalized to housekeeping gene BnGADPH. Expression levels were calculated at each time point relative to control treatment (water droplets). Each treatment consisted of 4–5 biological replicates and is reported as boxplots. The height of the boxes in represents the first to the third quartile of the range; the horizontal line within the box is the median; the whiskers indicate the data minimum and maximum; and dots represent data points. At each time point, gene expression between the two accessions was compared (Welch’s T-test). Frontiers in Ecology and Evolution frontiersin.org 4. Discussion In this study, we show that HR-like inducing EAMPs are in the egg-enveloping secretions that are produced in female ARGs. Our results indicate that the EAMP is unlikely of lipidic or proteinaceous nature but an organic compound of low-molecular weight. When plants are treated with egg wash, HR-like cell death is induced in responsive plant genotypes, together with upregulation of SA-responsive genes and elevated levels of ethylene suggesting that the HR-like cell death is mediated by one or both phytohormones. In addition, deposition of eggs leads to ROS and callose deposition, also in plant genotypes that do not show HR-like cell death. All phenotypes observed were shared between the two Brassica species that vary in cell death severity and are specific to eggs and egg wash of the specialist P. brassicae. Finally, both egg wash and wash of glue alone induced a severe HR-like cell death, and HR-like severity was significantly lower when B. nigra was treated with a wash of eggs from which the glue was removed (Kruskal–Wallis: H = 26.60, df = 3, p < 0.001; Figure  5D; Supplementary Table S6). 3.4. Responses to eggs and egg wash from Mamestra brassicae are dissimilar to Pieris brassicae in Brassica nigra Different letters indicate significant differences in mean PR1 expression (ANOVA followed by Tukey, p < 0.01). (D) Ethylene production in B. nigra leaf in response to egg washes. The height of the boxes in C,D represents the first to the third quartile of the range; the horizontal line within the box is the median; the whiskers indicate the data minimum and maximum; and dots represent data points. Values that have no accompanying letters in common differ significantly in production of ethylene (Kruskal–Wallis followed by pairwise Wilcoxon test, p < 0.001). Scale bars = 1 mm. cell death upon treatment of B. nigra with neither PC16:1/PC16:1 nor PC18:1/PC18:1 (Figure 7; Supplementary Table S8). A wash from unfertilized, deposited eggs (containing secretions from the ARG) from virgin butterflies induced a similar response as wash from fertilized, deposited eggs of mated females (Figure 5C). There was no significant effect of the mating status of the female (mated or virgin) on the frequency of HR-like cell death elicited or on HR severity (Kruskal–Wallis: χ2 = 2.61, df = 1, p > 0.1). In addition, the wash of ARGs from virgin females induced strong symptoms similar to those of mated females (Figure 5B). These results show that egg fertilization is not necessary for the induction of the HR-like cell death in B. nigra, and that an EAMP resides in the ARG, and is female-derived. 3.4. Responses to eggs and egg wash from Mamestra brassicae are dissimilar to Pieris brassicae in Brassica nigra A wash of dissected accessory reproductive glands (ARGs) induced HR-like cell death, similar to the positive control P. brassicae egg wash (Figure 5B). On the contrary, neither a wash of mature but unfertilized eggs dissected from the ovary (“ovarian eggs”) nor a wash of the sperm- containing bursa copulatrix (BC) induced symptoms (Figure  5B). HR-like severity was significantly higher in plants treated with egg wash or a wash of ARG, compared to wash of ovarian eggs or bursa copulatrix (Kruskal–Wallis: χ2 = 19.833, df = 4, p < 0.001; Supplementary Table S6). Staining of leaves showed that plant cells did not die underneath M. brassicae eggs as leaves did not stain with trypan blue (Figures 4A,B). Further, M. brassicae eggs induced O2 •− production in some plants, but weaker than Pieris eggs (Figures  4C,D). While P. brassicae egg wash induced the expression of PR1 after 24 h (ANOVA 07 frontiersin.org 10.3389/fevo.2022.1070859 Caarls et al. A B C D FIGURE 4 Responses to eggs and egg wash of M. brassicae in B. nigra. (A) Leaf of B. nigra oviposited on by M. brassicae moth by NBT showing light O2•− accumulation underneath M. brassicae eggs. (B) Leaf stained by trypan blue showing no cell death underneath M. brassicae eggs. (C) BnPR1 expression in leaf tissue treated with control solution, P. brassicae wash or M. brassicae wash. Different letters indicate significant differences in mean PR1 expression (ANOVA followed by Tukey, p < 0.01). (D) Ethylene production in B. nigra leaf in response to egg washes. The height of the boxes in C,D represents the first to the third quartile of the range; the horizontal line within the box is the median; the whiskers indicate the data minimum and maximum; and dots represent data points. Values that have no accompanying letters in common differ significantly in production of ethylene (Kruskal–Wallis followed by pairwise Wilcoxon test, p < 0.001). Scale bars = 1 mm. A B B D B A C D D D C FIGURE 4 Responses to eggs and egg wash of M. brassicae in B. nigra. (A) Leaf of B. nigra oviposited on by M. brassicae moth by NBT showing light O2•− accumulation underneath M. brassicae eggs. (B) Leaf stained by trypan blue showing no cell death underneath M. brassicae eggs. (C) BnPR1 expression in leaf tissue treated with control solution, P. brassicae wash or M. brassicae wash. Frontiers in Ecology and Evolution frontiersin.org 3.6. EAMP is neither a protein nor lipid The increasing knowledge on plant defense mechanisms to eggs of herbivores suggests that plants can specifically recognize and respond to egg deposition, presumably via the detection of EAMPs. However, very few EAMPs have been identified, and those that have so far, are organic compounds of low-molecular weight associated with the eggs (Reymond, 2013; Hilker and Fatouros, 2015; Stahl et al., 2018). Only recently a novel type of EAMP, a proteinaceous compound, diprionin, was found as an elicitor of pine defense None of the egg wash treatments conducted, i.e., freezing and thawing, proteinase K, or boiling, had an effect on its bioactivity. That the elicitor does not lose the capability to induce HR in B. nigra leaves after these treatments, indicates that it is a small, stable, water-soluble molecule that is likely not a protein (Figure 6; Supplementary Table S7). Lastly, we tested whether two phosphatidylcholines (PC) could also induce a HR-like responses in B. nigra. We did not observe any visible 08 10.3389/fevo.2022.1070859 Caarls et al. A B D C FIGURE 5 Proportion of HR-like cell death induced by washes made of eggs, washes made of tissues of the reproductive tract of Pieris brassicae, and washes of eggs with and without glue, in Brassica nigra. (A–D) (A) Egg washes made of eggs of different ages or eggshells and egg glue remaining on filter paper. N plants = 13–26 (B) Egg wash of dissected structures of reproductive tract. N plants = 5. (C) Egg wash of eggs of mated females versus virgin females. N plants = 8–24 (D) Wash of eggs, glue alone (in filter paper) or wash of eggs with egg-enveloping secretions removed. For glue removal, two treatments were used, either a 1% bleach wash or wash with NaPO4. N plants = 8–16. Values that have no accompanying letters in common differ significantly (Kruskal– Wallis). A C C A D B B D Proportion of HR-like cell death induced by washes made of eggs, washes made of tissues of the reproductive tract of Pieris brassicae, and washes of eggs with and without glue, in Brassica nigra. (A–D) (A) Egg washes made of eggs of different ages or eggshells and egg glue remaining on filter paper. N plants = 13–26 (B) Egg wash of dissected structures of reproductive tract. N plants = 5. (C) Egg wash of eggs of mated females versus virgin females. Frontiers in Ecology and Evolution FIGURE 7 FIGURE 7 FIGURE 7 Phosphatidylcholines (PC) does not induce HR-like cell death in B. nigra plants unlike P. brassicae egg wash. Brassica nigra (SF48-O1) plants were treated with phosphatidylcholines PC(16:1/16:1) and PC(18:1/18:1) or with egg wash (EW). PCs were solubilized in 1% DMSO, 0.5% Glycerol and 0.1% Tween and were applied at different concentrations (1, 5, and 10 μg/μL). A solution of 1% DMSO, 0.5% Glycerol and 0.1% Tween was used as control (ctl). Egg wash400 was prepared with 400 eggs/mL, egg wash1000 was prepared with 1,000– 1,200 eggs/1 mL. All treatments were applied on the same leaf, with two leaves per plant, on 15 biological replicates. Scale bar = 1. FIGURE 7 Phosphatidylcholines (PC) does not induce HR-like cell death in B. nigra plants unlike P. brassicae egg wash. Brassica nigra (SF48-O1) plants were treated with phosphatidylcholines PC(16:1/16:1) and PC(18:1/18:1) or with egg wash (EW). PCs were solubilized in 1% DMSO, 0.5% Glycerol and 0.1% Tween and were applied at different concentrations (1, 5, and 10 μg/μL). A solution of 1% DMSO, 0.5% Glycerol and 0.1% Tween was used as control (ctl). Egg wash400 was prepared with 400 eggs/mL, egg wash1000 was prepared with 1,000– 1,200 eggs/1 mL. All treatments were applied on the same leaf, with two leaves per plant, on 15 biological replicates. Scale bar = 1. Our study reveals that the EAMP associated with P. brassicae eggs likely belongs to the first category of non-proteinaceous molecules. Neither boiling nor treatment of the egg wash with proteinase K had an effect on elicitation of HR-like. Recently, phosphatidylcholines (PCs) were identified as EAMPs of the P. brassicae egg-induced response in A. thaliana. Active PCs were mainly containing C16- and C18-fatty acyl chains and treatment with the latter resulted in SA, H2O2, induction of PR1 and trypan blue staining (Stahl et al., 2020). PCs are components of cell membranes. Earlier, phospholipids of Sogatella furcifera Horváth were also found to induce an ovicidal response in rice (Yang J. O. et al., 2014). Our results suggest that the EAMPs in P. brassicae eggs that induce HR-like cell death in B. nigra are other compounds than PCs. First, the B. nigra response is specific to Pieris eggs and egg wash and is absent in response to other lepidopteran eggs. FIGURE 7 Second, given our method used of washing eggs in a water-like buffer, lipids are not expected to be present in (high amounts in) the wash. Finally, as PCs are present in membranes, ovarian eggs should also induce the response. Indeed, ovarian eggs alone induced PR1 expression in A. thaliana (Little et al., 2007) while we showed that ovarian eggs did not induce HR-like cell death in B. nigra. In A. thaliana, PR1 expression was also found in response to crushed egg extracts of different insects (Bruessow et al., 2010; Stahl et  al., 2020). In B. nigra, there was no cell death underneath M. brassicae eggs, and neither induction of PR1 nor ethylene production in response to M. brassicae egg wash. Our results suggest that cell death, ethylene production and gene expression, at least in B. nigra, are specific to P. brassicae eggs, and we expect the response to be activated after detection of a Pierinae-specific elicitor. This hypothesis is strengthened by our previous work which showed Pierinae-specific induction of PR1 in B. nigra (Griese et al., 2021). It is possible that a mild cellular defense-like response is activated against a general insect-derived EAMP, for example PCs, while the strong HR-like is activated only by EAMPs specifically derived from Pieris spp. in those plants that can detect these. The production of ethylene after incubation with egg wash only in plants that show a strong HR-like cell death (and not in non-HR plants), points to this effect. Mapping efforts in B. nigra plants, can reveal whether the genetic variation in plants is for the HR-like cell death and/or for the detection of a Pieris-specific elicitor. g When washing eggs, mainly compounds from outside of the eggs and from egg-enveloping secretions are dissolved. We  present evidence that at least one Pieris-specific EAMP is in these secretions that envelop the eggs: (i) wash of glue on filter paper is sufficient to induce the HR-like cell death, (ii) when the secretions are removed from eggs, and eggs are then washed, HR-like cell death in plants is diminished, and (iii) a wash of ARGs, the organs where secretions are produced, is also sufficient to induce HR-like cell death. As the egg surface and egg-exterior associated secretions are in direct contact with the leaves, it could be expected that plants evolve to detect elicitors in egg-enveloping secretions. 3.6. EAMP is neither a protein nor lipid N plants = 8–24 (D) Wash of eggs, glue alone (in filter paper) or wash of eggs with egg-enveloping secretions removed. For glue removal, two treatments were used, either a 1% bleach wash or wash with NaPO4. N plants = 8–16. Values that have no accompanying letters in common differ significantly (Kruskal– Wallis). A B FIGURE 6 Proportion of HR-like cell death induced by differently treated egg washes made of Pieris brassicae eggs in Brassica nigra. (A,B) Severity of HR-like symptoms induced in plants by the different washes. (A) Egg washes that were subjected to increasing amount of freeze–thaw cycles. N plants/ treatment = 24–32. NS (B) Egg washes subjected to boiling (95°C for 30 min) or incubated with proteinase K. N plants/treatment = 9. NS, not significant, Kruskal–Wallis test. A B B FIGURE 6 Proportion of HR-like cell death induced by differently treated egg washes made of Pieris brassicae eggs in Brassica nigra. (A,B) Severity of HR-like symptoms induced in plants by the different washes. (A) Egg washes that were subjected to increasing amount of freeze–thaw cycles. N plants/ treatment = 24–32. NS (B) Egg washes subjected to boiling (95°C for 30 min) or incubated with proteinase K. N plants/treatment = 9. NS, not significant, Kruskal–Wallis test. treated with diprionin they emitted similar quantities of the sesquiterpene (E)-β-farnesene that is known to attract egg parasitoids like when laden with sawfly eggs (Hundacker et al., 2021). against sawfly eggs (Hundacker et al., 2021). Diprionin is an annexin- like protein which is released with the egg-associated secretions of the sawfly Diprion pini into needles of pines. When pine twigs were Frontiers in Ecology and Evolution 0 09 frontiersin.org 10.3389/fevo.2022.1070859 Caarls et al. FIGURE 7 Phosphatidylcholines (PC) does not induce HR-like cell death in B. nigra plants unlike P. brassicae egg wash. Brassica nigra (SF48-O1) plants were treated with phosphatidylcholines PC(16:1/16:1) and PC(18:1/18:1) or with egg wash (EW). PCs were solubilized in 1% DMSO, 0.5% Glycerol and 0.1% Tween and were applied at different concentrations (1, 5, and 10 μg/μL). A solution of 1% DMSO, 0.5% Glycerol and 0.1% Tween was used as control (ctl). Egg wash400 was prepared with 400 eggs/mL, egg wash1000 was prepared with 1,000– 1,200 eggs/1 mL. All treatments were applied on the same leaf, with two leaves per plant, on 15 biological replicates. Scale bar = 1. and ARGs of virgin P. frontiersin.org Frontiers in Ecology and Evolution 3.6. EAMP is neither a protein nor lipid brassicae butterflies induced HR-like cell death in an equal manner as mated butterflies. We thus hypothesize that at least one HR-inducing EAMP is present in the egg-enveloping secretions and ARGs and is female-derived. Chemical analysis of egg wash and glands is currently carried out to identify this EAMP. g y y Plant responses to insect eggs are similar to responses to (microbial) pathogens, and include SA and ROS accumulation, callose deposition, defense gene expression and cell death (Reymond, 2013). For example, callose deposition was shown to be  associated with lesions following pathogen invasion or autoimmune responses (Koga et al., 1988; Dietrich et al., 1994). We observed the deposition of callose in B. nigra associated with lesions induced by P. brassicae eggs. Natural variation in strength of egg-induced cell death was found in several brassicaceous species, including B. nigra (Pashalidou et al., 2015; Griese et al., 2021; Groux et al., 2021). Here, we show that plants that do not express a strong HR-like cell death, still responded with ROS accumulation and cell death as showed by trypan blue staining. Similarly, in S. dulcamara, variation exists for egg-induced chlorosis, and a genotype that did not respond with chlorosis and on which egg hatching rate of S. exigua was not reduced, still accumulated SA after oviposition (Geuss et al., 2017). We thus hypothesize that Pieris eggs generally induce an immune response in all plants of B. rapa and B. nigra, that is only in some plants accompanied by a stronger cell death response. In pathogen-induced HR, cell death can often be uncoupled from (preceding) biochemical and molecular changes and the two processes can be genetically dissected (Künstler et al., 2016). In that case, cell death is dispensable for resistance. However, in egg-induced HR, previous studies show that the stronger the HR-like cell death, the higher egg mortality (Fatouros et al., 2014; Griese et al., 2017, 2020, 2021). References Erb, M., and Reymond, P. (2019). Molecular interactions between plants and insect herbivores. Annu. Rev. Plant Biol. 70, 527–557. doi: 10.1146/annurev-arplant-050718-095910 Balint-Kurti, P. (2019). The plant hypersensitive response: concepts, control and consequences. Mol. Plant Pathol. 20, 1163–1178. doi: 10.1111/mpp.12821 Fan, L., Chae, E., Gust, A. A., and Nürnberger, T. (2017). Isolation of novel MAMP-like activities and identification of cognate pattern recognition receptors in Arabidopsis thaliana using next-generation sequencing (NGS)–based mapping. Current Protocols in Plant Biology 2, 173–189. doi: 10.1002/cppb.20056 Bassetti, N., Caarls, L., Bukovinszkine’kiss, G., El-Soda, M., Van Veen, J., Bouwmeester, K., et al. (2022). Genetic analysis reveals three novel QTLs underpinning a butterfly egg-induced hypersensitive response-like cell death in Brassica rapa. BMC Plant Biol. 22:140. doi: 10.1186/s12870-022-03522-y Beament, J. W. L., and Lal, R. (1957). Penetration through the egg-shell of Pieris brassicae (L.). Bull. Entomol. Res. 48, 109–125. doi: 10.1017/S0007485300054134 Beament, J. W. L., and Lal, R. (1957). Penetration through the egg-shell of Pieris brassicae (L.). Bull. Entomol. Res. 48, 109–125. doi: 10.1017/S0007485300054134 Fatouros, N. E., Broekgaarden, C., Bukovinszkine'kiss, G., Van Loon, J. J. A., Mumm, R., Huigens, M. E., et al. (2008). Male-derived butterfly anti-aphrodisiac mediates induced indirect plant defense. Proc. Natl. Acad. Sci. 105, 10033–10038. doi: 10.1073/ pnas.0707809105 Bruessow, F., Gouhier-Darimont, C., Buchala, A., Metraux, J.-P., and Reymond, P. (2010). Insect eggs suppress plant defence against chewing herbivores. Plant J. 62, 876–885. doi: 10.1111/j.1365-313X.2010.04200.x Fatouros, N. E., Lucas-Barbosa, D., Weldegergis, B. T., Pashalidou, F. G., Van Loon, J. J. A., Dicke, M., et al. (2012). Plant volatiles induced by herbivore egg deposition affect insects of different trophic levels. PLoS One 7:e43607. doi: 10.1371/journal.pone.0043607 Campos, M. L., De Souza, C. M., De Oliveira, K. B. S., Dias, S. C., and Franco, O. L. (2018). The role of antimicrobial peptides in plant immunity. J. Exp. Bot. 69, 4997–5011. doi: 10.1093/jxb/ery294 Fatouros, N. E., Paniagua Voirol, L. R., Drizou, F., Doan, Q. T., Pineda, A., Frago, E., et al. (2015). Role of large cabbage white butterfly male-derived compounds in elicitation of direct and indirect egg-killing defenses in the black mustard. Front. Plant Sci. 6:794. doi: 10.3389/fpls.2015.00794 Couto, D., and Zipfel, C. (2016). Regulation of pattern recognition receptor signalling in plants. Nat. Rev. Immunol. 16, 537–552. doi: 10.1038/nri.2016.77 Cui, H., Tsuda, K., and Parker, J. E. (2015). Effector-triggered immunity: from pathogen perception to robust defense. Annu. Rev. Plant Biol. 66, 487–511. doi: 10.1146/annurev- arplant-050213-040012 Fatouros, N. E., Pashalidou, F. FIGURE 7 It resembles the natural situation of leaf-egg interaction, more so than, for example, crushing of eggs (Little et al., 2007; Bruessow et al., 2010) or crushing of adults (Doss et al., 2000; Yang Y. et al., 2014). Previously, a male-derived anti-aphrodisiac compound transferred during mating to the female ARG, benzyl cyanide, was suggested as potential elicitor (Fatouros et al., 2008). We find no evidence for a male-derived elicitor: eggs Molecular patterns that are detected by plants are thought to be structurally conserved molecules (Van der Burgh and Joosten, 2019). The presence of EAMPs in the glue suggests that their function could be a structural component of the glue, or a compound with an essential function to the fertilized eggs. Many proteins are found in glue of insect eggs (Li et al., 2008), and egg glue of P. brassicae was described to consist of proteins and unsaturated lipoids (Beament and Lal, 1957). In addition, the ARG secretions could contain molecules that are produced by the parents and/or microbial symbionts to protect the vulnerable egg, Frontiers in Ecology and Evolution 10 frontiersin.org Caarls et al. 10.3389/fevo.2022.1070859 Supplementary material This research was made possible by support of the Dutch Technology Foundation TTW, which is part of Netherlands Organisation for Scientific Research (NWO) (NWO/TTW VIDI grant 14854 to N.E.F.). The Supplementary material for this article can be  found at: https://www.frontiersin.org/articles/10.3389/fevo.2022.1070859/full# Supplementary-material Acknowledgments for example compounds with antimicrobial activity (Flórez et al., 2018). Further identification of EAMPs can lead to new research in this direction. We are grateful to the employees of Unifarm (WUR) for rearing and caring of the plants used in the experiment. We thank Pieter Rouweler, André Gidding, and the late Frans van Aggelen for rearing of Pieris brassicae and Mamestra brassicae. We thank Martijn Flipsen, Gabriella Bukovinszkine’Kiss, Klaas Bouwmeester, Femke van Doesburg, Ewan van Eijden and Arjen van der Peppel for assistance with some of the experiments. Guusje Bonnema and Erik Poelman are acknowledged for seeds of B. rapa and B. nigra, respectively. In summary, we present a method to obtain EAMPs from insect eggs, and using this method, show that the EAMP inducing the HR-like cell death response is in the egg glue, derived from the female ARG. We furthermore assess the chemical nature and specificity of these elicitors and the molecular response of Brassica plants to Pieris and other eggs. The obtained knowledge paves the way for future studies on identification of EAMPs in Pieris egg glue, and the corresponding receptor genes in Brassica plants. Data availability statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The datasets presented in this study can be  found in the data repository Zenodo at doi.org/10.5281/zenodo.7194716. Publisher’s note LC, NB, JL, RM, and NF planned and designed the research. LC, NB, and PV performed the experiments and/or analyzed the data. 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BMC Plant Biol. 14:145. doi: 10.1186/1471-2229-14-145 Lortzing, T., Kunze, R., Steppuhn, A., Hilker, M., and Lortzing, V. (2020). Arabidopsis, tobacco, nightshade and elm take insect eggs as herbivore alarm and show similar transcriptomic alarm responses. Sci. Rep. 10:16281. doi: 10.1038/s41598-020-72955-y 12 frontiersin.org Frontiers in Ecology and Evolution
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A systematic approach for risk assessment and mitigation of wind integrated electricity markets
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*Corresponding author: naresh.2021ree07@mnnit.ac.in E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 https://doi.org/10.1051/e3sconf/202447201010 A systematic approach for risk assessment and mitigation of wind integrated electricity markets Naresh Boda1,*, Prashant Kumar Tiwari2, and Rajesh Panda3 1,2Motilal Nehru National Institute of Technology Allahabad, UP, India, 211014 3SSN College of Engineering Chennai, India Abstract. A risk-based double auction mechanism with dispatchable loads is one of the most cost-efficient trading mechanisms in the modern real-time electricity markets. Risks in the system due to various uncertainties, such as line failure, generator failure, load changes and other severe contingencies causes non-systematic execution of real-time electricity markets. This paper presents a simple and systematic procedure for reducing system risks in deregulated power markets considering wind power integrations. For maximizing the considerable system social welfare and minimizing the system's economic risk, the optimal placement and operation of wind power producers (WPP) have been suggested. The critical buses have been identified by accessing the different assessment tools e.g., Value at Risk (VaR), Conditional Value at Risk (CVaR), and locational marginal pricing (LMP) based Marginal Pricing Index (MPI). The presented approach has been evaluated and validated on modified IEEE 30-bus system. Keywords: Risk Assessment, Locational Marginal Price, Wind Power Producers, Real Time Electricity Market. Keywords: Risk Assessment, Locational Marginal Price, Wind Power Producers, Real Time Electricity Market. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 In this context, a strategic bidding model is used for different contingencies and uncertainties with reference to WPP, the market price, demand bids and competitor offers [3]. Determine the best offer price for WPP by using multistage risk restrained stochastic interrelated model is used in electricity market[4]. The stability and security of the restructured power system can be improved by reducing the system risk and to assess the risk using risk hedging tools are proposed[5]. To trade the future value of the wind power the WPP may take a part in the prediction market and by accepting a different stance from the electricity market, the imbalance costs will be compensated for by rewards in the prediction market [6]. The energy output of wind power producers could be increased, and the cost of producing energy might be decreased, with the use of wind power bidding techniques. When wind power penetration is minimal, it is fair to view WPP as price-takers, as stated in [7]. The producers of wind energy are strategic participants in DA and RT markets, and impact of WPP penetration on energy prices cannot be ignored. By using the EPEC method, market equilibrium was discovered, which maximized the sum of expected profits from all producers [8]. While maximize the profit of the DA and ancillary services a strategic bidding is used to reduce the generation cost, and improving the revenue of GenCos & DisCos in electricity market [9]. In order to increase the profit, a bidding technique is proposed for WPP and energy storage systems in DA and Real-time electricity markets [10–11]. The proper risk measure approach is suggested to find the best scheduling for various risk levels [12–13]. At GenCo’s bids are generated by using best bidding strategy, profit of the system is increased while reducing risk [14–15]. The risk is considered an impediment to the bid that would increase the profit while reducing the uneven prices of the optimal placement of WPP in spot market. A risk-averse trading mechanism is proposed and assessed against several risk indicators, and an investigation of systemic risk of the power market is assessed by using two indices, i.e., contagious VaR (CoVaR) and marginal contagious VaR (ΔCoVaR) [16]. By considering uncertainty and coupling of risk in restructured power system, the accuracy of risk evaluation to be increased while reducing financial losses by applying a proper hierarchical risk evaluation method[17]. 1 Introduction Renewable energy becomes optimistic in a deregulated power system in response to rising environmental perturbations. The most popular renewable energy source today is wind power, which is a great addition to the current energy grid. Because of its benefits, including quick building timelines, cost competitiveness, and environmental consequences, wind power has emerged as a strong contender in long-term system planning. As a result, the world's installed wind power capacity has rapidly increased, with China, the United States, Brazil, Germany, and Sweden leading the way. Currently, there has been 906 GW of installed wind capacity worldwide. This reflects 9% year-over-year growth. The addition of 78 GW globally in 2022 made it the third-biggest year ever for new capacity [1]. In India, wind capacity increased two times from 21 GW to 42.6 GW since 2014, and India is globally in the fourth position in renewable energy [2]. Looking towards the increased wind potential in the power system networks, there are several research findings have been reported by the researchers in the past to handle the integration issues, intermittency etc. related to wind power in conventional and competitive power markets. https://doi.org/10.1051/e3sconf/202447201010 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 2.1 Electricity Market Mechanism A market is a system that facilitates the exchange of related goods and services for economic benefit. Sellers and buyers are getting profit in an auction market. This is defined as a place to trade with the auctioneers, and it is classified into two types, i.e., single-sided, and double- sided auction markets. New uncertainties are introduced as a result of the deregulation of the power sector. Recently, deregulation has given generation firms (GenCo’s) and distribution entities (DisCo’s) a platform to investigate price potential in the universally competitive electricity markets. In power system deregulation, DA and RT are the two notable time periods for market settlement. The RT market utilization is calculated according to real time balance of supply and demand. Market clearance occurs in the DA market before 24 hours of the following trading day, where offers and bids are decided on one DA. Sellers in electrical market typically enter the DA for the following day’s trade to submit offers to the system operator. In double auction market both buyers and sellers submit their offers and bids to the ISO in real time[27]. To avoid market power during the multi-round auction procedure, both generation company and distribution company transmit offers and bids to the ISO at the same time, and ISO clear the market based on MCP. The MCP is determine the offers price and demand bids are interact at one point as submitted to the ISO. using specific protocols selected by the ISO based on their transmission grid restrictions and pricing methods. Uniform and discriminatory are the two different ways pricing mechanisms are classified. Seller and buyer pairs are matched individually in a discriminatory pricing scheme, and every time in a double auction market, buyer and seller are matched equally in a uniform pricing scheme. The bid and offer price-quantity are iteratively connected through buyers and sellers in a double auction model to be submitted to the ISO. The MCP is the interaction between the bid curve and offer curve and the nodal price given by the LMP at each node of the deregulated power system [28]. LMP can be maximized at the voltage controlled node by maximizing consumer excess, while LMP can be reduced at P-Q bus by reducing produced excess. Flexible loads and fixed loads are the two load models in a power system. Under uncertain conditions in the power system, fixed loads do not change. E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 https://doi.org/10.1051/e3sconf/202447201010 The primary contribution of this article is to assess the risk associated with the dispatchable loads are used in double auction strategy under various uncertainties, including line fault, generator fault, and load changing in the RT market using various risk assessment tools. By locating wind power producers, the risk-averse solution is attained, and the GenCo’s profits can be maximized in RT market. The remainder of this article is structured as follows: Section II initially describes the overview of the methodology. Section III indicates problem formulation of presented strategy. Numerical results and discussion of presented strategy describes in Section IV. Finally, Conclusion of the article describe in Section V. E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 A bidding strategy to minimize projected cost under risk constraint optimum rigid and malleable bids in Day ahead and Real time markets are evaluated in [18]. By considering sensitivity factor, the system profit increase while reducing the generation cost of an electricity market[19]. A regular scheduled market auction bidding method has been used to increase the profit of the GenCo by setting bid price as payas-bid [20-21]. To improve prediction models and represent various preferences on WPP models of a real-world system to propose a risk assessment based adaptive USTWPP frame work is evaluated[22]. While considering the uncertainty surrounding renewable generation, real- time price, and power consumption method ensures the robustness of the day-ahead (DA) bidding strategy are used to reduce predicted regret value over a group of worst-case situations whose combined probability is no greater than a certain threshold[23]. For optimal placement of WPP in DA and RT electricity market by using an optimum bidding strategy of various market players are considered[24]. Find optimum strategy to place WPP in day ahead and balancing market by using a multi-stage risk-constrained stochastic approach is proposed in [25]. A Systemic risk investigation of future electricity market to propose a theoretical model for systemic analysis of the power market[26]. 2 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 2.2 Risk Assessment The existence or severity of system risks in the evaluated networks in accordance with LMP is investigated using the concept of risk evaluation techniques (i.e., VaR and CVaR). Fig.1. shows that VaR & CVaR are risk evaluation indices, with VaR designating a small loss of (1-α) the loss distribution of the system [30]. loss of (1 α) the loss distribution of the system [30]. Fig.1. Illustration of VaR and CVaR[29] Fig.1. Illustration of VaR and CVaR[29] 2.1 Electricity Market Mechanism A relatively easy technique is to model price-sensitive loads accompanying negative charges with uncooperative real power 3 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 https://doi.org/10.1051/e3sconf/202447201010 injections. The negative output of the generator is described with injection levels, ranging from zero to highest negative conceivable loads[29]. injections. The negative output of the generator is described with injection levels, ranging from zero to highest negative conceivable loads[29]. 2.3 Marginal Pricing Index(MPI) The presented strategic index is used to determine which nodes are most price-sensitive after line interruptions and other contingency situations. The level of risk in the system is identified by using price sensitive buses.   , b a se A O i M a x A O a b s M P I      (1) (1) Where is MPIi at bus i. λBas and λAO base case LMP values at bus ‘i’ after line interruptions. After various contingency conditions, λMax,AO has the highest LMP value among all buses in the system [31]. 2.4 Proposed Strategy To find the optimal placement of WPP in deregulated power system by using a strategic risk assessment of the system and to assess the risk by using risk indices i.e., VaR and CVaR and to find most critical bus by using marginal pricing index(MPI). The application of these risk assessment methods and MPI has greatly reduced the area of search for the optimal spot, and it has been demonstrated that placement locations are important for managing economic risk. 4 4 4 https://doi.org/10.1051/e3sconf/202447201010 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 Fig.2. Flow Chart of presented strategy Fig.2. Flow Chart of presented strategy 3 Problem Formulation A method consists of a risk-constrained double auction strategy in RT electricity market under deregulated power system has been presented that evaluate cost minimization, profit maximization, and social welfare maximization for each GenCo’s and DisCo’s with ideal placement of WPP. Mathematically, objective function is defined as below: Objective Function-I Objective Function-I             2 1 2 1 gi dj j dj i gi ng i gi i i gi i i nd j dj j j dj j j MaxF B P C P C P a P b P c B P a P b P c             (2) j             2 1 2 1 gi dj j dj i gi ng i gi i i gi i i nd j dj j j dj j j MaxF B P C P C P a P b P c B P a P b P c             (2) Here, Ci(Pgi) is Power system generation cost and Bj(Pdj) is the profit of the pool users to pay from demand. Social welfare will be increased while reducing the generation costs of the system. Here, Ci(Pgi) is Power system generation cost and Bj(Pdj) is the profit of the pool users to pay from demand. Social welfare will be increased while reducing the generation costs of the system. 5 5 https://doi.org/10.1051/e3sconf/202447201010 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 Objective Function-II Risk of the system measured with the risk evaluation tools i.e., VaR and CVaR, with VaR designating a small loss of (1- α) the loss distribution of the system for a certain level [0,1], and VaR is less successful than CVaR in a scenario of continuous loss distribution. To conclude that CVaR is the average loss at the lower back end of the loss dispensation.         ( , ) , min : , g A Y a A x Y dY A R A             (3)     1 1 1 Ns s s s MinimizeCVaR          (4)         ( , ) , min : , g A Y a A x Y dY A R A             (3)     1 1 1 Ns s s s MinimizeCVaR          (4) subjected to , 1,2,3,...., S s EP s Ns      (5) 0, 1, 2,3,...., s s Ns   (6) (3) (4) subjected to j , 1,2,3,...., S s EP s Ns      (5) 0, 1, 2,3,...., s s Ns   (6) , 1,2,3,...., S s EP s Ns      0, 1, 2,3,...., s s Ns   (6) Here, β indicate the aide positive coefficient and distinguish between VaR(ξ) and scenario payoff. Equations (3) and (4) show the equations for VaR and CVaR, respectively. The optimal configuration of wind power producers (WPP) is used to fulfil the objective function concurrently. Here, β indicate the aide positive coefficient and distinguish between VaR(ξ) and scenario payoff. Equations (3) and (4) show the equations for VaR and CVaR, respectively. The optimal configuration of wind power producers (WPP) is used to fulfil the objective function concurrently. In this article, VaR, CVaR, and MPI are the risk assessment tools. Fig. 2 shows an example of VaR and CVaR with profit maximization. When the values of VaR and CVaR are negative maximums on left side of the chart tail end, then the loss is increased and profit of the system is reduced, respectively. Equality Constraints: 1 g N g W d Loss a P P P P      (11)   2 2 1 2 cos d N Loss j i j i j i j a P G V V V V               (12)   1 cos B N i i k ik ik i k k P VV Y         (13)   1 sin B N i i k ik ik i k k Q VV Y         (14) (12) (14) From equations (11) and (12), power balance equations are power from wind, Ploss is transmission loss, and Gj is conductance between node a and k. Vi and Vj are the magnitudes of voltages at node i and j. Equation (13) and (14), shows that active and reactive power of the system. Fig.1 illustrates that, for the negative greatest value of VaR and CVaR, the system loss is high and the profit of the system is decreased, while for the positive greatest value of VaR & CVaR, the system loss decreases and the profit of the system increases at the right-back end of the diagram. At higher system-risk conditions, maintaining the stability and security of the system required some further arrangements. So, at most severe risk conditions, the cost of generation is improved in addition to the actual cost of production and ancillary costs. According to the above explanation, it is clear that the system's risk has an inverse relationship with VaR and CVaR. It means that the system's risk will be higher for the lower (highest negative) values of VaR and CVaR. So, it is essential to reduce the risk of the system and shift the graph (Fig.1.) from the left back-end to the right back-end, i.e., the value of VaR and CVaR is at its low value in the positive direction. The main aim of the presented strategy is to reduce the generation cost of the system. Consequentially, the values of VaR and CVaR have an inverse relation to the objective function (system generation cost) of the presented strategy. Therefore, the generation cost of the system decreases while increasing the value of social welfare. So, VaR and CVaR are proportional to the social welfare of the system. Objective Function-II At the tail end of the histogram, the values of generation cost and social welfare are minimized and maximized, for the maximum(positive) value of VaR and CVaR. To maintain the system's stability and security, some additional arrangements are required for higher system risks. The actual generation cost increases with higher risk and additional costs. Therefore, in a higher-risk scenario, social welfare is minimized. Constraints: Ensuring equality & inequality constraints are contemplate while resolving OPF issues. Inequality Constraints: min max i i i V V V   1,2,3,....., B i N  (7) min max i i i      1,2,3,....., B i N  (8) min max gi gi gi P P P   1,2,3,....., B i N  (9) min max gi gi gi Q Q Q   1,2,3,....., B i N  (10) Inequality Constraints: min max i i i V V V   1,2,3,....., B i N  (7) min max i i i      1,2,3,....., B i N  (8) min max gi gi gi P P P   1,2,3,....., B i N  (9) min max gi gi gi Q Q Q   1,2,3,....., B i N  (10) (10) (10) Here min iV and max iV lower limit and upper limit of the voltage at node i. min i and max i are lower and upper limits of phase angle at node i. min gi P and min gi Q are lower limit of real and 6 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 https://doi.org/10.1051/e3sconf/202447201010 reactive power at node i. max gi P , and max gi Q upper limit of real and reactive power at node i. Pd & Ploss are the power demand and power loss of the system. reactive power at node i. max gi P , and max gi Q upper limit of real and reactive power at node i. Pd & Ploss are the power demand and power loss of the system. 4 Simulation Results and Discussion The customized IEEE 30-bus data system has been evaluated to demonstrate the proposed strategy. The customized data test system contains 41 transmission lines, 30 node, 6 thermal generators, and four dispatchable loads. A complete proposed strategy has been developed in MATPOWER software [29], and MILP based AC optimal power flow has been employed for optimization. Three separate case studies on the system were studied in the entire execution and evaluation of the presented strategy in three cases as shown below. 7 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 https://doi.org/10.1051/e3sconf/202447201010 1. Generate Scenarios by considering different contingencies and study the VaR, CVaR by using scenarios based LMP and calculated most critical buses by using marginal pricing index(MPI). 1. Generate Scenarios by considering different contingencies and study the VaR, CVaR by using scenarios based LMP and calculated most critical buses by using marginal pricing index(MPI). p g ( ) 2. Finest of the most endangered buses for placement of wind power producers(WPP). 2. Finest of the most endangered buses for placement of wind power producers(WPP). 3. After the placement of WPP, to calculate the profit of the system before and after placement of WPP. 3. After the placement of WPP, to calculate the profit of the system before and after placement of WPP. Case 1: Given this, 42 different contingency scenarios have been created based on generator outages, line outages, and changes in loads. To determine the value of VaR & CVaR based on LMP of the system at different contingency conditions are used to determine the risk level of the selected scenarios. Fig. 3 represents risk(VaR and CVaR) according to confidence level 95%. From Fig. 3, The VaR and CVaR that the system is inversely correlated to risk of the system. So, social welfare of the deregulated system is reduced when the VaR and CVaR values are maximised, and vice versa. maximised, and vice versa. (a) (b) Fig.3. a) VaR and Social welfare ($/hr) at 95% before WPP and b) CVaR and Social welfare($/hr) at 95 % before WPP To find the riskiest nodes in the deregulated power system by using marginal pricing index(MPI) for optimal placement of the WPP. The calculation of MPI is based on LMP of the system. The values of MPI are subsequently organised into descending order to create a list of things to prioritise (Table 1 illustrates this). 4 Simulation Results and Discussion The system's most sensitive bus is represented by MPI's highest value. For the installation of wind farms, the top 6 buses (aside from generator buses) with the highest MPI values (bus numbers 29, 30, 27, 26, and 25) have been selected. 500 550 600 -0.25 -0.15 -0.05 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Social Welfare ($/hr) VaR Scenario's VaR at 95% Social Welfare 500 550 600 650 -0.40 -0.20 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Social Welafare ($/hr) CVaR Scenario's CVaR at 95% Social Welfare (a) (b) Fig.3. a) VaR and Social welfare ($/hr) at 95% before WPP and b) CVaR and Social welfare($/hr) at 95 % before WPP 500 550 600 -0.25 -0.15 -0.05 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Social Welfare ($/hr) VaR Scenario's VaR at 95% Social Welfare 500 550 600 650 -0.40 -0.20 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Social Welafare ($/hr) CVaR Scenario's CVaR at 95% Social Welfare (b) Fig.3. a) VaR and Social welfare ($/hr) at 95% before WPP and b) CVaR and Social welfare($/hr) at 95 % before WPP 500 550 600 650 -0.40 -0.20 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 Social Welafare ($/hr) CVaR Scenario's CVaR at 95% Social Welfare ( ) Fig.3. a) VaR and Social welfare ($/hr) at 95% before WPP and b) CVaR and Social welfare($/hr) at 95 % before WPP To find the riskiest nodes in the deregulated power system by using marginal pricing index(MPI) for optimal placement of the WPP. The calculation of MPI is based on LMP of the system. The values of MPI are subsequently organised into descending order to create a list of things to prioritise (Table 1 illustrates this). The system's most sensitive bus is represented by MPI's highest value. For the installation of wind farms, the top 6 buses (aside from generator buses) with the highest MPI values (bus numbers 29, 30, 27, 26, and 25) have been selected. 4 Simulation Results and Discussion To find the riskiest nodes in the deregulated power system by using marginal pricing index(MPI) for optimal placement of the WPP. The calculation of MPI is based on LMP of the system. The values of MPI are subsequently organised into descending order to create a list of things to prioritise (Table 1 illustrates this). The system's most sensitive bus is represented by MPI's highest value. For the installation of wind farms, the top 6 buses (aside from generator buses) with the highest MPI values (bus numbers 29, 30, 27, 26, and 25) have been selected. 8 8 8 https://doi.org/10.1051/e3sconf/202447201010 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 Table 1: shows the MPI-based priority list. Bus No. Locational Marging Pricing Marginal Pricing Index(MPI) Rank Base Case (λbase) After Line ouatge (λAO) Difference (λbase-λAO) 29 4.075 8.573 4.498 0.52467 1 30 4.155 7.174 3.019 0.35215 2 27 3.960 4.456 0.496 0.05786 3 26 4.064 4.518 0.454 0.05296 4 25 3.988 4.386 0.398 0.04642 5 24 3.981 4.144 0.163 0.01901 6 23 3.994 4.094 0.100 0.01166 7 28 3.939 4.045 0.106 0.01236 8 22 3.966 4.041 0.075 0.00875 9 21 3.970 4.042 0.072 0.00840 10 19 4.029 4.101 0.072 0.00840 12 20 4.008 4.075 0.067 0.00782 11 Table 1: shows the MPI-based priority list. Case 2: From Fig.4&5, VaR and CVaR at each bus of the system shows before and after placement of WPP in existing system at different confidence levels i.e., 95% and 99%.According to the below shown figure before WPP, the most critical buses are 30, 29, 18, 20, and 25. Compared to 18, 20, and 25, the critical buses are 30 and 29. The optimal placement of wind power producers is between 30 and 29 buses. After the placement of WPP, the risk of the system is reduced due to the reduced generation cost and increase in social welfare. (a) -0.60 -0.30 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 VaR Bus No. VaR at 99% before WPP VaR at 99% after WPP (a) -0.60 -0.30 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 VaR Bus No. VaR at 99% before WPP VaR at 99% after WPP (a) 9 9 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 https://doi.org/10.1051/e3sconf/202447201010 (b) Fig.4. 4 Simulation Results and Discussion VaR at each bus of the system before and after placement of WPP at 95% confidence level b) at 99% confidence level (a) (b) Fig.5. VaR at each bus represents before and after placement of WPP a) at 95% confidence level b) at 99% confidence level ase 3: The social welfare of every seller and buyer under various scenarios is computed fore and after the placement of wind power producers. The system's social welfare with d without wind energy producers as compared to 42 different scenarios, as illustrated in g. 6. -0.40 -0.20 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 VaR Bus No. VaR at 95% before WPP VaR at 95% after WPP -1.60 -0.80 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 CVaR Bus No. CVaR at 99% before WPP CVaR at 99% after WPP -0.50 -0.25 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 CVaR Bus No. CVaR at 95% before WPP CVaR at 95% after WPP SD 2023 (b) Fig.4. VaR at each bus of the system before and after placement of WPP 95% fid l l b) 99% fid l l -0.40 -0.20 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 VaR Bus No. VaR at 95% before WPP VaR at 95% after WPP (b) Fig.4. VaR at each bus of the system before and after placement of WPP at 95% confidence level b) at 99% confidence level (b) ig.4. VaR at each bus of the system before and after placement of WPP at 95% confidence level b) at 99% confidence level (b) Fig.5. VaR at each bus represents before and after placement of WPP a) at 95% confidence level b) at 99% confidence level -0.50 -0.25 0.00 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 CVaR Bus No. CVaR at 95% before WPP CVaR at 95% after WPP Case 3: The social welfare of every seller and buyer under various scenarios is computed before and after the placement of wind power producers. The system's social welfare with and without wind energy producers as compared to 42 different scenarios, as illustrated in Fig. 6. 5 Conclusion Presented strategy is effective for minimizing system financial risks according to the LMP of the system and recognizing the riskiest bus for optimal placement of WPP in centralized double-auctioned restructured power system. A MPI also used to evolve and find out the more sensitive nodes with the help of LMP at different contingencies in deregulated system. To minimize economic risk, priority based MPI and risk assessment tools are used to position wind generators in a deregulated system. The test results illustrate the success of the recommended strategy with reference to risk mitigation, system social welfare enhancement, and total generation cost reduction. As a result, the suggested approach is demonstrating its robustness in terms of reducing economic risk in competitive power markets. This generalised technique can be relating any small-scale, medium-scale, and large scale integrated system. 4 Simulation Results and Discussion Case 3: The social welfare of every seller and buyer under various scenarios is computed before and after the placement of wind power producers. The system's social welfare with and without wind energy producers as compared to 42 different scenarios, as illustrated in Fig. 6. 10 E3S Web of Conferences 472, 01010 (2024) ICREGCSD 2023 https://doi.org/10.1051/e3sconf/202447201010 Fig.6. 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https://genomebiology.biomedcentral.com/counter/pdf/10.1186/s13059-022-02692-0
English
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De novo reconstruction of cell interaction landscapes from single-cell spatial transcriptome data with DeepLinc
Genome biology
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© The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. METHOD Open Access * Correspondence: yangxuerui@ tsinghua.edu.cn * Correspondence: yangxuerui@ tsinghua.edu.cn Abstract Based on a deep generative model of variational graph autoencoder (VGAE), we develop a new method, DeepLinc (deep learning framework for Landscapes of Interacting Cells), for the de novo reconstruction of cell interaction networks from single-cell spatial transcriptomic data. DeepLinc demonstrates high efficiency in learning from imperfect and incomplete spatial transcriptome data, filtering false interactions, and imputing missing distal and proximal interactions. The latent representations learned by DeepLinc are also used for inferring the signature genes contributing to the cell interaction landscapes, and for reclustering the cells based on the spatially coded cell heterogeneity in complex tissues at single-cell resolution. MOE Key Laboratory of Bioinformatics, Center for Synthetic & Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China Keywords: Single-cell spatial transcriptome, Cell interaction, Variational graph autoencoder, VGAE, Deep learning De novo reconstruction of cell interaction landscapes from single-cell spatial transcriptome data with DeepLinc Runze Li and Xuerui Yang* Background The physiological functions of multicellular tissues are not only defined by heteroge- neous cells forming these tissues but are also highly dependent on complicated local and distal cell-cell interactions [1, 2]. Furthermore, it is increasingly recognized that many of the intracellular activities of each single cell are closely related to its interac- tions with the multicellular context [3]. The intrinsic gene expression profile of each single cell is both a consequence and a defining factor of the complicated cell inter- action network in physiological contexts [4, 5]. Recent advances in various spatially resolved transcriptome profiling techniques have made it possible to measure gene expression profiles at single-cell or subcellular reso- lution while simultaneously retaining information on the spatial locations of cells. These techniques include in situ sequencing methods, such as FISSEQ [6] and STAR- map [7]; imaging methods based on fluorescence in situ hybridization (FISH), such as MERFISH [8], seqFISH [9], SPOTs [10], and osmFISH [11]; spatial barcoding tech- niques, such as Slide-seq [12], HDST [13], and DBiT-seq [14]; and laser capture micro- dissection (LCM) combined with flow cytometry methods, such as GEO-seq [15] and Li and Yang Genome Biology (2022) 23:124 https://doi.org/10.1186/s13059-022-02692-0 Li and Yang Genome Biology (2022) 23:124 https://doi.org/10.1186/s13059-022-02692-0 Li and Yang Genome Biology (2022) 23:124 Page 2 of 24 Page 2 of 24 Li and Yang Genome Biology TSCS [16]. Due to different experimental designs, the coverage of these transcriptome profiles ranges from tens to thousands of genes. Spatially resolved transcriptome profiles provide insightful resources for understand- ing the cellular organization patterns in multiple types of tissues and organs, such as the central nervous system [13, 17, 18], developing human heart [19], and tumors [13, 20]. However, these snapshots of tissue sections are still incomplete observations of the multicellular organization and the potential local interactions between geometrically adjacent cells. It remains a major challenge to infer full cell interaction networks, in- cluding distal cell-cell interactions, which are potentially mediated by a broad range of different mechanisms. Various methods are currently available for spatially resolving predefined cell types with bulk RNA-seq or scRNA-seq data and for inferring cell-cell interactions based on known ligand-receptor pairs or other predefined features and ref- erences [21–25]. However, methods for the de novo reconstruction of cell interaction networks in an unbiased and more comprehensive manner by taking full advantage of spatial transcriptome profiles at single-cell resolution are still lacking. In addition, due to various technical limitations, spatially resolved single-cell tran- scriptome profiles suffer to different extents from data imperfections such as too many missing values, batch effects, biased and low coverage, and high noise levels [26, 27], which necessitate methods with a sophisticated design for mining cell-cell interactions from such imperfect and incomplete snapshots. Ideally, such methods should be cap- able of reducing the noise of spatial transcriptome data, reconstructing existing interac- tions, restoring missing interactions (including distal interconnections), and mining latent features related to cell-cell interaction landscapes. The nonlinear, high-dimensional, sparse and multimodal features of single-cell spatial transcriptome data make it a proper and feasible target of the deep learning strategy. Multiple deep learning models have been applied for various tasks with single-cell spatial transcriptome data, for example, integration of histology to define spatial do- mains and predict local gene expressions with convolutional networks [28, 29], imput- ation of spatial transcriptome profiles by graph-regularized tensor completion [30], inference of gene-gene interactions with convolutional networks [31], and integration of sc/snRNA-seq data with nonconvex optimization to resolve in situ cell clusters [32]. These methods serve as powerful frameworks for learning from multimodal data and resolving the spatial cell and gene organizations in tissues [33]. However, none of them were designed to directly uncover the cell-cell interactions that shape the tissue organization and define tissue physiological functions. Deep generative models such as generative adversarial networks (GANs) and vari- ational autoencoders (VAEs) have been proven to be powerful tools for leveraging la- tent features and modeling high-dimensional scRNA-seq data for various tasks, such as denoising [34], clustering [35], dimension reduction [36], and missing value imputation [37]. In the present study, we adapted another type of deep generative model, vari- ational graph autoencoders (VGAEs) [38], for encoding cell-cell interaction features from spatial single-cell transcriptome data and eventually regenerating full cell-cell interaction landscapes. VGAE is a recently developed state-of-the-art machine learning algorithm in which the core of variational autoencoders has been adapted [39] and ex- tended for graph representation learning [40]. Our method, referred to as DeepLinc (deep learning framework for landscapes of interacting cells), uses the VGAE model to Li and Yang Genome Biology (2022) 23:124 Page 3 of 24 Page 3 of 24 integrate and learn from the two dimensions of information (i.e., cell interactions and gene expression profiles) during the encoding phase. Furthermore, the adversarial strat- egy was applied to force the encoder to explicitly approximate the Gaussian distribution [41]. The decoding phase then uses the latent representation learned during encoding to reconstruct a cell-cell interaction graph. Specifically, DeepLinc assumes that the neighboring cells should be much more likely to have some types of interactions than randomly picked non-neighboring cells that are far away from each other. Therefore, for a particular tissue region, the neighboring cell pairs comprise an incomplete and potentially noisy observation of a subset of the full cell-cell interaction network. The main task of DeepLinc is to learn from this subset of cell-cell interactions, extract the underlying features of single-cell transcriptome pro- files, and finally, regenerate a more unbiased and complete landscape of cell-cell inter- actions, which would include both proximal and distal interactions. To the best of our knowledge, DeepLinc is the first framework of its kind to apply a deep generative model for recovering cell interactions from spatial single-cell transcriptomic data. Based on a wide array of tests with real and simulated data, DeepLinc demonstrated its high efficiency in learning from imperfect and incomplete spatial transcriptome data, filtering false interactions, and inferring missing distal and proximal interactions. The reconstructed full networks of cell interactions exhibited high physiological relevance. Such de novo reconstructions of cell interaction networks do not depend on prior knowledge of cell types, ligand-receptor pairs, or cell interaction mechanisms. Further- more, the interrogation of the pipeline revealed signature genes that are potentially in- volved in shaping cell interaction landscapes. Finally, the reconstructed cell interaction landscapes, which are presumably more complete than the original snapshots of cell spatial organization, categorized hub cells and partitioned cells into further subclusters based on both the features of cell interaction landscapes and transcriptome profiles. These new insights could greatly aid in elucidating the biological relevance and poten- tial machinery underlying cell organization patterns in physiological contexts. In sum- mary, as a specially designed method based on deep learning, DeepLinc is of unique value for mining the latent features of cell interaction landscapes and, thus, for taking full advantage of previous and future spatial transcriptome profiling data at single-cell resolution. The DeepLinc model and the datasets used for testing The DeepLinc model and the datasets used for testing We assume that in a solid tissue the neighboring cells with direct contacts should be much more likely to have some types of interactions than randomly picked non- neighboring cells that are far away from each other. It is well recognized that single-cell transcriptome profiles represent both the driving force and consequences of cell-cell interaction landscapes [4, 5]. DeepLinc combines the VGAE and an adversarial network to learn from single-cell spatial transcriptome profiles and generate a latent distribution capturing the intrinsic associations between cell-cell interactions and the gene expres- sion patterns of single cells (Fig. 1A). Specifically, adjacent cell pairs defined simply by the geometric closeness between single cells were summarized as an undirected adjacency network in which the nodes Page 4 of 24 Page 4 of 24 Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology Fig. 1 Overview of DeepLinc and the datasets. A Schematic description of the DeepLinc pipeline. DeepLinc consists of an encoder, a decoder, and an adversarial regularization module in the VGAE framework. The encoder is a two-layer graph convolutional network (GCN) and the decoder is a Sigmoid function for the dot product of latent variables. The single-cell transcriptome profile and the cell adjacency matrix derived from a single-cell spatial transcriptome dataset serve as inputs of DeepLinc. The output of DeepLinc is a new matrix (A’) presenting the reconstructed cell-cell interaction network. In addition, DeepLinc also uses the latent information of cell interaction landscapes and gene expression profiles for the visualization and clustering of single cells. B Statistics of the 4 datasets used for reconstruction of cell interaction networks by DeepLinc represent the cells and the edges indicate the neighboring cell pairs. This network is represented by cell adjacency matrix A. The single-cell gene expression profiles, as fea- tures of the nodes in A, are presented as the matrix X (Fig. 1A). The graph-structured data (A) and the node features (X) are fed into the VGAE consisting of two graph con- volutional layers. As the output of this variational graph convolutional network (VGCN) encoder, the latent representation (H) captures the characteristics of a single cell itself and its neighboring cells. In addition, H is further constrained by an adversar- ial regularization module from a prior Gaussian distribution (Fig. 1A). Modeling of cell interactions by DeepLinc and the effects of denoising Modeling of cell interactions by DeepLinc and the effects of denoising Single-cell spatial transcriptome profiles provide snapshots of cell-cell interaction land- scapes, which are presumably just small subsets of interactions from complete cell-cell interaction networks and usually contaminated by high levels of noise. Therefore, the ability of DeepLinc to efficiently learn from the provided information on cell spatial organization and single-cell transcriptome profiles, filter out noise, correctly infer latent interactions, and eventually recover complete cell interaction landscapes is a critical benchmark. DeepLinc was applied to the 4 seqFISH, MERFISH, HDST olfactory bulb, and HDST breast cancer datasets (Fig. 1B). First, cell adjacency maps were defined by assembling the neighboring cells in the tissue sections. Specifically, the distances between each pair of cells (Additional file 1: Fig. S1A) or between each cell and its 3 closest neighbors (Additional file 1: Fig. S1B) were used to assess the overall distance distributions of ad- jacent cell pairs with potential direct interactions. The distance threshold for defining direct contacts in each tissue section was then determined based on the above distribu- tions (Additional file 1: Fig. S1B). For each cell, only the 3 closest neighbors falling under the distance threshold were defined as direct contacts. The union of the direct contacts of all the cells then constituted the full adjacency matrix. DeepLinc uses neigh- boring cells with direct contacts as the positive set for learning the transcriptome fea- tures related to cell-cell interactions. From a general biological point of view, we think it is reasonable to assume that in a solid tissue, most of the cells in 2-D could directly contact with 3 or more other cells. The edges of direct contacts were randomly divided into two groups for training (90%) and testing (10%). The negative set for testing, which was 100 times larger than the positive set, was composed of fake edges between two randomly picked non- neighboring cells on the tissue section. For all of the 4 datasets, DeepLinc showed high sensitivity and accuracy in recovering the originally annotated cell-cell interactions, with area under the receiver operating characteristic (AUROC) ranging from 0.8 to lar- ger than 0.9 (Fig. 2A) and false positive rate (FPR) below 5% (Additional file 1: Fig. S2). The DeepLinc model and the datasets used for testing Next, with the information learned above, the decoder performs a dot product operation on H to gen- erate a new adjacency matrix (A’) presenting the reconstructed cell-cell interaction net- work (Fig. 1A). On the other hand, the vectors of H, which represent the latent information of cell interaction landscapes and gene expression profiles, could be ex- tracted for the visualization and clustering of single cells. DeepLinc was applied to 4 published spatial transcriptomic datasets obtained from the mouse visual cortex [17], the preoptic region of the mouse hypothalamus [18], the mouse olfactory bulb [13], and human breast cancer [13] (Fig. 1B). Note that the spatial Li and Yang Genome Biology (2022) 23:124 Page 5 of 24 Page 5 of 24 Li and Yang Genome Biology transcriptome profiling techniques and the sparseness of the gene expression profiles vary greatly across these studies (Fig. 1B). In brief, the seqFISH and MERFISH datasets show much lower gene coverage (fewer than 200 genes) than the two HDST datasets (approximately 10,000 genes). However, the latter 2 high-throughput datasets are very sparse, and large numbers of genes were not detected in all the single cells in these datasets. Modeling of cell interactions by DeepLinc and the effects of denoising This process was repeated 30 times for each proportion of the missing edges to draw a boxplot. C The AUROCs between mixed-in fake edges and pre-existing real edges. Different numbers (1- to 10-fold of the size of the original cell adjacency map) of randomly generated fake edges were added into the original cell adjacency map, which was then fed to DeepLinc. This process was repeated 30 times for each level of fake edges to draw a boxplot Fig. 2 Performance of DeepLinc in reconstructing cell interaction networks. A The levels of AUROC at different training epochs of DeepLinc. The original data or data with different levels of random noise in the gene expression profiles were used. Larger σ values would result in higher levels of random noise in general (see “Methods” for details). B The AUROCs between the randomly removed real edges and originally nonexistent edges. Different proportions of the edges in the original cell adjacency maps were randomly picked and discarded. Then, the remaining edges were used for training the DeepLinc model. This process was repeated 30 times for each proportion of the missing edges to draw a boxplot. C The AUROCs between mixed-in fake edges and pre-existing real edges. Different numbers (1- to 10-fold of the size of the original cell adjacency map) of randomly generated fake edges were added into the original cell adjacency map, which was then fed to DeepLinc. This process was repeated 30 times for each level of fake edges to draw a boxplot Methods for details). B The AUROCs between the randomly removed real edges and originally nonexistent edges. Different proportions of the edges in the original cell adjacency maps were randomly picked and discarded. Then, the remaining edges were used for training the DeepLinc model. This process was repeated 30 times for each proportion of the missing edges to draw a boxplot. C The AUROCs between mixed-in fake edges and pre-existing real edges. Different numbers (1- to 10-fold of the size of the original cell adjacency map) of randomly generated fake edges were added into the original cell adjacency map, which was then fed to DeepLinc. This process was repeated 30 times for each level of fake edges to draw a boxplot transcriptome data. Modeling of cell interactions by DeepLinc and the effects of denoising Notably, DeepLinc modeled the cell interaction networks almost equally well with ei- ther the highly sparse high-throughput datasets (HDST olfactory bulb and HDST breast cancer) or the low-throughput datasets (seqFISH and MERFISH). Next, we tested the tolerance of DeepLinc to artificial noise in the single-cell gene ex- pression data. Specifically, different levels of random noise with Gaussian distributions were introduced on top of the original gene expression profiles of all the cells (Methods). As shown in Fig. 2A, performances of DeepLinc were not compromised by fairly high levels of noise, indicating the robustness of DeepLinc to noise in gene ex- pression data, which is critical when dealing with highly noisy single-cell spatial Page 6 of 24 Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology Fig. 2 Performance of DeepLinc in reconstructing cell interaction networks. A The levels of AUROC at different training epochs of DeepLinc. The original data or data with different levels of random noise in the gene expression profiles were used. Larger σ values would result in higher levels of random noise in general (see “Methods” for details). B The AUROCs between the randomly removed real edges and originally nonexistent edges. Different proportions of the edges in the original cell adjacency maps were randomly picked and discarded. Then, the remaining edges were used for training the DeepLinc model. This process was repeated 30 times for each proportion of the missing edges to draw a boxplot. C The AUROCs between mixed-in fake edges and pre-existing real edges. Different numbers (1- to 10-fold of the size of the original cell adjacency map) of randomly generated fake edges were added into the original cell adjacency map, which was then fed to DeepLinc. This process was repeated 30 times for each level of fake edges to draw a boxplot Fig. 2 Performance of DeepLinc in reconstructing cell interaction networks. A The levels of AUROC at different training epochs of DeepLinc. The original data or data with different levels of random noise in the gene expression profiles were used. Larger σ values would result in higher levels of random noise in general (see “Methods” for details). B The AUROCs between the randomly removed real edges and originally nonexistent edges. Different proportions of the edges in the original cell adjacency maps were randomly picked and discarded. Then, the remaining edges were used for training the DeepLinc model. Modeling of cell interactions by DeepLinc and the effects of denoising However, further corruption of the data dramatically impaired the performance of DeepLinc, illustrating the minimal gene expression profile information needed for DeepLinc to correctly rebuild the cell interaction landscapes. Single-cell spatial transcriptome data is largely suffering from high sparsity due to large numbers of dropouts, especially for the high-throughput data such as HDST. Therefore, we also generated two more noise models to simulate different types of dropouts (Additional file 1: Fig. S3). In the first model, different percentages of genes were randomly removed from the expression dataset to mimic dropouts of genes, and in the second model, different percentages of the non-zero values were randomly picked and forced to be zero, which mimics dropouts of individual datapoints in single cells. DeepLinc showed high tolerance to the noise from both types of dropouts (Add- itional file 1: Fig. S3). In general, the performances of DeepLinc were still fairly good with the dropout ratio of 50%. As well expected, more severe dropouts would strongly reduce the accuracy of DeepLinc. In summary, these tests confirm the advantage of DeepLinc in extracting the latent information of cell-cell interactions from the highly sparse and noisy spatial transcriptome profiles. Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology (2022) 23:124 Page 7 of 24 Page 7 of 24 DeepLinc also demonstrated relatively stable and high levels of fitting (AUC 85~95%) with different sizes of tissue sections that were randomly picked from the original frame of the tissue sections and therefore included fewer cells than the original data (Additional file 1: Fig. S4). The performance of DeepLinc was still fairly good with small tissue sections containing as few as 500 cells, indicating the efficient learning capability of DeepLinc. This is a useful feature for dissecting the cell organization of ultrafine tis- sue sections. We also tested DeepLinc for its capability to recover cell-cell interaction landscapes with different proportions of missing interactions. Specifically, certain percentages of the existing edges were randomly removed from the original cell-cell interaction net- work. The remaining network and the single-cell transcriptome data were then used for training the DeepLinc model. Ultimately, AUROC values were calculated to evalu- ate the performance of DeepLinc in correctly distinguishing the arbitrarily removed in- teractions (positives) and the originally nonexistent interactions (negatives). As shown in Fig. 2B, DeepLinc exhibited high precision in imputing the missing positive interac- tions. Modeling of cell interactions by DeepLinc and the effects of denoising For example, even when half of the interactions were lost in the input, DeepLinc still managed to recover these missing edges with 65–75% precision (Fig. 2B). This indi- cates that DeepLinc can indeed efficiently learn from limited and incomplete networks of cell interactions to reinstate missing edges. Such an imputation and prediction cap- ability is critical for applications of DeepLinc for the inference of full cell interaction landscapes from the incomplete snapshots generated from spatially resolved single-cell transcriptome profiles. In addition to the missing interactions (i.e., false negatives), false annotations of cell- cell interactions (i.e., false positives) that are fed to DeepLinc are another major issue to address. To test the robustness of DeepLinc in correctly recovering cell interactions with random false interactions (i.e., the denoising capability), we added fake edges be- tween randomly picked cell pairs into the original cell interaction network. Taking such highly noisy data as the input, DeepLinc can still nicely distinguish the originally non- existent edges and the real pre-existing edges (Fig. 2C). Even when fake edges were arti- ficially added in numbers as high as several-fold the original number of cell interactions, DeepLinc still managed to filter out the extensive noise and recover the predefined cell interactions with fairly good precision (Fig. 2C). This indicates the high tolerance of DeepLinc to random false interactions and its efficient denoising effects Reconstructed cell-cell interaction landscapes The landscapes of the reconstructed and the original cell-cell interaction networks were interrogated with a permutation test as described previously [21, 24, 42] to evaluate the enrichment or depletion of certain types of interactions in a network by comparison with randomly assembled networks with similar scales. As shown in Fig. 3A–D, the cell-cell interaction landscapes reconstructed by DeepLinc exhibited substantial differ- ences from the original cell adjacency networks composed of proximal cell pairs with direct contacts. For the visual cortex data of seqFISH, relative to the original network, the recon- structed network reinforced the interactions between the astrocytes and the two types of neurons (glutamatergic and GABAergic) (Fig. 3A). Interactions between epithelial Page 8 of 24 Page 8 of 24 Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology Fig. 3 Landscapes of cell interactions reconstructed by DeepLinc. A–D Evaluation of the over- or under- representation of particular types of interactions in the original cell adjacency networks (left) and in the reconstructed cell interaction networks (right) with the 4 datasets. The results are represented as dot heatmaps showing the interaction relationships between cell types. Dot size represents the connectivity between two cell types, calculated by the number of connections divided by the product of the cell numbers. The color of the dots represents significance (−log10P) determined by comparing with the permutation test, in which positive values indicate enrichment and negative values indicate depletion. E Original and reconstructed cell interaction networks in a representative tissue section from MERFISH dataset of hypothalamic preoptic area. The red box indicates a microglia cell with distal interactions with many other cells in the reconstructed networks Fi 3 L d f ll i i d b D Li A D E l i f h d Fig. 3 Landscapes of cell interactions reconstructed by DeepLinc. A–D Evaluation of the over- or under- representation of particular types of interactions in the original cell adjacency networks (left) and in the reconstructed cell interaction networks (right) with the 4 datasets. The results are represented as dot heatmaps showing the interaction relationships between cell types. Dot size represents the connectivity between two cell types, calculated by the number of connections divided by the product of the cell numbers. The color of the dots represents significance (−log10P) determined by comparing with the permutation test, in which positive values indicate enrichment and negative values indicate depletion. Reconstructed cell-cell interaction landscapes E Original and reconstructed cell interaction networks in a representative tissue section from MERFISH dataset of hypothalamic preoptic area. The red box indicates a microglia cell with distal interactions with many other cells in the reconstructed networks Fig. 3 Landscapes of cell interactions reconstructed by DeepLinc. A–D Evaluation of the over- or under- representation of particular types of interactions in the original cell adjacency networks (left) and in the reconstructed cell interaction networks (right) with the 4 datasets. The results are represented as dot heatmaps showing the interaction relationships between cell types. Dot size represents the connectivity between two cell types, calculated by the number of connections divided by the product of the cell numbers. The color of the dots represents significance (−log10P) determined by comparing with the permutation test, in which positive values indicate enrichment and negative values indicate depletion. E Original and reconstructed cell interaction networks in a representative tissue section from MERFISH dataset of hypothalamic preoptic area. The red box indicates a microglia cell with distal interactions with many other cells in the reconstructed networks cells and almost all the other cell types were significantly enriched in the reconstructed network. On the other hand, despite the high count, the interactions among the gluta- matergic neurons appeared depleted when compared to random networks (Fig. 3A), whereas such interactions were highly enriched in the original cell adjacency network. Therefore, the reconstructed network indicates high selectivity of the interactions among the excitatory glutamatergic neurons. For the MERFISH data of the mouse hypothalamic preoptic region, the reconstructed network strengthened the interactions within endothelial cells and between endothelial cells and astrocytes (Fig. 3B), similar to the observations based on the mouse visual cor- tex data discussed above. However, unlike the visual cortex, the mouse hypothalamic preoptic region showed enriched interactions between excitatory (mainly glutamatergic) and inhibitory (GABAergic) neurons in the reconstructed network. In addition, the in- teractions within excitatory neurons were strongly enriched (Fig. 3B), whereas the op- posite observation was obtained from the visual cortex data (Fig. 3A). Therefore, the above results revealed both common and tissue-specific cell interaction patterns in the two types of CNS systems. Reconstructed cell-cell interaction landscapes Finally, unlike the original cell adjacency network, the re- constructed network of the mouse hypothalamic preoptic region showed strong Li and Yang Genome Biology (2022) 23:124 Page 9 of 24 Page 9 of 24 enrichment according to the interactions among the mature oligodendrocytes, the in- teractions between microglia and many other cells, and the interactions between peri- cytes and other cells (Fig. 3B). The cell interaction network reconstructed from HDST data of the olfactory bulb showed more enriched interactions between oligodendrocytes and most of the other cell types, whereas the interactions between immune cells and other cells were reduced relative to the random networks (Fig. 3C). In addition, the interactions among vascular cells and glial cells (peripheral glia, astrocytes, and ependymal cells) were specifically in- tensified in the reconstructed network (Fig. 3C), indicating strong internal interactions within the complex blood–brain barrier composed of these cells [43–45]. Finally, for the breast cancer tissue profile data obtained with HDST, the recon- structed network exhibited strong interactions between lymphocytes (T cells and B cells) and almost all the other cell types (Fig. 3D). In contrast, the interactions between stromal cells and endothelial cells were enriched in the original network but depleted in the reconstructed network, suggesting high specificity of such interactions (Fig. 3D). In summary, by learning from the cell adjacency maps defined simply by the spatial organization of cells, which are incomplete, sparse, and noisy, DeepLinc performs the imputation of missing interactions and the filtration of potentially false connections. Ultimately, the reconstructed interaction networks are significantly enlarged and reshaped, which should recapitulate the cell interaction landscapes in a more compre- hensive and precise manner. As shown by the local regions from different tissues in Fig. 3E and Additional file 1: Fig. S5 as examples, small numbers of the originally defined direct cell interactions were removed, and new interactions were added by DeepLinc. The reconstructed net- works had more hierarchical and modularized structures. Interestingly, in the two ex- amples of visual cortex and hypothalamic preoptic area, microglia showed dense connections with many other cells, including neurons, astrocytes, endothelial cells, and oligodendrocytes, thereby serving as hub cells (Fig. 3E, Additional file 1: Fig. S5C), which nicely recapitulated the wide-ranging functions of microglia and their well- acknowledged interactions with other cell types in the CNS [46]. Such insight was miss- ing in the original cell-cell interaction network. Distal interactions in the reconstructed cell networks The probability of cell-cell interactions predicted by DeepLinc showed slightly negative correlations with geometric distances in general, and significant numbers of distal cell pairs presented high probabilities of interactions (Additional file 1: Fig. S6). Such distal interactions were missing in the original cell adjacency map, and DeepLinc demon- strated its capability to recover cell-cell interactions that are not restricted by spatial proximity. The newly recovered distal interactions showed distinct patterns of participating cell types. For example, in the seqFISH study of the mouse visual cortex, distal interactions were highly enriched between neurons (Fig. 4A). An example is provided in Fig. 4E to show the interactions between a glutamatergic neuron and other GABAergic neurons. By contrast, distal interactions were depleted between endothelial cells (Fig. 4A). This is in line with the notion that endothelial cells form a tight one-cell-thick interior Page 10 of 24 Page 10 of 24 Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology Fig. 4 Landscapes of distal interactions in the reconstructed cell networks. A–D Evaluation of the over- or under-representation of particular types of distal interactions in the reconstructed cell interaction networks with the 4 datasets. The results are represented as dot heatmaps showing the enrichment or depletion of the distal interactions between certain cell types. The color of the dots represents significance (−log10P) determined by comparing with the permutation test, in which positive values indicate enrichment, and negative values indicate depletion. E–G Examples of distal interactions between a glutamatergic neuron and GABAergic neurons (E), between an epithelial cell and stromal cells (F), and between a T cell and other T cells (G). The proximal interactions between neighboring cells are shown as backgrounds Fig. 4 Landscapes of distal interactions in the reconstructed cell networks. A–D Evaluation of the over- or under-representation of particular types of distal interactions in the reconstructed cell interaction networks with the 4 datasets. The results are represented as dot heatmaps showing the enrichment or depletion of the distal interactions between certain cell types. The color of the dots represents significance (−log10P) determined by comparing with the permutation test, in which positive values indicate enrichment, and negative values indicate depletion. E–G Examples of distal interactions between a glutamatergic neuron and GABAergic neurons (E), between an epithelial cell and stromal cells (F), and between a T cell and other T cells (G). Distal interactions in the reconstructed cell networks The proximal interactions between neighboring cells are shown as backgrounds Fig. 4 Landscapes of distal interactions in the reconstructed cell networks. A–D Evaluation of the over- or under-representation of particular types of distal interactions in the reconstructed cell interaction networks with the 4 datasets. The results are represented as dot heatmaps showing the enrichment or depletion of the distal interactions between certain cell types. The color of the dots represents significance (−log10P) determined by comparing with the permutation test, in which positive values indicate enrichment, and negative values indicate depletion. E–G Examples of distal interactions between a glutamatergic neuron and GABAergic neurons (E), between an epithelial cell and stromal cells (F), and between a T cell and other T cells (G). The proximal interactions between neighboring cells are shown as backgrounds interface in blood vessels, and it is not surprising to see a lack of distal interactions be- tween endothelial cells. In addition, a similar depletion of distal interactions between endothelial cells was observed in the HDST breast cancer data (Fig. 4D). In the hypothalamic preoptic region dataset obtained with MERFISH, the newly re- constructed distal interactions were depleted between pericytes and other cell types (Fig. 4B) relative to the expectation based simply on chance. Indeed, embedded in the basement membrane of blood capillaries, pericytes directly interlock and communicate with endothelial cells [47]. Long-distance interactions between pericytes and other cell types are indeed not expected. Being similar to the seqFISH study of the mouse visual cortex, an enrichment of the distal interactions between neurons was also observed in the MERFISH data of hypo- thalamic preoptic region and HDST olfactory bulb dataset (Fig. 4B, C). In addition, in both the hypothalamic preoptic region and the olfactory bulb, the newly reconstructed distal interactions were depleted between neurons and glial cells (Neu-Glial in Fig. 4B, and Neu-Epen/Astro/Per/Oligo in Fig. 4C) relative to the expectation based simply on chance. By contrast, in the olfactory bulb, immune cells showed a broad spectrum of long-range interactions with almost all cell types, especially with neurons, indicating abundant nerve-immune circuits (Fig. 4C). In the HDST breast cancer data, the newly recovered distal interactions were enriched with interactions between epithelial and stromal cells (Fig. 4D). An example is Li and Yang Genome Biology (2022) 23:124 Page 11 of 24 provided in Fig. 4F to show the interactions between an epithelial cell and multiple stromal cells. Distal interactions in the reconstructed cell networks This was not surprising given the frequent reports of stromal-epithelial cell interactions mediated by cytokines and extracellular vesicles [48–50], which confer high potential for remote interactions [51, 52]. Similarly, T cells or B cells heavily rely on long-range communication via cytokines, a notion reflected by the distal interac- tions enriched among T or B cells in the reconstructed network (Fig. 4D, and an ex- ample illustrating interactions between T cells is given in Fig. 4G). In contrast, the long-distance interactions were depleted in interactions that are highly dependent on direct cell contacts (for example, antigen presentation), such as the interactions be- tween T and B cells, T cells and epithelial cells, T cells and macrophages, B cells and epithelial cells, and B cells and macrophages (Fig. 4D). In the HDST breast cancer data, the epithelial cells were mostly tumor cells. Taking 4 randomly picked T cells and epithelial cells as examples, Additional file 1: Fig. S7 shows the distances between the interacting cell pairs, which were gener- ally much shorter than those from the randomly shuffled networks. Therefore, DeepLinc recovered the enriched interactions between T cells and tumor cells (Fig. 3D), which were strongly limited to proximal and not distal interactions (Fig. 4D, Additional file 1: Fig. S7). Interestingly, in contrast to epithelial cells, endothelial cells were more involved in distal interactions with immune cells (Fig. 4D). Indeed, it has been frequently reported that endothelial cells secrete chemokines to facilitate the transmigration and homing of immune cells into tumor tissues [53, 54]. DeepLinc identifies signature genes shaping the cell interaction landscapes Next, we further interrogated the DeepLinc model to infer the signature genes that play important roles in shaping the comprehensive landscapes of cell-cell interactions. In brief, the expression profile of a particular gene was shuffled across all the single cells, and the new transcriptome data and the cell adjacency network were then fed to Dee- pLinc. The AUPRC calculated from the cell network reconstructed by DeepLinc with these modified data was then compared to the original AUPRC with the unmodified gene expression data. The reduction, defined as the sensitivity score, indicates the sen- sitivity of the DeepLinc model to the particular gene being shuffled and therefore serves as an assessment of the potential contribution of the gene to the cell-cell interaction landscape. The same procedure was performed for each gene one at a time to obtain the sensitivity scores of all the genes (Additional file 1: Fig. S8). The top-rated genes with the highest sensitivity scores were indeed enriched by mul- tiple molecular and cellular processes related to cell-cell interactions (Fig. 5A–D). For example, 205 genes (Additional file 2: Table S1) showed outstanding sensitivity scores with the HDST breast cancer data, and these genes were involved in the processes of cell-substrate adhesion, junction formation, and protein targeting to the membrane (Fig. 5A, and the full list of functional categories supplied in Additional file 1: Fig. S9A). SPATA2, which showed the highest sensitivity score (Additional file 1: Fig. S8B), mediates the recruitment of CYLD to the LUBAC complex [55, 56], thereby playing a crucial role in the TNFR1- and NOD2-signaling pathways. The rest of the top 10 genes listed in Additional file 1: Fig. S8B include PKHD1 (transmembrane or secreted protein, Page 12 of 24 Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology Fig. 5 Biological processes enriched by the signature genes in shaping the cell interaction networks. A, B For the two high-dimensional datasets of HDST, the biological processes involving the top-ranked genes were obtained through Gene Ontology Enrichment Analysis. C, D For two low-throughput datasets (seqFISH and MERFISH), due to the limitation of gene numbers, all the genes were sorted by their sensitivity scores, and gene set enrichment analysis was performed. E Prediction of cell interactions with genes annotated as ligands or receptors and the same number of top-ranked signature genes identified by DeepLinc in the seqFISH (left) and MERFISH (right) datasets. DeepLinc identifies signature genes shaping the cell interaction landscapes Simple machine learning models, including support vector classification (SVC), K-nearest neighbor (KNN), and gradient boosting decision tree (GBDT) models, were used for this test. The originally defined cell adjacency map was used as a positive set of cell interactions, and randomly selected nonadjacent cell pairs with the same numbers were used as a negative set. This process was repeated 30 times with different training and testing set assignments. P-value were obtained with Student’s t test Fig. 5 Biological processes enriched by the signature genes in shaping the cell interaction networks. A, B For the two high-dimensional datasets of HDST, the biological processes involving the top-ranked genes were obtained through Gene Ontology Enrichment Analysis. C, D For two low-throughput datasets (seqFISH and MERFISH), due to the limitation of gene numbers, all the genes were sorted by their sensitivity scores, and gene set enrichment analysis was performed. E Prediction of cell interactions with genes annotated as ligands or receptors and the same number of top-ranked signature genes identified by DeepLinc in the seqFISH (left) and MERFISH (right) datasets. Simple machine learning models, including support vector classification (SVC), K-nearest neighbor (KNN), and gradient boosting decision tree (GBDT) models, were used for this test. The originally defined cell adjacency map was used as a positive set of cell interactions, and randomly selected nonadjacent cell pairs with the same numbers were used as a negative set. This process was repeated 30 times with different training and testing set assignments. P-value were obtained with Student’s t test involved in ciliogenesis, cell-cell and cell-matrix adhesion, and calcium ion homeosta- sis), S100PBP (binding partner of the calcium sensor S100P, involved in cell adhesion) [57], PSD4 (GEF for ARF6, involved in endocytosis), TRIM5 (capsid-specific restriction factor, involved in innate immune signaling and autophagy), ADAP2 (GTPase-activat- ing protein for ARFs, localizing to the plasma membrane, showing phospholipid bind- ing activity and mediating TCR signaling), and CCL26 (C-C motif chemokine ligand for CCR3). For the other HDST high-throughput spatial transcriptome dataset of the mouse ol- factory bulb, the top-rated genes (Additional file 3: Table S2) were more specifically Li and Yang Genome Biology (2022) 23:124 Page 13 of 24 Page 13 of 24 involved in the cell-cell interactions of the nervous system, such as synaptic transmis- sion, ion transmembrane transport, axonogenesis, synaptic vesicle activities, and trans- synaptic signaling (Fig. DeepLinc identifies signature genes shaping the cell interaction landscapes 5B, and the full list of functional categories supplied in Additional file 1: Fig. S9B). For the two low-throughput datasets of the mouse visual cortex (seqFISH) and mouse hypothalamic preoptic region (MERFISH), we performed gene set enrichment analysis (GSEA) to identify the processes involving more of the top-rated signature genes for the cell interaction landscapes (Fig. 5C, D, and the full lists in Additional file 1: Fig. S10). In the mouse visual cortex, these processes included the inflammatory response, response to external stimulus, defense response, extracellular structure organization, signal release, secretory vesicle processes, and peptide secretion (Fig. 5C). In the hypo- thalamic preoptic region, the identified processes included actin-based cell projection, axon, membrane organization, cell-cell adhesion, secretory granule, and peptide secre- tion (Fig. 5D). Together, the above results indicate the capability of the DeepLinc model to mine transcriptome signatures that are directly or indirectly associated with cell interaction landscapes. The high-impact genes revealed by the interrogation of the DeepLinc pipe- line provide insights into the key processes defining different types of cell interactions in specific tissue contexts. Interestingly, compared to the annotated ligand and receptor genes, equal numbers of the top-rated signature genes were much more efficient in dis- tinguishing interactive and noninteractive cell pairs with simple classifiers in both the mouse visual cortex and mouse hypothalamic preoptic region datasets (Fig. 5E). This once again suggests that the information on cell-cell interactions is largely coded in the transcriptome and not limited to specific ligand and receptor genes, a notion that lays the foundation of DeepLinc for the inference of cell interaction landscapes with spatial transcriptome data. DeepLinc identifies multicellular domains informing organizational tissue structures The dot size represents the significance of the one-versus- rest test (Mann–Whitney U test). The color indicates the median value of the normalized z-scores of gene expression in each cluster Fig. 6 Spatial domains defined by reclustering of cells based on latent vectors recovered by DeepLinc. A, B The UMAP graph and spatial locations of different spatial domain clusters and cell types. The spatial domain clusters were acquired by the k-means method with latent vectors of the single cells. C, D Spatially specific gene expression signatures of homologous cells reclustered by DeepLinc. The heatmaps show the differential expression of domain-specific genes. The dot size represents the significance of the one-versus- rest test (Mann–Whitney U test). The color indicates the median value of the normalized z-scores of gene expression in each cluster g p y g y p , The UMAP graph and spatial locations of different spatial domain clusters and cell types. The spatial domain clusters were acquired by the k-means method with latent vectors of the single cells. C, D Spatially specific gene expression signatures of homologous cells reclustered by DeepLinc. The heatmaps show the differential expression of domain-specific genes. The dot size represents the significance of the one-versus- rest test (Mann–Whitney U test). The color indicates the median value of the normalized z-scores of gene expression in each cluster Taking astrocytes and GABAergic and glutamatergic neurons in the mouse visual cor- tex as examples, the same types of cells allocated in the 6 domains showed significantly differential expression patterns of some signature genes (Fig. 6C). The details of these patterns were well in line with the inner-outer structure of the mouse visual cortex. However, the same types of cells located in the same layer were also partitioned into different cell clusters (e.g., Clusters D1 and D3) (Fig. 6A). Although the astrocytes, GABAergic neurons, and glutamatergic neurons in D1 and D3 showed similar patterns Taking astrocytes and GABAergic and glutamatergic neurons in the mouse visual cor- tex as examples, the same types of cells allocated in the 6 domains showed significantly differential expression patterns of some signature genes (Fig. 6C). The details of these patterns were well in line with the inner-outer structure of the mouse visual cortex. However, the same types of cells located in the same layer were also partitioned into different cell clusters (e.g., Clusters D1 and D3) (Fig. 6A). DeepLinc identifies multicellular domains informing organizational tissue structures As a deep data-generative model, DeepLinc generates a latent representation (H), which captures both the characteristics of the single-cell transcriptome profile and its position in the cell-cell interaction landscape. Therefore, we used this latent information of the cells learned by DeepLinc to redefine the multicellular spatial domains in heteroge- neous tissues. For example, in the seqFISH data of the mouse visual cortex, the embedding vectors in H for each cell were used for unsupervised K-means clustering, resulting in 6 subgroups of cells (Fig. 6A). The optimal cluster number was defined by Calinski-Harabasz scores [58] (see methods for details). These cell clusters were largely different from the previ- ously annotated cell types based only on known marker genes (Fig. 6A). Instead, the new cell groups resembled the multilayered organization of the heterogeneous cells in the mouse visual cortex [17] (Fig. 6A), which reflected the spatially restricted developmental trajectory from the inner to outer regions of the tissue. Similarly, for the MERFISH data- set of the hypothalamic preoptic area, the same analysis classified the tissue section into 7 subregions (Fig. 6B), which were again highly correlated with the original anatomy. Next, for the same types of cells that are allocated to these different spatially coded cell clusters, differential expression analysis identified domain-specific signature genes. Page 14 of 24 Li and Yang Genome Biology (2022) 23:1 Li and Yang Genome Biology Fig. 6 Spatial domains defined by reclustering of cells based on latent vectors recovered by DeepLinc. A, B The UMAP graph and spatial locations of different spatial domain clusters and cell types. The spatial domain clusters were acquired by the k-means method with latent vectors of the single cells. C, D Spatially specific gene expression signatures of homologous cells reclustered by DeepLinc. The heatmaps show the differential expression of domain-specific genes. The dot size represents the significance of the one-versus- rest test (Mann–Whitney U test). The color indicates the median value of the normalized z-scores of gene expression in each cluster Fig. 6 Spatial domains defined by reclustering of cells based on latent vectors recovered by DeepLinc. A, B The UMAP graph and spatial locations of different spatial domain clusters and cell types. The spatial domain clusters were acquired by the k-means method with latent vectors of the single cells. C, D Spatially specific gene expression signatures of homologous cells reclustered by DeepLinc. The heatmaps show the differential expression of domain-specific genes. DeepLinc identifies multicellular domains informing organizational tissue structures Although the astrocytes, GABAergic neurons, and glutamatergic neurons in D1 and D3 showed similar patterns Li and Yang Genome Biology (2022) 23:124 Page 15 of 24 Page 15 of 24 of gene expression in general, some genes were differentially expressed, indicating the differences between D1 and D3 (Fig. 6C). This again indicates that DeepLinc captures features from both gene expression profiles and spatially coded cell interaction land- scapes. Similarly, for the MERFISH dataset of the hypothalamic preoptic area, neurons allocated into different domains also showed differentially expressed signature genes (Fig. 6D). In summary, DeepLinc rebuilt more comprehensive cell interaction landscapes by learning from spatially resolved single-cell transcriptome profiles. The latent informa- tion on spatially dependent cellular features redefined the geometric domains of tissues composed of heterogeneous cells. These results therefore provide unique insights into the spatially coded cellular organization underlying the potential physiological relevance of cell interaction landscapes. Discussion As a major methodological breakthrough, technologies for profiling spatially resolved transcriptomes have provided unprecedented opportunities for the dissection of highly heterogeneous cellular organizations in physiological and pathophysiological contexts. However, it has been a major challenge to comprehensively and precisely infer cell-cell interactions based on spatial transcriptome profiles at single-cell resolution. First, spatial transcriptome profiles are random snapshots of very limited numbers of tissue sections. The cellular organization patterns contain information on cell-cell interactions and presumably represent only small collections sampled from the full landscape. Therefore, methods for correctly inferring the complete landscapes of cell interactions by taking advantage of the observed cell geometric distributions and gene expression profiles are urgently needed. Second, current spatial transcriptome data are limited by a series of technical issues, including biased and low gene coverage, low signal-to-noise ratios, high data sparsity under high-throughput techniques, and/or batch effects, thereby necessitating data-mining methods that are highly robust to these technical limitations. Powered by the strong data representation capacity of the VGAE model, DeepLinc was specifically designed and optimized to denoise and infer missing interactions by ef- ficiently learning from the incomplete sets of cell organization revealed by different methods of single-cell spatial transcriptome profiling. Further analysis of the features mined by DeepLinc helps identify the signature genes shaping cell interaction land- scapes and cells with special features, such as hub cells and cells in specific domains of the interaction landscape. Various methods have been available for inferring the cell-cell interaction landscapes utilizing bulk RNA-seq, scRNA-seq, or spatially resolved single-cell data. These methods are mostly based on predefined or previously annotated ligands, receptors, and downstream target genes [23, 25, 59–61]. These methods have mostly been applied to scRNA-seq data and are not well suited for single-cell spatial transcriptome data, which show very limited and uneven coverage of the ligand and receptor genes due to high sparsity and noise. This necessitates de novo methods, such as DeepLinc, for re- construction of cell-cell interaction networks in a more unbiased manner independent of prior knowledge such as ligand-receptor pairs. Instead, by assuming that cell inter- action potentials have been coded in single-cell transcriptome profiles, DeepLinc is Li and Yang Genome Biology (2022) 23:124 Page 16 of 24 trained by the observed cell-cell connections and then utilized to infer missing interac- tions. As shown in Fig. 4 and Additional file 1: Fig. Discussion S5, DeepLinc recovered great num- bers of distal interactions, and meanwhile, DeepLinc removed some of the predefined proximal interactions between neighboring cells. Therefore, DeepLinc is positioned as a data-mining and processing strategy tailored for single-cell spatial transcriptome profil- ing data, with the goal of recovering the full landscapes of cell interactions from limited observations of tissue sections. Specifically, DeepLinc uses VGAE to encode the intrinsic characteristics of spatial transcriptome data and decode cell interactions. The dual modal data (i.e., the graph- like cell adjacency map and the high- or mid-throughput transcriptome profile) are in- tegrated at the same time. Here, the design of VGAE constrains the model to put more weight on the key genes by force-fitting the global cell interaction graph signatures. To the best of our knowledge, DeepLinc is the first deep generative learning model for in- ferring cell interactions from spatial transcriptomic data. Based on a wide array of tests with real and simulated data, DeepLinc demonstrated high efficiency in learning from imperfect and incomplete spatial transcriptome data, filtering false interactions, and in- ferring missing distal and proximal interactions, thereby suggesting the reliability and effectiveness of the deep learning strategy. With the DeepLinc model, our sensitivity test of transcriptome profiles suggested sig- nature genes with potentially high impacts on the cell interaction landscape. Import- antly, compared to known ligand and receptor genes, these genes showing high sensitivities performed better in simple classification models of cell interactions. This supports our presumption that cell-cell interactions are coded in single-cell transcrip- tome profiles, rather than simply being defined by ligand and receptor genes. The iden- tification of these signature genes could facilitate the understanding of the various machineries underlying heterogeneous cell interactions and further suggests the possi- bility of directly predicting cell interactions simply from single-cell transcriptome pro- files. Further refined models would be needed for this purpose. Due to the lack of more detailed information in spatial transcriptome data, DeepLinc does not provide the directionality or strength of the cell interactions. Potentially different modes of cell interactions also cannot be differentiated by DeepLinc in its current form. Finally, similar to most of the deep learning models based on GAE, it is difficult to effi- ciently and directly backtrack the important features encoded by the graph convolutional networks used in DeepLinc. Conclusions DeepLinc is a novel computational method, with a strategy of deep learning, for de novo reconstruction of cell interaction landscapes from single-cell spatial transcriptome profiles. With simulated and real data, DeepLinc demonstrates high efficiency in learn- ing from imperfect and incomplete spatial transcriptome data, filtering false interac- tions, and inferring missing distal and proximal interactions. Interrogations of the DeepLinc pipeline reveal signature genes that are potentially involved in shaping cell interaction landscapes. In addition, clustering of cells based on the latent features of cell interactions and transcriptome profiles learned by DeepLinc indicates multicellular domains informing organizational tissue structures. Discussion Technical advances in spatial transcriptome profiling and breakthroughs in graph convolutional models would help further improve DeepLinc in the future. In addition, if more information becomes available due to future technological advances, DeepLinc could be further expanded to incorporate features such as cell morphology, surface markers, and metabolic profiles. Finally, although this is not the main target of DeepLinc, the framework can be used for inferring single-cell gene expression profiles by using information on cell interactions based on the assumption that the gene expression profiles are partly correlated with local and distal cell interaction patterns [62]. In summary, as a tool for deep data mining, DeepLinc further empowers rapidly emerging spatial transcriptome profiles for the de novo reconstruction of cell inter- action maps. This new strategy, facilitated by deep graph convolutional networks, does not rely on any prior knowledge of cells or gene functions. We anticipate that the combination of state-of-the-art spatial transcriptome profiling techniques and Li and Yang Genome Biology (2022) 23:124 Page 17 of 24 an efficient data deep mining framework will greatly facilitate the identification of the biological mechanisms underlying complex cell communication networks with physiological relevance. Datasets of single-cell spatial transcriptome profiles Four published datasets were used for the current study [13, 17, 18], including mouse visual cortex profiled by seqFISH [17], preoptic region of the mouse hypothalamus by MERFISH [18], mouse olfactory bulb, and human breast cancer by HDST [13] (Fig. 1B). Fewer than 200 genes were profiled by seqFISH and MERFISH, whereas HDST generates high-throughput transcriptome profiles coving around 10,000 genes (Fig. 1B). Four published datasets were used for the current study [13, 17, 18], including mouse visual cortex profiled by seqFISH [17], preoptic region of the mouse hypothalamus by MERFISH [18], mouse olfactory bulb, and human breast cancer by HDST [13] (Fig. 1B). Fewer than 200 genes were profiled by seqFISH and MERFISH, whereas HDST generates high-throughput transcriptome profiles coving around 10,000 genes (Fig. 1B). The seqFISH dataset was downloaded from the data portal at https://bitbucket.org/qzhu/ smfish-hmrf/src/master/. The data was processed according to the procedure described in the original study [17]. The final dataset covers 125 genes measured in 1597 cells. The seqFISH dataset was downloaded from the data portal at https://bitbucket.org/qzhu/ smfish-hmrf/src/master/. The data was processed according to the procedure described in the original study [17]. The final dataset covers 125 genes measured in 1597 cells. The MERFISH dataset of the mouse hypothalamic preoptic region was downloaded from https://datadryad.org/stash/dataset/10.5061/dryad.8t8s24818. We used the slice re- gion at Bregma+0.11 mm of the animal No. 18, as it contains the largest number of sin- gle cells. After removing the ambiguous cells, the final dataset contains 4975 cells, covering 160 genes, five of which were blank controls. The datasets of mouse olfactory bulb and human breast cancer profiled by HDST were downloaded from the data portal at https://portals.broadinstitute.org/single_cell/ study/SCP420 [13]. The olfactory bulb dataset contains 3 sections (CN13_D2, CN24_ D1, and CN24_E1) from two mice. We selected a field of CN13_D2 (x:8447~11447, y: 5447.5~6447.5), which consists of 1981 nuclei. In total, 14,909 genes were measured among these cells, although a majority of them were not detected in each cell. The breast cancer dataset also includes 3 tissue sections (CN21_E2, CN21_C1, and CN21_ D1), which were obtained from a histological grade 3 HER2+ patient. We selected a field of CN21_E2 (x:8464~9664, y:5000~6500) consisting of 1765 nuclei. This data, be- ing very sparse as well, covers 8336 genes. The algorithm of DeepLinc DeepLinc assumes that the neighboring cells in a solid tissue should be much more likely to have some types of interactions than randomly picked non-neighboring cells Li and Yang Genome Biology (2022) 23:124 Page 18 of 24 that are far away from each other. Therefore, for a particular tissue region, the neigh- boring cell pairs comprise an incomplete and potentially noisy observation of cell-cell interactions sampled from the comprehensive cell interaction landscape. In other words, neighboring cell pairs in tissue sections reveal some cell-cell interactions, whereas large numbers of other interactions are not explicitly shown. We then assume that the features related to cell interactions are encoded in the gene expression profile of each singe cell. DeepLinc uses neighboring cells with direct contacts as the positive set for learning the transcriptome features related to cell-cell interactions. From a general biological point of view, it is reasonable to assume that in a solid tissue, most of the cells in 2-D could directly contact with 3 or more other cells. From a technical point of view, for the strategies of machine learning, it is critical to minimize the potential false positives in the predefined positive sets for training. Therefore, for each cell, we only used the 3 nearest neighbors to define direct contacts, which we believe is a balanced choice gen- erating enough number of direct contacts for training the DeepLinc pipeline, and at the same time, ensuring few false positives to contaminate the positive training set. DeepLinc leverages a variational graph autoencoder (VGAE) with an adversarial net- work for regularization, to infer the unobserved interactions between cells. The entire architecture includes three parts: a variational graph convolutional network (VGCN) encoder, an inner product decoder, and an adversarial module. Given a cell adjacency matrix A and a gene expression matrix X, the VGAE learns the embedded features H of the cell graph. A two-layer graph convolutional network (GCN) (250-125) is used as the encoder module. q HjX; A ð Þ ¼ Yn i¼1q hijX; A ð Þ q hijX; A ð Þ ¼ Ν hijμi; diag σ2     Similar to variational autoencoder, μ = GCNμ(X, A) is the matrix of mean vectors and logσ = GCNσ(X, A). Implementation of DeepLinc DeepLinc is implemented as a Python package, which is available at https://github. com/xryanglab/DeepLinc. Two files are needed as inputs of the pipeline, one for the cells-by-genes matrix of gene expression data and the other for the predefined cell adja- cency matrix. If an enrichment analysis of the interactions between cell populations, an annotation file defining the cell types would be needed. DeepLinc allows users to specify the node numbers. For datasets with more cells, lar- ger numbers of nodes are recommended to capture the complexity of data better. Al- though the number of hidden layers for the encoder is also adjustable, extra caution has to be taken, since deeper graph convolutional networks are prone to over- smoothing [64]. In general, the two-layer graph convolutional network has been widely used and proven suitable for most of the tasks. The Adam optimizer [65] with 4e-4 learning rate is used for optimization. The dropout method is also used to ease the overfitting effect. A new cell interaction matrix is the main output of DeepLinc. The latent representa- tion output by DeepLinc can also be used for unsupervised clustering of the single cells. Implementation of DeepLinc requires the following packages: Numpy v1.16.4, Scipy v1.2.1, Pandas v0.20.3, Matplotlib v3.0.2, Seaborn v0.8.1, Networkx v2.1, Scikit-learn v0.21.2, Tensorflow v1.4.0, Python 3.5.2 (Anaconda3-4.2.0). The DeepLinc model was trained on a single GPU of GTX 1050 or Tesla K80. The algorithm of DeepLinc As a typical structure, the GCN is defined as GCNðX; AÞ ¼ ~AReLUð ~AXW 0ÞW 1 with weight Wi and normalized adjacency matrix ~A ¼ D−1 2ðA þ IÞD−1 2 to realize layer-to-layer information transmission. We use ReLU as the activation function of the first layer instead of the second layer. The purpose of this step is to learn mean- ingful embedded features supporting the interaction formation by aggregating informa- tion of each cell itself and its interacting neighbor cells. The inner product decoder p(A| H) = σ(H · HT) is employed to reconstruct a new cell interaction matrix, which reports probability scores of the interactions between each pair of single cells. VGAE has exhibited outstanding performance in learning the embedded features of graph elements [38]. However, for single-cell spatial transcriptome data, a major chal- lenge is its high sparsity and data noise. For such non-ideal situations, the standard variational encoder-decoder structure adopted by VGAE does not make more con- straints on latent variables, often resulting in underfitting of the sparse data [63]. Here we implement strong regularization on the latent distribution with the adversarial network. Specifically, the adversarial module is built on a multilayer perceptron (MLP). The architecture is similar to the discriminator of generative adversarial networks (GAN). Li and Yang Genome Biology (2022) 23:124 Page 19 of 24 Page 19 of 24 The adversarial network has two fully-connected hidden layers with ReLU activation (125–150). The output layer uses sigmoid function to judge whether a latent sample hi is from a prior Gaussian distribution or from VGAE. Eventually, the DeepLinc pipeline was optimized to maximize the log-likelihood of cell-cell interaction network. The objective of VGAE is to minimize its cost function by optimizing the evidence lower bound (ELBO): ζELBO ¼ Eq HjX;A ð Þ logp AjH ð Þ ½ −DKL q HjX; A ð Þ p H ð Þ k ð Þ For the adversarial module, the loss function is binary cross-entropy. For the adversarial module, the loss function is binary cross-entropy. Reconstruction of the cell interaction networks As introduced above, DeepLinc inferred interaction probability scores for all the cell pairs. Next, cell interaction networks were assembled with the cell pairs with high probability scores. Specifically, different thresholds of the probability scores were tested for identifications of cell interactions. The optimal threshold was then determined so that the newly assembled cell interaction network on the testing set has the highest ac- curacy for recovering the predefined cell adjacency maps. Evaluating the over- or under-representation of particular types of interactions A previously described strategy [21, 24, 42] was used to evaluate the enrichment or de- pletion of the interactions between different cell types or within a cell type. In brief, we randomly shuffled the reconstructed networks for 1000 times. From these 1000 net- works, the numbers of cell interactions of a certain type, e.g., interactions between two types of cells, were used to generate a null distribution. The observed interaction num- ber was then compared to this null distribution, generating a P-value indicating how often the random values were higher or lower than the observed ones, which corre- sponds to either enrichment or depletion of the particular type of interactions in the network. Two-tail test was done for such analysis. Evaluation of the enrichment or depletion of the distal interactions between certain cell types was done with a similar strategy. For each dataset, we first generate a distance distribution with all the edges in the reconstructed cell interaction network, from which a threshold was determined to classify the distal interactions (Additional file 1: Fig. S6). The same number of interactions was established between randomly picked distal cell pairs. Such procedure was repeated for 1000 times, generating a null background of the distal cell interaction landscapes. Next, the observed number of distal interactions was compared to this null distribution, generating a P-value indicating how often the ran- dom values were higher or lower than the observed one, which corresponds to either enrichment or depletion of the particular type of distal interactions in the network. Two-tail test was done for such analysis. Benchmark of DeepLinc for recovering missing interactions Different proportions of the edges in the original cell adjacency maps were randomly picked and discarded. The remaining edges were used for training the DeepLinc model. The area under the receiver operating characteristic (AUROC) was calculated to assess the accuracy of the reconstructed interaction networks for recovering the discarded edges. Here equal numbers of the originally non-existing edges were used as true nega- tive sets. Page 20 of 24 Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology Benchmark of DeepLinc for differentiating real and fake interactions Different numbers (1 to 10 folds of the size of the original cell adjacency map) of ran- domly generated fake edges were added into the original cell adjacency map, which was then fed to DeepLinc. AUROC of the reconstructed network was calculated by taking the pre-existing real edges as positives and the mixed-in fake edges as negatives. Benchmark of DeepLinc for the tolerance to random noise in gene expression profiles Random noise was independently generated and implemented to each gene in each sin- gle cell. Specifically, for each gene, the new expression level was regenerated by adding random noise as follows: Expnoiseþ i; j ð Þ Expnormal i; j ð Þ ¼ 2rand; rand  Ν 0; σ ð Þ Expnoiseþ i; j ð Þ Expnormal i; j ð Þ ¼ 2rand; Among them, Expnoise+(i, j) represents the regenerated expression value of gene j in sample i, and Expnormal(i, j) represents the original expression level. The fold change used to simulate noise, after log2 transformation, follows the normal distribution N(0, σ), whereas different σ values would result in different levels of random noise in general. Sensitivity analysis for each gene Sensitivity analysis was performed to evaluate the potential contribution of a gene to shaping the reconstructed cell interaction landscape. In brief, for a particular gene in Page 21 of 24 Li and Yang Genome Biology (2022) 23:124 Page 21 of 24 the transcriptome profile, the expression profile was shuffled among all the single cells. This new data, with the expression of one gene shuffled at a time, was then fed to Dee- pLinc, which then generates a new cell interaction network. ΔAUPRC was then calcu- lated by comparing the AUPRC values of the two cell interaction networks built by DeepLinc with the original unperturbed gene expression data and the data with the ex- pression of one gene perturbed. For each gene, the process above was repeated for 30 times, and the average ΔAUPRC was used to quantitatively evaluate the sensitivity of the reconstructed cell interaction network to the gene, thereby defined as the sensitivity score. For the two high-throughput HDST datasets, distributions of the sensitivity scores of all the genes were prepared, and the biological processes involving the top-ranked genes were obtained with the Gene Ontology enrichment analysis [66]. For two low- throughput datasets of seqFISH and MERFISH, all the genes were sorted by their sensi- tivity scores, and the Gene Set Enrichment Analysis [67] was performed to acquire the biological processes enriched by the top-ranked genes. The biological processes were filtered by gene numbers (min size=3 and max size=500), resulting in 1240 and 1691 gene sets for the GSEA analysis with the seqFISH and MERFISH data, respectively. Prediction of cell interactions with simple machine learning models For the seqFISH and MERFISH datasets, simple machine learning models including Support Vector Classification (SVC), K-nearest neighbor (KNN), and Gradient Boosting Decision Tree (GBDT) were used to test the capability of specified genes for classifica- tions of the interacting and non-interacting cell pairs. The sklearn package in Python was used to build these models. The originally defined cell adjacency map was used as positive sets of cell interactions, and randomly selected unadjacent cell pairs, with the same numbers, were used as negatives. Sixteen genes annotated as ligands or receptors and the same number of top-ranked signature genes identified by DeepLinc were used for the classification models. Their expression vectors of a pair of cells were concatenated and used as the feature vectors. The combined set of positive and negative cell interactions were randomly divided into 4:1, serving as the training and testing sets, respectively. The three classification models were then applied on the data to predict the interacting cell pairs with the testing set. This process was repeated for 30 times with different training and testing set assign- ments. The AUROC values were calculated based on the predictions. Clustering of the single cells based on the latent representation learned by DeepLinc Dimension of the latent representation H was set as 10 for clustering of the single cells with the k-means method. The Calinski-Harabasz score was used to determine the op- timal cluster number. The “umap” package in Python was used for dimension reduction and visualization of the latent vectors of the single cells. Review history Review history is available as additional file 4. Supplementary Information pp y The online version contains supplementary material available at https://doi.org/10.1186/s13059-022-02692-0. The online version contains supplementary material available at https://doi.org/10.1186/s13059 022 02692 0. Additional file 1: Figures S1-S10 Additional file 2: Table S1. Signature genes of the HDST breast cancer data. Additional file 3: Table S2. Signature genes of the HDST mouse olfactory bulb data. Additional file 4: Availability of data and materials y The DeepLinc pipeline has been deposited in github (https://github.com/xryanglab/DeepLinc) [68] and Zenodo (https://doi.org/10.5281/zenodo.6564143) [69]. The source code is released under MIT License (http://opensource.org/ licenses). Four published datasets were used for the current study [13, 17, 18], including mouse visual cortex profiled by seqFISH [17, 70], preoptic region of the mouse hypothalamus by MERFISH [18, 71], mouse olfactory bulb and human breast cancer by HDST [13, 72]. Funding h k g This work was funded by the National key research and development program, Precision Medicine Project (2016YFC0906001), the Tsinghua University Spring Breeze Fund, the Tsinghua University Initiative Scientific Research Program (2019Z06QCX01), and the National Natural Science Foundation of China (81972912 and 31671381). Peer review information Stephanie McClelland was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Authors’ contributions R L d X Y d R.L. and X.Y. conceived and designed the project. R.L. developed the DeepLinc pipeline and conducted the bioinformatics analyses. X.Y. supervised the whole project. R.L. and X.Y. wrote the manuscript. All author(s) read and approved the final manuscript. Acknowledgements The authors would like to thank the supports from the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) and Tsinghua University Technology Center for Protein Research, including the core facilities of Biocomputing, Genome Sequencing and Analysis at Tsinghua University. Competing interests Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 7 November 2021 Accepted: 20 May 2022 Received: 7 November 2021 Accepted: 20 May 2022 Received: 7 November 2021 Accepted: 20 May 2022 Ethics approval and consent to participate Ethical approval is not needed for this study Ethical approval is not needed for this study. Differential gene expression analysis between single-cell groups For the cells of the same type but distributed in different spatial domains, differential expression analysis was performed to identify the upregulated genes specifically in each spatial domain. For each domain, the genes that are significantly upregulated in both Page 22 of 24 Li and Yang Genome Biology (2022) 23:124 Li and Yang Genome Biology one-versus-one and one-versus-rest tests (Mann–Whitney U test, P < 0.01) were deemed as domain-specific high-expression genes. one-versus-one and one-versus-rest tests (Mann–Whitney U test, P < 0.01) were deemed as domain-specific high-expression genes. References Asp M, et al. 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https://openalex.org/W3164371899
https://pubs.rsc.org/en/content/articlepdf/2021/md/d1md00138h
English
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Will the chemical probes please stand up?
RSC medicinal chemistry
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a CZ-OPENSCREEN, National Infrastructure for Chemical Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20, Prague 4, Czech Republic. E-mail: ctibor.skuta@img.cas.cz b Deanery of Biomedical Sciences, University of Edinburgh, Edinburgh EH8 9XD, UK † Electronic supplementary information (ESI) available: Full data set used for the study. See DOI: 10.1039/d1md00138h ‡ Current address: Medicines Discovery Catapult, Alderley Park, Macclesfield SK10 4TG, UK. Introduction contemporary efforts. These include considerations of the MLSMR fitness-for-purpose as a library accrued in an academic context (compared to arguably better-resourced pharmaceutical company screening collections), persistent probe quality issues and remaining confusion on exactly how many probe compounds the program generated.6–8 In 2005, the NIH Molecular Libraries Program (MLP) undertook the first large-scale identification of tool compounds to expand biological insights, now termed small- molecule chemical probes.1,2 Their systematic generation against a range of molecular targets was a key driver for the establishment of the PubChem database in order to collate structures and data from the initial ten funded screening centres.3 The concomitant screening compound collection was established as the Molecular Libraries Small Molecule Repository (MLSMR) for which PubChem had hosted 255 000 compounds by the end of 2005 and since expanded to 406 000 by 2015 (but updates have ceased). Although 25 of the 64 early compounds were judged to be of equivocal quality by a crowdsourcing assessment in 2009 (ref. 4) the program progressed to 375 probes (see data section below) before ending in 2014. As conceived from the outset, the availability of these compounds and, crucially, their associated characterisation data, have facilitated the exploration of new targets, pathways and therapeutic hypotheses.5 Notwithstanding these success stories, the MLP undertaking has been subject to criticisms that remain relevant to A less tangible but equally important success of the MLP is that, from approximately 2010 onwards, it inspired other organisations to also initiate probe discovery with open dissemination. The three most recent announcements (but not yet surfacing data) are EU-OPENSCREEN9 with probe development as one of their main objectives, the EUbOPEN10 consortium aiming to synthesize at least 100 new chemical probes and the Target 2035 initiative to accrue probes for all human targets by 2035.11 As of June 2021, we were able to collect probe data from the sources listed below. • MLP probes (NIH screening initiative) • MLP probes (NIH screening initiative) • Structural Genomics Consortium (SGC, 3D structure- based)12 • Nathanael Gray Laboratory (cancer research focused)13 • Chemical Probes Portal (literature curation, expert opinion)14 • Pharmaceutical companies (offering in-house compounds)15 • Probe Miner (data filtration for putative probes)16 • Probes & Drugs (comprehensive collation of probe data)17 Detailed descriptions of these sources and their individual approaches to probe development are available from their websites and related publications. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54 This article is licensed under a Creative Commons Attribution 3.0 U Received 23rd April 2021, Accepted 28th June 2021 Received 23rd April 2021, Accepted 28th June 2021 DOI: 10.1039/d1md00138h rsc.li/medchem This journal is © The Royal Society of Chemistry 2021 Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Ctibor Škuta, *a Christopher Southan ‡b and Petr Bartůněk a In 2005, the NIH Molecular Libraries Program (MLP) undertook the identification of tool compounds to expand biological insights, now termed small-molecule chemical probes. This inspired other organisations to initiate similar efforts from 2010 onwards. As a central focus of the Probes & Drugs portal (P&D), we have standardised, integrated and compared sets of declared probe compounds harvested from 12 different sources. This turned out to be challenging and revealed unexpected anomalies. Results in this work address key questions including; a) individual and total structure counts, b) overlaps between sources, c) comparisons with selected PubChem sources and d) investigating the probe coverage of druggable targets. In addition, we developed new high-level scoring schemes to filter collections down to probes of higher quality. This generated 548 high-quality chemical probes (HQCP) covering 447 distinct protein targets. This HQCP collection has been added to the P&D portal and will be regularly updated as established sources expand and new ones release data. Experimental vs. calculated We chose to internally partition P&D probes into two main categories; experimental and calculated. Experimental denotes compounds from published probe characterisation experiments. These papers include profiling data for target modulation potency, selectivity, and possible secondary targets. Also important to note is that the experimental data largely originate from a single laboratory and are thus likely to have more consistent and reproducible data (e.g. where intra-laboratory assay variation is controlled via sufficient replicates and internal standards). Examples of such experimental probe sets include; bromodomains chemical toolbox,37 Chemical Probes Portal,14 Gray Laboratory Probes,13 Nature Chemical Biology Probes, Open Science Probes,15 opnMe Portal,38 Protein methyltransferases chemical toolbox,39 and SGC Probes.12 We use the term calculated here to denote in silico evaluation using the combination of public data with a custom scoring function (we intentionally avoided the term predicted in this context because machine learning methods were not used).16,40 The evaluation of probes at scale involves comparing public data at different stringencies according to availability. The seven key criteria are: 1) <100 nM target potency in vitro, 2) <1 uM for cell-based assays with evidence of direct target engagement, 3) target selectivity >100-fold, 4) absence of structural alerts indicating chemical liabilities 5) identification of an inactive analogue as control with significantly lower potency or inactive against the primary target,41 6) an orthogonal probe with a different chemotype against the same primary target, and 7) SAR data to increases confidence in specific target modulation. The simplest approach to assigning a compound as a probe is to use these criteria (or a subset thereof). However, this can be nuanced by weightings (e.g. for potency and selectivity) as well as more complex scoring (e.g. functions favouring compounds with a wider range of target profiling data). In contrast to experimental probes, the data for the evaluation of the calculated probes from different sources can be less consistent and may not be acquired with the objective of developing and validating a probe per se. The calculated sources included here are from Probe Miner16 and the tool compound set.40 P&D has additional advantages for this study. An important one is that chemical structures from all sources are standardised after importation. This means that our internal comparisons are as rigorous as we can make them (notwithstanding cheminformatic nuances that preclude this from being perfect). Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Addressing these questions is specifically enabled by the Probes & Drugs portal (P&D, https://probes-drugs.org).17 P&D was designed as a hub for the integration of high-quality bioactive compound sets enabling their analysis and comparison. As the name indicates, the main focus is on probes and drugs but includes additional relevant sets extracted from, or supplied by, recently published specialist databases (e.g. BiasDB19), vendor sets, ESI† from papers (e.g. kinase inhibitors) or harvested from publication out-links (e.g. the British Journal of Pharmacology “Concise Guide” series20–26). Other high-quality curated bioactive chemistry sources, including ChEMBL,27 BindingDB,28 Guide To Pharmacology (GtoPdb),29 DrugCentral30 and DrugBank31 are utilized for compound biological annotation (the latter 3 are also P&D compound sets). The P&D compound database is currently composed of 69 sources including 12 probe-related, 7 drug compilations, 36 academic (non-commercial) sets and 14 precompiled sets from bioactive compound vendors (suggestions for expansion are welcome). Experimental vs. calculated We have used the standardiser python package32 for salt-striping, charge neutralization, standardizing common functional groups, preserving stereochemistry and identification of the main active pharmaceutical ingredients (API) in mixtures. Structural uniqueness is based on the InChIKey, a hashed version of the International Chemical Identifier, InChI.33 Within the text of this article, we have used InChIKeys to designate mentioned compounds. These can be searched not only against P&D and all major databases but also in Google.34 On our website, users can choose to browse and compare the structures in three different forms: 1) standardized; 2) original (i.e. as imported from the source) and 3) non- isomeric (i.e. core connectivity without stereochemistry). Importantly, we made the considered decision not to submit our entire content to PubChem for two main reasons. The first is to reduce non-obvious circularity which can confound database users.35,36 The second is that this enables informative Boolean query combinations to be made between P&D sets and essentially any PubChem source or filtration selects. This journal is © The Royal Society of Chemistry 2021 Introduction These are expanded in a recent review18 as well as articles in this special issue. This work was conceived to answer the following questions that can not be answered via the individual sources: • How many declared chemical probe structures are there? 1428 | RSC Med. Chem., 2021, 12, 1428–1441 This journal is © The Royal Society of Chemistry 2021 View Article Online RSC Medicinal Chemistry RSC Medicinal Chemistry Research Article Research Article • What is the distribution of their physicochemical properties? • What is the distribution of their physicochemical properties? • What is the distribution of their physicochemical properties? considered equivalent in a canonical sense because they have been generated by divergent approaches. By analysing the structures and associated metadata, we have tried to establish a high-quality subset based on the probe origin and by the application of scoring schemes. In addition, where the data allow plausible assignments, we have compiled target coverage. • What are the differences between experimental and calculated probes? • What is their representation in PubChem sources? • What are their intersections with each other? • What are their individual targets? • What is their combined human proteome coverage? • What is their combined human prot Historical compounds The use of these obsolete compounds is no longer recommended by the CP portal because new data indicates promiscuity or displacement by better tool compounds.42 These structures include the well-known and notorious medicinal chemistry time-wasters of staurosporine (HKSZLNNOFSGOKW- FYTWVXJKSA-N), quercetin (REFJWTPEDVJJIY-UHFFFAOYSA-N), resveratrol (LUKBXSAWLPMMSZ-OWOJBTEDSA-N), and curcumin (VFLDPWHFBUODDF-FCXRPNKRSA-N). Set comparisons The sets are compared by exact matches in Table 2. The resulting matrix is unique in that no individual source has published a comparable analysis. However, some results were unexpected. The first surprise was the low intersection between MLP and the calculated sets. We attribute this to the 80 compounds from the MLP set without bioactivity data on P&D. In addition, 203 have a primary target potency above the 100 nM threshold. The unexpectedly high overlap between the Chemical Probes Portal and tool compound sets is a consequence of the (already mentioned) inclusion of the former in the latter but without data-supported evaluation. Also surprising is the low overlap between the two calculated sets, even though the data sources and selection criteria were conceptually similar. However, the main goal of the tool compound set was to select effective agonists or antagonists with a high stringency for target selection and cell potency. Bromodomains and protein methyltransferases chemical toolboxes These compounds were extracted from publications focused on the study of bromodomains and methyltransferases. Except for four bromodomain probes developed elsewhere, these also belong to the SGC set. MLP and Nature Chemical Biology Probes MLP and Nature Chemical Biology Probes The former has been outlined in the introduction. The latter was extracted from articles published in Nature Chemical Biology (although since 2018 this dedicated section of the Journal is no longer available) As legacy collections, probes from these two sources may lack the stringent characterisation of maintained collections. This is reflected in some cases by a) the lack of controls or orthogonal probes, b) unclear potency and selectivity criteria or c) no target annotation. SGC Probes, Open Science Probes, opnMe Portal, and Gray Laboratory Probes These organisations apply the currently accepted probe quality criteria and are maintained sets in that P&D has picked up at least some new compounds since their initial release (even if at a variable frequency). Tool compound set and Probe Miner These are calculated selections, mainly from ChEMBL, selected via probe-likeness criteria. While the tool compound set is a one-off extraction from the publication, Probe Miner is regularly updated. The tool compound set independently includes more than 100 compounds available from the Chemical Probes Portal at the time of its publication. Another surprising observation was that, while overlap with historical compounds is reassuringly low, Table 2 indicates there are still nine of these undesirables in Probe Miner, four in the MLP, and two in the high-quality SGC Probes. The first of these, bromosporine43 (UYBRROMMFMPJAN-UHFFFAOYSA-N), was designed to be a pan-bromodomain inhibitor and could usefully be family- selective. The second, GSK-J1 (ref. 44) (AVZCPICCWKMZDT- UHFFFAOYSA-N), an inhibitor of the KDM protein family is This journal is © The Royal Society of Chemistry 2021 Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:5 This article is licensed under a Creative Commons Attribution 3.0 U lists historical probes (these are also captured as a P&D set, see next section). n Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Medicinal Chemistry View Article Online View Article Online Research Article RSC Medicinal Chemistry Table 1 Sources with their compound numbers and probe type. “E” refers to experimental and “C” to calculated probe type. The targets column counts distinct probe-target annotations. The total number of compounds/targets represents a distinct number of compounds/targets for all sets combined Table 1 Sources with their compound numbers and probe type. E refers to experimental and C to calculated probe type. The targets column counts distinct probe-target annotations. The total number of compounds/targets represents a distinct number of compounds/targets for all sets combined Set Probe type Set class Compounds Targets 1 Bromodomains toolbox E High-quality 25 26 2 Chemical Probes.org E High-quality 362 322 3 Gray Laboratory E High-quality 53 56 4 MLP E Legacy 375 156 5 Nature Chemical Biology E Legacy 58 51 6 Open Science Probes E High-quality 83 95 7 opnMe Portal E High-quality 55 57 8 Probe Miner C Calculated 3187 326 9 Methyltransferases toolbox E High-quality 19 20 10 SGC Probes E High-quality 81 97 11 Tool compound set C Calculated 515 392 12 Historical compounds E Historical/obsolete 239 — Total (Not historical) 4466 819 Source descriptions and counts As of version 02.2021 (March 2021), P&D contains 77 130 compounds with 4466 labelled as chemical probes by their sources used for this study. However, these should not be Those compared in this study are listed in Table 1 with brief descriptions below. This journal is © The Royal Society of Chemistry 2021 RSC Med. Chem., 2021, 12, 1428–1441 | 1429 RSC Medicinal Chemistry Research Article View Article Online Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54 This article is licensed under a Creative Commons Attribution 3.0 Un not cell-permeable. The SGC Probes resource has noted this and consequently now recommends a pro-drug of GSK-J1, GSK-J4 (WBKCKEHGXNWYMO-UHFFFAOYSA-N) for cell- based assays. Fig. 1 Physico-chemical properties distribution (top: molecular weight, middle: calculated log P, bottom: TPSA) for experimental probes (blue), calculated probes (orange) and small-molecule approved drugs set from ChEMBL (green) (x: property range, y: compounds percentage). The properties were calculated by RDKit. Dataset compilation Merging individual sets resulted in 4466 structurally distinct probe compounds (i.e. as unique InChIKeys). This includes 940 (21%) experimental plus 3670 (82.2%) calculated probes including 3178 from Probe Miner. The overlap of 143 (3.2%) compounds is mainly due to the inclusion of the Chemical Probes Portal in the tool compound set. The full 4466 set includes 275 labelled as drugs (reported to be in clinical phases) with 103 labelled as approved by FDA, EMA and other agencies. The full set also includes 29 PROTACs (Proteolysis Targeting Chimeras) from PROTAC-DB45 and Chemical Probes Portal, 60 covalent binders from CovalentInDB,46 and 21 biased GPCR ligands from BiasDB.19 Our analysis also established that 132 compounds were flagged with one or more structural alerts from either a) PAINS filters,47,48 b) aggregators,49 c) cellular assay nuisance compounds50 or d) historical compounds. Of the 60 stereoisomers, 54 originate from the Probe Miner set. Compared to small-molecule approved drugs extracted from ChEMBL (as a set in P&D) probes are generally larger and more complex (Fig. 1). This correlates with higher target selectivity that, in turn, is reflected in the number of associated targets (Fig. 2). However, these values could be biased by approved drugs accumulating more cross-screening data and hence a wider range of secondary targets. Fig. 1 Physico-chemical properties distribution (top: molecular weight, middle: calculated log P, bottom: TPSA) for experimental probes (blue), calculated probes (orange) and small-molecule approved drugs set from ChEMBL (green) (x: property range, y: compounds percentage). The properties were calculated by RDKit. This journal is © The Royal Society of Chemistry 2021 Chemical Probes Portal (CP portal) This provides expert usage recommendations based on publication evaluations from a Scientific Advisory Board (SAB, of which one of us, CS, is a member). While content had languished below 200 compounds for some time, this has recently expanded to 362. Importantly, this portal also This journal is © The Royal Society of Chemistry 2021 1430 | RSC Med. Chem., 2021, 12, 1428–1441 1430 View Article Online RSC Medicinal Chemistry Table 2 A matrix showing the intersections between 12 sources. This was computed using the InChIKey exact match for the standardised structures from the P&D portal. The diagonal figures in white represent the source counts in Table 1 Table 2 A matrix showing the intersections between 12 sources. This was computed using the InChIKey exact match for the standardised structures from the P&D portal. The diagonal figures in white represent the source counts in Table 1 Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. RSC Medicinal Chemistry Ki, IC50 or Kd). Some may also have secondary targets with a data-supported potency below that against the primary target (we have avoided using the term “off-target” since there are few cases where secondary targets have been mechanistically assigned as a side-effect or toxicity liabilities). The 132 probes without primary target annotation were, in most cases, directed against viruses, bacteria, cell lines or pathways. They also predominantly belonged to the MLP and Nature Chemical Biology legacy sets. For the remainder, we collated 819 single and multi-component protein targets with 549 for both experimental and calculated with an overlap of 279. In total these constituted 807 distinct single protein identifiers (i.e. UniProt IDs51), 544 for experimental and 535 for calculated probes with 272 in- common. The human Swiss-Prot target count was 796. The differences in Fig. 3 reflect inherent biases. For example, predicted probes have almost double the number of GPCRs52 but, compared to predicted probes, proportionally fewer kinases and epigenetic regulators. This is likely to be due to the challenges of optimising single-target selective ligands within these target families.37,53 However, probes with intra-family selectivity can also be experimentally useful but run the risk of being rejected by quality scoring weighted towards single-target selectivity. RSC Medicinal Chemistry RSC Medicinal Chemistry there are several length forms in TrEMBL but not Swiss-Prot) or ABL1 (P00519) or, in the case of ∼20 probes from the Probe Miner set, BCR (P11274). Other complicating examples are the cyclin-dependent kinases (CDKs), where the probes may be annotated by sources with one of the human CDKs, a CDK in complex with a specific cyclin, or both. The highest probe target numbers (5 experimental and 225 calculated) have been assigned against mTOR (MTOR, P42345). Next in rank are histone deacetylase 1 (HDAC1, Q13547) with 2/161 experimental/calculated, epidermal growth factor receptor (EGFR, P00533) with 7/103 and estrogen receptor (ESR1, P03372) with 1/92. The larger number for calculated probes reflects their more frequent origin from panel screening papers and consequent higher average compounds: target ratio of 9.8 for the Probe Miner set compared to ∼1.0 for experimental probes. For these, the highest assigned target numbers are BRD4 (O60885) and the BRD3/BRD2 (Q15059/P25440) subfamily pair with 18 and 15 probes, respectively. This is a consequence of the declared SGC Probes focus on epigenetic regulators. The family distribution of all annotated targets is shown in Fig. 3. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 The number of associated targets for probes (blue) and the small-molecule approved drugs set from ChEMBL (green) (x: axis number of targets, y: compounds percentage). Only compounds with at least one associated target were included encompassing 4257 probes and 2005 approved drugs. in vitro binding data (e.g. Ki, IC50 or Kd). Some may also have secondary targets with a data-supported potency below that against the primary target (we have avoided using the term “off-target” since there are few cases where secondary targets have been mechanistically assigned as a side-effect or toxicity liabilities). The 132 probes without primary target annotation were, in most cases, directed against viruses, bacteria, cell lines or pathways. They also predominantly belonged to the MLP and Nature Chemical Biology legacy sets. For the remainder, we collated 819 single and multi-component protein targets with 549 for both experimental and calculated with an overlap of 279. In total these constituted 807 distinct single protein identifiers (i.e. UniProt IDs51), 544 for experimental and 535 for calculated probes with 272 in- common. The human Swiss-Prot target count was 796. in vitro binding data (e.g. RSC Medicinal Chemistry View Article Online View Article Online View Article Online Fig. 2 The number of associated targets for probes (blue) and the small-molecule approved drugs set from ChEMBL (green) (x: axis number of targets, y: compounds percentage). Only compounds with at least one associated target were included encompassing 4257 probes and 2005 approved drugs. Research Article RSC Medicinal Chemistry Target mapping The majority of probes have primary targets specified in their sources. In most cases these are supported by quantitative RSC Med. Chem., 2021, 12, 1428–1441 | 1431 This journal is © The Royal Society of Chemistry 2021 Target intersections in UniProt In practice, the number of protein targets is below 819, since multi-component targets may be variably annotated against either a protein subunit, the complex target, or both. For example, probes directed against the BCR–ABL1 fusion protein may be annotated with the fusion protein (of which Having assigned target IDs to both probe sets we compared these with informative cross-references in UniProt.51 We selected 4213 protein IDs (as human Swiss-Prot entries) based on the union (OR operator) of the four high-quality Fig. 3 A bar chart showing the target families distribution separately for all (green), experimental (blue) and calculated (orange) probes (x: target family, y: number of targets). The assignments are based on the ChEMBL and Guide to Pharmacology target classification. Fig. 3 A bar chart showing the target families distribution separately for all (green), experimental (blue) and calculated (orange) probes (x: target family, y: number of targets). The assignments are based on the ChEMBL and Guide to Pharmacology target classification. 1432 | RSC Med. Chem., 2021, 12, 1428–1441 This journal is © The Royal Society of Chemistry 2021 This journal is © The Royal Society of Chemistry 2021 Research Article View Article Online View Article Online RSC Medicinal Chemistry Research Article Research Article curated chemistry-to-target databases (already mentioned) of ChEMBL, BindingDB, GtoPdb and DrugCentral. These IDs represent liganded targets for 21% of the UniProt proteome of 20 395 (although this drops to 19 205 for HUGO Gene Nomenclature Committee annotation). The comparative protein sets we also selected were from the four target development levels (TDLs) of the Pharos resource for Illuminating the Druggable Genome (IDG).54 This facilitates exploration of both the characterised and the understudied (or “dark”) regions of the human proteome with a view to expanding functional insights and finding new drug targets. We initially selected the combination of the Tchem54 (1593 proteins) known to bind small molecules (other than approved drugs) with target-class specific potency thresholds plus the Tclin54 (659 proteins) as targets of approved drugs. The union of these two is 2221 proteins. The intersections of these four lists are shown in Fig. 4. • Concentration unit errors for secondary target activity (i.e. the compound was thus not selective). • Erroneous target annotations that falsely indicate potent secondary target activity. • Potency values assigned to only a subunit in a multi- component target (i.e. thus technically without supporting bioactivity data). ess Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. As an operating principle P&D fixes any unequivocal errors we spot. At the same time, we notify the originating sources about these errors that could otherwise persist and proliferate between databases. However, we have found the speed with which these are fixed at source has been variable (although this is clearly dependent on build cycle times and release versions). The two notable features are: The two notable features are: Probe scoring schemes 1. Probes have activity against 53 proteins not in Tclin or Tchem. We optimised four different scoring schemes to support users for probe triage and selection. As explained, the Probe Miner (PMIS) and P&D probe-likeness scores (PDPS) are data- supported. The other two scores are expert opinion-based and thus more subjective. These are abstracted from the Chemical Probes Portal rating for use in cells (CPOC) and in organisms (CPOO). These represent a summed rating of chemical properties, primary targets, secondary targets and in most cases, expert judgments. The conceptual difference with data- supported scores is that these are calculated for compound- target pairs. Thus, a single compound can have multiple scores against each of its assigned targets. Users may thus select the most suitable of these pairings but the probes can also be used for intra- or inter-family selectivity. For comparability, all scores are normalized to between 0 and 100%. 2. The Swiss-Prot liganded proteome includes 743 probe targets. Analysis with other TDL sets established that of the 659 approved targets 265 were also covered by probes. However, there were no intersections between probes and the 6368 Tdark proteins. This implies that the current probes may have already expanded Tchem but there is no overlap with dark targets. This journal is © The Royal Society of Chemistry 2021 RSC Med. Chem., 2021, 12, 1428–1441 | 1433 Target intersections in UniProt • Some sources had incorrectly assigned human TrEMBL partial sequence entries as targets rather than the human Swiss-Prot IDs (although only three cases were found). Source errors Despite curatorial diligence, low levels of annotation errors, including the transitive inheritance of author mistakes extracted from papers, inevitably creep into the bioactivity databases we have used as sources.55 During manual cross- checking we identified the following error types affecting approximately 40 probe entries: The PMIS, ranging from 0 to 1, combines partial scores for 1) potency, 2) selectivity 3) activity in cells, 4) SAR data 5) availability of an inactive analogue and 6) a PAINS score. Nevertheless, probe suitability is not prescribed by the score value but by so-called minimum quality criteria. These include 100 nM potency in biochemical assays, 10-fold target selectivity, and 10 μM potency in cell-based assays (but not necessarily evidence of intra-cellular primary target engagement). The Probe Miner set of 3187 compounds meeting these criteria thus have a PMIS between 0.38 and 0.85. A more detailed description is given in the Probe Miner publication.16 • Substitution of a biochemical for a cell-based assay as well as vice versa cases (the most common problem). Fig. 4 Venn diagram of P&D targets against selected human UniProt cross-references and two Pharos TDLs. The PDPS, scaled from 0 to 1, incorporates partial scores in common with PMIS but adds in orthogonal probes. The comparison of both scoring schemes is shown in Table 3. Unlike the Probe Miner selectivity score, P&D also highlights target sub-family selectivity beyond just single proteins. The probe-likeness of a compound is closely related to the PDPS value. Each compound with a score above 0.7 is labelled as P&D-approved based on the available data. The score is capped to not exceed 0.7 unless it passes all three core criteria (i.e., in vitro potency, cell potency and selectivity). Fig. 4 Venn diagram of P&D targets against selected human UniProt cross-references and two Pharos TDLs. RSC Med. Chem., 2021, 12, 1428–1441 | 1433 This journal is © The Royal Society of Chemistry 2021 Table 3 Comparison of PMIS and PDPS. For parameters with a defined range, the score is 0% for values below the minimum and 100% for values greater than the maximum. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. — Binary (1 and −3 for historical compounds) PAINS, aggregators + other nuisance compounds in cellular assays, historical compounds Compounds labelled as P&D approved for PDPS >70% 1109 probe with low ratings from the Chemical Probes Portal (not scored by Probe Miner), AGI-5198 (ref. 59) (FNYGWXSATBUBER-UHFFFAOYSA-N), was proposed as a prototypical IDH-1 R132H inhibitor. However, this was surpassed in potency and characterization details by the more recent GSK864 (ref. 60) (DUCNNEYLFOQFSW- PMERELPUSA-N) which also has an inactive control for proof of target involvement. However, as a second, distinct chemotype, this probe could be used for corroborative phenotypic assays. Compounds labelled as historical are down-weighted by subtracting 0.3 and thus cannot be labelled as P&D-approved. Currently, there are 1109 probes labelled as P&D approved. More details on this are given on the P&D FAQ page.56 The CPOC and CPOO scores (from 0 to 4 stars) are based on Scientific Advisory Board (SAB) reviews. These may be accompanied by comments and usage recommendations. However, there is currently a review backlog in that out of 362 compounds, 274 have been rated for use in cells and 225 for use in model organisms. Compounds labelled as historical are down-weighted by subtracting 0.3 and thus cannot be labelled as P&D-approved. Currently, there are 1109 probes labelled as P&D approved. More details on this are given on the P&D FAQ page.56 The CPOC and CPOO scores (from 0 to 4 stars) are based on Scientific Advisory Board (SAB) reviews. These may be accompanied by comments and usage recommendations. However, there is currently a review backlog in that out of 362 compounds, 274 have been rated for use in cells and 225 for use in model organisms. In Table 4, we review three examples of compounds with assigned probe scores. The first is a selective RIPK1 inhibitor, GSK2982772 (ref. 57) (LYPAFUINURXJSG-AWEZNQCLSA-N), highly scored by all three probe sources. The second is BET family bromodomain inhibitor from SGC Probes, JQ-1 (ref. 58) (DNVXATUJJDPFDM-KRWDZBQOSA-N), scored highly by P&D and Chemical Probes Portal, but as a family-selective probe with lower PMIS. The latter is the only P&D approved Source errors Within this range, there is a linear relationship between the value and the score Parameter PMIS value range (weight) note PDPS value range (weight) note Potency (biochemical) 5–10 [−log(M)] (4) 6.5–7 [−log(M)] (2) Selectivity Complex selectivity score normalized per target (8) 10–30-Fold (2) Potency (cell-based) 5 [−log(M)] (2) without the evidence of primary target engagement 5.5–6 [−log(M)] (2) with the evidence of primary target engagement Inactive analogue Binary (1) Binary (1) Orthogonal probe — Binary (1) SAR Binary (1) — Structural alert Binary (1) PAINS Binary (1 and −3 for historical compounds) PAINS, aggregators + other nuisance compounds in cellular assays, historical compounds Probe-likeness determination Independent of the score value, compounds labelled as possible suitable probes if they meet minimum quality criteria (100 nM potency, 10 μM cell potency, 10-fold selectivity) Compounds labelled as P&D approved for PDPS >70% Probe-like compounds count 3187 1109 RSC Medicinal Chemistry Research Article View Article Online Research Article RSC Medicinal Chemistry Table 3 Comparison of PMIS and PDPS. For parameters with a defined range, the score is 0% for values below the minimum and 100% for values greater than the maximum. Within this range, there is a linear relationship between the value and the score High-quality chemical probes set Table 5 Matrix showing the intersections between six different probe scores and probe types. This was computed using the InChIKey exact match for the standardised structures from the P&D portal As an outcome of this study, we have compiled a high-quality chemical probes subset (HQCP). We have used the PDPS for the addition of P&D approved experimental probes plus those P&D approved calculated probes that are in at least one established tool compound set. We have thus partitioned four compound sets from P&D: 1. Concise Guide to Pharmacology 2019/20 is a set extracted from a biennial series of publications providing concise overviews of the key properties of ∼1800 human drug targets with an emphasis on selective pharmacology.20–26 Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2. Kinase chemogenomics set is a collection of narrow- spectrum small molecule kinase inhibitors assembled by the SGC-UNC to study the biology of dark kinases. This is the most diverse and highly annotated public collection of kinase inhibitors.61 3. Kinase inhibitors were extracted from a series of Molecular Cell papers by Wang and Gray summarising recently-reported kinase inhibitors.62,63 since the highest value is 85%, a setting of 75% would leave only 54 compounds from more than 3000. We thus chose to set the Probe Miner threshold at 60%, thus leaving 1282 compounds, a similar number to P&D approved probes. 3. Kinase inhibitors were extracted from a series of Molecular Cell papers by Wang and Gray summarising recently-reported kinase inhibitors.62,63 4. Novartis Chemogenomic Library – NIBR MoA Box was compiled via data mining and institutional crowdsourcing. It is regularly updated and used widely both within Novartis and by their external collaborators.64 4. Novartis Chemogenomic Library – NIBR MoA Box was compiled via data mining and institutional crowdsourcing. It is regularly updated and used widely both within Novartis and by their external collaborators.64 The comparison between different scoring schemes (Table 5) highlights differences between judgment-based and calculated scores. One of the reasons for these differences is data incompleteness, for example where affinity data is only for the presumed primary target thereby precluding selectivity assessment. We used the quality criteria in Table 6 to select 548 probes for HQCP (451 experimental, 208 calculated with 114 in common). Score comparisons To extend our systematic comparison of scoring, we introduced quality thresholds. PDPS was set at 70% as used for P&D approved probes. For CPOC and CPOO, we raised this to 75% (equivalent to 3 out of 4 stars in the original rating system). For the PMIS, there is no clear threshold and Table 4 Three selected chemical probes with assigned probe scores from P&D, Probe Miner and Chemical Probes Portal Three selected chemical probes with assigned probe scores from P&D, Probe Miner and Chemical Probes Portal Table 4 Three selected chemical probes with assigned probe scores from P&D, Probe Miner and Chemical Probes P probes with assigned probe scores from P&D, Probe Miner and Chemical Probes Portal Table 4 Three selected chemical probes with assigned probe scores from P&D, Probe Miner and Chemical Probes Portal Name GSK2982772 JQ-1 AGI-5198 PDPS 100% 100% 86% PMIS 70% (in Probe Miner set) 48% (not in Probe Miner set) — CPOC 100% 100% 50% CPOO 83% 75% 42% 1434 | RSC Med Chem 2021 12 1428–1441 This journal is © The Royal Society of Chemistry 2021 Table 4 Three selected chemical probes with assigned probe scores from P&D, Probe Miner and Chemical Probes Portal Name GSK2982772 JQ-1 AGI-5198 PDPS 100% 100% 86% PMIS 70% (in Probe Miner set) 48% (not in Probe Miner set) — CPOC 100% 100% 50% CPOO 83% 75% 42% This journal is © The Royal Society of Chemistry 2021 Research Article View Article Online Research Article View Article Online View Article Online RSC Medicinal Chemistry Research Article Table 6 The criteria used for the selection of HQCP. The count column contains the number of compounds matched by the criterion. The total number represents the union of all criteria number represents the union of all criteria Criterion Count 1 Belong to one of the high-quality probe sets (except Chemical Probes Portal) 256 2 CPOC or CPOO score at least 75% (i.e. three out of four stars in the original Chemical Probes Portal rating system) 208 3 P&D approved experimental probes 150 4 P&D approved probes belonging to one of the non-commercial high-quality sets (Concise Guide To Pharmacology, Kinase Chemogenomic Set, Kinase Inhibitors, and Novartis Chemogenetic Library) 177 5 Not labelled as a historical compound −2 Total 548 This journal is © The Royal Society of Chemistry 2021 High-quality chemical probes set The intersections are shown in Table 7 including the EU-OPENSCREEN Bioactive Compound Library and Drug Repurposing Hub65 set. As non-commercial bioactive libraries, these are included in P&D as relevant for probe research. Even the experimental probes included 195 compounds without bioactivity data. In addition, we found 142 compounds annotated against single targets without selectivity data. The union of these represents 36% of the experimental probes that cannot thus be properly scored. Another reason for differences arises between stringent criteria-based evaluation and expert judgment. The HQCP set contains 42 approved drugs with 102 clinical candidates, 27 PROTACs, 15 covalent binders and 10 compounds tagged with a structural alert (four for aggregation and six for PAINS). The overlap between HQCP and the calculated sets is largest for the P&D approved probes with 244 out of 1109 compounds, but these were also partly used for the HQCP selection. From the complete Probe Miner set (i.e. without the PMIS threshold applied), there are 66 compounds from nearly 3200 meeting the Probe Miner minimum quality criteria. On the other hand, there are 153 compounds from 515 in the tool compound set, mainly from the inclusion of the Chemical Probes Portal compounds in the tool compound set. We also found differences between calculated scores for the 265 compounds-in-common between the 1109 (P&D) and 1282 (Probe Miner) sets. This could be attributed to the differences in the scoring methodology but also the associated data (i.e. not all experimental probes have PMIS). While Probe Miner currently employs bioactivity data from ChEMBL and BindingDB, P&D uses more recent versions and complements these with smaller data sources such GtoPdb. This is reflected in a high P&D score for 150 experimental probes (with 55 highly-rated by CPOC) while Probe Miner detects 20 (including 10 based on the CPOC). The intersections between the three bioactive screening libraries (Novartis Chemogenetic Library, EU-OPENSCREEN RSC Med. Chem., 2021, 12, 1428–1441 | 1435 This journal is © The Royal Society of Chemistry 2021 View Article Online Research Article RSC Medicinal Chemistry Table 7 Matrix showing the intersections between HQCP and other selected sets. This was computed using the InChIKey exact match for the standardised structures from the P&D portal Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. High-quality chemical probes set first part of this necessitated the mapping of all P&D probe structures to PubChem CIDs via InChIKey matches and SMILES strings for cross-corroboration. This was done using the PubChem Identifier Exchange Service.67 We expected high coverage from the probe sources which we knew to have entered PubChem by various routes. From the 940 experimental probes, we recorded 915 CID matches (910 from IKs plus five more from SMILES). Inspection of the unmapped probes confirmed that most from SGC Probes, opnMe Portal and Gray Laboratory had no submission path into PubChem (directly, or via other source). In addition, we found the three unmatched MLP probes had different or flattened stereochemistry in PubChem (i.e. matched different non-isomeric CIDs). The corresponding 3670 calculated probes matched 3557 CIDs. While the mismatches were still only 3%, the reasons behind these are (again) differences in the handling of stereochemistry between PubChem and P&D (the latter uses RDKit as its main cheminformatics framework68). We also discovered that the links to some compounds are missing from ChEMBL because of InChIKey differences (ChEMBL is also using the RDKit framework69). Bioactive Compound Library, Drug Repurposing Hub) are 5.0%, 7.5% and 2.8%, respectively. For target assessment, the HQCP covers 447 distinct proteins. The distribution of target families for all, experimental, calculated and HQCP probes is shown in Table 8. The HQCP was added as a separate compound set to the P&D portal and will be updated regularly. As new bioactivity data and new versions of compound sets are integrated we expect the number to increase. This journal is © The Royal Society of Chemistry 2021 1436 | RSC Med. Chem., 2021, 12, 1428–1441 Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The 67% inclusion in the MLSMR means these particular probes may have expanded profiling data from unpublished assays not captured by ChEMBL, including testing against malaria, other disease parasites and cancer cell lines (this is particularly the case for the older MLP compounds). The extensive data overlap between ChEMBL and BindingDB arises from their mirroring collaboration but the latter has unique content from patent SAR extractions. For some years GtoPdb has included probe curation from papers selected for their pharmacological relevance and this is reflected in the capture of 305 probes.29 The availability of a PDB ligand structure for 242 probes is clearly enabling for many reasons but note these may not all be for the probe-primary target pair or species. The explanation for the low hits to the Chemical Probes Portal was the inclusion of historical probes in their 2017 PubChem submission (we suggest the separate submission of this cautionary subset in the future). The last row in the table presents two anomalies. As discussed above, at least 100 additional nominal MLP probes can be found in various lists beyond the 223 in the PubChem CID select for “BioAssay, Probes”.7 While reasons for the low match in P&D are being investigated the historical confusion associated with legacy MLP compounds may confound explanation. PubChem (note also the opnMe probes are free upon application). The 770 matches in ChEMBL indicate high levels of probe-target activity data extracted from papers. However, there is an unexpected shortfall of 115 probes without any active results in BioAssay. The explanations are either the probe generators have not published in their assay results or these were not in journals that ChEMBL, BindingDB or the Guide to Pharmacology would have extracted and then submitted to PubChem. PubChem (note also the opnMe probes are free upon application). The 770 matches in ChEMBL indicate high levels of probe-target activity data extracted from papers. However, there is an unexpected shortfall of 115 probes without any active results in BioAssay. The explanations are either the probe generators have not published in their assay results or these were not in journals that ChEMBL, BindingDB or the Guide to Pharmacology would have extracted and then submitted to PubChem. The fact that 71% of the experimental probes have patent matches was a surprise since the impact of potential Intellectual Property (IP) issues on probe usage has not been widely discussed. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. While this high proportion seems at odds with the Open Science context that probe development teams espouse, the matches only mean the structures are specified in patent documents rather than necessarily being within the scope of allowed claims. Many of the automated extractions may merely represent prior-art mentions including where applicants have exploited analogue expansions from existing probe structures as drug discovery starting points. Notwithstanding, some probe structures may be explicitly claimed in maintained and granted patents (although precisely how many is difficult to assess). However, open patent information has become increasingly available and compound-to-patent document mappings are now indexed for nearly 40 million PubChem CIDs.70 An interesting example is the Boehringer opnMe GPR142 agonist BI-1046 (MLOGCHDCTRINMU-UHFFFAOYSA-N). Two sources in PubChem have extracted the structure from Boehringer's WO2020007729 “Triazole benzamide derivatives as GPR142 agonists”. From CID 146293963 (via SureChEMBL SID 405725530), we can map the structure to example 2 and a table of low nM IC50 SAR values for 20 analogues (with synthesis details) that can also be found in the PubChem “Similar Compounds” section. The explanations for the calculated probes are the same as for the experimental but show a different pattern in the 11 rows of Table 9. Since these are predominantly derived from ChEMBL, the matches against this source, BioAssay active and BindingDB are all high. In contrast, vendor matches are proportionally much lower. While the patent intersection drops to 61% this still impacts 2227 CIDs. The explanation lies in the fact that many of the organisations (academic or commercial) generating the medicinal chemistry papers that ChEMBL curates (and Probe Miner selects) also file patents on their characterised compounds in advance of publication. Notwithstanding potential IP complications, it is important to note that patent matches are potentially advantageous for probe evaluation because they may well contain unpublished selectivity and SAR data not captured in probe sources.70 This journal is © The Royal Society of Chemistry 2021 PubChem intersections As the de facto global hub for chemical structures, associated bioactivity data and a massive range of informatic connectivity it was of considerable interest to profile probe sets against the 110 million compounds in PubChem.66 The 1436 | RSC Med. Chem., 2021, 12, 1428–1441 Table 8 The target families distribution separately for all, experimental, calculated and HQCP probes Target family All Experimental Calculated HQCP Kinase 201 179 146 152 GPCR 146 65 115 70 Hydrolase 98 59 54 36 Epigenetic regulator 75 73 44 68 Transferase 39 28 23 26 Ion channel 34 22 21 16 Oxidoreductase 33 10 27 8 Transporter 29 11 22 12 Nuclear receptor 26 14 20 15 Cytochrome P450 9 2 9 3 Isomerase 8 6 3 2 Ligase 5 3 5 2 Lyase 5 0 5 0 Other 111 77 55 37 Total 819 549 549 447 Table 8 The target families distribution separately for all, experimental, calculated and HQCP probes The second part of this analysis compared the two probe sets with selected PubChem sources to give additional insights. The numbers, shown in Table 9, are, again, a mixture of the expected and unexpected. We can propose explanations, starting with the experimental probes. The high level of BioAssay positive results is expected but does not establish whether those are the same probe-target pairs annotated in P&D. From the 915 CID matches in PubChem, 784 (85%) include vendors submissions, indicating a high availability for purchase. However, this expands slightly since additional vendor matches internal to P&D may not be indexed in This journal is © The Royal Society of Chemistry 2021 View Article Online RSC Medicinal Chemistry Research Article Table 9 CID intersections between experimental and calculated probes for selected PubChem sources, ranked by the number of experimental probe matches. Note that most of these results can alternatively be read off directly from the P&D portal and give the same or close numbers. The total column represents a number of distinct compounds in the respective sources patent holders can only be addressed on a case-by-case basis. The assumption of Research Use Exemption should apply to US academics but the position of commercial institutions is less clear.71 Note, however, despite the detailed data package in the opnMe portal, the absence of a publication (BI-1046 is PubMed- negative but has over 40 false positives in Google Scholar because of an HIV clinical trial designation BI 1046) means that CID 146293963 has neither ChEMBL nor BioAssay links. Source Total Experimental Calculated PubChem 109 818 005 915 3557 BioAssays – active 1 457 929 800 3487 Vendors 59 867 622 784 810 ChEMBL 2 067 192 770 3519 Patents 39 401 959 652 2227 MLSMR 406 097 622 416 BindingDB 975 228 608 3331 GtoPdb 8705 305 335 PDBe 33 543 242 287 Chemical Probes Portal 467 186 131 BioAssays Probes 223 152 2 Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Discussion This work provides a uniquely comprehensive and comparative overview of probe sources and targets. This will be maintained and expanded for experts and non- informaticians seeking probes to use in their work. Although our results are presented in good faith, we understand the causes of fuzziness (some of which have been discussed) that caution against these numbers being taken as ground truth. While the opnMe portal magnanimously declares that results generated with their molecules belong to the ordering scientists, the IP situation regarding other probe structure RSC Med. Chem., 2021, 12, 1428–1441 | 1437 This journal is © The Royal Society of Chemistry 2021 RSC Medicinal Chemistry View Article Online View Article Online View Article Online Author contributions CŠ collated the data with CŠ and CS being responsible for the analysis. All authors edited and approved the final manuscript. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 16 July 2021. Downloaded on 10/24/2024 4:54:43 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 5. There are many selective and potent compounds appearing in the recent medicinal chemistry and chemical biology literature that, while not officially yet declared in probe sources, include sufficient characterisation for useful probe criteria scoring. However, the rate of data extraction from these publications and flow into databases remains slow.72 6. We are considering how to address the mismatched and missing probes in PubChem but we need to iterate with the originating sources in the first instance. FAIR and reproducible This situation can obviously be ameliorated by generating more data but, going forward, it is not clear how the existing data gaps can best be backfilled. 2. Comprehensive characterisation of a probe, including the necessary broad cross-screening, requires extensive experimental work. In addition, the results need to be accessible (ideally in an open-access text-minable publication), reproducible and easily captured for transfer into database records. This situation can obviously be ameliorated by generating more data but, going forward, it is not clear how the existing data gaps can best be backfilled. FAIR and reproducible We have endeavoured to make this work findable, accessible, interoperable and reusable, according to Open Science principles.73 As for P&D itself, the licence is CC BY-SA 4.0. We have submitted a ESI† sheet that includes compound names, SMILES, InChIKeys, target identifiers, source assignments as well as other data used for the study. For interoperability, this will be deposited into Figshare in .xlsx format. No proprietary software has been used in this work and we thus expect any analysis reported here to be reproducible (if users encounter difficulties, they are welcome to contact us). As mentioned, all the intersections between sources inside the P&D database can simply be read off, combined, downloaded and users' own sets uploaded for further intersection analysis. Also as described, we recommend the PubChem Identifier Exchange Service for casting SMILES or InChIKeys at a medium scale against PubChem in total or selected sources within it. Many of the data sources used in this work (and consequently P&D) will expand with new releases so we expect numbers to change within a few months of these compilations made in April 2021. During the course of this work and the preceding years of P&D operation, the team has encountered a range of technical challenges most of which have been alluded to above. In this regard, while most stand-alone probe sources are designed with the needs of their users in mind, it is important for scientists to be able to navigate across multiple sources to obtain an overview of all potential probes in advance of experimental planning. This presents a particular challenge for non-informaticians and for which we needed much data-wrangling effort to complete the overview that P&D now offers. During this work, we also detected problematic anomalies, some of which are listed below. These are not presented as criticisms but more as pointers towards what could be improved. 1. The current probe data landscape is particularly patchy. This means for many compounds their associated data falls short of the well-publicised criteria and thus compromises the utility of scoring. 2. Comprehensive characterisation of a probe, including the necessary broad cross-screening, requires extensive experimental work. In addition, the results need to be accessible (ideally in an open-access text-minable publication), reproducible and easily captured for transfer into database records. Conflicts of interest 3. The bias towards known targets seems counter-intuitive. Given that mTOR has 38 020 PubMed hits and ChEMBL has 4557 compounds aligned against P42345 (including 20 clinical candidates), the need for 5 experimental and 225 calculated probes is not obvious (although new highly selective and potent allosteric modulators of old targets could provide new insights). As the Pharos TLD categories indicate, probe development that would broaden Tchem and make There are no conflicts to declare. 3. The bias towards known targets seems counter-intuitive. Given that mTOR has 38 020 PubMed hits and ChEMBL has 4557 compounds aligned against P42345 (including 20 clinical candidates), the need for 5 experimental and 225 calculated probes is not obvious (although new highly selective and potent allosteric modulators of old targets could provide new insights). As the Pharos TLD categories indicate, probe development that would broaden Tchem and make This journal is © The Royal Society of Chemistry 2021 Research Article RSC Medicinal Chemistry Notwithstanding, we have analysed 940 experimental and 3670 calculated probe candidates. Together these provide evidence of specific binding for 796 human proteins across the target classes. We have flagged unsuitable (i.e. potentially misleading and resource-wasting) compounds from both probe groups. Compared to ChEMBL approved drugs, probes tend to be larger and more complex structures. inroads into Tdark could lead to functional illumination (but with the caveat of the obvious paucity of assays for understudied proteins). 4. The identification and provision of the crucial control compounds lag behind probe availability. Notably, a recent analysis of negative controls extols the council of perfection in that a quartet of compounds is needed to maximise interpretation (i.e. two probes of different chemotypes and two negative controls, also matched as different chemotypes).41 Although calculated probes are in a large majority, we established that their scoring is influenced by methodology and biases in data sources. Consequently, the application of PMIS and PDPS scoring retrieves different numbers of quality-rated probes from the Chemical Probes Portal set (i.e. 6 : 1 in favour of PDPS). We thus support scoring as a pragmatically useful means of compound prioritisation. By combining established criteria, we developed this further to delineate 548 high-quality chemical probes (HQCP) covering just under 450 targets. As we shown above, the Swiss-Prot bioactive chemistry cross-references indicate a data- supported druggable proteome of 20%. The current “probe proteome” targets would reach only 4% dropping to half of that for the HQCP set. Notes and references Bunnage, C. Buser-Doepner, R. M. Campbell, A. J. Carter, P. Cohen, R. A. Copeland, B. Cravatt, J. L. Dahlin, D. Dhanak, A. M. Edwards, M. Frederiksen, S. V. Frye, N. Gray, C. E. Grimshaw, D. Hepworth, T. Howe, K. V. M. Huber, J. Jin, S. Knapp, J. D. Kotz, R. G. Kruger, D. Lowe, M. M. Mader, B. Marsden, A. Mueller-Fahrnow, S. Müller, R. C. O'Hagan, J. P. Overington, D. R. Owen, S. H. Rosenberg, R. Ross, B. Roth, M. Schapira, S. L. Schreiber, B. Shoichet, M. Sundström, G. Superti-Furga, J. Taunton, L. Toledo-Sherman, C. Walpole, M. A. Walters, T. M. Willson, P. Workman, R. N. Young and W. J. 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RSC Medicinal Chemistry Research Article Acknowledgements This work was supported by the MEYS grant LM2018130 and by RVO: 68378050-KAV-NPUI. Useful comments from the two referees and discussions with Prof. Michal Gilson were much appreciated. This journal is © The Royal Society of Chemistry 2021 1438 | RSC Med. Chem., 2021, 12, 1428–1441 View Article Online RSC Medicinal Chemistry Notes and references Grimshaw, A. IJzerman, T. Hanke, I. V. Hartung, S. Hitchcock, T. Howe, T. V. Hughes, S. Laufer, V. M. Li, S. Liras, B. D. Marsden, H. Matsui, J. Mathias, R. C. O'Hagan, D. R. Owen, V. Pande, D. Rauh, S. H. Rosenberg, B. L. Roth, N. S. Schneider, C. Scholten, K. Singh Saikatendu, A. Simeonov, M. Takizawa, C. Tse, P. R. Thompson, D. K. Treiber, A. Y. Viana, C. I. Wells, T. M. Willson, W. J. Zuercher, S. Knapp and A. Mueller-Fahrnow, eLife, 2018, 7, e34311. Open Access Article. Published on 16 July 2021. 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PHAGOCYTOSIS ACTIVITY OF BINAHONG (Anredera cordifolia (Tenore.) Steenis) FROM SECANG, MAGELANG, CENTRAL JAVA, INDONESIA
Jurnal farmasi sains dan komunitas
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*Corresponding author: Nunung Yuniarti Email: nunung@ugm.ac.id, nunzyuniarti@gmail.com Vol. 16 No. 1 Vol. 16 No. 1 JURNAL FARMASI SAINS DAN KOMUNITAS, May 2019, 7-13 p-ISSN 1693-5683; e-ISSN 2527-7146 doi: http://dx.doi.org/10.24071/jpsc.001693 PHAGOCYTOSIS ACTIVITY OF BINAHONG (Anredera cordifolia (Tenore.) Steenis) FROM SECANG, MAGELANG, CENTRAL JAVA, INDONESIA Dika Sotya Sakti, Perdana Priya Haresmita, Nunung Yuniarti*), Subagus Wahyuono Faculty of Pharmacy, Universitas Gadjah Mada, Bulaksumur, Yogyakarta, Indonesia Received January 14, 2019; Accepted March 18, 2019 ABSTRACT The use of medicinal plants is increasing due to the search for alternative resources to treat diseases such as hypertensions and infection. Along with the development in science, preventive action should take place to prevent our body from suffering from these diseases. This can be done by increasing the human immune status with immunomodulatory agents. Binahong empirically have benefits for wound healing. The purpose of this research was to investigate the immunomodulatory effect of ethanolic extract of binahong leaves. The non-specific modulatory effects of the ethanolic extracts of binahong leaves on the immune systems were measured based on phagocytosis index and phagocytosis capacity. Tests were conducted on strain Balb/C male mice at the age of 6-8 weeks. Mice were administered orally with the extract of binahong leaves (doses of 25, 50, 75 mg/kgBW) for 14 days. The test results with the index parameters and macrophages phagocytosis capacity at doses of 50 and 75 mg/kgBW did not significantly increased when compared with the controls. From these results, we concluded that the ethanolic extract of binahong leaves with a dose of 25, 50 and 75 mg/kgBW cannot significantly the activity of macrophages by phagocytosis index parameters and phagocytosis capacity significantly. Keywords: Anredera cordifolia; immunomodulatory agent; macrophage; phagocytosis capacity; phagocytosis index Preparation of macrophage cells CMC-Na, Distilled water, alcohol 70%, ethanol 96% (General Labora), methanol, Giemsa 20%, latex beads (Sigma™), RPMI medium 1640 (Gibco™), FBS (Sigma™), Fungizone (Gibco™), pen-strep (Gibco™), PBS (Sigma™), hepatitis B vaccine (Euvax™). Macrophage cells were isolated from BALB/c mice (6–8 weeks old). Mice were euthanized with neck dislocation and 10 mL of cold RPMI medium were then injected inside the stomach. After 3-5 minutes, the RPMI was withdrawn from the stomach using syringe and put into a conical flask, centrifuged at 1,500 rpm (4oC, for 10 minutes). The supernatant was removed and the residue resuspended with RPMI (80% FBS). The numbers of cells was calculated with hemocytometer, diluted with RPMI (80% FBS) till 2.5 x 106/mL cells density was obtained. The cells suspension that had been cultured on 24 wells (200 µL/well, 5x105 cells/well) plate for 24 hours was put in round coverslips, incubated in a CO2 (5%) incubator, at 37oC for 30 minutes and then complete medium, containing 10% FBS, fungizone 0.5% and penicillin streptomycin 2% (1.0 mL) was added to each well and then incubated for another 2 hours. The cells were washed twice with RPMI, and then complete medium (1.0 mL) was added to each well and incubated for the next 24 hours (Hartini et al., 2013). Preparation of animal test Male mice, strain Balb/c aged 6-7 weeks weighing 25-35 g were obtained from the Animal Cage Test Faculty of Pharmacy, Universitas Gadjah Mada. The protocol of the study was approved by the Ethics Commission for Preclinical Trials of Integrated Research and Testing Laboratory (LPPT), Universitas Gadjah Mada with certificate number 00123/04/LPPT/X/2017. Extraction of binahong leaves infected by Pseudomonas aeruginosa (Sukrama et al., 2017). Binahong leaves were obtained from Secang, Magelang Regency, Central Java Province, Indonesia. Powdered leaves material of Anredera cordifolia was macerated with ethanol 96% (2x) for 24 hours each, filtered and the filtrates obtained were combined and evaporated in vacuum to give thick liquid material of ethanol extract. The ethanol extract with the dose of 25, 50 and 75 mg/kgBW were given orally to assess its immunomodulatory effects by measuring macrophage activity using phagocytosis index and phagocytosis capacity and compared with normal and CMC-Na groups. We therefore designed this study to determine the immunomodulatory effects of binahong leaves extract by measuring phagocytosis activity and phagocytosis index. The antimicrobial-antioxidant activity was correlated with immunomodulatory effects (Umar et al., 2012; Yuniarti and Lukiswanto, 2017). Phagocytosis ratio indicates the percentage of active macrophage to 100 macrophages, and phagocytosis index indicates the number of latex able to be consumed by active macrophages. These number are compared to the controls. INTRODUCTION medicinal plant in Indonesia. Binahong leaves are used for treatment of wounds, refreshing the body, headache and lowering blood pressure. Ether fraction of binahong leaves extract exhibited antioxidant activity measured by DPPH (1,1-diphehyl-2-picrylhydrazyl) (Ardianti and Guntarti, 2014). Binahong leaves extract accelerated wound healing infected by Staphylococcus aureus in mice. Binahong leaves extract also inhibited the growth of Staphylococcus aureus and as a result, the healing process the wounds is faster than wound healing without binahong leaves extract (Umar et al., 2012). Topical application of binahong leaves extract makes wound healing process faster, IL-6 level higher and increases vascular endothelial growth factor (VEGF) production in burns Medicinal plants have been used as an alternative to synthetic drugs, for a long time. Medicinal plants are potential sources of drugs and are widely used to gain health benefit empirically (Rodríguez et al., 2018). Medicinal plants are used because they are easier to obtain, cheaper than synthetic drugs and have less side effect (Wardhani and Sulistyani, 2013). Various diseases such as hypertension, diabetes mellitus, headache, inflammation and modulation of the immune system have been widely treated with medicinal plants (Leliqia et al., 2017; Putri et al., 2017). Binahong (Anredera cordifolia (Ten.) Steenis) is one of the species from the family of Basellaceae, which has fleshy leaves and thick aerial tubers. It is widely used as a Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 Preparation of sample Binahong plant leaves were obtained from the District Secang, Magelang, Central Java. The identify was authenticated by the Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada in certificate number UGM/FA/2413/M/03/02. Based on the results, the plants studied is binahong (Anredera cordifolia (Tenore) Steenis). Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 added into each well containing peritoneum macrophages, then incubated for another 60 minutes in a CO2 incubator. The cells suspension was washed 3 times with PBS in order to remove particles. The cells suspension was dried at room temperature and then fixed with methanol. Coverslips were dyed with Giemsa 20% for 20 minutes, washed with aquadest, lifted up from the wells and re-dried at room temperature. The macrophages activity was calculated as the number (%) of consumed latex (substrates), visualized by light microscope (magnified 400x) as seen as Figure 1. Phagocytosis ratio was indicated by the percentage of active macrophage in 100 macrophages, and phagocytosis index was indicated by the number of latex able to be consumed by active macrophages (Hartini et al., 2014). These data were compared to the controls. obtained by calculating the percentage of the number of active macrophages phagocyted latex beads per 100 macrophages observed, whereas macrophage phagocytosis index was obtained by counting the number of latex beads phagocyted per 100 macrophages (Jensch-Junior et al., 2006). These parameters would be able to show immunostimulatory effects of ethanolic extract of binahong leaves. Macrophages were isolated from the peritoneal cavity of mice because the number of macrophages in the peritoneal cavity is larger than the other organs and easy to obtain from the peritoneal fluid. The medium used was RPMI because this medium can attract macrophages in the peritoneal cavity and provide nutrition such as vitamins, amino acids, and essential materials required for macrophage cell culture processes. Liquids that have been isolated from the peritoneal cavity contains not only macrophage cells but also granulocytes and lymphocytes. Peritoneal fluid that was place into a conical flasks was centrifuged to separate macrophage cells from other cells such as lymphocytes and granulocytes (Hay and Westwood, 2002). Macrophages activity measurement Macrophages activity measurement was done involving latex (2 µm in diameter) as substrates (suspended in PBS, at 2.5 x 107/mL). The cells suspension (200 µL) was 8 Nunung Yuniarti et al. Nunung Yuniarti et al. Phagocytosis Activity of Binahong … Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 Hepatitis B vaccine was administered intraperitoneally on day 5 and 12 as an immune booster. The first vaccination was done to activate the non-specific and specific immune system and the second vaccination was done to increase the expression of the immune system (as a booster) so it is easier to analyze. Surgery was performed on the 15th day because the immune system will be activated optimally up to 3 days after induction of the vaccine and gradually decline thereafter (Abbas et al., 2014). macrophages. Other studies found that binahong leaves extract can also increase the production of interleukin-6 in the blood plasma of mice during the healing of burns. Interleukin-6 is a cytokine produced by T-cells and macrophages to stimulate the immune response during an infection or during the healing process (Sukrama et al., 2017). From the previous research of Sukrama et al., (2017) and Wijayanti et al., (2018), ethanolic extract of binahong leaves has the effect to increase the response of the immune system, but in the present study, the increasing phagocytosis capacity and phagocytosis index were not significant when compared to the control group. Quantification of macrophages was performed using latex beads with a size of 3 μm which were resuspended in the serum of test animals. The size of the latex beads that resemble the size of bacteria could trigger macrophage phagocytosis by being perceived as foreign particles. The addition of serum of the test animals into the latex suspension would help the macrophage phagocytosis process as serum would facilitate the introduction of the antigen by macrophages (Harvath and Terle, 1999). Research by Wijayanti et al., (2018) was using a guinea pig test animals while this study used Balb/c strain mice. Previous research was conducted to see the effect of binahong leaves extract on blood leukocytes profile and the average number of descendants of the test animals. The increase in the number of monocytes as one of the parameters of blood leukocytes profile in these studies was not always followed by an increase in activity of macrophages in the tissue. Monocytes from the blood can differentiate into multiple cell types of the immune system and in such a network of dendritic cells, osteoclasts and macrophages depends on the existing stimulus inside the body. Monocyte activity in the network tends to be more specific and different in each tissue. Phagocytosis Activity of Binahong … RESULTS AND DISCUSSION The parameters of the observed activity of macrophages were phagocytosis capacity of macrophages and macrophage phagocytosis index. Macrophage phagocytosis capacity was Figure 1. Macrophage observation under microscope magnification 400x. A: active macrophage; B: inactive macrophage. B A Figure 1. Macrophage observation under microscope magnification 400x. A: active macrophage; B: inactive macrophage. 9 Phagocytosis Activity of Binahong … Phagocytosis Activity of Binahong … Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 A Figure 2. Results of statistical analysis of phagocytosis index (A) and phagocytosis capacity (B). B A B A A B Figure 2. Results of statistical analysis of phagocytosis index (A) and phagocytosis capacity (B). Table I. Phagocytosis Capacity and Phagocytosis Index of Binahong Leaves Ethanolic Extracts Phagocytosis Capacity Phagocytosis Index Results Mean ±SD Results Mean ±SD Normal Group 44.00% 46.20% ± 0.0414 1.97 2.07 ± 0.1521 46.67% 1.92 40.33% 2.32 50.33% 2.04 49.67% 2.08 CMC-Na + Vaccine Group 48.67% 49.87% ± 0.0899 242 2.70 ± 0.4076 65.33% 3.02 42.00% 3.08 47.33% 284 46.00% 2.13 Dose 25 mg/kgBW + Vaccine 63.33% 49.33% ± 0.1269 2.88 2.59 ± 0.2267 33.67% 2.65 54.67% 2.35 45.67% 2.48 Dose 50 mg/kgBW + Vaccine 40.00% 58.86% ± 0.1324 2.81 2.55 ± 0.3002 59.67% 2.88 53.00% 2.76 74.33% 2.25 67.33% 3.06 Dose 75 mg/kgBW + Vaccine 61.00% 62.33% ± 0.0610 3.39 3.12 ± 0.6526 61.67% 2.20 56.00% 3.72 70.67% 3.17 Table I. Phagocytosis Capacity and Phagocytosis Index of Binahong Leaves Ethanolic Extra Table I. Phagocytosis Capacity and Phagocytosis Index of Binahong Leaves Ethanolic Extracts Phagocytosis Capacity Phagocytosis Index Table I. Phagocytosis Capacity and Phagocytosis Index of Binahong Leaves Ethanolic Extracts Phagocytosis Capacity Phagocytosis Index 10 Nunung Yuniarti et al. Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 (33), 1582–1614. (33), 1582–1614. (33), 1582–1614. has a lower bioavailability compared with administration by injection and the drug can be metabolized by gastrointestinal fluids or often called first-past experience effect (Atanasov et al., 2015). Hartini, Y.S., Wahyuono, S., Widyarini, S., and Yuswanto, A., 2013. Phagocytic Macrophage Activity of Fractions from Methanolic Leaf Extract of Red Betel (Piper crocatum Ruiz & Pav.) In Vitro. Jurnal Ilmu Kefarmasian Indonesia, (11), 108–115. Another factor that could affect the results when compared with previous studies was the source of the sample. Wahyukundari and Praharani (2016) collected the binahong leaves from Jember, East Java whereas in this study the binahong leaves were collected from Secang, district of Magelang, Central Java. Different areas could cause differences in the content of secondary metabolites in plants because each area has different soil nutrients. Differences in soil nutrients would affect the availability of nutrients and plant precursor to form secondary metabolites (Salim et al., 2017). Hartini, Y.S., Wahyuono, S., Widyarini, S., and Yuswanto, A., 2014. In vivo Immunomodulatory Effect and Histopathological Features of Mouse Liver and Kidney Treated With Neolignans Isolated from Red Betel (Piper crocatum Ruiz & Pav) Leaf. Tropical Journal of Pharmaceutical Research. (13), 1609–1614. Harvath, L. and Terle, D.A., 1999. Assay for Phagocytosis, In: Javois, L.C., Immunocytochemical Methods and Protocols. New York: Humana Press, 281-290. CONCLUSION Ethanolic extract of binahong leaves at dose of 25, 50 and 75 mg/kgBW cannot significantly increase the activity of macrophages by phagocytosis index parameters and phagocytic capacity. Ethanolic extract of binahong leaves at dose of 25, 50 and 75 mg/kgBW cannot significantly increase the activity of macrophages by phagocytosis index parameters and phagocytic capacity. Hay, F.C. dan Westwood, O.M.R., 2002. Practical Immunology, Fourth edition, Blackwell Science, United Kingdom. Hulin, I., Jakubovsky, J., and Viera, S., 1995. Inflammation and Fever, in: Hulin, I., Jakubovsky, J., and Viera, S., Pathophysiology Principles of Diseases. Bratislava: Comenius University, 65-72. Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 The difference is caused by different stimuli derived from the macrophage microenvironment (Hulin et al., 1995). Based on our results which was presented in Table I, after examining macrophage phagocytic capacity and macrophage phagocytosis index, the ethanolic extract of binahong leaves at dose of 25, 50 and 75 mg/kgBW did not significantly enhance the ability of macrophage through an increase of both parameters compared with the control group 0.5% CMC-Na. A significant difference was found only in the phagocytosis index data for the dose group 75 mg/kgBW when compared with the normal group, as seen as in Figure 2. Research conducted by Wahyukundari et al. (2016) was performed in vitro by taking blood monocytes. Experiments in vitro have less variables that cannot be controlled as compared to experiments in vivo. In vivo experiments involve factors including pharmacokinetics (absorption, distribution, metabolism and excretion) and the first-past effect that could affect the availability of the extract in the body. In addition, the physiological state of the test animals such as hormones could also influence the effect of the test sample. Absorption through the oral drug According to previous research, 70% ethanolic extract of binahong leaves dose of 50 mg/ kgBW could raise the profile of leukocytes as an increase in total leukocytes, neutrophils and total monocytes in guinea pigs (Wijayanti et al., 2018). In addition, the ethanolic extract of binahong leaves at concentrations of 50% and 100% could increase the phagocytic monocytes with in vitro method (Wahyukundari and Praharani, 2016). Monocytes are produced by the bone marrow which would circulate in the blood before it becomes differentiated in the tissue to 11 Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 Phagocytosis Activity of Binahong … REFERENCES Abbas, A.K., Lichtman, A.H., and Pillai, S., 2014. Cellular and Molecular Immunology, Eighth edition, Elsevier Saunders. Jensch-Junior, B.E., Pressinotti, L.N., Borges, J.C.S., and da Silva, J.R.M.C., 2006. Characterization of macrophage phagocytosis of the tropical fish Prochilodus scrofa (Steindachner, 1881). Aquaculture, (251), 509–515. Ardianti, A. and Guntarti, A., 2014. Uji Aktivitas Antioksidan Fraksi Eter Hasil Hidrolisis Infusa Daun Binahong (Anredera cordifolia (Ten.) Steenis) dengan Metode DPPH (1,1-diphenil-2- picrylhydrazyl). Pharmaciana, (4), 1– 8. Leliqia, N.P.E., Sukandar, E.Y., and Fidrianny, I., 2017. Overview of Efficacy, Safety and Phytochemical Study of Anredera cordifolia (Ten.) Steenis. Pharmacology Online, (1), 124–131. Atanasov, A.G., Waltenberger, B., Pferschy- Wenzig, E., Linder, T., Wawrosch, C., Uhrin, P., Temml, V., Wang, L., Schwaiger, S., Heiss, E.H., Rollinger, J.M., Schuster, D., Breuss, J.M., Bochkov, V.N., Mihovilovic, M.D., Kopp, B., Bauer, R., Dirsch, V.M., and Stuppner, H., 2015. Discovery and Resupply of Pharmacologically Active Plant-Derived Natural Products: A review. Biotechnological Advance, Putri, D.U., Rintiswati, N., Soesatyo, M.H., and Haryana, S.M., 2017. Immune Modulation Properties of Herbal Plant Leaves: Phyllanthus niruri Aqueous Extract on Immune Cells of Tuberculosis Patient - in vitro Study. Natural Product Research, (32), 463– 12 Nunung Yuniarti et al. Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 Phagocytosis Activity of Binahong … Jurnal Farmasi Sains dan Komunitas, 2019, 16(1), 7-13 Ekstrak Daun Binahong (Anredera cordifolia (Ten.) Steenis) terhadap Kesembuhan Luka Infeksi Staphylococcus aureus pada Mencit. Analis Kesehatan Sains, 1 (2), 1–8. Ekstrak Daun Binahong (Anredera cordifolia (Ten.) Steenis) terhadap Kesembuhan Luka Infeksi Staphylococcus aureus pada Mencit. Analis Kesehatan Sains, 1 (2), 1–8. 467. Rodríguez, E.T., Frias, M. de la C., Galardis, M.B., and Leon, J.A.M., 2018. In Vitro Antibacterial Activity of Dried Extract from Anredera vesicaria Rhizomes. Journal Of Advance in Plants and Agricultural Research, (3), 237–239. Wahyukundari, M. and Praharani, D., 2016. Pengaruh Ekstrak Daun Binahong (Anredera cordifolia (Tenore) Steen.) terhadap Aktivitas Fagositosis Monosit. In: Proceeding Books Forkinas VI FKG Unej, 416–425. Salim, M., Yahya, Y., Sitorus, H., Ni’mah, T., and Marini, M., 2017. Hubungan Kandungan Hara Tanah dengan Produksi Senyawa Metabolit Sekunder pada Tanaman Duku (Lansium domesticum Corr var Duku) dan Potensinya sebagai Larvasida. Jurnal Vektor Penyakit, (10), 11–18. Wijayanti, D., Setiatin, E.T., and Kurnianto, E., 2018. Leukocyte Profile and Offspring Production of Guinea Pig (Cavia cobaya) Given Anredera cordifolia Leaf Extract. Journal of the Indonesian Tropical Animal Agriculture, (43), 18–25. Sukrama, D.M., Wihandani, D.M., and Manuaba, A.P., 2017. Topical Binahong (Anredera cordifolia) Leaf Extract Increases Interleukin-6 and VEGF (Vascular Endothelial Growth Factor) during Burn Wound Healing in Wistar Rats Infected with Pseudomonas aeruginosa. Biology and Medicine, (9), 1–6. Yuniarti, W.M. and Lukiswanto, B.S., 2017. Effects of Herbal Ointment Containing The Leaf Extracts of Madeira Vine (Anredera cordifolia (Ten.) Steenis) for Burn Wound Healing Process on Albino Rats. Veterinary World, (10), 808–813. Umar, A., Krihariyani, D., and Mutiarawati, D.T., 2012. Pengaruh Pemberian 13 Phagocytosis Activity of Binahong …
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Histopathology of broiler chickens fed diets supplemented with Prosopis africana (African mesquite) essential oil
Brazilian Journal of Science
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Abstract This study was carried out to investigate the hepatic histopathology of broiler chickens fed diets supplemented with Prosopis africana (African mesquite) essential oil (PRSO). 300, 1-day old broiler chicks were randomly distributed into six treatments with 5 replicates consisting of 10 birds each. Birds in diet 1 (D1) was fed basal diet supplemented with 0 % PRSO, D2: basal diet with 1.0 g/kg-1 oxytetracycline, D3, D4, D5 and D6 were fed basal diet supplemented with PRSO at 200 mg, 400 mg, 600 mg and 800 mg/kg-1 respectively. The experiment lasted for 56 days and a completely randomized design was adopted. Phyto-constituents of PRSO revealed the presence of tannins (201.21 mg/g-1), flavonoids (677.83 mg/g-1), alkaloids (405.90 mg/g-1), saponins (75.62 mg/g-1), phenols (508.28 mg/g-1), phytates (20.10 mg/g-1) and oxalates (5.26 mg/g-1). Hepatic histopathology showed that tissues from D1, D3, D4, D5 and D6 showed no signs of any damage; the central vein, blood portal vessels showed no congestion. Mild hepatocellular damage was recorded among liver of birds in D2. It can be concluded that Prosopis africana essential oil are among the key alternative solutions to antibiotics because they contain bioactive compounds that are safe, effective and performs multiple biological functions in the body of animals. Keywords: Prosopis africana oil, phytochemicals, broilers, liver, histopathology. Resumo Este estudo foi realizado para investigar a histopatologia hepática de frangos de corte alimentados com dietas suplementadas com óleo essencial de Prosopis africana (mesquita africana) (PRSO). 300 pintos de corte de 1 dia de idade foram distribuídos aleatoriamente em seis tratamentos com 5 repetições de 10 aves cada. Aves na dieta 1 (D1) receberam dieta basal suplementada com 0 % de PRSO, D2: dieta basal com 1,0 g/kg-1 de oxitetraciclina, D3, D4, D5 e D6 receberam dieta basal suplementada com PRSO a 200 mg, 400 mg, 600 mg e 800 mg/kg-1, respectivamente. O experimento teve duração de 56 dias e foi adotado o delineamento inteiramente casualizado. Os fitoconstituintes do PRSO revelaram a presença de taninos (201,21 mg/g-1), flavonoides (677,83 mg/g-1), alcaloides (405,90 mg/g-1), saponinas (75,62 mg/g-1), fenóis (508,28 mg/g-1), fitatos (20,10 mg/g-1) e oxalatos (5,26 mg/g-1). A histopatologia hepática mostrou que os tecidos de D1, D3, D4, D5 e D6 não apresentavam sinais de danos; a veia central, os vasos sanguíneos portais não apresentaram congestão. Danos hepatocelulares leves foram registrados entre fígados de aves em D2. Pode-se concluir que o óleo essencial de Prosopis africana está entre as principais soluções alternativas aos antibióticos porque contém compostos bioativos que são seguros, eficazes e desempenham múltiplas funções biológicas no corpo dos animais. Palavras-chave: óleo de Prosopis africana, fitoquímicos, frangos de corte, fígado, histopatologia. Histopathology of broiler chickens fed diets supplemented with Prosopis africana (African mesquite) essential oil Olujimi John Alagbe1, Oluchi C. P. Agubosi1 & Rufus Adebisi Oluwafemi1 1 Department of Animal Science, University of Abuja, Gwagwalada, Nigeria Correspondence: Olujimi John Alagbe, Department of Animal Science, University of Abuja, Gwagwalada, Nigeria. E-mail: dralagbe@outlook.com Accepted: April 25, 2023 Publi URL: https://doi.org/10.14295/bjs.v2i9.385 Published: September 01, 2023 9.385 Received: April 05, 2023 DOI: 10.14295/bjs.v2i9.385 Received: April 05, 2023 DOI: 10.14295/bjs.v2i9.385 Published: September 01, 2023 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 Histopathology of broiler chickens fed diets supplemented with Prosopis africana (African mesquite) essential oil Olujimi John Alagbe1, Oluchi C. P. Agubosi1 & Rufus Adebisi Oluwafemi1 1 Department of Animal Science, University of Abuja, Gwagwalada, Nigeria Correspondence: Olujimi John Alagbe, Department of Animal Science, University of Abuja, Gwagwalada, Nigeria. E-mail: dralagbe@outlook.com 1. Introduction Nowadays, there is an increasing awareness on the need to maintain good health via the use of natural products from plants because they are efficient, cheap, easily metabolized with minimum health hazards and do not pose 49 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 any environmental pollution (Alagbe, 2022; Wallace et al., 2011). Medicinal plants are also known as phytogenic feed additives are regarded as complex mixtures of bioactive components or phytochemicals found in varying amount depending on geographical location, stage of growth or harvesting, parts used (leaf, seeds, bark, buds, flowers and twig) as well as method of processing (Shittu; Alagbe, 2020; Simitzis, 2017). any environmental pollution (Alagbe, 2022; Wallace et al., 2011). Medicinal plants are also known as phytogenic feed additives are regarded as complex mixtures of bioactive components or phytochemicals found in varying amount depending on geographical location, stage of growth or harvesting, parts used (leaf, seeds, bark, buds, flowers and twig) as well as method of processing (Shittu; Alagbe, 2020; Simitzis, 2017). Bioactive compounds or phytochemicals from plants includes: phenolics, flavonoids, terpenoids, carotenoids, saponins, alkaloids, flavours and fragrances. Plant extracts (essential oils) with these bioactive molecules are widely used in the food, pharmaceutical and cosmetic industries (Lewis et al., 2003; Alagbe, 2017). They are also capable of enhancing livestock productivity through the improvement of digestibility, nutrient absorption and elimination of pathogens residents in the animal gut (Denli et al., 2004). Medicinal plants had possessed antimicrobial (Agubosi et al., 2022; Nursoy et al., 2011), antioxidants (Adewale et al., 2021), antibacterial (Dhama et al., 2015), antiviral (Mehr et al., 2014), immune-modulatory (Krishan; Narang, 2014; Olorunmaiye et al., 2019), hepato-protective, antifungal and cytotoxic (Rostagno et al., 2011; Souza et al., 2015), antifungal, anti-proliferative, anti-helmithic, antiviral and anti-tumor (Souza et al., 2015), anti-cancer (Assis et al., 2017) and anti-protozoal, anti-proliferative, anticoagulant, anticonvulsant, antipsychotic (Brenes; Roura, 2010; Cho et al., 2014). Essential oils (EOs) has shown positive effect in livestock by promoting growth, relieving the host animals from immune defense stress during critical situations, scavenging free radicals capable of causing diseases and increase the intestinal availability of essential nutrients for absorption, thereby helping animals to grow better within the framework of their genetic potential (Lee et al., 2003; Bozkurt et al., 2016). 1. Introduction EOs have been demonstrated to work through the antioxidative mechanisms serving as free radical scavengers promoted by polyphenols, flavonoids and terpenoids which possess the ability to protect the cell organelles from damage caused by free radicals induced oxidative stress either by inhibiting the initiation or propagation of oxidative chain reactions (Sevim et al., 2017; Zeng et al., 2015). Studies have shown that the plants active principle agents in the herbs have a strong capability for scavenging superoxide radicals, hydrogen peroxide and nitric oxide from activated macrophages, reducing iron complex and inhibiting lipid peroxidation (Stef et al., 2009; Umashanker et al., 2011). Prosopis africana (African mesquite) is a leguminous multipurpose tree that belongs to the family Fabaceae and subfamily Mimosoideae (Ogunshe et al., 2017). It is known as Iron wood in English, Kiriya in Hausa, Ubwa in Igbo and it consist of 14 species and widely grown in tropical, arid and semi arid regions of the world (Olorunmaiye et al., 2019). Essential oils can be extracted mostly from the seed which contains compounds with therapeutic properties (alkaloids, saponins, tannins, phenols, cardiac glycosides, flavonoids and terpenoids) (Olorunmaiye et al., 2019; Gberikon et al., 2015; Ugwoke et al., 2019). Traditionally, the roots and stem bark extracts are used for the treatment and prevention of fever, skin diseases, diarrhea, dysentery, respiratory disorders, rheumatism, pneumonia and ulcer (Gberikon et al., 2015; Oguntoyinbo et al., 2017). Prosopis africana oil can also inhibit the growth of pathogenic microorganisms; Staphyllococcus spp, Bacillus spp, Escherichia coli, Enterobacter spp, Enterococcus spp, Pseudomonas aeruginosa, Shigella spp, Klebsiella spp and Proteus spp (Alagbe, 2023; Oguntoyinbo et al., 2007; Gordon et al., 1973). There is no justification for assuming that just because essential oils are natural, they must therefore be safe; certain phytochemicals or bioactive compounds can interfere with the body’s system for metabolizing medications in the liver, which can have a chain reaction of negative effects. Therefore, this experiment was designed to investigate the histopathology of broiler chickens fed diets supplemented with Prosopis africana (African mesquite) essential oil. 2. Materials and Methods 2.1 Ethical approval, collection and processing of Prosopis africana oil (PRSO) 2.4 Determination of phytochemical components of Prosopis africana oil 2.4 Determination of phytochemical components of Prosopis africana oil Phenols and total tannins in PSO were evaluated using Folin-Ciocalteau method outlined by Otles and Yacin (2012). Total flavonoids, alkaloids, phytates and oxalates were determined using the methods described by Harborne (1973); Odebiyi and Sofowora (1978). Saponins was quantified using the protocols used by Brunner (1984). Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 Prosopis africana oil was extracted employing the steam-distillation method, which also calls for the use of a digital scale, a round bottom flask, distilled water, a heating mantle made of glass yarn, a measuring cylinder, and a separatory funnel. 250 mL of distilled water was added to a round-bottom flask along with 50 grams of ground Prosopis seed. The mixture was added to a glass yarn heating mantle, warmed to a temperature of 80 °C, and then the condenser was positioned above the flask with a round bottom. The combination is forcefully boiled for 15 min, the distillate is collected in a beaker until no more oil droplets flow over, 5 grams of sodium chloride are added, and the mixture is constantly agitated. To get Prosopis africana oil, the distillate was then put into the separatory funnel and evaporated in a steam bath. 2.2 Management of experimental animals and design 2.2 Management of experimental animals and design 300, 1-day old broiler chicks of mixed sex (Arbo Acres) were purchased from a reputable commercial hatchery in Oyo State, Nigeria and randomly distributed into six treatments with 5 replicates consisting of 10 birds each. A galvanized battery cage measuring (100 cm × 75 cm × 50 cm) (length × depth × height) was equipped with automatic nipple drinkers and galvanized manual feeder in a semi open pens. Chicks were weighed on arrival to determine their average initial body weight and thereafter on weekly basis and given anti-stress (glucose + water) at the rate of 2 grams of glucose to 10 liters of water. Completely randomized experimental design was used in this study. 2.3 Experimental diets Diets were compounded based on the nutrient requirements for broilers according to Olomu (2021) as presented in (Table 1). Experimental diet at the starter phase (1-28 days) and finisher phase (29-56 days) contains 23.06% crude protein (CP), 21.40% CP and metabolizable energy content (ME) of 2991.5 Kcal/kg-1 and 3108.1 Kcal/kg-1 dry matter respectively. All birds had unrestricted access to feed and water throughout the entire experimental period (8 weeks). Experimental set-up is shown below: Experimental set-up is shown below: Diet 1 (D1): Basal diet with no Prosopis africana oil (Negative control) Diet 2 (D2): Basal diet + Oxytetracycline 1.00 g/kg-1 (Positive control) Diet 3 (D3): Basal diet + 200 mg/kg-1 Prosopis africana oil (PRSO) Diet 4 (D4): Basal diet + 400 mg/kg-1 PRSO Diet 5 (D5): Basal diet + 600 mg/kg-1 PRSO Diet 6 (D6): Basal diet + 800 mg/kg-1 PRSO Diet 1 (D1): Basal diet with no Prosopis africana oil (Negative control) 2.1 Ethical approval, collection and processing of Prosopis africana oil (PRSO) The study was carried out at the University of Abuja Teaching and Research Farm in the month of April - June, 2022 according to the guidelines of animal protocol agreed by the Research and Ethic committee (ANSJ/004) of the Department of Animal Science, University of Abuja, Gwagwalada, Nigeria. Fresh seeds of Prosopis africana were purchased from a local market in Gwagwalada, authenticated at the Department of Biological Sciences, University of Abuja by a certified taxonomist where a Voucher specimen number PRO/ANS/004 was deposited at the herbarium of the same department. Air dried on a clean metallic plate for 11 days and grinded into powder using a blender thereafter poured into a well labeled zip log before it was transported to a laboratory for further laboratory examination. 50 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 2.5 Histopathological evaluation **Premix supplied per kg diet: - vit A, 8,500 I.U; vit E, 10 mg; vit D3, 1500 I.U, vit K, 3.8 mg; vit B2, 10 mg; Niacin, 15 mg; vit B12, 10 mg; choline chloride, 250 mg; Mn, 5.0 mg; Zn, 56 mg; Cu, 1.6 g; folic acid, 2.8 mg; Fe, 5.1 g; pantothenic acid, 10 mg; biotin, 30.5 g; antioxidant, 56 mg (finisher’s mash). Source: Autors, 2023. 2.5 Histopathological evaluation Liver specimens (1 cubic cm) were taken on the 56th day of the experiment and placed in a well-sealed leak proof labelled container containing 10% formalin. The 10% formalin solution prevents the tissues from degeneration. Thereafter, specimens were transported at room temperature to the laboratory for further examination. All tissues were stained with Hematoxylin and Eosin (H & E) stain and the hsitopathological changes were observed under a microscope. 51 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 Table 1. Ingredient composition of the experimental diets. Materials Starters mash (1-28 days) Finishers mash (29-56 days) Maize 52.55 57.55 Wheat offal 2.00 5.00 Soya bean meal 29.00 22.45 Groundnut cake 8.00 5.00 Fish meal (72%) 3.00 3.00 Limestone 1.50 2.00 Bone meal 3.00 4.00 Lysine 0.20 0.20 Methionine 0.20 0.20 **Premix 0.25 0.25 Salt 0.30 0.35 Total 100.0 100.0 Determined analysis (% DM) Crude protein 23.06 21.40 Crude fibre 4.00 5.30 Ether extract 4.03 4.47 Calcium 1.66 1.83 Phosphorus 0.78 0.99 Energy (Kcal/kg-1) 2991.5 3108.1 Note: *Premix supplied per kg diet: - vit A, 10,000 I.U; vit E, 5 mg; vit D3, 3000 I.U, vit K, 3 mg; vit B2, 5.5 mg; Niacin, 25 mg; vit B12, 16 mg; choline chloride, 120 mg; Mn, 5.2 mg; Zn, 25 mg; Cu, 2.6 g; folic acid, 2 mg; Fe, 5g; pantothenic acid, 10 mg; biotin, 30.5 g; antioxidant, 56 mg (starter’s mash). **Premix supplied per kg diet: - vit A, 8,500 I.U; vit E, 10 mg; vit D3, 1500 I.U, vit K, 3.8 mg; vit B2, 10 mg; Niacin, 15 mg; vit B12, 10 mg; choline chloride, 250 mg; Mn, 5.0 mg; Zn, 56 mg; Cu, 1.6 g; folic acid, 2.8 mg; Fe, 5.1 g; pantothenic acid, 10 mg; biotin, 30.5 g; antioxidant, 56 mg (finisher’s mash). Source: Autors, 2023. Note: *Premix supplied per kg diet: - vit A, 10,000 I.U; vit E, 5 mg; vit D3, 3000 I.U, vit K, 3 mg; vit B2, 5.5 mg; Niacin, 25 mg; vit B12, 16 mg; choline chloride, 120 mg; Mn, 5.2 mg; Zn, 25 mg; Cu, 2.6 g; folic acid, 2 mg; Fe, 5g; pantothenic acid, 10 mg; biotin, 30.5 g; antioxidant, 56 mg (starter’s mash). 3.1 Phytochemical constituents of Prosopis africana oil Constituents Concentrations (mg/g-1) Tannins 201.21 Flavonoids 677.83 Alkaloids 405.90 Saponins 72.62 Phenols 508.28 Phytates 20.10 Oxalates 5.26 Source: Authors, 2023. Figure 1. Phyto-constituents in Prosopis africana oil. Source: Authors, 2023. 3.2 Histopathology examination of birds fed Prosopis africana oil (PRSO) The examined histopathogical liver sections is presented in (Board 1 (Figure 1 at 6) Histopathology) The liver is Table 2. Phytochemical constituents of Prosopis africana oil. Constituents Concentrations (mg/g-1) Tannins 201.21 Flavonoids 677.83 Alkaloids 405.90 Saponins 72.62 Phenols 508.28 Phytates 20.10 Oxalates 5.26 Source: Authors, 2023. Table 2. Phytochemical constituents of Prosopis africana oil. Figure 1. Phyto-constituents in Prosopis africana oil. Source: Authors, 2023. Figure 1. Phyto-constituents in Prosopis africana oil. Source: Authors, 2023. Figure 1. Phyto-constituents in Prosopis africana oil. Source: Authors, 2023. 3.1 Phytochemical constituents of Prosopis africana oil Phytochemical constituents of Prosopis africana oil is summarized in (Table 2 and Figure 1). Flavonoids had the highest concentration (677.83 mg/g-1) followed by phenols (508.28 mg/g-1), alkaloids (405.90 mg/g-1), tannins (201.21 mg/g-1), saponins (72.62 mg/g-1), phytates (20.10 mg/g-1) and oxalates (5.26 mg/g-1) respectively. All the phytochemicals have stated above have different medicinal and pharmacological importance. For instance, flavonoids therapeutically function as strong antimicrobial against pathogenic organisms (Alagbe, 2020), antioxidants against reactive oxygen species which are generated from free radicals (Oluwafemi et al., 2021). Alkaloids have several pharmacological properties on human and livestock health including anti-inflammatory, analgesics, antifungal, antimalarial, anti-hypertensive, neuro-protective, anti-parasitic and anti-tumor (Xie et al., 2021; Roy, 2017). High concentrations of alkaloids in the body system can lead to chronic toxicity and other health complications such as abdominal pains, nausea, increased cardiac rates, neuro-toxicity, genotoxicity and liver diseases (Ma et al., 2007; Adibah et al., 2019). Tannins exhibits strong biological activities (anti-bacterial, antimicrobial, hepato-protective, antiarrhythmic and immune-modulatory) (Adewale et al., 2021; Singh et al., 2021). Phenols have strong antioxidant properties and are capable of preventing oxidative damage to biomolecules such as DNA, lipids and proteins (Hassan et al., 2012; Singh et al., 2023). Saponins are immune stimulators (Hassan et al., 2012), antimicrobials (Soetan et al., 2006). Researchers have reported its hypocholesterolemic properties (Cheeke, 2000). Phytates have been reported to be effective in the treatment of wounds and haemorrage 52 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 (Oluwafemi et al., 2021). They are antioxidants capable of reducing the risk of cardiovascular disease, kidney stones, osteoporosis and iron absorption (Shittu; Alagbe, 2021). Excessive ingestion of oxalates can interfere with the absorption of calcium in the body (Varadharajan et al., 2012); Sombié et al. (2011). The results obtained in this research is in consonance with previous findings of Ogbeba et al. (2017); Singh & Bhat (2013). Tobias (2010) reported that the composition of essential oils is influenced by cultivar within the plant variety, climatic conditions and time of harvest. The presence of multiple phyto-constituents in Prosopis africana shows that it has notable beneficial effects in maintaining animal’s performance, antioxidant and osmotic balance, palatability, retention time of feed, modulating fatty acid in the muscle as well as supporting the intestinal integrity (Singh et al., 2023). Table 2. Phytochemical constituents of Prosopis africana oil. 3.2 Histopathology examination of birds fed Prosopis africana oil (PRSO) The examined histopathogical liver sections is presented in (Board 1 (Figure 1 at 6) Histopathology). The liver is the largest gland in the body. It is made up of hepatic lobules which are classically described as been hexagonal in structure (Dooley et al., 2002). Birds in Figure 1 (fed 0% PRSO) showed no histopathological signs of any damage; the central vein, blood portal vessels showed no congestion. Figure 2 (fed oxytetracycline) showed mild perivascular lymphocytic aggregation and moderate hepatic sinusoids. Figure 3, 4, 5 and 6 (fed PRSO) revealed normal tissue achietecture with normal hepatic lobules, central vein and hepatic sinusoids. 53 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 Board 1. Figure 1. Basal diet with no Prosopis africana oil (Negative control). Figure 2. Basal diet + Neomycin 1.00 g/kg-1 (Positive control). Figure 3. Basal diet + 200 mg/kg-1 Prosopis africana oil (PRSO). Figure 4. Basal diet + 400 mg/kg-1 PRSO. Figure 5. Basal diet + 600 mg/kg-1 PRSO. Figure 6. Basal diet + 800 mg/kg-1 PRSO. Source: Authors, 2023. Board 1. Figure 1. Basal diet with no Prosopis africana oil (Negative control). Figure 2. Basal diet + Neomycin 1.00 g/kg-1 (Positive control). Figure 3. Basal diet + 200 mg/kg-1 Prosopis africana oil (PRSO). Figure 4. Basal diet + 400 mg/kg-1 PRSO. Figure 5. Basal diet + 600 mg/kg-1 PRSO. Figure 6. Basal diet + 800 mg/kg-1 PRSO. Source: Authors, 2023. According to Harold et al. (2008); Plinio & Broglia (2000), the liver help to detoxify toxic substances, metabolism of micro-nutrients, excretion of bilirubin and synthesis of bile salts. The healthy tissue architecture of birds given Prosopis africana oil is evident that both their bioactive components and inclusion levels are safe. PRSO are well known for their beneficial effects on animals, from anti-inflammatory, antioxidant and antimicrobial properties (Agubosi et al., 2022; Oluwafemi et al., 2020). The outcome of this investigation is consistent with the findings of Oloruntola (2019) when phytogenics (pawpaw leaf and seed meal) were supplemented in the diet of broiler chickens. Similar findings were made by Samantha et al. (2019) when essential oils were given to broilers that were coccidiosis-challenged. Omokore & Alagbe (2019); Alagbe & Oluwafemi (2019) claimed that bioactive substances found in essential oils may inhibit the pathogenicity features in bacteria, such as flagella, which are important for bacterial adhesion or adhesion to any mammalian preference sites. 5. Acknowledgments We will like to acknowledge the efforts of Mrs. Mercy Cincinsoko Kadiri in sourcing Prosopis seeds. 3.2 Histopathology examination of birds fed Prosopis africana oil (PRSO) Alagbe (2022) asserts that the liver is crucial for the creation of bile, the metabolism of carbohydrates, and the removal of toxins from an animal's system. The abundance of flavonoids, a significant phytogeenic compound could significantly decrease liver tissue degenerative conditions particularly blockages in the liver of animals when compared to the other treatments. Antibiotics like oxytetracycline are widely used to prevent, control, and treat disease in animals; however, due to their uncontrolled usage and insufficient sensitivity testing before treatment, there are a rising number of incidences of resistance (Adewale et al., 2021). Alagbe et al. (2019) claim that bioactive substances found in essential oils may inhibit the pathogenicity features in bacteria, such as flagella, which are important for bacterial adhesion or adhesion to any mammalian preference sites. Alagbe (2022) asserts that the liver is crucial for the creation of bile, the metabolism of carbohydrates, and the removal of toxins from an animal's system. The abundance of flavonoids, a significant botanical compound could significantly decrease liver tissue degenerative conditions particularly blockages in the liver of animals. Degeneration in liver histopathology has also been observed to be enhanced by contaminants (Soetan; Oyewole, 2009). Infections with bacteria may have caused the severe and mild hepatic damage seen in birds in T2. Despite the fact that antibiotics were administered to the birds in T2, this finding suggests that there may be an antimicrobial resistance issue. 4. Conclusions 4. Conclusions 54 Brazilian Journal of Science, 2(9), 49-59, 2023. ISSN: 2764-3417 It can be concluded that the more antibiotics (oxytetracycline) fed to animals the greater the selective pressure that favours resistant strains. However, Prosopis africana essential oil possess several properties – antioxidant, antiviral, anti-inflammatory, anti-depressant, immune-stimulatory, antimicrobial, hepato-protective amongst others, all of which are vital for the performance enhancing effects in livestock’s. 8. Ethics Approval Yes applicable. 6. Auhors’ Contributions Olujimi John Alagbe: field work and compilation of results, subimission, corrections and publication. O. C. P Agubosi: field work and compilation of results. R. A. Oluwafemi: field work and compilation of results. 7. Conflicts of Interest No conflicts of interest. http://dx.doi.org/10.32861/ajls.511.107.111 http://dx.doi.org/10.32861/ajls.511.107.111 Alagbe, J. O. (2020). Caecal microbial population of growing grass cutters fed Phyllantus amarus and Piliostigma thonngii leaf meal mixture as partial replacement for soya meal. Concept of Dairy and Veterinary Sciences, 3(5), 350-355. http://dx.doi.org/10.32474/CDVS.2020.03.000174 Alagbe, J. O. (2021). Prosopis africana stem bark as an alternative to antibiotic feed additives in broiler chicks diets: Performance and carcass characteristics. Journal of Multidimensional Research and Reviews, 2(1), 64-77. https://jmrr.org/V2I1/V2I1P05.pdf Alagbe, J. O. (2021). Daniellia oliveri leaf extracts as an alternative to antibiotic feed additives in broiler chicken diets: Meat quality and fatty acid composition. 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Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, 67, 940-944. https://doi.org/10.1590/1678- 4162-7580 Tajudeen, A. I., Kayode, C. O., Yahaya, A., & Mohammed, S. A. (2011). Development and testing of a Prosopis pod thresher. Australian Journal of Basic and Applied Sciences, 5(5), 759-767. Tobias, S. (2010). Producers can benefit from essential oil in pig finishing diets. International Poultry Science Magazine, 26(5), 19-21. http://www.positiveaction.info/pdfs/articles/pt26.5p19.pdf Ugwoke, A. O., Oluka, S. I., & Eze, P. C. (2019). Physicomechanical properties of iron tree seed (Prosopis africana) from South eastern Nigeria. Journal of Engineering and Technology, 24(1), 13-18. http://jaet.com.ng/index.php/Jaet/article/view/72 Varadharajan, V., Janarthanan, U. K., & Vijayalakshmi K. (2012). Physicochemical, phytochemical screening and profiling of secondary metabolites of Annona squamosa leaf extract. World Journal of pharmaceutical research, 1(4), 1143-1164. Xie, Z., Cao, N., & Wang, C. (2021). A review on beta carboline alkaloids and their distribution in feedstuffs: A class of potential functional components. Food Chemistry, 348. https://doi.org/10.1016/j.foodchem.2021.129067 Zeng, Z., Zhang, S., Wang, H., & Piao, X. (2015). Essential oil and aromatic plants as feed additives in non-ruminant nutrition: a review. Journal of Animal Science Biotechnology, 6(1), 2-10. https://doi.org/10.1186/s40104-015-0004-5 Copyrights py g Copyright for this article is retained by the author(s), with first publication rights granted to the journal. Copyright for this article is retained by the author(s), with first publication rights granted to the journal This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). 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Ueber gemischte, Aethylendiamin und Ammoniak enthaltende Triamminkobaltsalze
Berichte der Deutschen Chemischen Gesellschaft
1,905
public-domain
4,031
das komtnt bei Gleicbgewicbtastudien, wie diejenigen von S c h e n c k iind H e l l e r , vallkommen auf da-selbe hinaos. das komtnt bei Gleicbgewicbtastudien, wie diejenigen von S c h e n c k iind H e l l e r , vallkommen auf da-selbe hinaos. studirt, man bei Renutzurig von amorpher Kolrle resp. Diamant co*+c+2co . . . . . . (1) P a w PCC), fur dae Verhaltniss Werihe bekomrnen muss. welche, z. B. bei 64 I " , 5.4 resp. 4.2 hlal so gross sind als bei Graphit. Wir durfen hier aber nicht vergessen, dass wir zu diesem Rtwltate gelangt sind ntiter der Annahme, dass der Quotient bei dem Versuclir mit den verscbiedenen Kohlenstotrmodificationen denselben Werth bat, und dieses iet sicher nicht der Fall. Jedenfalls wird also dns Studium des (;leichgewichtes ( I ) ons mit grnsserer Sicherheit etwas iiber die Ddntpfspannungen der Kohlettstoffmoclificatiouert aussagen. als es die Untersuchung von S c h e n c k und H e l l e r vermag. Das Gleichgewicht ( 1 ) w i d von mir in dieser Richtung untersucht. Ptw I P 1.' Q verscbiedenen Kohlenstotrmodificationen denselben Werth bat, und dieses iet sicher nicht der Fall. Jedenfalls wird also dns Studium des (;leichgewichtes ( I ) ons mit grnsserer Sicherheit etwas iiber die Ddntpfspannungen der Kohlettstoffmoclificatiouert aussagen. als es die Untersuchung von S c h e n c k und H e l l e r vermag. Das Gleichgewicht ( 1 ) w i d von mir in dieser Richtung untersucht. A. rns t e r d a m . Aoorg.-chem. Laboratarium der Univereitat. A. rns t e r d a m . Aoorg.-chem. Laboratarium der Univereitat. A. rns t e r d a m . Aoorg.-chem. Laboratarium der Univereitat. 703. A. Werner und A. Griin: Ueber gemischte, Aethylen- diamin und Ammoniak enthaltende TI-iamminkobaltsalae. (Eingegangen am 30. Novemb1.r 1905) Zur Klarung bestimmter structureller und stereachemischer Fragen bei Coordiriationsrerbindungen erschien es wunschenswerth , an Stelle der nicht sehr bestfindigen, nur Ammonia% enthaltenden. reinen Tri- :imminsalze analoge. virlleicht bestandigere, aminsubstititirte Verbin- dungen zu gewinnen. Wir hahen deshalb einige athylendiatninbaltige Vertreter der Triamminkobaltreihe dargestellt, die wir im Folgenden beschreibcn wollen. Die Unterruchung hat aber gezeigt, dass der Ein- tritt von Aetbylendiarnin den Chemismus und die physikalischen Eigen- schaften van Triamminsalzen mehr andpi t, als es bei Tetraamminsalzen der Fall ist, nnd dass, inr Gegensatz zum Verhalten der Tetrammin- salze, die Hestandigkeit des Kobaltiakrndica!s der Triamminsalze dnrch den Eintritt vou hethylendiamin nicht erbiiht, soodern erniedrigt wird. das komtnt bei Gleicbgewicbtastudien, wie diejenigen von S c h e n c k iind H e l l e r , vallkommen auf da-selbe hinaos. I) Zeitscbr. fiir anorg. Chem. 13, 1SO [189G]. 2, Ztsitscbr. fiir anorg. Chem. 8, 171 [1S95]. das komtnt bei Gleicbgewicbtastudien, wie diejenigen von S c h e n c k iind H e l l e r , vallkommen auf da-selbe hinaos. Als Ausgsngsmnterial zur Dnrstellung iinserer Verbindungen diente ein Tr i n i tri t o - a t h y I e n d i am in-a mrni t i - ko b al t , das sic+, neben anderen Producten, bei der Einwirkung ron Aethylendi:iinin auf tetra- 1034 iiitritodiamminkobaltsaure Salze: [(HJN)~CO(SC~)~]R ( E r d m a n n - sches Salz) bildet. Es liornmt ihm die Forniel zu, uud seine Bilduug aus dern Erdmanii’schen Salz lasst sich fol- eendermanssen forniuliren: Dab ‘L’rinitritoatliylet diarninamminkobalt ahnelt in seinen Eigen- schaften dem ’~ririitritotii;~mmi~iknbalt, deniles m c h constitutionell an die Seite zu srrllen ist: Triammin kobalt. l’rinitritoathylendiamin:rulminkobalt. Wie das Trianiminkobalt zeigt e~ die Eigenschaft. in zwei so sehr yon einander verschiedenen und bestgudigen Krystallgestalteri aufzu- trrten, dass man :LU isomere Formen denken kiinnte. Da nber ein- schlagige Versuche geieigt hat~en, dass die Ansiclit \-on J i i r g e n s e n l ) , die von dem Eiiien von uns [We r n e r 2)] a18 isornrr bezeichneteo Formen des Trinitritotriamriiinko~~:ilts seien auf Lhnorphie zurlckzufuhren, wahr- sctieinlich riclitig ist, s o sollen nrich die beiden Pormen des Trinitri- toathylendiaminamniinkob:~lts vord+i hand niir als dirnnrphe Modifica- tionen au fgefasst w erd en. Durch Einwirkung von kochender Salzsiiure verwandelt sich die neue Trioitritoverbindung in riii schwarzes Salz, welches nach Kry- atallisation aus s:ilzsiiurehalti~erii Wasser grlnschwaree Nadeln bildet cind sich durch seiue Zusarnrnmsetzung uiid sein Verhalten als athylen- diaminheltiges Dichrosalz erweist. g Es konimt ihm folgende Constitutii,nJforineI zu: Nur e i n Chloratorn dieses Salies zeigt den gewiihnlichen doppelten Umsatz, wie die Einwit kung von Salpetersaure letlrt, welche folgendes Nitrat lielei t : I) Zeitscbr. fiir anorg. Chem. 13, 1SO [189G]. I) Zeitscbr. fiir anorg. Chem. 13, 1SO [189G]. 2, Ztsitscbr. fiir anorg. Chem. 8, 171 [1S95]. 2, Ztsitscbr. fiir anorg. Chem. 8, 171 [1S95]. In wassriger Losung rrfahrt e i ti intraradicales Cbloratorn, infolge VOII Hydratation, sehr rasch rlektrolptische Dissociation, sodass die anfanglich griine Losung blao wird. I) Dieser Niederschlag besteht aus : [(Oa N)g Co :en)a]. [(OS N)dCo (K tj;>j], wic in einer spLteren Abhatodlung gezoigt wird das komtnt bei Gleicbgewicbtastudien, wie diejenigen von S c h e n c k iind H e l l e r , vallkommen auf da-selbe hinaos. Ein Salz des dann in der Lo- sung vorhandenen C h lor o- d i a q uo -a t h y I end i a m i n-a m rn i n - k o - b a l t r a d i c a l e , namlich das Oxalat: C1 CO (NHj) & 0 4 , [ (O;::;] , C1 CO (NHj) & 0 4 , [ (O;::;] [ in welchem sich Oxalsiiure durch analytische Reagentien sofort, Chlor dagegen erst uach IangeIeiii Kochen nachweisen lasst, ist schwer 16s- lich und kann deshalb leicht isolirt werden. Wird die blaue Liisung des Chlorids [nit RrornwasserstoffsZur~ versetzt, so scheiden sich olivengriine h'adehl aus. Die so gebildete Verbindung zeigt iu ihreni complexen positiven Radical die gr6sste. bis jrtzt i n complexen Ioneri brobacbtete Variation von Componmten, denn sie ist als Chloro-bromo-aquo-Bthylendiamin-ammin- k o b ; i l ~ i b r o n i i d zu bezeichnen, d. h. in directer Bindung mit dem Rob;, ltatoni befinden sich: eiu Chloratorn, ein Bromatoin, ein Wasser-, ein Animoniak- und ein Aethylendiamin-Molekul. Die Forrnel der Verbindung ist folgendermaassen 711 schreibm: W irkt KromwasseratoHbIure auf eine ill der WIrme bereitete Lo- rung von Dichloroaqu~ithylendiaminarnminkobaltchlorid ein, so werdeii siimmtliche Chloratonie durch Brom ersetzt, und man gelangt zu dem stark dicbroytischen D i brorno-:tcl uo - a t h y le ndiamin - a m m in- ko- bnlt tlromid: 1)ie Erystalle dieser Verbinduilg erscheiueu, je nach der Krystall- axe, blauschwarz oder braun; eerdruckt man sie, so grben sie eii~ griines Pulver. Sowohl die beschriebene leichte Ersetzbarkeit der Chloratome als anch die leichte Bildung von Diaquooxalat u. s. w. zeigm, dass der Eintritt ron Aethylendiamin in das Triamminkobaliradical die Beweg- lichkeit der an Kobalt gebundenen Saurereste begiinstigt, sodas8 die innere Stabilitiit der iii Form von Salzen nachgewieseiien complexen' Kobaltradicale wesentlicli grringer ibt 11s bei den gawiihnlicheii Tri- arnminsalzeii. Infolgedessen sind auch die eiiizelneii VerLinduugstypen bei den ithylei~diaminhaltigen Verbindungen weniger charakteristisch :rls bei den gew6hnlichen Triamminsalzen. Es wird aber erst durch 4036 eine eingehendere Untersucbung der einzeluen Salze , specie11 in phy- sikalisch chernischer Richtung, rniiglich sein, den Unterwhied in den Htabilitatsoerhaltniesen genauer festzustrllen. eine eingehendere Untersucbung der einzeluen Salze , specie11 in phy- sikalisch chernischer Richtung, rniiglich sein, den Unterwhied in den Htabilitatsoerhaltniesen genauer festzustrllen. T ri n i t r i to-ath y 1 end i am i n- a n i niin- ko b a It , [(OrN)sCo (NH3) (en) I . hei der Modification in Blattchen: Co 21.94, N 29.27. D )) ,) n D Nadeln: )) 21.83, )) 29 15. Di c h 1 o r o- aq u 0-9 t ti y 1 e n d i a m in - a m ni i 11 - li o b a1 t c h 1 o r i d , Die Darstellung dieses Salzes aus dem Nitritos.ilz ert'olgt :iiialog der von S. M. J o r g e n s e n ' ) aogegebenen Darstelluug von Dichloro- aquotriamminkobaltchlorid aus Tiinitritotriamminkobalt. 10 g Nitrit werden mit 80 ccm 1,oncentrirter Salzsiiure verrieben and kurze Zeit (3 Minuteii) au1' dein Wavserbade erhiczt, wobei leb- hafte Entwickelurrg von salpetriger Same eintritt. Sofort beginnt die Ansscheidung eiiies schwarzen Salzes, weiche bcim Stehen des Re- actionsgemisches in der Kiilte nach einigen Stunden beendet ist. Deri abgesaugten, durch Waschen mit Alkohol von Saurr be- freiten Niederschlag 16st man in so wenig Wasser, dasa ein kleiner Theil ungeliist bleibt, und filtrirt. Das Filtrat versetzt man sofort mit 25 cc'm concentrirter Salzsaure und fugt nach Ablauf einer Stunde ooch einaial 25 ccm und nach 2 Stunden noch 50 ccrn hinzu. Das Salz scheidet sic11 dabei in Form kleiner, griinschwarzer Nadeln ans, welcbe scliwachen Dichroismus zeigen. Gewinnt man das Salz durch Einleiten von Salzsauregas in die wassrige Losung, so bildet es ein graues Pulver, aber die Ausbeuten sind grBsser. Die auf einem Saugfilter gesammelten und mit saurehaltigeni Wasser und Alkohol gewaschenen Krystalle ergaben bei der Analyse: 0.1774 g Sbst.: 0.0826 g HsO. - 0.2305 g Shst.: 03845 g AgCl. - 0.1274 p Sbst.: 18.7 ccm N (15O, 720mm). 0.0886 g Sbst.: 0.0532 g COSO~. - 0.1774 g Sbst.: 0.0505 g CO,. - 0.1774 g Sbst.: 0.0826 g HsO. - 0.2305 g Shst.: 03845 g AgCl. - 0.1274 p Sbst.: 18.7 ccm N (15O, 720mm). 0.0886 g Sbst.: 0.0532 g COSO~. - 0.1774 g Sbst.: 0.0505 g CO,. - C O N ~ G C I ~ H I S O . Ber. Co 22.64, N I G . l G , C 9.20, H 5.04, C1 40.81. Gef. )) 22.84, )) 16.25, 7.76, )) 5.17, )) 41.17. C O N ~ G C I ~ H I S O . Ber. Co 22.64, N I G . l G , C 9.20, H 5.04, C1 40.81. Gef. )) 22.84, )) 16.25, 7.76, )) 5.17, )) 41.17. I ) Zeitsclir. f. anorg. Chem. 14, 418-419. T ri n i t r i to-ath y 1 end i am i n- a n i niin- ko b a It , [(OrN)sCo (NH3) (en) I . Fiigt man zu einer Liisung YOU Tetranitritodiarnminkobaltnatrium bei 60'' Aethylendiamin (Monohydrat) und zwar 1.4 g auf 5 g des Yalzes, so scheidet sich ein gelber, k rystallinisrher Niederschlag ') ails, wHhrend in der braungefarbten Mutterlauge eine Anzahl leichter 16s- licher Reactionsproducte enthalten ist. Dampft man die Muiterlauge auf ungefahr die Halfte itirrs ur- spriinglichen Volumens ein , so scheidet sich nach dem Erkalteri Tri- ~iitritoathylendiarninarnmiokobalt als gelber Kryetallbrei aue. dem aber iioch andere Salze heigemisclit sind. Nach drei- bis vier-maligem Umkrystallisiren ails essigaaurehaltigrrn Wabser erhalt man ea rein, in Form hellbrauner Blattchen, welche in Wasser schwer liislich sind. 0.0578 g Sbst.: 0.0508 g CoSO,. - 0.07i3 g Sbst.: 0.0417 g CoSOh. - 0.0787 g Sbst.: 22.2 ccm N (l3.So, 720 mrn). - 0.07f3 g Sbst.: 20 2 CCIU N # 1 3 O , 722 mm). - 0.1518 g Sbs:.: 0.04s; g COT. 0.0765 g Sbst.: 0.0442 a CO SO&. - 0.0974 R Sbst.: 0.0562 g C O S O ~ . - , ) g ; g (JoNsC.rOaH11. Ber. Co B l . i S , N 3O.ti5, c 8.84. Gcf. 21.94, 21.98, 21.99, 22.00, n 31.45, 29.95, D 8.66, , , g ; g (JoNsC.rOaH11. Ber. Co B l . i S , N 3O.ti5, c 8.84. , Gcf. 21.94, 21.98, 21.99, 22.00, n 31.45, 29.95, D 8.66, , Dieser Zusarnmrnsetzung entepricht folgende Formel: Dieser Zusarnmrnsetzung entepricht folgende Formel: Die Nitritgruppen verhalten sich nicht als Iorieu, iind dement eprechend kann man das Salz mit kalter Salzeiiure iiberschichten, ( h e dass vine Entwickelung von salpetriger Saure bemerkbar wire. Natiirlich wirkt die Salzsiiure ill der Warrne daranf ein. Hierbei entsteht ein schwarzes Salz, welches, nach allen seinen Eigenschaften zu schliessen, dae Analogon des Dichloroaquotriarnminkobaltchlorids (Dichroaalz) ist. Damit ist bewiesen, dass nnsere Verbindung einer Triarnminreihe angehort. Dae Trinitritoathylendiaminarnminkobalt wurde gelegentlich auch i n einer anderen Krystallform erhalten, obwohl die Daretellungaweise i n keiner Weiee von der bereits angegebenen abwich. 4037 Es krystallisirte in ungefiihr 2 cm langen, breiten, federartig gestreiften Nadeln. Nach Analyse und Verhalten hat dieae Modifi- cation dieselbe Zusammensetzung wie die Rlattchenform. g 0.0807 g Shst.: 0.0464 g CoSO,. - 0.0978 g Shst.: 28.4 ccm N (14O, 713mm). Gef. hei der Modification in Blattchen: Co 21.94, N 29.27. D )) ,) n D Nadeln: )) 21.83, )) 29 15. g 0.0807 g Shst.: 0.0464 g CoSO,. - 0.0978 g Shst.: 28.4 ccm N (14O, 713mm). Gef. T ri n i t r i to-ath y 1 end i am i n- a n i niin- ko b a It , [(OrN)sCo (NH3) (en) I . In eiskaltem Wasser 16st pith das Salz mit griiner Farbe, welche bald in blan umschlagt; bei weiterem Verdiinnen wird die LBsuog roth, und dann tallt auf Zusatz von Silbernitrat sammtliches Chlor als Ghlorsilber nus. Erhitzt man die neutrale Losung des Salzes, so tritt Zersvtzung unter Abscheidung von Kobalti hydroxyd ein. Wird mit I ) Zeitsclir. f. anorg. Chem. 14, 418-419. 4038 Salzsaure erhitzt, so erhalt man als Zersetzungsproduct blaue blattrige Krystalle, welcbe sich in Wasser mit rother Farbe 18sen. Diese LB- sung wird bei Zusatz eines Tropfens Salzsaure blau und ecbeidet heirn Umriihren salzsaures Aethylendiarnin aus, wiihrend Kobaltchloriir i n Lijsung hleibt. Die hlauen Krystallc sind dernnnch das bpkannte Chlorokobaltoat des Aethylendiamins. Auf Ziisatz der berechneten Menge Natriumnitrii wird die schwach saure L8sung des Cblorids gelb gefiirlit und scheidet nach einiger Zeit Trinitritoathylendiaminamrninkobxlt in den charakteristischeir Formen aus. Nitrat, CIzCo(NH3) NO?. i “I Wird fein gepulvertes, angefeuchtetes Chlorid rnit Salpetersaure vorn spec. Gewicht 1.4 in solcher Menge verrieben, dass ein diinn- Hiissiger Brei entstebt, so wird die Maese nach einiger Zeit ziernlich zahfliissig. Man wascht sie dann rnit Wasser, um die Salpetersanre zn entfernen, liierauf rnit Alkotiol und trocknet. Der Process wird dreimal wiederholt, worauf das Nitrat rein ist. Es wird in wenig kaltern Wasser geltist und die filtrirte Lasung mit einigen Tropfen concentrirter Salpetersaure versetzt. Rei laugsamem Verdonste‘n der LBsung krystallisirt das Salz in g r h e n , metallisch glaozenden Flittern. In Wasser ist es ausser~t leicht ldslich. In Wasser ist es ausser~t leicht ldslich. 01021 Sb t 0 0553 CoSOa 0 2045 Sh t 0 2015 g A CI - 0.1021 g Sbst.: 0.0553 g CoSOa. - 0.2045 g Shst.: 0.2015 g AgCI. - 0.1485 g Sbbt.: 26.2 ccrn N (1.50, 723 mm). CoClNdC12HtoO. Ber. Co 20.58, K 19.59, C1 24.73. Gef. n 20.61, I) 19.61, D 24.36. CoClNdC12HtoO. Ber. Co 20.58, K 19.59, C1 24.73. Gef. n 20.61, I) 19.61, D 24.36. C b lo ro-d i aq uo-a t h y 1 e n d i a rn i n- a m m i II - k o b a 1 to x a1 a t . C b lo ro-d i aq uo-a t h y 1 e n d i a rn i n- a m m i II - k o b a 1 to x a1 a t . ClCO (NHB) God. T ri n i t r i to-ath y 1 end i am i n- a n i niin- ko b a It , [(OrN)sCo (NH3) (en) I . Erhitzcn auf 105O nurmehr O.lti17 g. g p Ber. fur obige Formel: 1 Mol. HsO -= 6.05 pCt. Ch I o r n - b r o m 0- a q u o-at 11 y 1 end i a min- a m m i 11 - k o b a 1 t b r o m id , Ch I o r n - b r o m 0- a q u o-at 11 y 1 end i a min- a m m i 11 - k o b a 1 t b r o m id , Lost u~aii das Chlorid in der eben nothwendigen Menge Wasser auf iind setzt das gleiche Volurnen Bromwasserstoffsaure vom spec. Gewicht 1.49 zii, so scheiden sich sofort kleine, olivengriin gefarbte Nadeln am, die rnit Alkohol gewaschen und neben Natronkalk ge- trocknet werden. Die Zusammensetzuog entspricht dem aiich in der reineo Triarnminreihe sich bildenden Chlorobromobromid. Chlor und Brom wurden in dcr Weiw heetimmt, dass ein Theil des Halogensilbrrs eine Stunde lang im Chlorstrorn erhitzt nnd die in Folge der Ueberfiihrung des Bromsilbers in Chlorsilber entstandene Gewichtsverminde- rung auf die ganzc! Halogensilbermenge umgerechnet wurde. g g g g g 0.2033 g Sbst.: 0.3021 g Halogensilber. - 0.2034 g Sbst.: 0.3022 g Ha- logensilber. - 0.2845 g Halogensilber crgabeii, im Chlorstrom erhitzt, eine Gewichtsabnahme von 0.0479 8; den, entspricht fur 0.3022 g eine Abnabme von 0.0513 g, woraus sicb 0.21584 g Bg Br und 0.08636 g AgCl berecbnen. - 0.106 g Sbst.: 0.0468 g COSOJ. - 0.115 g Sbst.: 12.4 ccm N (13q 703 mm). CoBr2ClC?NgH130. Ber. Co 16.88, N 12.05, Br 45.74, C1 10.14. Gef. n 16.80, D 11.75, )) 45.14, * 10.49. g g g g g 0.2033 g Sbst.: 0.3021 g Halogensilber. - 0.2034 g Sbst.: 0.3022 g Ha- logensilber. - 0.2845 g Halogensilber crgabeii, im Chlorstrom erhitzt, eine Gewichtsabnahme von 0.0479 8; den, entspricht fur 0.3022 g eine Abnabme von 0.0513 g, woraus sicb 0.21584 g Bg Br und 0.08636 g AgCl berecbnen. - 0.106 g Sbst.: 0.0468 g COSOJ. - 0.115 g Sbst.: 12.4 ccm N (13q 703 mm). CoBr2ClC?NgH130. Ber. Co 16.88, N 12.05, Br 45.74, C1 10.14. Gef. n 16.80, D 11.75, )) 45.14, * 10.49. Eine noch nicht naher untersuchte Verhderung erleidet die Ver- binduug durch Erhitzen: g 0.1518 g Sbst. wogen nach einstiindigem Erhitzen iin Trockenschrd 0.1437 8 ; Abnahmc = 0.0081 g = 5.34 pCt. Bw. T ri n i t r i to-ath y 1 end i am i n- a n i niin- ko b a It , [(OrN)sCo (NH3) (en) I . [ (:e:)al [ 5 g Chlorid werden mit eiuer Loaung von 5 g Oxalsaure iii 50 ccm, Wasser; sowie rnit 50 ccrn Alkohol versetzt und eine Minute erhitzt. Reim Erkalteu scheidet sich das Salz nus. Es bildet blaue, silber- gliinzende Krystallblattcben, die auf dern Filter durch Waschen rnit salzsaurehaltigern Wasser und Alkohol gereinigt werden. In kaltem Wasser ist es sehr schwer, in heissem leichter Ioslicb; in Alkohol ist es fast uulrislich. Die wassrige Losung giebt rnit Salzsaure wieder Dichlorochlorid. Aus arnrnouiakhaltiger Liisung entsteht auf Zusatz \-on Chiorcalcium ao fort ein weisser Niederschlag von Calciurnoxalat. Mit Silbwnitrat giebt die wassrige Lijsung anfanglich nor pine schwacbe Triibung, und erst nacb Iangerem Steheu fallt d:is Cblor rollstindig aus -- ein Bewris dafiir. dass das Chlor nicht als Ion wirkt. Triibung, und erst nacb Iangerem Steheu fallt d:is Cblor rollstindig 0.1063 g Sbst.: 13.7 ccm N (170, 723 mm). - 0.2834 g Sbst.: 0.13YY g AgCI. - 0.1260 g Sbst.: 0.0585 g H20. - 0.1260 g Sbst.: 0.0766 g Con. - 0.08li4 p Sbst.: 00150 g CoSOi. 0.1063 g Sbst.: 13.7 ccm N (170, 723 mm). - 0.2834 g Sbst.: 0.13YY g AgCI. - 0.1260 g Sbst.: 0.0585 g H20. - 0.1260 g Sbst.: 0.0766 g Con. - 0.08li4 p Sbst.: 00150 g CoSOi. 0.1063 g Sbst.: 13.7 ccm N (170, 723 mm). - 0.2834 g Sbst.: 0.13YY g AgCI. - 0.1260 g Sbst.: 0.0585 g H20. - 0.1260 g Sbst.: 0.0766 g Con. - 0.08li4 p Sbst.: 00150 g CoSOi. p g C O C ~ N B C I O ~ H ~ ~ , . Uer Co l!l.Y6, I\i 14.02, Cl 11.97, H 5.11, C 16.23. Gef. )) 19.81, I) 14.30, 8 12.19, )) 4.63. 16.56. p g C O C ~ N B C I O ~ H ~ ~ , . Uer Co l!l.Y6, I\i 14.02, Cl 11.97, H 5.11, C 16.23. Gef. )) 19.81, I) 14.30, 8 12.19, )) 4.63. 16.56. stanz eineii Gewichtsverlust, der eiriem MolekLil Wasser entspricht. Gewichtsvcrliist = 0.01 g = 5.82 pct. RrO. Heini Erhitzeri erleidet die iiber Schwefelsaure getrocknete Sub- 0.1717 g Shst. wogen nach I-stdg. Erhitzcn auf 105O nurmehr O.lti17 g. Ber. fur obige Formel: 1 Mol. HsO -=6.05 pCt. stanz eineii Gewichtsverlust, der eiriem MolekLil Wasser entspricht. Heini Erhitzeri erleidet die iiber Schwefelsaure getrocknete Sub- , p Gewichtsvcrliist = 0.01 g = 5.82 pct. RrO. 0.1717 g Shst. wogen nach I-stdg. D i b r o m o - a q u o - 6: I I , y I end i a rn in- a m m in- k o b a 1 t b r o m i d , D i b r o m o - a q u o - 6: I I , y I end i a rn in- a m m in- k o b a 1 t b r o m i d , D i b r o m o - a q u o - 6: I I , y I end i a rn in- a m m in- k o b a 1 t b r o m i d , D i b r o m o - a q u o - 6: I I , y I end i a rn in- a m m in- k o b a 1 t b r o m i d , Zur Darstelluug dieses Salzes liist m:in das soeben beschriebenc Chlorobromobromid in etwas mehr als der nothigen Menge IV'asser :tiif, erwarmt die Losung schwech :tiif dem Wasserbade und setzt nach erfolgter Abkiihlung daa gleiche Volumen Kromwasserstoffsiiure (spec. Grwicht 1.49) hiinu. Nach 12 Stdn. ist das Salz augkrystalli- sirt. Es bildet kleine. aber wohlansgebildete, sternformige Nadel- :iggregate und zeigt einen Y lei ausgepriigteren Dichroismus als das entsprechende Dichloroch!orid. Die Krystalle erscheinen blauscbwarz oder braun gefiirbt uud geben beini Zerreiben ein griines Pulver. In Wasser lost sich das Salz mit brauner Farbe. 0.1102 g Sbst.: 0.0443 g CoSO,. - 0.1060 g Sbst.: I0 ccm N (ll*,#. 711 mm). - 0.1466 g Sbst.: 14.2 ccm N (141/2", 724 mm). - 0.1003 g Sbst.: 0.2872 g AgBr. CoBr3N3C2H130. Ber. Co 14.92, N 10.69, Br 60.91. Gef. D 15.'?Y, 10.47, 10.80, D FI.01. Ziirich, Unirersitatslaboi atorium, December 1905. Ziirich, Unirersitatslaboi atorium, December 1905. I) Diese Berichte 82, 2197 [l899]. 2, Arch. f. Pharm. 234, 487 [1896l. 704. H. K i l i a n i : Ueber Digitoxose. [Aus der medic. Abth. des Univer3.-Laboratoriums Freiburg i. B.; (Eingegsngen am 5. December 1905.) (Eingegsngen am 5. December 1905 Meine friihereli Beobaclitungen hatten es zweifelhaft gelassen, ob die Digitoxose, CsH1201, ale Aldeliyd oder als Keton zu betrachten sei I). Jetzt habe ich sicher festgestellt, dn53 der Zucker eine Aldose ist. Diese enthalt ein CHS (Y. 1. c.), folglicli muss, w e n n eine normale Kohlenstoff-Kette vorliegt, geniiiss obiger Formcl ein CH2 vorhanden sein, das w a h r s c h e i n l i c h die u-Stellung zur Aldehydgruppe ein- nimmt, weil es nicht gelungeu war, ein Osazon ZII gewinnen2). Da- mit ergab sich aber sofort Eiir die drei Hydroxyle die Stellung ani $-, y- und 8-Kohlenstoff, d. h. die C o n s t i t u t i o n d e r D i g i t o x o s r rnusste sein: Ziirich, Unirersitatslaboi atorium, December 1905. T ri n i t r i to-ath y 1 end i am i n- a n i niin- ko b a It , [(OrN)sCo (NH3) (en) I . fiir 1 Mol. HnO = 5.15 pCt 0.1518 g Sbst. wogen nach einstiindigem Erhitzen iin Trockenschrd 0.1437 8 ; Abnahmc = 0.0081 g = 5.34 pCt. Bw. fiir 1 Mol. HnO = 5.15 pCt Dabei war die Substanz zusarnmengrsintert und bildete e k e gelbe Kriistp. die sich ill Wasser leicht mit gelber Farbe liiste. Dabei war die Substanz zusarnmengrsintert und bildete e k e gelbe Kriistp. die sich ill Wasser leicht mit gelber Farbe liiste. A 118 der Liisung des Clilorobroniobromids fiillt Jodkaliumliisung ein scliwer IiislicheR Jodid, und diirch Vcrreiben des trocknen Salzes rnit conceriti irter Salpetereaure bildet sich ein sehr leicht lijslicheq hellgrCines Sitrat. 4040 CHx.CH(OH) .CH (OH) .CH(OH) .CX2 CHO. CHx.CH(OH) .CH (OH) .CH(OH) .CX2 CHO. CHx.CH(OH) .CH (OH) .CH(OH) .CX2 CHO.
https://openalex.org/W4230099459
https://escholarship.org/content/qt4cr9d8d3/qt4cr9d8d3.pdf?t=rh0mie
English
null
Association of Intradialytic Hypotension Events With All-Cause Mortality in Hemodialysis Patients
American journal of kidney diseases
2,016
cc-by
347
UC Irvine UC Irvine Previously Published Works Title Association of Intradialytic Hypotension Events With All-Cause Mortality in Hemodialysis Patients Permalink https://escholarship.org/uc/item/4cr9d8d3 Journal American Journal of Kidney Diseases, 67(5) ISSN 0272-6386 Authors Chou, Jason Obi, Yoshitsugu Kim, Tae Hee et al. Publication Date 2016-05-01 DOI 10.1053/j.ajkd.2016.03.075 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Association of Intradialytic Hypotension Events With All-Cause Mortality in Hemodialysis Patients Permalink https://escholarship.org/uc/item/4cr9d8d3 Journal American Journal of Kidney Diseases, 67(5) ISSN 0272-6386 Authors Chou, Jason Obi, Yoshitsugu Kim, Tae Hee et al. Publication Date 2016-05-01 DOI 10.1053/j.ajkd.2016.03.075 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Am J Kidney Dis. 2016;67(5):A1-A118 Am J Kidney Dis. 2016;67(5):A1-A118 A35 NKF 2016 Spring Clinical Meetings Abstracts NKF 2016 Spring Clinical Meetings Abstracts Powered by the California Digital Library University of California eScholarship.org eScholarship.org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¶V QDGLU6%3ZDV OHVVWKDQ  ZLWKLQWKHVW GD\VRI+' WUHDWPHQW EDVHOLQHTXDUWHU ZLWKDOOFDXVHPRUWDOLW\LQDVWXG\FRKRUWRI LQFLGHQWDGXOW+'SDWLHQWVUHFHLYLQJFDUHIURPD86EDVHGODUJH GLDO\VLVRUJDQL]DWLRQEHWZHHQ±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±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
https://openalex.org/W4241539654
https://journals.iium.edu.my/ejournal/index.php/iiumej/article/download/1706/818
English
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MODELLING A 1-DOF FINGER EXTENSOR MACHINE FOR HAND REHABILITATION
IIUM engineering journal
2,021
cc-by
5,742
KEYWORDS: modelling; simulation; experimental validation; hand rehabilitation; hardware-in-the-loop IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. 1. INTRODUCTION Every year, up to 15 million people suffer from stroke, making it one of the leading causes of severe disability in the world [1]. After a neurological injury such as stroke, patients suffer from several impairments, of which the most common and impeding is the impairment to the hand sensorimotor function. The finger extensor muscles develop weakness during voluntary movements as a result of activity deficit. This leads to spasticity which is a velocity-dependent increase in muscle tone. Spasticity causes intense muscle contractions and stiffness that manifest in the form of a clenched fist, tensed fingers, a curled wrist and other deformities. These impairments lead to a decreased ability to open and close the hand, control individual finger movements and perform force coordination, thereby severely affecting the quality of life of stroke patients [2]. Patients undergo intense physical therapy under the guidance of an occupational therapist to regain functionality in the affected limb. Activity-based exercises are known to be effective in helping overcome the motor deficits induced by post-stroke spasticity [3]. However, occupational therapy is expensive and as a result, a vast majority of the patients do not have continuous access to it. Besides, conventional therapy is monotonous, repetitive and difficult to be sustained for prolonged periods of time, leading to high drop- out rates. Furthermore, with conventional therapy, it is not possible to measure the exact progress of patients over time and provide them with well-regulated and a wide range of rehabilitative forces. Due to these factors, robot-assisted rehabilitation has seen a growing interest in the last few decades. Robotic devices, due to their features of high repeatability, interactivity, ability to provide a wide range of accurate forces and automatic measurement of patient progress, are becoming increasingly popular. The interactive nature of robot- aided therapy not only increases physical engagement but also encourages cognitive engagement of the patient through immersive and challenging exercises. Patient engagement in rehabilitation is vital because encouraging human users to perform self- initiated movements is an essential requirement to achieve motor learning and neuroplasticity [4]. Furthermore, robot-aided rehabilitation is capable of producing assistive as well as resistive forces that can be tailored to the specific impairment of the patient. When patients gain some amount of motor function in the impaired part, they are subjected to active- resistive therapy whereby, forces are applied on the patient in a direction opposite to the one they are tracing. *Corresponding author: sinnira@iium.edu.my (Received: 24th November 2020; Accepted: 22nd December 2020; Published on-line: 4th July 2021) ABSTRACT: It is essential to have an accurate representation of a robotic rehabilitation device in the form of a system model in order to design a robust controller for it. This paper presents mathematical modelling and validation through simulation and experimentation of the 1-DOF Finger Extensor rehabilitation machine. The machine’s design is based on an iris mechanism, built specifically for training open and close movements of the hand. The goal of this research is to provide an accurate model for the Finger Extensor by taking into consideration various factors affecting its dynamics and to present an experimental validation of the devised model. Dynamic system modelling of the machine is performed using Lagrangian formulation and the involved physical parameters are obtained experimentally. To validate the developed model and demonstrate its effectiveness, hardware-in-the-loop experiments are conducted in the Simulink-MATLAB environment. Mean absolute error between the simulated and experimental response is 1.38° and the relative error is 1.13%. The results obtained are found to be within the human motion resolution limits of 5 mm or 5º and exhibit suitability of the model for application in robotic rehabilitation systems. The model accurately replicates the actual behavior of the machine and is suitable for use in controller design. ABSTRAK: Gambaran tepat mengenai model sistem peranti rehabilitasi robotik adalah sangat penting bagi pembangunan sesebuah reka bentuk alat kawalan tahan lasak. Kajian mengenai model matematik dan pengesahan melalui simulasi dan eksperimentasi mesin pemulihan 1-DOF ‘Finger Extensor’. Mesin ini direka bentuk berdasarkan mekanisme iris, dibangunkan khusus bagi melatih gerakan buka dan tutup tangan. Tujuan kajian ini adalah bagi menyediakan model Finger Extensor yang tepat dengan mengambil kira faktor mempengaruhi dinamik dan pengesahan model eksperimen yang dirancang. Model sistem dinamik mesin ini diuji menggunakan formula Lagrangian dan parameter fizikal yang terlibat diperoleh melalui eksperimen. Model ini disahkan dan diuji keberkesanannya menggunakan eksperimen Perkakasan-dalam-gelung melalui MATLAB-Simulink. Purata ralat mutlak antara dapatan simulasi dan respon eksperimen adalah 1.38° dan ralat relatif 1.13%. Dapatan kajian adalah dalam had resolusi gerakan tangan manusia iaitu 5 mm atau 5º dan didapati model ini sesuai bagi aplikasi sistem rehabilitasi robotik. Model ini tepat dalam mereplikasi kelakuan sebenar mesin dan sesuai digunakan bagi reka bentuk kawalan. 384 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. Before the implementation of these control strategies, dynamic and kinematic modelling of the rehabilitation machine is necessary. Modelling is the mathematical representation of a physical system [14]. A system model is developed from mathematical equations and then represented graphically in software such as MATLAB, Simulink and StateFlow, etc. The developed model is then validated through simulations, reducing the cost associated with experimental validation. During the process of model development, trade-offs have to be made between the fidelity and simplicity of the model. Fidelity implies the extent to which the system model mimics the behavior of the real system and simplicity ensures that the model remains simple enough for smooth controller development in later stages. Thus, there are some deviations between the behavior of the actual system and its model. After balancing simplicity and fidelity, the model is validated using a Hardware-in-the-Loop (HiL) approach. In this approach, one or more real subsystems interact in a closed loop with the virtual subsystems of the model. A comparison between the response of such a closed-loop interaction with the simulation output reveals performance of the developed model [15]. In [16], dynamic and kinematic modelling of the multipurpose rehabilitation robot, the Universal Haptic Pantograph, was performed. Validation of the model was carried out through experiments with healthy subjects reflecting its performance. Dynamic modelling and experimental validation of a parallel robot for upper limb rehabilitation was presented in [17]. Approximately zero steady-state errors points to a high degree of accuracy and suitability of the developed model. Dynamic modelling of a haptic finger actuated by a McKibben artificial muscle built for tele-operated rehabilitation was performed in [18]. Upon experimental validation, the model demonstrated a satisfactory performance. In this work, mathematical modelling and system simulation of a 1 degree-of-freedom (DOF) Finger Extensor rehabilitation machine was performed. A Model-Based Development (MBD) [15] approach to controller design was taken. Such an approach to design enabled verification and validation at each stage. Based on simulation results, modifications to the system could be made easily without incurring high costs. As a first step, mathematical modelling was done, followed by simulation of the system behavior. Simulation results were then compared to hardware measurements which demonstrated the accuracy of the realized model. Hardware-in-the-Loop testing of the model carried out in this study ensures minimization of risks and costs associated with experimental validation. IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 The developed model aids in the design and implementation of a control algorithm for the Finger Extensor machine. 1. INTRODUCTION Resistive strategies enhance patient performance as they require additional effort from the patient to resist opposite forces. Such efforts induce neuroplasticity and help in development of neural pathways from the impaired muscles to the brain which aids in regaining of motor function [5]. However, all these features of robotic rehabilitation devices are possible only when efficient controllers are developed [6]. A variety of control strategies have been proposed for rehabilitation robots over the past few decades. These control schemes are designed around the central idea of modulating assistance/resistance provided to the patient based on various factors such as force exerted by the patient, limb velocity, tracking error, interaction force/torque, EEG/EMG activity [7]. In recent literature, approaches such as force/stiffness control [8] Assist-as-Needed control (AAN) [9-11] and performance-based impedance control [7,12,13] have been implemented on rehabilitation robots. AAN control aims at providing minimal intervention in patient recovery. Performance-based impedance control modulates the assistance provided, based on real-time measurements of the patient's biomechanical characteristics. 385 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 3.1 Mechanical Model Newton-Euler and the Lagrange-Euler methods are the most commonly used approaches for deriving dynamic equations of mechanical parts of robots [20]. The former is ideal for on-line control applications due to reduced computational time. However, it falls short in providing an adequate insight into control design due to recursive computation. The Lagrange-Euler method is simple and systematic, providing dynamic equations in a matrix form and was chosen for deriving the mechanical model for the Finger Extensor. Dynamic equation describing motion of the Finger Extensor was written as 𝑀(𝜃(𝑡), 𝜉)𝜃̈(𝑡) + 𝐷(𝜃(𝑡), 𝜃̇(𝑡), 𝜉) + 𝐺(𝜃(𝑡), 𝜉) = 𝑇(𝑡) (1) where, 𝑀(𝜃(𝑡), 𝜉)𝜃̈(𝑡) + 𝐷(𝜃(𝑡), 𝜃̇(𝑡), 𝜉) + 𝐺(𝜃(𝑡), 𝜉) = 𝑇(𝑡) (1) where, (1) 𝑀(θ(𝑡), ξ) is the N x N inertia matrix. 𝑀(θ(𝑡), ξ) is the N x N inertia matrix. 𝐷(𝜃(𝑡), 𝜃̇(𝑡), 𝜉) is the N x 1 vector of coriolis and centrifugal forces. 𝐺(𝜃(𝑡), 𝜉) is the N x 1 vector of gravitational forces. 𝑇(𝑡) is the N x 1 vector of torques applied by the actuators. 𝐷(𝜃(𝑡), 𝜃̇(𝑡), 𝜉) is the N x 1 vector of coriolis and centrifugal forces. 𝐺(𝜃(𝑡) 𝜉) is the N x 1 vector of gravitational forces 𝐷(𝜃(𝑡), 𝜃̇(𝑡), 𝜉) is the N x 1 vector of coriolis and centrifugal forces. 𝐺(𝜃(𝑡), 𝜉) is the N x 1 vector of gravitational forces. 𝐺(𝜃(𝑡), 𝜉) is the N x 1 vector of gravitational forces. 𝑇(𝑡) is the N x 1 vector of torques applied by the actuators. 𝜃(𝑡), 𝜃̇(𝑡), 𝜃̈(𝑡) are the N x 1 vectors of joint displacements, velocities and accelerations respectively. 𝜉 is a vector (with appropriate dimension) of parameters of the mechanism such as payload. For the Finger Extensor, 𝑀(𝜃(𝑡), 𝜉) is a 1x1 matrix, with one element equal to the moment of inertia of a solid cylinder with iris radius of 0.055m. 𝐷(𝜃(𝑡), 𝜃̇(𝑡), 𝜉) and 𝐺(θ(𝑡), ξ) for a 1-DOF machine were null matrices. 𝑇(𝑡) for the Finger Extensor is calculated in the subsequent sections. For developing the mechanical model of the Finger Extensor, the next step was to derive equations of motion of the moving parts of the mechanism. These were represented by flywheel and spring mechanical elements as shown in Fig. 4. 𝐽1, 𝐽2 represent inertia elements of the motor and iris mechanisms respectively, 𝑘1, 𝑏1 represent the spring stiffness effect and viscous and coulomb friction. Fig. 4: Block diagram representation. Fig. 4: Block diagram representation. 2. THE FINGER EXTENSOR MACHINE The Finger Extensor rehabilitation machine is a 1-DOF device with an iris mechanism lying at the heart of its dynamics. The design of the iris or diaphragm is inspired by the constriction and dilation of the iris of the human eye, which varies size of the pupil, adjusting the aperture of the eye. The iris mechanism built into the Finger Extensor constricts and dilates the poles at the top of its surface, to allow openings of variable sizes. These poles, as shown in Fig. 1 and Fig. 2 are grasped by the patient during therapy and enable him/her to practice finger flexion and extension [19]. There are six poles in the current Finger Extensor design and each pole has a blade associated with it. On the upper end, each blade is connected to a slider and at the bottom, each blade goes into a slot in a disc using a bearing, as illustrated in Fig. 2. The disc with slots is in turn connected to the motor actuation system through a chain and sprocket mechanism. As the motor turns, the sprocket induces motion into the slotted disc. This 386 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. https://doi.org/10.31436/iiumej.v22i2.1706 causes the blades to slide and move the poles in their own respective slots at the top of the machine. Fig. 1: 1-DOF Finger Extensor machine. Fig. 1: 1-DOF Finger Extensor machine. Fig. 2: CAD drawing of the side view of the iris mechanism. Fig. 1: 1-DOF Finger Extensor machine. Fig. 1: 1-DOF Finger Extensor machine. Fig. 2: CAD drawing of the side view of the iris mechanism. Fig. 2: CAD drawing of the side view of the iris mechanism. Fig. 3: Inner view of the machine. Fig. 3: Inner view of the machine. Fig. 3: Inner view of the machine. Figure 3 shows the actuation system of the machine comprising of a brushed planetary geared DC motor (Gear Ratio- 32.5:1). Rotation of the motor is recorded by a rotary encoder by ESB electronics, Japan (Counts per Revolution- 500) connected to the sprocket using a flexible shaft coupling. A torque sensor, connected close to the chain-sprocket mechanism provides the torque applied by the machine on to the patient. Actuation system and sensors are controlled by Arduino microcontroller board which is programmed in the Simulink environment. 387 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. 3.1 Mechanical Model (5), 𝑇𝑔= 1 𝑁𝐽2𝜃2̈ + 1 𝑁𝑇𝑒+ 𝑁𝑘1𝜃2 + 𝑉𝑐(𝜃2̇ ) + 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) (6 𝑇𝑔= 1 𝑁𝐽2𝜃2̈ + 1 𝑁𝑇𝑒+ 𝑁𝑘1𝜃2 + 𝑉𝑐(𝜃2̇ ) + 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) (6) where, 𝑉𝑐(𝜃2̇ ) and 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) are the viscous and coulomb friction terms. Therefore, dynamic equation of the 1 DOF Finger Extensor was represented as (6) 𝑇𝑔= 𝑁𝐽2𝜃2 + 𝑁𝑇𝑒+ 𝑁𝑘1𝜃2 + 𝑉𝑐(𝜃2) + 𝐹𝑐𝑆𝑔𝑛(𝜃2) (6) where, 𝑉𝑐(𝜃2̇ ) and 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) are the viscous and coulomb friction terms. Therefore, dynamic equation of the 1-DOF Finger Extensor was represented as where, 𝑉𝑐(𝜃2̇ ) and 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) are the viscous and coulomb friction terms. Therefore, dynamic equation of the 1-DOF Finger Extensor was represented as where, 𝑉𝑐(𝜃2̇ ) and 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) are the viscous and coulomb friction terms. Therefore, dynamic equation of the 1-DOF Finger Extensor was represented as 𝑇𝑔(𝑡) = 1 𝑁𝐽2(𝜃2)𝜃2̈ (𝑡) + 1 𝑁𝑇𝑒(𝑡) + 𝑁𝑘1𝜃2(𝑡) + 𝑉𝑐(𝜃2̇ )(𝑡) + 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) (7) 𝑇𝑔(𝑡) = 1 𝑁𝐽2(𝜃2)𝜃2̈ (𝑡) + 1 𝑁𝑇𝑒(𝑡) + 𝑁𝑘1𝜃2(𝑡) + 𝑉𝑐(𝜃2̇ )(𝑡) + 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) (7 𝑇𝑔(𝑡) = 1 𝑁𝐽2(𝜃2)𝜃2̈ (𝑡) + 1 𝑁𝑇𝑒(𝑡) + 𝑁𝑘1𝜃2(𝑡) + 𝑉𝑐(𝜃2̇ )(𝑡) + 𝐹𝑐𝑆𝑔𝑛(𝜃2̇ ) (7) (7) 3.1 Mechanical Model Forces acting on the motor and iris sprockets are shown in Fig. 5, where 𝜃1 is the angle of rotation of the motor sprocket, 𝜃2 is the angle of rotation of the iris mechanism, 𝑇𝑔 is the torque on the driver sprocket, 𝑇𝑒 is the torque exerted by the user, 𝑓𝑐𝑟1 and 𝑓𝑐𝑟2 388 eering Journal, Vol. 22, No. 2, 2021 Shahdad et al. g/10.31436/iiumej.v22i2.1706 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. are the contact force torques. Figures 4 and 5, hence represent Eq. (1) in terms of sum of forces (inertial, gravitational, frictional) acting on the machine. are the contact force torques. Figures 4 and 5, hence represent Eq. (1) in terms of sum of forces (inertial, gravitational, frictional) acting on the machine. As per D’Alembert’s law, sum of torques on both flywheels was given by the equations 𝑇𝑔= 𝐽1𝜃1̈ + 𝑘1𝜃1 + 𝑏1(𝜃1̇ −𝜃2̇ ) −𝑓𝑐𝑟1 (2) −𝑓𝑐𝑟2 −𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒= 0 (3) From Eq. (3), value of 𝑓𝑐 is calculated as 𝑓𝑐= 1 𝑟2 [−𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒] (4) 𝑇𝑔= 𝐽1𝜃1̈ + 𝑘1𝜃1 + 𝑏1(𝜃1̇ −𝜃2̇ ) −𝑓𝑐𝑟1 (2) −𝑓𝑐𝑟2 −𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒= 0 (3) From Eq. (3), value of 𝑓𝑐 is calculated as 𝑓𝑐= 1 𝑟2 [−𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒] (4) 𝑇𝑔= 𝐽1𝜃1̈ + 𝑘1𝜃1 + 𝑏1(𝜃1̇ −𝜃2̇ ) −𝑓𝑐𝑟1 −𝑓𝑐𝑟2 −𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒= 0 From Eq. (3), value of 𝑓𝑐 is calculated as 𝑓𝑐= 1 𝑟2 [−𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒] (2) (3) 𝑓𝑐= 1 𝑟2 [−𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒] (4) Fig. 5: Forces acting on mechanism. Fig. 5: Forces acting on mechanism. Substituting value of 𝑓𝑐 in Eq. (2) Substituting value of 𝑓𝑐 in Eq. (2) Substituting value of 𝑓𝑐 in Eq. (2) 𝑇𝑔= 𝑘1𝜃1 + 𝑏1(𝜃1̇ −𝜃2̇ ) − 𝑟1 𝑟2 [−𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒] (5) 𝑇𝑔= 𝑘1𝜃1 + 𝑏1(𝜃1̇ −𝜃2̇ ) − 𝑟1 𝑟2 [−𝐽2𝜃2̈ −𝑏1(𝜃2̇ −𝜃1̇ ) −𝑇𝑒] (5) (5) Using the inverse gear ratio, 𝑟2 𝑟1 = N and the equality of arc lengths, 𝑟1𝜃1 = 𝑟2𝜃2, it follows that 𝜃1 = 𝑟2 𝑟1 𝜃2 = 𝑁𝜃2. Substituting in Eq. (5), Using the inverse gear ratio, 𝑟2 𝑟1 = N and the equality of arc lengths, 𝑟1𝜃1 = 𝑟2𝜃2, it follows that 𝜃1 = 𝑟2 𝑟1 𝜃2 = 𝑁𝜃2. Substituting in Eq. 3.2 Actuator Model The Finger Extensor is powered by a planetary-geared, 24 V brushed DC motor. A schematic of the motor is shown in Fig. 6. Using Kirchhoff's voltage law and Newton's law of motion, dynamic equations of the actuator were written as 𝐽𝑚𝜃𝑚̈ (𝑡) = −𝐵𝑣𝜃𝑚̇ (𝑡) + 𝑘𝑡𝑖𝑎(𝑡) −𝑇𝐿(𝑡) (8) 𝐿𝑖𝑎(𝑡) = −𝑘𝑣𝜃𝑚̇ (𝑡) −𝑅𝑖𝑎(𝑡) + 𝑉(𝑡) (9) (8) (9) 𝐿𝑖𝑎(𝑡) = −𝑘𝑣𝜃𝑚(𝑡) −𝑅𝑖𝑎(𝑡) + 𝑉(𝑡) (9) where, 𝐽𝑚 is the moment of inertia of the motor (kg. m2) 𝐽𝑚 is the moment of inertia of the motor (kg. m2) 389 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. 𝜃𝑚(𝑡) is the angular displacement of the motor (rad) 𝐵𝑣 is the viscous friction coefficient (Nm/rad/s) 𝑘𝑡, 𝑘𝑣 are the torque and back emf constants (Nm/A, V/rad/s) L, R are the armature inductance and resistance (H, Ω) 𝑖𝑎(𝑡) is the armature current for the motor (A) 𝑇𝐿(𝑡) is the load torque for the motor (Nm) 𝑉(𝑡) is the voltage input (V) Fig. 6: Motor schematic. Fig. 6: Motor schematic. Fig. 6: Motor schematic. Utilizing the relationship between loading torque 𝑇(𝑡) (from the motor) and load torque 𝑇𝐿(𝑡) (on the motor shaft) and the relationship between the motor's angular position and the machine's angular position, we formulated 𝑇𝐿(𝑡) = 𝑇(𝑡) 𝑁, 𝑁≥1 𝜃𝑚(𝑡) = 𝑁𝜃(𝑡) 𝑇𝐿(𝑡) = 𝑇(𝑡) 𝑁, 𝑁≥1 𝑇𝐿(𝑡) = 𝑇(𝑡) 𝑁, 𝑁≥1 (10) 𝜃𝑚(𝑡) = 𝑁𝜃(𝑡) (11) 𝑇𝐿(𝑡) = 𝑇(𝑡) 𝑁, 𝑁≥1 (10) 𝜃𝑚(𝑡) = 𝑁𝜃(𝑡) (11) (10) (11) where N is the inverse gear ratio. 3.3 Integrated Model For developing the integrated model of the machine, a time derivative of the dynamic equation of the machine was found. Differentiating Eq. (7) gave 𝑇̇𝑔(𝑡) = { 𝐽2(𝜃2) 𝑁} 𝜃2⃛(𝑡) + { 𝑇̇𝑒(𝑡) 𝑁} + 𝑁𝑘𝜃2̇ (𝑡) + 𝐹𝑐{2𝛿(𝜃2̇ )𝜃2̈ } + 𝑉𝑐𝜃2̈ (𝑡) (15) Substituting Eq. (15) in Eq. (12), (15) 𝜃2⃛(𝑡) {𝐽𝑚𝑁3 + 𝐽2(𝜃2) 𝐽𝑚𝑁3 } + 𝜃2̈ (𝑡) {𝑁2(𝐵𝑣𝐿+ 𝐽𝑚𝑅) + 2𝐹𝑐𝐿𝛿(𝜃2̇ ) + 𝑉𝑐𝐿 𝐽𝑚𝑁2𝐿 } + 𝜃2̇ (𝑡) {𝑁(𝑘𝑣𝑘𝑡+ 𝐵𝑣𝑅) + 𝐿𝑘1 𝐽𝑚𝑁𝐿 } + 𝑇𝑔(𝑡) { 𝑅 𝑁2𝐽𝑚𝐿} + 𝑇̇𝑒(𝑡) { 1 𝑁3𝐽𝑚 } = { 𝑘𝑡 𝑁𝐽𝑚𝐿} 𝑉(𝑡) (16) (16) Equation (16) was solved for 𝜃 and put in state-space form, the integrated model of the Finger Extensor was obtained as Equation (16) was solved for 𝜃 and put in state-space form, the integrated model of the Finger Extensor was obtained as 𝑋̇𝐵(𝑡) = 𝐴𝐵(𝑋𝐵, 𝑡)𝑋𝐵(𝑡) + 𝐵𝐵(𝑋𝐵, 𝑡)𝑈(𝑡) + 𝐹𝐵𝑇𝑔(𝑡) + 𝑄𝐵𝑇̇𝑒(𝑡) (17) (17) where 𝑋𝐵= [𝜃2 𝜃2̇ 𝜃2̈ ] 𝑇 where 𝑋𝐵= [𝜃2 𝜃2̇ 𝜃2̈ ] 𝑇 where 𝑋𝐵= [𝜃2 𝜃2̇ 𝜃2̈ ] 𝑇 𝐴𝐵, 𝐵𝐵, 𝐹𝐵, 𝑄𝐵 are the system, input, load distribution and rate of load distribution respectively. 𝑈(𝑡), 𝑇𝑔(𝑡), 𝑇𝑒(𝑡) are the input vector, mechanical link torque and toque exerted by the patient. 𝐴𝐵(𝑋𝐵, 𝑡) = [ 0 0 1 0 1 0 0 𝑎32 𝑎33 ] , 𝐵𝐵(𝑋𝐵, 𝑡) = [ 0 0 𝑏31 ] , 𝐹𝐵(𝑋𝐵, 𝑡) = [ 0 0 𝑓31 ] , 𝑄𝐵(𝑋𝐵, 𝑡) = [ 0 0 𝑞31 ] Equation (17) was put in the state-space form and solved for 𝑋̇𝐵. Equation (17) was put in the state-space form and solved for 𝑋̇𝐵. 𝑎32 = −{𝑁2(𝑁𝑘𝑣𝑘𝑡+ 𝑁𝐵𝑣𝑅+ 𝐿𝑘1) 𝐿(𝐽𝑚𝑁3 + 𝐽2(𝜃2)) } , 𝑎33 = −{𝑁(𝑁2𝐵𝑣𝐿+ 𝑁2𝐽𝑚𝑅+ 2𝐹𝑐𝐿𝛿(𝜃2 ˙ ) + 𝑉𝑐𝐿 𝐿(𝐽𝑚𝑁3 + 𝐽2(𝜃2)) } 𝑏31 = { 𝑘𝑡𝑁2 𝐿(𝐽𝑚𝑁3 + 𝐽2(𝜃2))} , 𝑓31 = −{ 𝑅𝑁 𝐿(𝐽𝑚𝑁3 + 𝐽2(𝜃2))} , 𝑞31 = −{ 1 (𝐽𝑚𝑁3 + 𝐽2(𝜃2))} The state-space model derived in Eq. (17) represents the integrated dynamics of the Finger Extensor and was utilized in the simulation of the developed system. The state-space model derived in Eq. (17) represents the integrated dynamics of the Finger Extensor and was utilized in the simulation of the developed system. where N is the inverse gear ratio. where N is the inverse gear ratio. Substituting Eq. (10) and Eq. (11) in Eq. (8) and Eq. (9), the otherwise linear, time- invariant actuator dynamic was converted into a non-linear time-variant one. The actuator model in the form of a third-order differential equation was represented as: 𝜃2⃛(𝑡) + { 𝐵𝑣𝐿+𝐽𝑚𝑅 𝐽𝑚𝐿 } 𝜃2̈ (𝑡) + { 𝑘𝑣𝑘𝑡+𝐵𝑣𝑅 𝐽𝑚𝐿 } 𝜃2̇ (𝑡) + { 𝑅 𝑁2𝐽𝑚𝐿} 𝑇𝑔(𝑡) + { 1 𝑁2𝐽𝑚} 𝑇̇𝑔= { 𝑘𝑡 𝑁𝐽𝑚𝐿} 𝑉(𝑡) (12) To represent the actuator dynamic model in the state variable form, 𝜃, 𝜃̇, 𝜃̈ were chosen as state variables and the state vector for the actuator was given by To represent the actuator dynamic model in the state variable form, 𝜃, 𝜃̇, 𝜃̈ were chosen as state variables and the state vector for the actuator was given by 𝑋𝐵(𝑡) = [𝜃(𝑡) 𝜃̇(𝑡) 𝜃̈(𝑡)] 𝑇 (13) 𝑋𝐵(𝑡) = [𝜃(𝑡) 𝜃̇(𝑡) 𝜃̈(𝑡)] 𝑇 (13 (13) The state equation was formulated as The state equation was formulated as 𝑋𝐵̇ (𝑡) = 𝐴𝐵𝑋𝐵(𝑡) + 𝐵𝐵𝑈(𝑡) + 𝐹𝐵𝑇𝑔(𝑡) + 𝑄𝐵𝑇̇𝑒(𝑡) (14) 𝑋𝐵̇ (𝑡) = 𝐴𝐵𝑋𝐵(𝑡) + 𝐵𝐵𝑈(𝑡) + 𝐹𝐵𝑇𝑔(𝑡) + 𝑄𝐵𝑇̇𝑒(𝑡) (14) where (14) 𝐴𝐵(𝑋𝐵, 𝑡) = [ 0 0 1 0 1 0 0 𝑎32 𝑎33 ] , 𝐵𝐵(𝑋𝐵, 𝑡) = [ 0 0 𝑏31 ] , 𝐹𝐵(𝑋𝐵, 𝑡) = [ 0 0 𝑓31 ] , 𝑄𝐵(𝑋𝐵, 𝑡) = [ 0 0 𝑞31 ] 390 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. Here, 𝐴𝐵, 𝐵𝐵, 𝐹𝐵, 𝑄𝐵 are the system, input, load distribution and rate of load distribution respectively. 𝑈(𝑡), 𝑇𝑔(𝑡), 𝑇𝑒(𝑡) are the input vector, mechanical link torque and toque exerted by the patient. The values of elements 𝑎32, 𝑎33, 𝑏31, 𝑓31, and 𝑞31 are 𝑎32 = {−𝑘𝑣𝑘𝑡+ 𝐵𝑣𝑅 𝐽𝑚𝐿 }; 𝑎33 = {−𝐵𝑣𝐿+ 𝐽𝑚𝑅 𝐽𝑚𝐿 }; 𝑏31 = { 𝑘𝑡 𝑁𝐽𝑚𝐿} ; 𝑓31 = −{ 𝑅 𝑁2𝐽𝑚𝐿} ; 𝑞31 = −{ 1 𝑁2𝐽𝑚 } 4. MODEL VALIDATION Using the MBD approach for designing a controller for the Finger Extensor, dynamic system modelling was followed by graphical representation of the mathematical formulation in the MATLAB-Simulink environment. Simulation results were validated through hardware-in-the-loop experimentation. The same input was provided to both the developed model as well as the machine and comparisons were drawn between resultant outputs. 3.4 Mechanical and Electrical Parameter Determination The development of the integrated model for the Finger Extensor was followed by determination of mechanical and electrical parameters involved. This step aids in enhancing the fidelity of the developed model. Mechanical parameters such as radius of 391 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. the iris and moment of inertia of the inner mechanism were adapted from [20]. Electrical parameters associated with the actuator model were experimentally found from the motor characteristic curve as shown in Fig. 7. Torque and back EMF constants 𝑘𝑡, 𝑘𝑣 were found from the torque-current and torque-speed curves with similar values of 0.791 and 0.886, respectively. Other parameters such as the viscous friction constant 𝐵𝑣, armature resistance R and inductance L were adapted from [20]. the iris and moment of inertia of the inner mechanism were adapted from [20]. Electrical parameters associated with the actuator model were experimentally found from the motor characteristic curve as shown in Fig. 7. Torque and back EMF constants 𝑘𝑡, 𝑘𝑣 were found from the torque-current and torque-speed curves with similar values of 0.791 and 0.886, respectively. Other parameters such as the viscous friction constant 𝐵𝑣, armature resistance R and inductance L were adapted from [20]. Fig. 7: Motor characteristic curves. Fig. 7: Motor characteristic curves. 4.1 Simulation For carrying out simulation of the mathematical model of the Finger Extensor, the voltage and torques from the motor and user acted as inputs to the Finger Extensor plant while the angle of rotation of the iris was the output. A PID controller was used for optimal position control. Rotational position output and its derivatives were used to calculate velocity and acceleration. These values helped in calculating the force acting on the patient. A desired angle of 𝜃 = 121° (2.11 radians) was fed into the plant and the simulated position of the iris was plotted as shown in Fig. 8. Fig. 8: Comparison between target and simulated angular position of the iris. Fig. 8: Comparison between target and simulated angular position of the iris. 392 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. The figure shows a complete open and close cycle of the Finger Extensor for 30 seconds. The poles of the Extensor open to their maximum rotation angle of 121° followed by closing motion to 7.89°. From the figure, it is clear that the hardware had a slower response than the simulation and took about 11.20 seconds to sync with the simulation results. However, beyond that time, error was negligible and actual behavior of the Finger Extensor overlapped with the simulated, modeled behavior. From experimental data, it was observed that the mean absolute error was 1.38° and the relative error was 1.13%. However, for rehabilitation applications, movement accuracy is less critical as compared to surgical applications. Also, position resolution of human arm movement is 5 mm or 5º [21], which indicates that a mean absolute error of 1.38° is likely to be acceptable for rehabilitation purposes. Figure 11 shows the comparison between the simulated result and the hardware-in- the-loop response, when the Finger Extensor was programmed to open to an angle of 59.58° (1.04 radians). Simulation and experimental curves took 0.3 seconds to reach the desired angle. The Root Mean Squared Error (RMSE) was calculated as 0.9875. There was a slight overshoot of 2.86º (0.05 radians) which caused the poles of the Finger Extensor to open to an angle 2.86º greater than the target. However, this negligible overshoot did not inflict any discomfort on the subject. Also, the response settled down to its desired value within 0.08 seconds. Fig. 11: Simulated and experimental responses for target angle experiment. Fig. 11: Simulated and experimental responses for target angle experiment. 4.2 Hardware-in-the-Loop Experiments To validate the developed dynamic model and demonstrate its effectiveness, experiments were conducted making the Finger Extensor a part of the simulation loop. These experiments were conducted with a healthy subject, a female of 29 years of age. A simple open-close movement of the machine was performed. Results from the simulations run in MATLAB were compared with the experimental tests with the Finger Extensor. The experimental set-up as shown in Fig. 9 consisted of the Finger Extensor connected to an Intel Core i5 PC by means of an Arduino Uno microprocessor board. This board received sensory data from the encoder as well as the torque sensor. Communication between the Arduino and MATLAB 2018b software, running on the PC was established using a MATLAB s-function. This function read data from the encoder i.e. the position sensor of the machine and converted the encoder counts to angular displacement of the iris. Fig. 9: Experimental setup for hardware-in-the-loop simulation. Fig. 9: Experimental setup for hardware-in-the-loop simulation. The movement commands to the motor and consequently to the encoder were provided using Simulink's support package for Arduino which allowed Simulink blocks to control hardware connected to the Arduino using PWM signals. In this case, hardware controlled was the motor driver connected to the motor. The desired input angle was fed into the hardware and the Finger Extensor was run for about 30 seconds, performing open- close movements. The comparison between the simulated and the experimental angular displacement of the machine is shown in Fig. 10. Fig. 10: Simulated and experimental responses during open-close movements. Fig. 10: Simulated and experimental responses during open-close movements. 393 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 5. CONCLUSION This paper presented a mathematical model for the 1-DOF Finger Extensor rehabilitation machine, taking into consideration the dynamics of the mechanism as well as that of the actuation system. Simulation results show that the developed model tracks the desired position efficiently with minimal error. Hardware-in-the-Loop experiments carried out with the machine demonstrate the validity of the model. System response using the developed model was compared with that obtained through hardware experimentation. The results illustrate the accuracy of the model to the actual behavior of the rehabilitation system and make it suitable for use in the design of a control scheme for it. Obtained results with mean absolute error of only 1.38° are within the human motion resolution limits and exhibit suitability of the model for application in robotic rehabilitation systems. Future research with the Finger Extensor and the developed model will be directed towards development of a control scheme for the machine. This will be aimed at modulating assistance/resistance provided to the patient based on his/her performance using various metrics such as limb velocity, applied force, position error etc. Considering patient's strength and residual ability, primary goal of the controller would be to increase 394 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. https://doi.org/10.31436/iiumej.v22i2.1706 self-initiated movements and patient engagement in training exercises which would lead to an increase in neuroplasticity. As part of future study, clinical testing of the Finger Extensor will also be carried out with stroke patients. This will include a detailed study of the level of comfort of the device for use with such patients. self-initiated movements and patient engagement in training exercises which would lead to an increase in neuroplasticity. As part of future study, clinical testing of the Finger Extensor will also be carried out with stroke patients. This will include a detailed study of the level of comfort of the device for use with such patients. REFERENCES [1] Khor KX, Chin PJH, Rahman HA, Yeong CF, Su ELM, Narayanan ALT. (2014) A novel haptic interface and control algorithm for robotic rehabilitation of stoke patients. In 2014 IEEE Haptics Symposium (HAPTICS), Houston, Texas-USA; pp. 421-426. [2] Plantin J, Pennati GV, Roca P, Baron JC, Laurencikas E, Weber K, Godbolt AK, Borg J, Lindberg PG. (2019) Quantitative assessment of hand spasticity after stroke: imaging correlates and impact on motor recovery. Frontiers in neurology, 10:1-11. p y gy, [3] Cramer SC. (2019) Intense rehabilitation therapy produces very large gains in chronic stroke. Journal of Neurology, Neurosurgery & Psychiatry, 90: 497. [4] Lambercy O, Ranzani R, Gassert R. (2018) Robot-assisted rehabilitation of hand function. In Rehabilitation Robotics. Volume 1. 1st edition. Academic Press; pp 205–225. [5] Popovic MB. (2019) Biomechatronics. Academic Press. [5] Popovic MB. (2019) Biomechatronics. Academic Press. [6] Miao Q, Zhang M, Cao J, Xie SQ. (2018) Reviewing high-level control techniques on robot- assisted upper-limb rehabilitation. Advanced Robotics, 32(24): 1253-1268. [7] Marini F, Hughes CM, Squeri V, Doglio L, Moretti P, Morasso P, Masia L. (2017) Robotic wrist training after stroke: Adaptive modulation of assistance in pediatric rehabilitation. Robotics and Autonomous Systems, 91: 169-178. [8] Su YY, Yu YL, Lin CH, Lan CC. (2019) A compact wrist rehabilitation robot with accurate force/stiffness control and misalignment adaptation. International Journal of Intelligent Robotics and Applications, 3(1): 45-58. [9] Luo L, Peng L, Wang C, Hou ZG. (2019) A greedy assist-as-needed controller for upper limb rehabilitation. IEEE Transactions on Neural Networks and Learning Systems, 30(11): 3433-3443 [10] Pehlivan AU, Losey DP, O’Malley MK. (2015) Minimal assist-as needed controller for upper limb robotic rehabilitation. IEEE Transactions on Robotics; 32(1): 113-124. [11] Pehlivan AU, Losey DP, Rose CG, O'Malley MK. (2017) Maintaining subject engagement during robotic rehabilitation with a minimal assist-as-needed (mAAN) controller. In 2017 International Conference on Rehabilitation Robotics (ICORR): 2017; London; pp. 62–67. [12] Fricke SS, Bayon C, Rocon E, van der Kooij H, van Asseldonk EH. (2018) Pilot study of a performance based adaptive assistance controller for stroke survivors. In International Conference on Neuro Rehabilitation. Springer, pp. 302–306. [13] Stroppa F, Loconsole C, Marcheschi S, Mastronicola N, Frisoli A. (2018) An improved adaptive robotic assistance methodology for upper-limb rehabilitation. In International Conference on Human Haptic Sensing and Touch Enabled Computer Applications. Springer; pp. 513–525. [14] Bringmann E, Krämer A. (2008). Model-based testing of automotive systems. ACKNOWLEDGEMENTS The authors would like to acknowledge International Islamic University Malaysia (IIUM) for supporting this research under the grant number: P-RIGS18-019-0019. The authors would like to acknowledge International Islamic University Malaysia (IIUM) for supporting this research under the grant number: P-RIGS18-019-0019. REFERENCES in 1st international conference on software testing, verification, and validation. IEEE; pp. 485-493. 15] Soltani A, Assadian F. (2016) A hardware-in-the-loop facility for integrated vehic dynamics control system design and validation. IFAC-Papers Online, 49(21): 32-38. [16] Mancisidor A, Zubizarreta A, Cabanes I, Bengoa P, Brull A, Jung JH. (2019) Inclusive and seamless control framework for safe robot-mediated therapy for upper limbs rehabilitation. Mechatronics, 58: 70-79. [17] Guang H, Ji L, Shi Y, Misgeld BJ. (2018) Dynamic modeling and interactive performance 395 IIUM Engineering Journal, Vol. 22, No. 2, 2021 https://doi.org/10.31436/iiumej.v22i2.1706 Shahdad et al. of PARM: A parallel upper-limb rehabilitation robot using impedance control for patients after stroke. Journal of Healthcare Engineering, 2018: 1-11. of PARM: A parallel upper-limb rehabilitation robot using impedance control for patients after stroke. Journal of Healthcare Engineering, 2018: 1-11. [18] Franco W, Maffiodo D, De Benedictis C, Ferraresi C. (2018) Dynamic modeling and experimental validation of a haptic finger based on a mckibben muscle. in IFToMM Symposium on Mechanism Design for Robotics. Springer; pp. 251–259. [19] Ali MAA, Azlan NZ. (2006) Design of iris mechanism for flexion and extension training in hand rehabilitation. ARPN Journal of Engineering and Applied Sciences; 11: 4115-4122. [20] Osman JHS. (1992) Integrated Model of Industrial Robot for Control Applications. Jurnal Teknologi, 19(1): 27-41. [21] Germanotta M, Vasco G, Petrarca M, Rossi S, Carniel S, Bertini E, Cappa P, Castelli E. (2015) Robotic and clinical evaluation of upper limb motor performance in patients with Friedreich’s ataxia: an observational study. Journal of Neuroengineering and Rehabilitation, 12(1): 1-3. 396
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https://www.econstor.eu/bitstream/10419/179788/1/jefas_v22_i43_p131-153.pdf
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Efectos de la realización de un megaevento deportivo sobre una economía local. El caso de los Juegos Mundiales 2013 Cali
Journal of economics finance and administrative science/Journal of economics, finance and administrative science
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Article Efectos De La Realización De Un Megaevento Deportivo Sobre Una Economía Local. El Caso De Los Juegos Mundiales 2013 Cali Journal of Economics, Finance and Administrative Science Journal of Economics, Finance and Administrative Science Provided in Cooperation with: Universidad ESAN, Lima Suggested Citation: Aguado, Luis F.; Osorio Mejía, Ana M.; Arbona, Alexei; Peña-Vinces, Jesús C. (2017) : Efectos De La Realización De Un Megaevento Deportivo Sobre Una Economía Local. El Caso De Los Juegos Mundiales 2013 Cali, Journal of Economics, Finance and Administrative Science, ISSN 2218-0648, Emerald Publishing Limited, Bingley, Vol. 22, Iss. 43, pp. 131-153, https://doi.org/10.1108/JEFAS-01-2017-0012 Aguado, Luis F.; Osorio Mejía, Ana M.; Arbona, Alexei; Peña-Vinces, Jesús C. Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. 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The full terms of this licence may be seen at http:// creativecommons.org/licenses/by/4.0/legalcode Resumen Propósito – En el artículo se presenta una síntesis del impacto sobre la producción y el empleo generado por la realización de un megaevento deportivo en la ciudad de Cali, Colombia. Diseño/metodología/enfoque – El impacto se estima a través de los efectos sistemáticos producidos por las actividades de inversión/gasto de los agentes identificados y los consecuentes efectos multiplicadores capturados a partir de un modelo insumo-producto. Hallazgos – La realización de los Juegos Mundiales significó un nuevo flujo de ingreso monetario a la ciudad de Cali, de $101.036 millones de pesos (=53,4 millones de dólares) y se generaron 9.598 puestos de trabajo (=7.711 empleos de tiempo completo). Adicionalmente, se atrajo a la ciudad a 2.174 turistas internacionales y 11.250 turistas nacionales. Originalidad/valor – El artículo aporta aspectos innovadores metodológicamente útiles para futuro estudios de impacto económico en ciudades poco turísticas situadas en países en vías desarrollo. Palabras clave – Cali, Colombia, Impacto económico, Los Juegos Mundiales, Megaeventos deportivos Tipo de artículo – Artículo de investigación Efectos de la realización de un megaevento deportivo sobre una economía local. El caso de los Juegos Mundiales 2013 Cali Luis F. Aguado, Ana M. Osorio and Alexei Arbona Departamento de Economía, Pontificia Universidad Javeriana, Cali, Colombia, y Efectos de la realización de un megaevento deportivo 131 Received 25 January 2017 Accepted 8 February 2017 Jesús C. Peña-Vinces Business Management, Universidad de Sevilla, Sevilla, España JEL classification – L83, C67 Journal of Economics, Finance and Administrative Science Vol. 22 No. 43, 2017 pp. 131-153 EmeraldPublishingLimited 2077-1886 DOI 10.1108/JEFAS-01-2017-0012 1. Introducción En el artículo se presentan los resultados del impacto económico a corto plazo, sobre la ciudad de Santiago de Cali[1] y su área metropolitana (en adelante AM), asociado a la celebración de la IX edición de los Juegos Mundiales (World Games). Los Juegos Mundiales están constituidos por las federaciones de disciplinas deportivas que no participan en el programa de los Juegos Olímpicos. La IX edición de los Juegos Mundiales se llevó a cabo entre el 25 de julio y el 4 de agosto de 2013 y ha sido la primera organizada por una ciudad de habla hispana y situada en un país de América del Sur[2]. Los Juegos Mundiales, como megaevento deportivo, involucraron la competición simultánea en 31 disciplinas deportivas[3], el desplazamiento de 2.929 deportistas y de 1.903 delegados de 97 países de los cinco continentes. Adicionalmente, la novedad y la diversidad que implica la presencia de atletas de élite en deportes poco conocidos y practicados como el sumo, el wushu y el korfball, por mencionar algunos en el contexto de América Latina. El artículo se centra en los impactos producidos por las actividades de inversión/gasto de los agentes identificados en la celebración de los Juegos Mundiales: Comité Local Organizador de Cali (inversión en infraestructura, gasto en logística) y espectadores (gasto en consumo), y los consecuentes efectos multiplicadores capturados a partir de un modelo insumo-producto. Estos impactos se sintetizan en los efectos sobre la producción y el empleo en el área metropolitana de la ciudad, definida como área de impacto. p , p La inversión para garantizar la infraestructura física deportiva y la organización y logística de los Juegos Mundiales más el gasto de los espectadores ascendió a $140.357 millones de pesos (:74,2 mill. US$)[4]. De este total, $101.036 millones de pesos (:53,4 mill. US$) significaron un nuevo flujo de ingreso monetario derivado de agentes de fuera de Cali y A.M. (Gobierno Nacional; Turistas Extranjeros y Nacionales) que de no haberse realizado Los Juegos Mundiales no hubiese llegado a la ciudad. Adicionalmente, los Juegos Mundiales atrajeron a 2,174 turistas internacionales y 11,250 turistas nacionales. Asimismo, generaron 9.598 puestos de trabajo (:7.711 empleos de tiempo completo) en sectores vinculados con la logística de la organización de los Juegos y con la “turbina de gasto” de los turistas. g Los resultados lucen interesantes para la literatura empírica sobre los estudios de impacto económico de megaeventos deportivos. Abstract Purpose – A summary of the economic impact resulting from the celebration of a sporting mega-event in the city of Cali, Colombia, is carried out in this article. Design/methodology/approach – The impacts are estimated by means of the systematic effects produced by the activities of investment/expenditure of the identified agents and the consequent multiplying effects captured from an input-output model. Findings – The World Games represented a new income flow to the city of Cali of $101.036 million pesos (: US$53.4 mill.) and 9.598 jobs were created (:7,711 full-time jobs). Additionally, 2,174 foreign tourists and 11,250 national tourists were attracted to the city. Journal of Economics, Finance and Administrative Science Vol. 22 No. 43, 2017 pp. 131-153 EmeraldPublishingLimited 2077-1886 DOI 10.1108/JEFAS-01-2017-0012 Originality/value – The article contributes with innovative aspects methodologically speaking, useful for future studies of economic impact in cities with low tourism located in developing countries. Keywords Economic impact, Sporting mega-events, World Games, Cali Paper type Research paper Originality/value – The article contributes with innovative aspects methodologically speaking, useful for future studies of economic impact in cities with low tourism located in developing countries. Keywords Economic impact, Sporting mega-events, World Games, Cali Originality/value – The article contributes with innovative aspects methodologically speaking, useful for uture studies of economic impact in cities with low tourism located in developing countries. Keywords Economic impact, Sporting mega-events, World Games, Cali Keywords Economic impact, Sporting mega-events, World Games, Cali Paper type Research paper Keywords Economic impact, Sporting mega-events, World Games, Cali Paper type Research paper 132 Efectos de la realización de un megaevento deportivo 2. Los Juegos Mundiales como megaevento deportivo D d l li d l í d l d (R h De acuerdo con la literatura de la economía del deporte (Roche, 1994; Llopis-Goig, 2012), los megaeventos deportivos se caracterizan por ser eventos a gran escala con un profundo significado internacional; tener una duración breve y perfectamente acotada; estar organizados por un país o ciudad que generalmente compite por ser sede; atraer un importante número de participantes (deportistas) y espectadores, tanto locales como extranjeros; ser cubiertos por un amplio número de medios internacionales de comunicación; requerir alta inversión en infraestructura, logística y seguridad; y ser regentados por un Comité Local Organizador integrado por autoridades de gobiernos nacionales y locales y organizaciones y federaciones deportivas internacionales. Los Juegos Mundiales encajan dentro de la tipología de megaevento deportivo (ver Tabla I). En efecto, estos tienen carácter multideportivo, un gran número de deportistas y países participantes, se celebran cada cuatro años, son regentados por la Asociación Internacional de los Juegos Mundiales (IWGA)[5]. La literatura de la economía del deporte identifica como megaeventos deportivos a los Juegos Olímpicos, la Copa Mundial de Fútbol (FIFA) y los Juegos Olímpicos de Verano e Invierno y en menor escala los Juegos Panamericanos. Los Juegos Mundiales constituyen un megaevento impulsado por los resultados deportivos —“competitor driven”— que alcancen los deportistas de élite en competencia, frente a aquellos megaeventos impulsados por su alto número de seguidores y espectadores —“spectator driven”— (Gratton et al., 2006, p. 51). 1. Introducción Por un lado, en el contexto latinoamericano es muy limitada la información de los estudios de esta naturaleza, exceptuando la investigación ex-post para los Juegos Panamericanos de Río de Janeiro 2007, el estudio ex- ante de los Juegos Panamericanos de Guadalajara 2011 y el más reciente para el pasado mundial de fútbol de Brasil. Por otro lado, se presentan elementos innovadores en la metodología seguida. Así, la metodología para determinar los turistas internacionales por vía administrativa ofrece un cálculo directo y, por tanto, realista del flujo de visitantes extranjeros, usualmente sobreestimados en los estudios de impacto económico. En el caso de la estimación de los turistas nacionales, se combina información de una encuesta cara a cara aplicada a los asistentes con información de las entradas vendidas por el Comité Organizador Local. En ambos casos (turistas nacionales e internacionales), se tiene en cuenta el tamaño de los escenarios, la frecuencia de asistencia y la rotación de entradas, lo que hace más precisas las estimaciones. Adicionalmente, se plantea el limitado alcance de los efectos “expulsión” y de “cancelación de reservas” en ciudades poco turísticas situadas en países en vías desarrollo, lo que incrementa para este tipo de ciudades los beneficios económicos de realizar este tipo de eventos internacionales. El artículo se organiza en siete secciones, la primera es esta introducción. En la segunda y tercera secciones, se caracterizan los Juegos Mundiales como megaevento deportivo y se detalla la metodología empleada en la estimación del impacto económico. En la cuarta sección se presenta la estimación del flujo de espectadores, tanto nacionales como extranjeros. En quinto lugar se estima el impacto económico de los Juegos Mundiales a través de un modelo insumo-producto. En la sexta sección se discute el legado de los Juegos Mundiales para Cali, es decir los efectos de largo plazo esperados por la celebración del megaevento deportivo, los cuales no necesariamente se capturan por las relaciones monetarias implícitas en los modelos insumo-producto. Por último, se presentan las conclusiones. 133 3. Metodología l l En el artículo se utiliza la metodología de efectos, propia de los Estudios de Impacto Económico —en adelante EIE—. La literatura empírica de estudios de impacto económico aplicada al sector deportivo está concentrada en países desarrollados situados en Europa, Estados Unidos y Asia. Una aproximación detallada de la metodología empleada, sus limitaciones y aplicaciones se encuentra en: (Baade and Matheson, 2016; Zimbalist, 2015; Barajas et al., 2012; Matheson, 2012; Porter and Chin, 2012; Barget and Gouguet, 2008; Gratton et al., 2006; Matheson, 2006). Para el contexto de América Latina (Devesa et al., 2012; Ávalos, 2011). El análisis del impacto económico resulta útil para ofrecer una imagen cuantitativa de la actividad económica adicional generada en la ciudad por la celebración de los Juegos Mundiales. Por un lado, referida a la focalización de las inversiones —públicas y privadas— en infraestructura deportiva y urbana y el gasto en gestión y logística para llevar a cabo el Tabla I. Perfil de los Juegos Mundiales 2013 - Cali Características Descripción Gobierno del evento International World Games Association (IWGA) Comité Local Organizador de Cali (CLOC) Comité Nacional Organizador Ámbito geográfico Internacional 97 países: 40 (Europa) þ 23 (América) þ 23 (Asia) þ 2 (Oceanía) þ 9 (África) Frecuencia de realización Cada 4 años con cambio de sede Duración 11 días (10 de competiciones þ 1 de inauguración) Competidores 2.929 deportistas: 1.511 (Europa) þ 823 (América) þ 407 (Asia) þ 110 (Oceanía) þ 78 (África) Delegaciones 1.903 delegados de las federaciones deportivas, jueces internacionales, delegaciones nacionales —entrenadores, médicos, ayudantes—, incluidos los representantes de los medios internacionales Deportes 31 (26 oficiales þ 5 invitados) Interés mediático Cali compitió por ser sede con las ciudades de Johannesburgo (Sudáfrica) y San Petersburgo (Rusia) 96 periodistas de 34 países Escenarios 25 (20 cerrados þ 5 al aire libre) Voluntariado 2.539 voluntarios Novedad en América Latina Primera edición organizada por una ciudad de habla hispana y situada en un país de América del Sur Nota: Elaboración propia con información del Comité Organizador Local Cali JEFAS 22,43 134 Nota: Elaboración propia con información del Comité Organizador Local Cali megaevento deportivo. De otro lado, referida a la generación de gasto de los asistentes — turistas nacionales y extranjeros— en bienes y servicios relacionados con su estadía en la ciudad. 3. Metodología l l Desde esta perspectiva, el gasto de los residentes de Cali y AM no constituye una entrada neta de dinero a la economía local, sino una reasignación de su gasto. Este supuesto señala que los residentes en Cali y AM disponen de un presupuesto fijo para sus gastos de recreación y ocio. Por tanto, incluir este gasto puede conducir a sobrestimar los efectos indirectos, error muy común en los estudios de impacto económico (Siegfried and Zimbalist, 2006). De igual forma, se debe tener presente el posible efecto expulsión provocado por el desplazamiento de público local residente, y/o la cancelación de visitas a la ciudad de turistas (extranjeros/nacionales), para evitar la congestión y los altos precios que supone el exceso de demanda provocado por los espectadores atraídos por la celebración del megaevento deportivo. En el primer caso, la celebración de los Juegos Mundiales representó Figura 1. Metodología impacto económico Juegos Mundiales 2013 Cali IMPACTOS Fuentes de demanda Corto plazo Comité Local Organizador Inversión en infraestructura deportiva Construcción y remodelación de escenarios deportivos. Inversión en gestión y logística Preparación y gestión por parte del equipo que integra el CLOC. Equipamiento de las instalaciones deportivas; transporte y alimentación de deportistas, jueces y delegados, uniformes de los voluntarios, etc. Gasto visitantes/espectadores Gasto en bienes y servicios por parte de los visitantes/espectadores no locales Encuesta cara/cara asistentes Efectos directos Efectos indirectos Producción y empleo adicionales generados por los Juegos Mundiales 2013 Cali Comité Local Organizador de Cali, CLOC [Presupuesto/Gastos] Fuentes de información Efectos inducidos : Matriz insumo producto adaptada par el Valle del Cauca FLUJO MONETARIO DE GASTO Elaboración propia Efectos de la realización de un megaevento deportivo 135 Figura 1. Metodología impacto económico Juegos Mundiales 2013 Cali IMPACTOS Fuentes de demanda Corto plazo Comité Local Organizador Inversión en infraestructura deportiva Construcción y remodelación de escenarios deportivos. Inversión en gestión y logística Preparación y gestión por parte del equipo que integra el CLOC. Equipamiento de las instalaciones deportivas; transporte y alimentación de deportistas, jueces y delegados, uniformes de los voluntarios, etc. 3. Metodología l l Ambas fuentes de nueva demanda generan un efecto multiplicador sobre la economía de la ciudad a través de la interrelación sectorial capturada a partir de un modelo insumo-producto (Schuschny, 2005). La metodología se centra en los efectos sistemáticos producidos por las actividades de inversión/gasto de los agentes identificados (ver Figura 1). megaevento deportivo. De otro lado, referida a la generación de gasto de los asistentes — turistas nacionales y extranjeros— en bienes y servicios relacionados con su estadía en la ciudad. Ambas fuentes de nueva demanda generan un efecto multiplicador sobre la economía de la ciudad a través de la interrelación sectorial capturada a partir de un modelo insumo-producto (Schuschny, 2005). La metodología se centra en los efectos sistemáticos producidos por las actividades de inversión/gasto de los agentes identificados (ver Figura 1). Es así como la estimación del impacto económico de los Juegos Mundiales 2013 Cali se lleva a cabo a partir de la suma de tres efectos: directo, indirecto e inducido. 3.1 Efectos directos Los efectos directos miden los gastos/inversión totales en que incurre el Comité Local Organizador de Cali, CLOC, nómina de sueldos, bienes y servicios comprados, construcción y adecuación de infraestructura física deportiva, logística, alquileres, etc. Son obtenidos a partir de la información suministrada por el Comité Local Organizador discriminada por la fuente de financiación, gobierno local o entidades del gobierno regional/nacional. Estos últimos constituyen la inyección de gasto nuevo en el área metropolitana de Santiago de Cali y no una recomposición del gasto del municipio. Asimismo, un aspecto a considerar en el análisis del impacto económico de los megaeventos deportivos es el tipo de financiación usado (Zimbalist, 2010). La financiación vía deuda pública deja compromisos en el mediano y largo plazo asumibles mediante mayores impuestos y/o recortes del gasto en otros servicios prestados por el gobierno (local/nacional). Si bien este estudio se enfoca en el corto plazo, es importante señalar que la financiación de los Juegos Mundiales provino en mayor medida de recursos del gobierno nacional y no requirió contraer deuda pública por parte del gobierno local[6]. 3.2 Efectos indirectos Los efectos indirectos miden los gastos que realizan los espectadores en bienes y servicios — compra de entradas, alojamiento, alimentación, transporte local, recuerdos—. Los gastos relevantes son los realizados por los espectadores de fuera del área metropolitana de Santiago de Cali. 3. Metodología l l Gasto visitantes/espectadores Gasto en bienes y servicios por parte de los visitantes/espectadores no locales Encuesta cara/cara asistentes Efectos directos Efectos indirectos Producción y empleo adicionales generados por los Juegos Mundiales 2013 Cali Comité Local Organizador de Cali, CLOC [Presupuesto/Gastos] Fuentes de información Efectos inducidos : Matriz insumo producto adaptada par el Valle del Cauca FLUJO MONETARIO DE GASTO Elaboración propia Efectos de la realización de un megaevento deportivo 135 3.2 Efectos indirectos 3.2 Efectos indirectos f Los efectos indirectos miden los gastos que realizan los espectadores en bienes y servicios — compra de entradas, alojamiento, alimentación, transporte local, recuerdos—. Los gastos relevantes son los realizados por los espectadores de fuera del área metropolitana de Santiago de Cali. Desde esta perspectiva, el gasto de los residentes de Cali y AM no constituye una entrada neta de dinero a la economía local, sino una reasignación de su gasto. Este supuesto señala que los residentes en Cali y AM disponen de un presupuesto fijo para sus gastos de recreación y ocio. Por tanto, incluir este gasto puede conducir a sobrestimar los efectos indirectos, error muy común en los estudios de impacto económico (Siegfried and Zimbalist, 2006). , ) De igual forma, se debe tener presente el posible efecto expulsión provocado por el desplazamiento de público local residente, y/o la cancelación de visitas a la ciudad de turistas (extranjeros/nacionales), para evitar la congestión y los altos precios que supone el exceso de demanda provocado por los espectadores atraídos por la celebración del megaevento deportivo. En el primer caso, la celebración de los Juegos Mundiales representó un evento extraordinario que motivó a los locales a participar como asistentes a las competiciones y a demostrar su hospitalidad a los visitantes internacionales. El segundo caso parece ajustarse más a la realidad de grandes ciudades turísticas en países desarrollados. En la Figura 2 se observa que la demanda adicional de hospedaje provocada por la celebración de los Juegos Mundiales fue perfectamente absorbida por la capacidad hotelera disponible en la ciudad, para los mismos meses de 2014 la tasa de ocupación estuvo alrededor del 50 per cent, por tanto no sería válido concluir que se presentó un efecto desplazamiento y de “cancelación de reservas”. Para la estimación del número de espectadores y la descripción de su perfil sociodemográfico y de gasto se requirió el uso de varias fuentes de información, entre ellas la colaboración de Migración Colombia[7] y la realización de una encuesta cara a cara aplicada a los espectadores en la entrada/salida y los alrededores de los escenarios deportivos. Figura 2. Tasa de ocupación hotelera en Cali 2010-2013 43.8% 41.0% 44.0% 92.0% 47.8% 42.0% 49.0% 78.0% 0% 20% 40% 60% 80% 100% 2010 2011 2012 2013* Agosto Julio Nota: *Corresponde al período del 1 al 4 de agosto y al período del 24 al 31 de julio. . Estimación del número de espectadores y de su gasto 4. Estimación del número de espectadores y de su gasto Los Juegos Mundiales se desarrollaron durante 10 días, los seis últimos del mes de julio y los primeros cuatro del mes de agosto de 2013. Las competiciones tuvieron lugar en 25 escenarios con capacidad para albergar a 108.259 espectadores[9]. Los tres escenarios de mayor capacidad son el Estadio Olímpico Pascual Guerrero (25.486)[10], la Plaza de Toros de Cañaveralejo (19.000) y el Coliseo El Pueblo (18.700) que representan el 58 per cent del total de la capacidad. El 25 de julio se llevó a cabo la ceremonia de inauguración (no hubo competencias) y el 4 de agosto se realizó la ceremonia de clausura. El cálculo de los espectadores requirió usar distintas fuentes de información, a través de un conjunto de matices y especificaciones que procedemos a explicar. 4.1 La encuesta Asistentes a los Juegos Mundiales 2013 Cali 3.2 Efectos indirectos Fuente: Asociación Hotelera del Valle del Cauca, COTELVALLE JEFAS 22,43 136 136 Figura 2. Tasa de ocupación hotelera en Cali 2010-2013 Nota: *Corresponde al período del 1 al 4 de agosto y al período del 24 al 31 de julio. Fuente: Asociación Hotelera del Valle del Cauca, COTELVALLE 3.3 Efecto inducido El efecto inducido es el efecto combinado de los dos gastos anteriores (Comité Organizador Local y espectadores) sobre el conjunto de la economía local, a través de dos canales, el canal de relaciones intersectoriales (por ejemplo, la construcción de un nuevo escenario deportivo significa un aumento en la demanda de insumos —ladrillo, aluminio, acero— a otros sectores) y el canal de consumo asociado a los nuevos ingresos que la actividad económica genera (por ejemplo, la contratación de mano de obra —ingenieros, obreros de construcción—, utilidades del sector de obras civiles). Los efectos inducidos son calculados a través de multiplicadores derivados de la Matriz insumo-producto (MIP) para el Valle del Cauca, elaborada por Duque et al. (2013). Esta matriz es la más reciente disponible en la región para este tipo de estudios a falta de una MIP para Cali y AM[8]. p y [ ] La matriz parte de información del valor bruto de la producción, del valor agregado y de la demanda final, y utiliza el método RAS para calcular el cuadrante de consumo intermedio (Duque et al., 2013). La matriz calculada es de tipo producto por producto, simétrica, a valores básicos y presentados en pesos constantes de 2005. Se encuentra desagregada en 37 sectores de la economía del Valle del Cauca; sin embargo, para los fines de este artículo se ha agregado en 18 sectores de acuerdo con las dos grandes fuentes de demanda identificadas. 4.2 El cálculo de los turistas extranjeros Gracias al apoyo de Migración Colombia se habilitó temporalmente en el sistema de registro de los diferentes puestos de control migratorio en el país la categoría “Juegos Mundiales” entre el 18 de julio y el 4 de agosto de 2013[13]. Los resultados muestran que en el período ingresaron 10.481 extranjeros al país con destino a la ciudad de Cali, de los cuales 7.006 manifestaron que la principal razón para visitar Cali era participar de las actividades asociadas a los Juegos Mundiales, distribuidos así: 4.832 visitantes en calidad de deportistas, jueces, miembros de federaciones deportivas, prensa, invitados especiales, etc. y 2.174 como espectadores/turistas extranjeros. Esta estrategia para determinar el número de turistas extranjeros resulta novedosa en este tipo de estudios, en la medida en que implica un cálculo directo y preciso del grupo de interés. Usualmente, en la literatura de evaluación de impacto económico se utilizan estimaciones indirectas basadas en encuestas al público asistente y en la ocupación hotelera. Otra ventaja de esta estrategia es que elimina el sesgo de los visitantes casuales, es decir, los espectadores extranjeros que asisten a los Juegos pero su visita a la ciudad es explicada por otra razón distinta a la celebración de los Juegos (visita a familiares, razones de estudio/ trabajo). 4.1 La encuesta Asistentes a los Juegos Mundiales 2013 Cali J g Se diseñó una encuesta cara a cara[11] aplicada a los espectadores en los sitios de entrada/ salida y alrededores de los escenarios de competición, para ello se requirió el apoyo de 56 voluntarios/as de los Juegos que a través de tres reuniones recibieron capacitación en el desarrollo de encuestas cara a cara, un conocimiento profundo de los cuestionarios y de los objetivos de la investigación. Los voluntarios/as se distribuyeron en función de la programación de las competiciones y la capacidad de los escenarios. Asimismo, se diseñaron dos modelos de cuestionario: uno dirigido a los turistas extranjeros (en inglés) y otro para los locales, tanto para residentes como para no residentes de Cali y AM. En total, se aplicaron 1.299 encuestas: 508 a turistas extranjeros, 282 a turistas nacionales y 509 a residentes del área metropolitana de Santiago de Cali[12]. voluntarios/as de los Juegos que a través de tres reuniones recibieron capacitación en el desarrollo de encuestas cara a cara, un conocimiento profundo de los cuestionarios y de los objetivos de la investigación. Los voluntarios/as se distribuyeron en función de la programación de las competiciones y la capacidad de los escenarios. Asimismo, se diseñaron dos modelos de cuestionario: uno dirigido a los turistas extranjeros (en inglés) y otro para los locales, tanto para residentes como para no residentes de Cali y AM. En total, se aplicaron 1.299 encuestas: 508 a turistas extranjeros, 282 a turistas nacionales y 509 a residentes del área metropolitana de Santiago de Cali[12]. 137 La encuesta permitió capturar:  las características generales de los asistentes, tanto demográficas com socioeconómicas;  la frecuencia de asistencia a las competiciones; es decir, para un mismo individuo, el número medio de competiciones y el número medio de días que asiste;  la estructura del gasto derivado de la asistencia a las competiciones; y  el motivo de asistencia y grado de satisfacción. Para cada uno de los anteriores apartados la información distingue entre los tres tipos de espectadores identificados: locales residentes en Cali y AM, turistas nacionales y turistas extranjeros. Efectos de la realización de un megaevento deportivo Figura 3. Número de espectadores* que presenciaron los Juegos Mundiales 2013 Cali 4.3 Otros aspectos metodológicos clave La empresa encargada por el Comité Local Organizador de Cali para la venta de las entradas proporcionó el número total de entradas disponibles, las efectivamente vendidas y las de cortesía por día, escenario y disciplina deportiva, incluyendo las ceremonias de inauguración y clausura. Si bien en principio resulta claro que incluir el gasto del público local residente en Cali y AM representa una recomposición de su gasto en el tiempo y en la misma área geográfica, se hace necesario incorporar a aquellos espectadores locales residentes en el área de impacto que pudiendo salir de la ciudad (fuga de gasto) se quedaron, producto de una elección racional, para participar como espectadores por la novedad que significaban los Juegos Mundiales. Este público asistente es capturado a través de la siguiente pregunta de la encuesta: >Ha asistido a otras competiciones internacionales en los últimos tres años? Asimismo, se tiene en cuenta el fenómeno observado de rotación de entradas entre familiares/amigos de la población residente en Santiago de Cali. Este fenómeno es explicado en parte por las largas jornadas de las competiciones y porque la entrada permitía el acceso durante toda la jornada. Así, por ejemplo, una persona usaba la misma entrada en la mañana y otra la usaba en la tarde, a lo largo del mismo día. 138 4.4. >Cuántos espectadores presenciaron los juegos mundiales 2013 cali? La anterior información y refinamientos metodológicos permitieron convertir el número total de entradas (vendidas þ cortesía) en número total de asistentes por tipo de residencia que se muestran en el Figura 3. Esta metodología de estimación del número de espectadores también se puede calificar de novedosa en los estudios de impacto económico. En efecto, conociendo el número de espectadores internacionales, a partir de la encuesta se puede estimar el número de turistas nacionales y de espectadores residentes en Cali y AM, a través de la pregunta sobre el “motivo de asistencia”. Además, la información de la encuesta permite corregir el número de entradas vendidas por la frecuencia de asistencia, es decir, por el número medio de competiciones vista por cada tipo de espectador. De esta forma, se convierte el número de entradas en número de espectadores, lo que asegura evitar cometer un error muy común en los estudios de impacto económico de sobreestimar el número de asistentes, que en muchos estudios rebasa la capacidad de los escenarios de competencia y/o la capacidad hotelera local. JEFAS 22,43 4.3 Otros aspectos metodológicos clave 2,174 11,250 90,249 50,540 0 20,000 40,000 60,000 80,000100,000 Turistas Internacionales Turistas Nacionales Residentes en Cali y área metropolitana** Ceremonia Inaugural y Clausura*** Notas: *Corresponde al número de espectadores corregido por la frecuencia de asistencia. Es decir, teniendo presente que un mismo espectador asiste a distintas competiciones a lo largo de los días de celebración de los Juegos; **incluye corrección por el número de residentes de Cali que compartieron la entrada, con ello se aumentó el número de personas que disfrutaron de las competiciones; ***incluye corrección para evitar la duplicación de asistentes que presenciaron ambas ceremonias Fuente: Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali e información de Migración Colombia 2,174 11,250 90,249 50,540 0 20,000 40,000 60,000 80,000100,000 Turistas Internacionales Turistas Nacionales Residentes en Cali y área metropolitana** Ceremonia Inaugural y Clausura*** Figura 3. Número de espectadores* que presenciaron los Juegos Mundiales 2013 Cali 2,174 11,250 90,249 50,540 0 20,000 40,000 60,000 80,000100,000 Turistas Internacionales Turistas Nacionales Residentes en Cali y área metropolitana** Ceremonia Inaugural y Clausura*** Notas: *Corresponde al número de espectadores corregido por la frecuencia de asistencia. Es decir, teniendo presente que un mismo espectador asiste a distintas competiciones a lo largo de los días de celebración de los Juegos; **incluye corrección por el número de residentes de Cali que compartieron la entrada, con ello se aumentó el número de personas que disfrutaron de las competiciones; ***incluye corrección para evitar la duplicación de asistentes que presenciaron ambas ceremonias Fuente: Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali e información de Migración Colombia Notas: *Corresponde al número de espectadores corregido por la frecuencia de asistencia. Es decir, teniendo presente que un mismo espectador asiste a distintas competiciones a lo largo de los días de celebración de los Juegos; **incluye corrección por el número de residentes de Cali que compartieron la entrada, con ello se aumentó el número de personas que disfrutaron de las competiciones; ***incluye corrección para evitar la duplicación de asistentes que presenciaron ambas ceremonias Fuente: Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali e información de Migración Colombia Figura 3. Efectos de la realización de un megaevento deportivo 4.5 Estimación del gasto de los espectadores La estructura de gasto de los espectadores, en especial de aquellos que vienen de fuera de la ciudad atraídos por la celebración de los Juegos, depende de un conjunto de factores, entre ellos: la duración de la estancia en la ciudad y del número de competiciones a las que asista, del tipo de alojamiento y el sistema de transporte que utilicen en la ciudad. La duración de la estadía en la ciudad por parte de los turistas extranjeros es en promedio de 4,8 días y asisten en promedio a 4,4 competiciones en los diez días de duración de los Juegos (Figura 4). En el caso de los turistas nacionales la estancia media es de 4,1 días y una media de asistencia a 4,0 competiciones. La participación como asistente a eventos deportivos dinamiza la interacción social que se refleja en estilos de vida compartidos. En efecto, los turistas extranjeros asisten con un promedio de 4.5 acompañantes frente a 3,9 y 3,6 de los turistas nacionales y asistentes locales, respectivamente. El 58,3 per cent de los turistas extranjeros se hospeda en hoteles, cerca del 13 per cent en pensiones/hostales y el 23,3 per cent en casa de familiares/amigos (Figura 5). Por su parte, el 58,4 per cent de los turistas nacionales usó como principal forma de alojamiento la casa de familiares/amigos y el 30,6 per cent se aloja bien sea en hoteles o pensiones/hostales. Las anteriores cifras permiten ilustrar cómo cada subgrupo de espectadores tiene una estructura de gasto diferente. Para los efectos de la estimación tomamos como criterio de clasificación del gasto el lugar de residencia. La Tabla III muestra el gasto total por tipo de espectador, excluyendo el gasto en entradas, que fue suministrado por la empresa encargada de su venta ($4.137 mill. de pesos), lo que permite identificar las actividades económicas más beneficiadas por la “turbina de gasto” asociada a los Juegos. Este gasto constituye la inyección total de recursos a través del consumo que dinamiza los sectores vinculados al turismo como la alimentación, la hotelería, el transporte y el comercio local. De igual forma se beneficia el Estado a través de una mayor recaudación de impuestos derivados de las distintas transacciones. 4.3 Otros aspectos metodológicos clave Número de espectadores* que presenciaron los Juegos Mundiales 2013 Cali En resumen, los Juegos Mundiales 2013 Cali fueron vistos en vivo en los distintos escenarios deportivos por 103.673 personas (ver Figura 3), de las cuales 2.174 fueron turistas internacionales, 11.250 turistas nacionales y 90.249 residentes en Cali y AM. Asimismo, 50.540 espectadores presenciaron las ceremonias de inauguración y clausura. p p g y El perfil de los asistentes a los Juegos Mundiales 2013 Cali indica que se trata de un espectador de mediana edad, en su mayoría hombres, casados o en unión libre y con educación universitaria (ver Tabla II). En el caso de los turistas extranjeros, el rango de edad que más asistió fue entre los 30 y 39 años (29,6 per cent), el 64,8 per cent son hombres, el 51,7 per cent de estado civil con algún vínculo formal (casado/unión libre) y el 80 per cent cuenta con titulación universitaria. Asimismo se trata, en el caso del turista internacional, de un espectador especializado y con experiencia. En efecto, para el caso del 41,3 per cent de los turistas extranjeros su trabajo/estudio está vinculado con actividades deportivas y el 71,1 per cent ha asistido a eventos deportivos internacionales en los últimos tres años. Para los turistas nacionales y los residentes en Cali estos porcentajes caen fuertemente, indicando con ello, en parte, que se trata de espectadores más motivados por la novedad de las disciplinas deportivas y la experiencia de asistir a un evento deportivo de escala mundial. Por nivel de ingresos mensuales, la mayoría de los espectadores tiene ingresos de entre $1.200.000 [US $635] y $3.600.000 [US$1,904]. 139 Tabla II. Tabla II. Figure 5. Tipo de hospedaje de los asistentes a los Juegos Mundiales 2013 Cali 5. Impacto económico p El objetivo de este apartado es presentar los resultados de la estimación del impacto económico de los Juegos Mundiales sobre la economía de Cali y AM. El impacto Variables Turista Extranjero Turista Nacional Residente en Cali y área metropolitana Rango de Edad (años) 18-24 25-29 30-39 40-49 >50 18-24 25-29 30-39 40-49 >50 18-24 25-29 30-39 40-49 >50 15.2% 17.2% 29.6% 17.8% 20.2% 13.9% 13.1% 25.3% 22.0% 25.7% 12.5% 9.7% 26.3% 27.9% 23.6% Sexo Hombre 64.8 61.6 56.2 Mujer 35.2 38.4 43.8 Estado civil Soltero 44.1 44.1 37.0 Casado/unión libre 51.7 49.0 56.3 Viudo/divorciado 4.1 6.9 6.7 Nivel educativo Ninguno 0.2 0.0 0.4 Primaria 0.4 1.2 0.8 Secundaria 10.2 6.9 11.5 Técnico 9.1 11.8 12.7 Universitaria/Postgrado 80.0 80.0 74.6 Su trabajo/estudios están relacionados con la actividad deportiva Si 41.3 22.9 12.7 Ha asistidio a otras competiciones internacionales en los últimos 3 años Si 71.1 46.1 40.2 Actividad principal Estudiar 9.6 13.5 12.1 Trabajar 81.1 77.1 74.3 Oficios del hogar 0.7 4.1 6.5 Buscar trabajo 1.1 0.0 1.0 Jubilado 3.7 3.3 4.4 Otra 3.9 2.0 1.6 (continued) Tabla II. Perfil sociodemográfico de los asistentes a los Juegos Mundiales 2013 Cali JEFAS 22,43 140 140 Variables Turista Extranjero Turista Nacional Residente en Cali y área metropolitana Nivel de ingresos mensuales (pesos colombianos) <$600.000 10.3 8.2 11.4 $601.000-$1.200.000 15.7 15.5 17.7 $1.201.000-$2.400.000 19.0 23.7 23.3 $2.401.000-$3.600.000 19.7 21.6 16.8 $3.601.000-$4.800.000 16.2 13.5 11.0 $4.801.000-$6.000.000 7.9 8.6 8.9 >$6.001.000 11.4 9.0 11.0 Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali. Elaboración propia Tabla II. Efectos de la realización de un megaevento deportivo 141 Variables Turista Extranjero Turista Nacional Residente en Cali y área metropolitana Nivel de ingresos mensuales (pesos colombianos) <$600.000 10.3 8.2 11.4 $601.000-$1.200.000 15.7 15.5 17.7 $1.201.000-$2.400.000 19.0 23.7 23.3 $2.401.000-$3.600.000 19.7 21.6 16.8 $3.601.000-$4.800.000 16.2 13.5 11.0 $4.801.000-$6.000.000 7.9 8.6 8.9 >$6.001.000 11.4 9.0 11.0 Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali. Elaboración propia Efectos de la realización de un megaevento deportivo 141 141 económico se mide como la suma de tres efectos. En primer lugar, los efectos directos derivados del gasto/inversión en que incurrió el Comité Local Organizador de Cali para garantizar la celebración de los Juegos. 5. Impacto económico Tipo de hospedaje de los asistentes a los Juegos Mundiales 2013 Cali Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali p p Mundiales 2013 Cali  la inversión en construcción de nuevos escenarios deportivos y la adecuación y remodelación de los ya existentes;  la inversión en construcción de nuevos escenarios deportivos y la adecuación y remodelación de los ya existentes;  el gasto en preparación, gestión y logística. La inversión en construcción y adecuación de infraestructura deportiva para llevar a cabo los Juegos Mundiales fue un esfuerzo tripartito entre los gobiernos local, departamental y nacional. El esfuerzo del gobierno local corresponde al 44,2 per cent del total de la inversión (Tabla IV), el gobierno nacional apoyó con el 48 per cent y el gobierno departamental con cerca del 8 per cent. 143 Siguiendo la metodología adoptada en este estudio, para evitar cometer un error ampliamente documentado en los estudios de impacto económico, la inversión de la Alcaldía de Santiago de Cali en escenarios deportivos se excluye del efecto directo, dado que no puede ser considerada como una nueva inyección de recursos en el área de impacto de los Juegos. Esta constituye una transferencia de los mismos residentes locales en forma de gasto público local, frente a otros usos potenciales de estos recursos. Finalmente, como inyección de nuevos recursos financieros al área de impacto, en este caso Cali y AM, ingresaron $24.175 millones de pesos correspondientes al apoyo de los gobiernos nacional y departamental. p p p y g y p El gasto en operación y logística para la realización de los Juegos se muestra en el Tabla V. La información se presenta en las partidas que utiliza el Comité Olímpico Colombiano (COC). En total los recursos ascienden a $66.255 millones de pesos financiados en su totalidad por el gobierno nacional en cabeza de Coldeportes[15]. El gasto en operación y logística de acuerdo con la metodología adoptada, para tener en cuenta solo la inyección de recursos provenientes de fuera del área de impacto y que beneficien a los agentes locales (sectores productivos locales), requiere ajustes en tres partidas:  contratos internacionales, puesto que corresponden a equipos importados alquilados para los procesos de medición y cronometraje en las competencias; Tabla III. Efectos de la realización de un megaevento deportivo 5. Impacto económico En segundo lugar, los efectos indirectos, derivados del gasto que los espectadores de los Juegos realizan en la ciudad, en la medida en que son inherentes al hecho de su celebración. Por último, los efectos inducidos derivados de la dinámica que genera el impulso de las dos demandas anteriores, vía relaciones intersectoriales (compras a otros sectores) y de consumo (nuevos ingresos), sobre toda la economía local. 142 5.1 Efecto directo El efecto directo incluye todos los gastos/inversiones necesarios para poner en marcha los Juegos Mundiales. En este caso se consideran dos grandes fuentes de gasto/inversión por parte del Comité Local Organizador de Cali[14]: 5.1 Efecto directo El efecto directo incluye todos los gastos/inversiones necesarios para poner en marcha los Juegos Mundiales. En este caso se consideran dos grandes fuentes de gasto/inversión por parte del Comité Local Organizador de Cali[14]: 4.8 4.1 4.3 4.4 4.0 4.5 4.5 3.9 3.6 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Turista Extranjero Turista Nacional Residente en Cali y A.M. Número medio de acompañantes Número medio de competiciones vistas Número medio de días asistencia Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali 4.8 4.1 4.3 4.4 4.0 4.5 4.5 3.9 3.6 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Turista Extranjero Turista Nacional Residente en Cali y A.M. Número medio de acompañantes Número medio de competiciones vistas Número medio de días asistencia Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali Figure 4. Duración media de la estancia, número medio de competiciones y acompañantes de los asistentes a los Juegos Mundiales 2013 Cali Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali 58.3% 22.0% 12.8% 8.6% 23.3% 58.4% 5.7% 11.0% 0% 10% 20% 30% 40% 50% 60% 70% Turista Extranjero Turista Nacional Otro En casa de familiar/amigo Pensión/Hostal Hotel Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali Turista Nacional Turista Extranjero Figure 5. 5. Impacto económico Gasto total de los espectadores [cifras en pesos 2013] Tipo de Consumo Turista Extranjero Turista Nacional Residente Cali y A.M. Total Alojamiento $ 1,248,745,461 $ 3,154,258,391 $ 0 $ 4,403,003,852 14.3% Alimentación $ 713,253,218 $ 1,900,533,906 $ 7,738,784,649 $ 10,352,571,773 33.6% Transporte Local $ 534,951,751 $ 1,252,874,373 $ 5,101,579,580 $ 6,889,405,703 22.4% Compras/Recuerdos $ 574,170,087 $ 1,015,911,937 $ 4,136,692,156 $ 5,726,774,180 18.6% Ocio/Diversión $ 852,810,232 $ 509,430,261 $ 2,074,349,251 $ 3,436,589,745 11.2% Total $ 3,923,930,749 $ 7,833,008,867 $ 19,051,405,636 $ 30,808,345,252 100% Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali Elaboración propia sobre la base de la Encuesta Asistentes a los Juegos Mundiales 2013 Cali Tabla IV. Inversión en infraestructura deportiva (cifras en pesos de 2013) Entidad Obra civil Interventoria Total (%) Alcaldía Santiago de Cali $ 18,163,169,691 $ 955,956,300 $ 19,119,125,990 44.2 Gobernación del Valle del Cauca $ 3,230,000,000 $ 170,000,000 $ 3,400,000,000 7.9 Gobierno Nacional [Coldeportes] $ 19,735,863,587 $ 1,038,729,662 $ 20,774,593,249 48.0 Total $ 41,129,033,277 $ 2,164,685,962 $ 43,293,719,239 100 Fuente: Comité Local Organizador de Cali - Secretaria del Deporte, Alcaldía de Santiago de Cali Tabla V. Gasto en gestión, logística y organización (cifras en pesos de 2013) Concepto Valor (%) Implementación $ 11.962.898.607 18.1 Contratos internacionales $ 8.376.422.862 12.6 Tecnología $ 5.862.516.211 8.8 Hoteles $ 5.422.557.519 8.2 Gastos administrativos $ 4.573.791.131 6.9 Alimentación $ 4.461.985.231 6.7 Promoción $ 3.866.146.939 5.8 Recurso humano $ 3.820.796.721 5.8 Montaje $ 3.329.738.754 5.0 Manejo administrativo $ 3.286.534.087 5.0 Transporte $ 2.594.802.981 3.9 Plan de medios $ 2.025.572.397 3.1 Inauguración/clausura $ 2.000.000.000 3.0 Otras \bacute\aéreas logísticas $ 1.892.106.901 2.9 Protocolo $ 672.045.813 1.0 Voluntariado $ 551.180.928 0.8 Deportes $ 541.774.066 0.8 Dopaje $ 430.857.684 0.7 Aérea médica $ 363.569.969 0.5 Medio ambiente $ 219.828.700 0.3 Total $ 66.255.127.503 100 Fuente: Comité Local Organizador de Cali JEFAS 22,43 144  tecnología y servicios de transmisión de televisión, en ambos casos solo se incluye el gasto realizado en el área de impacto (gastos locales por parte de los técnicos), y  tecnología y servicios de transmisión de televisión, en ambos casos solo se incluye el gasto realizado en el área de impacto (gastos locales por parte de los técnicos), y  manejo administrativo, en lo correspondiente a la devolución de los derechos de inscripción del equipo de Gimnasia de Israel que no se presentó. 5. Impacto económico  manejo administrativo, en lo correspondiente a la devolución de los derechos de inscripción del equipo de Gimnasia de Israel que no se presentó. Asimismo, se excluyeron los convenios —canje— con el Masivo Integrado de Occidente – MIO– para el transporte de los voluntarios y con EMCALI para suministro de servicios públicos y comunicaciones. Asimismo, se excluyeron los convenios —canje— con el Masivo Integrado de Occidente – MIO– para el transporte de los voluntarios y con EMCALI para suministro de servicios públicos y comunicaciones. Una vez realizados los ajustes, como inyección de nuevos recursos financieros al área de impacto ingresaron $57.445 millones de pesos. En las Tablas 8, 9 y 10 se presenta la información homologada con los sectores de la Matriz insumo-producto (MIP) para el Valle del Cauca. En resumen, el efecto directo asociado a la celebración de los Juegos Mundiales 2013 Cali significó una inyección de $81.620 millones de pesos a la economía de Cali y AM, representados por $24.175 millones de pesos de inversión en infraestructura deportiva y $57.445 millones de pesos en gastos de operación, gestión y logística[16]. 5.2 Efecto indirecto i i d l La estimación del número de espectadores efectuada en el apartado 4.5. se refina al incluir residentes locales que no se desplazaron a otro lugar debido precisamente a la celebración de los Juegos (ver la Figura 6). Se trata de una equivalencia entre individuos que decidieron quedarse en su casa y asistir a los eventos programados en lugar de desplazarse de vacaciones a otro lugar, producto de una elección racional. El cálculo se ha realizado con ayuda de la pregunta consignada en la Encuesta a Asistentes sobre si ha asistido a otras competiciones internacionales en los últimos tres años: Figure 6. Espectadores de fuera del área metropolitana de Santiago de Cali Atracción de visitantes de fuera de Cali y área metropolitana Turistas extranjeros 11.250 Turistas nacionales 2.174 36.280 Residentes locales Gasto en: entradas, alojamiento, alimentación, transporte local, compras/recuerdos, ocio/diversión Impacto en la producción de bienes y servicios y sobre el empleo en Cali y AM Elaboración propia Efectos de la realización de un megaevento deportivo 145 Efectos de la realización de un megaevento deportivo Atracción de visitantes de fuera de Cali y área metropolitana Gasto en: entradas, alojamiento, alimentación, transporte local, / d i /di ió Figure 6. Espectadores de fuera del área metropolitana de Santiago de Cali Figure 6. Espectadores de fuera del área metropolitana de Santiago de Cali Impacto en la producción de bienes y servicios y sobre el empleo en Cali y AM Elaboración propia Elaboración propia  El supuesto subyacente indica que quienes responden afirmativamente siguen la actividad deportiva, disponen de los recursos financieros para desplazarse pero deciden quedarse en la ciudad para seguir las competiciones del deporte de su interés y/o por la novedad que implican los Juegos Mundiales.  El supuesto subyacente indica que quienes responden afirmativamente siguen la actividad deportiva, disponen de los recursos financieros para desplazarse pero deciden quedarse en la ciudad para seguir las competiciones del deporte de su interés y/o por la novedad que implican los Juegos Mundiales.  Adicionalmente, el 97 per cent de los espectadores residentes en Cali y AM considera que la celebración de los Juegos Mundiales 2013 Cali generó un impacto positivo sobre el “orgullo y sentido de pertenencia de los caleños por su ciudad”. Figure 6. Espectadores de fuera del área metropolitana de Santiago de Cali 5.2 Efecto indirecto i i d l Tal como lo ha puesto de manifiesto Zimbalist (2010) para los residentes locales este tipo de celebraciones es un momento “corto pero intenso” a través del cual “la atención del mundo se centrará en sus hogares” lo que a corto plazo genera “una gran sensación de logro”.  Adicionalmente, el 97 per cent de los espectadores residentes en Cali y AM considera que la celebración de los Juegos Mundiales 2013 Cali generó un impacto positivo sobre el “orgullo y sentido de pertenencia de los caleños por su ciudad”. Tal como lo ha puesto de manifiesto Zimbalist (2010) para los residentes locales este tipo de celebraciones es un momento “corto pero intenso” a través del cual “la atención del mundo se centrará en sus hogares” lo que a corto plazo genera “una gran sensación de logro”. La Tabla VI muestra el efecto indirecto asociado a la inyección de gasto adicional en Cali y AM derivado de los espectadores de los Juegos Mundiales. Un total de $19.416 millones de pesos que resultan de la suma del gasto de los turistas extranjeros y nacionales, y el gasto de los residentes locales que se quedaron en la ciudad para presenciar las competiciones. Tabla VI. Estimación del efecto indirecto (cifras en pesos de 2013) Tipo de espectadores Número de espectadores Gasto medio por persona Gasto total Turistas extranjeros 2.174 $ 1.805.237 $ 3.924.586.128.93 Turistas nacionales 11.250 $ 696.242 $ 7.832.724.858.48 Residentes locales 36.280 $ 211.099 $ 7.658.665.065.52 Elaboración propia 5.3 Efecto total sobre la producción Una vez descritos y estimados los efectos directos e indirectos, resulta clave analizar cómo estas fuentes de demanda inicial se irrigan hacia los distintos sectores productivos para conocer y determinar el efecto o impacto total sobre el sistema económico local. Una parte de esa demanda directa que perciben las empresas locales constituye fuente de nueva demanda en forma de compras a otras empresas e individuos, que les proveen los insumos necesarios para realizar su producción (efecto indirecto), este aumento de la producción a su vez genera nuevos ingresos, que inducen nuevas rondas de gasto y aumento de la producción y así sucesivamente (efecto inducido), hasta llegar al efecto total sobre el sistema económico. JEFAS 22,43 5.2 Efecto indirecto i i d l 146 El resultado generado en la simulación, al introducir el cambio en la demanda final por $101.036 millones de pesos derivados de las dos fuentes de demanda identificadas: $81.620 millones de pesos (infraestructura y logística) más $19.416 millones de pesos (gasto de espectadores) generó un impacto total acumulado de $569.493 millones de pesos sobre la economía de la ciudad de Cali y AM medido a través del valor bruto de la producción y utilizando los multiplicadores de producción tipo II (ver Tabla VII). Como puede apreciarse en la Tabla VII, el impacto sobre la producción recae sobre tres sectores (72,9 per cent) donde se concentra el gasto en logística de los Juegos (servicios a las empresas, 28,01 per cent), la inversión en infraestructura deportiva (construcción, 23,38 per cent) y la “turbina de gasto” de los espectadores (hostelería, 21,55 per cent). s Sector Descripción Inyección de nuevos recursos [Mill. $] Impacto sobre la producción Mill. $ (%) Servicios a las empresas [p.e. gastos en gestión del CLOC; montaje y adecuación de escenarios; interventoria obras civiles; alquiler de equipos de medición y cronometraje; transport e de equipos; servicios de vigilancia] $27.803 $159.497 28.01 Construcción y reparación de edificaciones [p.e. inversión en infraestructura de escenarios] $22.966 $133.122 23.38 Alojamiento, suministro de comidas y bebidas [p.e. gasto de los espectadores en alojamiento y alimentación] $20.110 $122.739 21.55 Otra industria manufacturera [p.e. gasto en uniformes de voluntarios] $11.748 $46.899 8.24 Servicios de transporte [p.e. gasto en transporte local de los turistas y delegaciones] $6.563 $38.807 6.81 Servicios de esparcimiento [p.e. gasto de los espectadores en ocio/diversión, boletería] $4.249 $25.839 4.54 Comercio y servicios de reparación [p.e. gasto de los espectadores en compras de recuerdos] $3.253 $19.536 3.43 Servicios financieros e inmobiliarios [p.e. gasto en arrendamiento sedes, bodegas] $1.808 $10.873 1.91 Correo y telecomuncaciones [p.e. gasto en promoción y medios de comunicación] $1.115 $6.755 1.19 Maquinaria y equipo de transporte [p.e. gasto en compra PC, impresoras, celulares, etc.] $1.421 $5.428 0.95 Total $101.036 $569.493 100 Elaboración propia Sector Tabla VIII. Impacto sobre el empleo por sectores económicos Número de empleos Sector Descripción Ocupados Equivalente de tiempo completo Comercio y servicios de reparación [p.e. gasto de los espectadores en compras de recuerdos] 3.085 2.479 32.14% Servicios de transporte [p.e. gasto en transporte local de los turistas y delegaciones] 1.355 1.089 14.12% Producción agropecuaria, silvicultura y pesca [p.e. demanda derivada] 935 751 9.74% Alojamiento, suministro de comidas y bebidas [p.e. gasto de los espectadores en alojamiento y alimentación] 772 620 8.04% Construcción y reparación de edificaciones [p.e. gastos en infraestructura de escenarios] 636 511 6.63% Servicios de enseñanza y salud de mercado [p.e. gasto de logistica en el \bacute \aérea médica y manejo de dopaje] 558 448 5.82% Servicios de esparcimiento [p.e. gasto de los espectadores en ocio/ diversión, boletería] 482 387 5.02% Servicios a las empresas [p.e. gastos en gestión del CLOC; montaje y adecuación de escenarios; interventoria obras civiles; alquiler de equipos de medición y cronometraje; transporte de equipos; servicios de vigilancia] 481 387 5.01% Otra industria manufacturera [p.e. gasto en uniformes de voluntarios] 383 307 3.99% Servicios del gobierno [p. ej. demanda derivada] 240 193 2.50% Correo y telecomuncaciones [p.e. gasto en promoción y medios de comunicación] 234 188 2.44% Industria alimenticia [p. ej. demanda derivada] 200 161 2.09% Servicios financieros e inmobiliarios [p.e. gasto en arrendamiento sedes, bodegas] 143 115 1.49% Maquinaria y equipo de transporte [p.e. gasto en compra PC, impresoras, celulares, etc.] 42 34 0.44% Trabajos de construcción, construcción de obras civiles y servicios de arrendamiento de equipo con operario [p.e. demanda derivada] 28 22 0.29% Electricidad, gas, agua y saneamiento b\bacute\aésico [p.e. demanda derivada] 23 19 0.24% Productos de minería [p.e. demanda derivada] 1 1 0.01% Total 9.598 7.711 100% Elaboración propia Efectos de la realización de un megaevento deportivo 147 Elaboración propia 5.4 Efecto sobre el empleo 5.4 Efecto sobre el empleo En cuanto al empleo, el cambio en la demanda final por $101.036 millones de pesos creó un total de 9.598 puestos de trabajo (ocupados), equivalentes a 7.711 empleos de tiempo completo (ver Figura 7)[17]. De estos últimos, 1.775 son causa directa del impulso inicial de demanda (empleos directos), 1.013 son empleos indirectos, derivados de los incrementos en la demanda de otros sectores productivos y 4.923 inducidos asociados a los nuevos efectos sobre los propios proveedores vía ingresos y consumo. La Tabla VIII muestra el empleo por sectores económicos. Figure 7. Impacto agregado sobre el empleo 6. El legado de los Juegos Mundiales para Cali D d R F hli h id d I 6. El legado de los Juegos Mundiales para Cali De acuerdo con Ron Froehlich, presidente de International World Games Association (IWGA) los pasados Juegos Mundiales 2013 Cali son “la mejor de las nueve versiones de los Juegos, por eso la medalla de oro es para Cali y Colombia[18]”. Este éxito inicial de los Juegos Mundiales 2013 Cali no garantiza que en el largo plazo los efectos positivos se mantengan. Lo que podemos denominar “el legado de los Juegos”, depende de un conjunto complejo de factores e interacciones que no son capturados por las relaciones monetarias entre agentes y sectores que se describen en la Figura 4. Con el objetivo de que perdure la ventaja competitiva generada por la realización del megaevento deportivo, se requiere de una adecuada gestión por parte de los actores clave (sector público —local, regional, nacional—, sector deportivo, las empresas privadas y la comunidad) que mitigue los riesgos identificados (ver la Tabla IX). ( ) La Figura 8 propone una interacción estratégica entre los actores clave (stakeholders) que pueden dinamizar “el legado de los Juegos”. Los actores clave arriba identificados a través de una adecuada interacción y adopción de un conjunto de acciones son quienes garantizan el sostenimiento en el largo plazo de los impactos positivos de los Juegos. En la Figura 4 se presentan en forma sucinta algunas de las acciones relevantes, que implican algo más allá de una actitud de colaboración entre los actores locales clave, significa que realmente se comparten experiencias, recursos y capacidades para adquirir, desarrollar y consolidar ventajas competitivas en la organización de megaeventos deportivos dentro de una visión estratégica global de gestión del territorio; al respecto, ver Radicchi (2012, pp. 38-39). Sector Los sectores que más empleo generaron, en términos relativos, fueron los vinculados con la logística de la organización de los Juegos y la “turbina de gasto” de los turistas. Es así como en el sector comercio y servicios de reparación se generaron 2.479 empleos equivalentes de tiempo completo, seguidos del sector de servicios de transporte con 1.089, el sector de producción agropecuaria, silvicultura y pesca con 751 y el sector de alojamiento, suministro de comidas y bebidas con 620. 148 6. El legado de los Juegos Mundiales para Cali JEFAS 22,43 RIESGOS Capital Físico/Infraestructura: infraestructura física para la realización de eventos deportivos de escala mundial Los escenarios deportivos se deterioran físicamente; las adecuaciones y remodelaciones sin mantenimiento y actualización permanente se vuelven obsoletas. Adem\bacute\aés de que representan una carga para el presupuesto público local/nacional 149 Capital humano/social: profesionales capacitados en áreas relacionadas con la realización de grandes eventos deportivos. Beneficios sociales del Programa de Voluntarios*. Beneficios sociales de la nueva infraestructura deportiva y la diversidad en la práctica deportiva que posibilitan** “Marca de Ciudad”: mejora en la percepción y posicionamiento de la ciudad en el contexto nacional e internacional como ciudad “deportiva, moderna y competitiva” Capital humano/social: profesionales capacitados en áreas relacionadas con la realización de grandes eventos deportivos. Beneficios sociales del Programa de Voluntarios*. Beneficios sociales de la nueva infraestructura deportiva y la diversidad en la práctica deportiva que posibilitan** La experiencia de los profesionales vinculados con la organización y logística, asimismo de los voluntarios, se puede subutilizar en la medida en que no puedan aplicar su experiencia con la organización y oferta de otros eventos de gran escala “Marca de Ciudad”: mejora en la percepción y posicionamiento de la ciudad en el contexto nacional e internacional como ciudad “deportiva, moderna y competitiva” Sin promoción y apoyo a la práctica del deporte recreativo y competitivo el uso de las instalaciones deportivas se puede desviar para otros fines. Sin que ello implique que la nueva infraestructura deportiva no se incorpore dentro del equipamiento cultural y social de la ciudad para el uso y disfrute del tiempo libre de los residentes y visitantes de Cali Sin una gestión adecuada de la Marca de Ciudad se pueden perder los beneficios positivos asociados con la buena imagen para la ciudad que deja el éxito deportivo y de asistencia a los Juegos Mundiales como potencial destino turístico de futuros visitantes Intangible: Aumento del sentido de identidad, pertenencia, autoestima y cohesión social Notas: *La convocatoria para voluntarios recibió la inscripción de 5.872 personas, se capacitaron 5.366 y finalmente se seleccionaron 2.539; **Por ejemplo, como legado de los Juegos Mundiales quedan nuevos deportes incluidos en el sistema nacional, entre ellos: batalla de fuerza; baile deportivo y escalada. Elaboración propia conocimiento de los autores, este sería el primer estudio de esta naturaleza aplicado a un megaevento deportivo en Colombia. Tabla IX. El legado de los Juegos Mundiales 2013 Cali 7. Conclusiones l En este artículo se estimó el impacto económico a corto plazo asociado a la celebración de la IX edición de los Juegos Mundiales sobre la ciudad de Cali y su área metropolitana. En Figure 7. Impacto agregado sobre el empleo 2,209 1,261 6,128 9,598 1,775 1,013 4,923 7,711 Directo Indirecto Inducido Total Directo Indirecto Inducido Total Ocupados Empleo Tiempo Completo Elaboración propia 2,209 1,261 6,128 9,598 1,775 1,013 4,923 7,711 Directo Indirecto Inducido Total Directo Indirecto Inducido Total Ocupados Empleo Tiempo Completo Elaboración propia Tabla IX. El legado de los Juegos Mundiales 2013 Cali RIESGOS Los escenarios deportivos se deterioran físicamente; las adecuaciones y remodelaciones sin mantenimiento y actualización permanente se vuelven obsoletas. Adem\bacute\aés de que representan una carga para el presupuesto público local/nacional La experiencia de los profesionales vinculados con la organización y logística, asimismo de los voluntarios, se puede subutilizar en la medida en que no puedan aplicar su experiencia con la organización y oferta de otros eventos de gran escala Sin promoción y apoyo a la práctica del deporte recreativo y competitivo el uso de las instalaciones deportivas se puede desviar para otros fines. Sin que ello implique que la nueva infraestructura deportiva no se incorpore dentro del equipamiento cultural y social de la ciudad para el uso y disfrute del tiempo libre de los residentes y visitantes de Cali Sin una gestión adecuada de la Marca de Ciudad se pueden perder los beneficios positivos asociados con la buena imagen para la ciudad que deja el éxito deportivo y de asistencia a los Juegos Mundiales como potencial destino turístico de futuros visitantes ripción de 5.872 personas, se capacitaron 5.366 y legado de los Juegos Mundiales quedan nuevos batalla de fuerza; baile deportivo y escalada. Efectos de la realización de un megaevento deportivo 149 Efectos de la realización de un megaevento deportivo RIESGOS En resumen, la celebración de los Juegos Mundiales significó para la ciudad de Cali:  En lo económico, la movilización de recursos que dinamizó diversos sectores de la economía local: 2.174 turistas internacionales, 11.250 turistas nacionales, 9.598 puestos de trabajo equivalentes a 7.711 empleos de tiempo completo.  En lo deportivo, una oportunidad para continuar consolidando y explotando las ventajas competitivas que ha adquirido en la realización de eventos deportivos de escala mundial.  En lo deportivo, una oportunidad para continuar consolidando y explotando las ventajas competitivas que ha adquirido en la realización de eventos deportivos de escala mundial.  En lo social, exaltar el sentido de pertenencia y el orgullo de los residentes locales por su ciudad, a través de la celebración de un megaevento deportivo excepcional, inédito en América Latina. Noventa y siete de cada 100 espectadores residentes en Cali y AM consideraron que la celebración de los Juegos Mundiales tuvo un efecto positivo sobre el “orgullo y sentido de pertenencia de los caleños por su ciudad”. Figure 8. Actores y acciones para sostener “el legado de los Juegos” JEFAS 22,43 150 150 150 Figure 8. Actores y acciones para sostener “el legado de los Juegos” Efectos de la realización de un megaevento deportivo Figure 8. El principal aporte del artículo se centra en el desarrollo de algunos puntos que resultan metodológicamente útiles en la elaboración de estudios de impacto económico en ciudades de países en desarrollo. En efecto, en el cálculo del flujo de espectadores (nacionales y extranjeros) se refina la estimación teniendo presente: El principal aporte del artículo se centra en el desarrollo de algunos puntos que resultan metodológicamente útiles en la elaboración de estudios de impacto económico en ciudades de países en desarrollo. En efecto, en el cálculo del flujo de espectadores (nacionales y extranjeros) se refina la estimación teniendo presente: El principal aporte del artículo se centra en el desarrollo de algunos puntos que resultan metodológicamente útiles en la elaboración de estudios de impacto económico en ciudades de países en desarrollo. En efecto, en el cálculo del flujo de espectadores (nacionales y extranjeros) se refina la estimación teniendo presente:  El cálculo directo vía administrativa de los turistas extranjeros, a través de un acuerdo con la autoridad migratoria colombiana, lo que implica un cálculo preciso del grupo de interés.  Se corrige el posible “efecto expulsión” y de “cancelación de reservas”.  Se incluyeron aquellos espectadores locales, residentes en el área de impacto, que ante el costo de oportunidad de salir de la ciudad (fuga de gasto) optó por quedarse y presenciar los Juegos.  Se corrige el número de espectadores al incluir el fenómeno observado de rotación de entradas entre familiares/amigos de la población residente en Santiago de Cali. Por último, desde una perspectiva de largo plazo, el principal legado de los Juegos se dirige a consolidar las ventajas competitivas que ha adquirido la ciudad en la organización de megaeventos deportivos. En el marco de una visión estratégica global de gestión del territorio, resulta necesaria una fluida interrelación entre los actores locales clave (sector público, sector privado, sector deportivo y comunidad). Notas El aporte del gobierno de Cali se estima en un 44,2 per cent para la infraestructura construida. 7. La Unidad Administrativa Especial Migración Colombia es la entidad del Estado Colombiano encargada de “ejercer control como autoridad migratoria a ciudadanos nacionales y extranjeros en el territorio colombiano”, ver www.migracioncolombia.gov.co/index.php/entidad/la–unidad/ mision–vision.html 8. La elaboración de una MIP para Cali y AM constituiría un esfuerzo que sobrepasa los alcances de este artículo por los altos requerimientos de información y tiempo necesarios para su construcción, y revela la falta de una contabilidad regional y local en Colombia. La utilización de la MIP actualizada para el Valle del Cauca constituye la mejor aproximación disponible para el cálculo de los multiplicadores, puesto que la estructura económica de Cali refleja la estructura económica del Valle del Cauca. Lo anterior se observa en el peso relativo de Cali y AM en el PIB del Valle del Cauca, que asciende al 67,9 per cent (DANE, 2013). 9. Los escenarios se utilizaron en forma simultánea, aunque no todos los días. Por ejemplo, el Estadio Olímpico Pascual Guerrero se utilizó del 28 al 30 de julio para las competencias de disco volador y del 1 al 2 de agosto para rugby 7. 10. Corresponde a las tribunas habilitadas para presenciar las competiciones y no a la capacidad total del estadio. 11. El sistema de encuesta elegido fue la entrevista personal cara a cara. Este sistema tiene sus ventajas; con un entrevistador entrenado, una vez que se aborda al entrevistado y este acepta la realización de la misma, es difícil que la encuesta no sea válida ya que el número de preguntas contestadas es prácticamente del 100 per cent. No obstante, tiene el inconveniente de realizarse en un momento próximo al disfrute del evento por parte del entrevistado, lo que la hace difícil y costosa. 12. El diseño muestral siguió el procedimiento sugerido por Levy and Lameshow (1991) aceptando un error muestral del 5 per cent y un nivel de confianza del 95 per cent. Las 1.299 encuestas constituyen una muestra representativa del total de espectadores de los Juegos Mundiales 2013 Cali, estratificada según el origen (turistas extranjeros, turistas nacionales y residentes en Cali y AM). 12. El diseño muestral siguió el procedimiento sugerido por Levy and Lameshow (1991) aceptando un error muestral del 5 per cent y un nivel de confianza del 95 per cent. Notas 1. La ciudad de Santiago de Cali tiene amplia tradición en la celebración de eventos deportivos de gran escala, por citar algunos: VI Juegos Panamericanos en 1971, I Juegos del Océano Pacífico en 1995, Campeonato del Mundo de Patinaje de Velocidad en 2007, Copa Mundo de Ciclismo de Pista en 2008, XVIII Campeonato Mundial de Futbol Sub-20 en 2011 (sede grupo B). 1. La ciudad de Santiago de Cali tiene amplia tradición en la celebración de eventos deportivos de gran escala, por citar algunos: VI Juegos Panamericanos en 1971, I Juegos del Océano Pacífico en 1995, Campeonato del Mundo de Patinaje de Velocidad en 2007, Copa Mundo de Ciclismo de Pista en 2008, XVIII Campeonato Mundial de Futbol Sub-20 en 2011 (sede grupo B). 2. Los primeros Juegos Mundiales se realizaron en 1981 en la ciudad de Santa Clara (Estados Unidos). Desde entonces ocho ciudades han sido sede: Londres (Reino Unido), Karlsruhe (Alemania), La Haya (Países Bajos), Lahti (Finlandia), Akita (Japón), Duisbur (Alemania) y Kaohsiung (China). La próxima edición en 2017 se llevará a cabo en la ciudad de Wroclaw (Polonia). 151 3. Adicionalmente, cinco disciplinas deportivas participaron como invitadas. 4. Tasa de cambio representativa del mercado (TCRM) $/US$1,890.33, fin de mes (julio de 2013). Fuente: Banco de la República/Superintendencia Financiera de Colombia. (www.banrep.gov.co/ es/series–estadisticas/see_ts_trm.htm). 4. Tasa de cambio representativa del mercado (TCRM) $/US$1,890.33, fin de mes (julio de 2013). Fuente: Banco de la República/Superintendencia Financiera de Colombia. (www.banrep.gov.co/ es/series–estadisticas/see_ts_trm.htm). 5. Por sus siglas en inglés: International World Games Association, IWGA (www.theworldgames. org/). 5. Por sus siglas en inglés: International World Games Association, IWGA (www.theworldgames. org/). 5. Por sus siglas en inglés: International World Games Association, IWGA (www.theworldgames org/). 6. La estrategia de financiación de los Juegos Mundiales implicó la construcción de cinco nuevos escenarios en terrenos del municipio y/o públicos; los demás escenarios fueron adaptados y remodelados para las competiciones. La financiación recayó sobre el gobierno nacional (Coldeportes) tanto para infraestructura como para la logística. El aporte del gobierno de Cali se estima en un 44,2 per cent para la infraestructura construida. 6. La estrategia de financiación de los Juegos Mundiales implicó la construcción de cinco nuevos escenarios en terrenos del municipio y/o públicos; los demás escenarios fueron adaptados y remodelados para las competiciones. La financiación recayó sobre el gobierno nacional (Coldeportes) tanto para infraestructura como para la logística. Notas De acuerdo con el DANE, los empleos (u ocupados) hacen referencia al contrato explícito o implícito entre una persona y una unidad institucional para realizar un trabajo a cambio de remuneración, durante un período definido o hasta nuevo aviso, y se dividen en dos grandes grupos: los asalariados y los independientes. Estos trabajos pueden ser de tiempo completo, medio tiempo o empleos ocasionales. 17. Los empleos de tiempo completo equivalente se calculan como el total de horas trabajadas dividido el promedio anual de horas trabajadas en puestos de trabajo considerados de tiempo completo. De acuerdo con el DANE, los empleos (u ocupados) hacen referencia al contrato explícito o implícito entre una persona y una unidad institucional para realizar un trabajo a cambio de remuneración, durante un período definido o hasta nuevo aviso, y se dividen en dos grandes grupos: los asalariados y los independientes. Estos trabajos pueden ser de tiempo completo, medio tiempo o empleos ocasionales. 17. Los empleos de tiempo completo equivalente se calculan como el total de horas trabajadas dividido el promedio anual de horas trabajadas en puestos de trabajo considerados de tiempo completo. De acuerdo con el DANE, los empleos (u ocupados) hacen referencia al contrato explícito o implícito entre una persona y una unidad institucional para realizar un trabajo a cambio de remuneración, durante un período definido o hasta nuevo aviso, y se dividen en dos grandes grupos: los asalariados y los independientes. Estos trabajos pueden ser de tiempo completo, medio tiempo o empleos ocasionales. 18. Ver: www.ntn24.com/noticias/italia–se–consagra–campeon–de–los–juegos–mundiales–en–la– ciudad–colombiana–de–cali–101438 18. Ver: www.ntn24.com/noticias/italia–se–consagra–campeon–de–los–juegos–mundiales–en–la– ciudad–colombiana–de–cali–101438 Notas Las 1.299 encuestas constituyen una muestra representativa del total de espectadores de los Juegos Mundiales 2013 Cali, estratificada según el origen (turistas extranjeros, turistas nacionales y residentes en Cali y AM). 13. Por razones de deficiente infraestructura de transporte terrestre, ferroviario y fluvial en el país y la topografía del territorio se estima como mínima y no significativa la llegada de turistas extranjeros por otra vía distinta del trasporte aéreo. 14. Las diferentes partidas de la inversión en infraestructura, los gastos de organización y logística y el gasto de los espectadores se homologa en los sectores económicos en los que se desagrega la matriz insumo producto del Valle del Cauca, utilizada en la simulación. Se emplean deflactores implícitos calculados a partir de las Cuentas Departamentales y de las Cuentas Trimestrales del DANE. 15. El Departamento Administrativo del Deporte, la Recreación, la Actividad Física y el Aprovechamiento del Tiempo Libre –Coldeportes– es el ente oficial del Estado colombiano “encargado de formular, coordinar y vigilar la práctica del deporte, la recreación, la educación física, el aprovechamiento del tiempo libre y la actividad física, orientados al mejoramiento de la calidad de vida de la sociedad colombiana”, ver [www.coldeportes.gov.co/]. 16. En este total se incluye el ingreso por venta de entradas que de acuerdo con la contabilidad del CLOC se incorporó en el presupuesto general de los Juegos. 17. Los empleos de tiempo completo equivalente se calculan como el total de horas trabajadas dividido el promedio anual de horas trabajadas en puestos de trabajo considerados de tiempo completo. De acuerdo con el DANE, los empleos (u ocupados) hacen referencia al contrato explícito o implícito entre una persona y una unidad institucional para realizar un trabajo a cambio de remuneración, durante un período definido o hasta nuevo aviso, y se dividen en dos grandes grupos: los asalariados y los independientes. Estos trabajos pueden ser de tiempo completo, medio tiempo o empleos ocasionales. 18. Ver: www.ntn24.com/noticias/italia–se–consagra–campeon–de–los–juegos–mundiales–en–la– ciudad–colombiana–de–cali–101438 JEFAS 22,43 152 13. Por razones de deficiente infraestructura de transporte terrestre, ferroviario y fluvial en el país y la topografía del territorio se estima como mínima y no significativa la llegada de turistas extranjeros por otra vía distinta del trasporte aéreo. 13. Notas Por razones de deficiente infraestructura de transporte terrestre, ferroviario y fluvial en el país y la topografía del territorio se estima como mínima y no significativa la llegada de turistas extranjeros por otra vía distinta del trasporte aéreo. 14. Las diferentes partidas de la inversión en infraestructura, los gastos de organización y logística y el gasto de los espectadores se homologa en los sectores económicos en los que se desagrega la matriz insumo producto del Valle del Cauca, utilizada en la simulación. Se emplean deflactores implícitos calculados a partir de las Cuentas Departamentales y de las Cuentas Trimestrales del DANE. 14. Las diferentes partidas de la inversión en infraestructura, los gastos de organización y logística y el gasto de los espectadores se homologa en los sectores económicos en los que se desagrega la matriz insumo producto del Valle del Cauca, utilizada en la simulación. Se emplean deflactores implícitos calculados a partir de las Cuentas Departamentales y de las Cuentas Trimestrales del DANE. 152 15. El Departamento Administrativo del Deporte, la Recreación, la Actividad Física y el Aprovechamiento del Tiempo Libre –Coldeportes– es el ente oficial del Estado colombiano “encargado de formular, coordinar y vigilar la práctica del deporte, la recreación, la educación física, el aprovechamiento del tiempo libre y la actividad física, orientados al mejoramiento de la calidad de vida de la sociedad colombiana”, ver [www.coldeportes.gov.co/]. 15. El Departamento Administrativo del Deporte, la Recreación, la Actividad Física y el Aprovechamiento del Tiempo Libre –Coldeportes– es el ente oficial del Estado colombiano “encargado de formular, coordinar y vigilar la práctica del deporte, la recreación, la educación física, el aprovechamiento del tiempo libre y la actividad física, orientados al mejoramiento de la calidad de vida de la sociedad colombiana”, ver [www.coldeportes.gov.co/]. 16. En este total se incluye el ingreso por venta de entradas que de acuerdo con la contabilidad del CLOC se incorporó en el presupuesto general de los Juegos. 16. En este total se incluye el ingreso por venta de entradas que de acuerdo con la contabilidad del CLOC se incorporó en el presupuesto general de los Juegos. 17. Los empleos de tiempo completo equivalente se calculan como el total de horas trabajadas dividido el promedio anual de horas trabajadas en puestos de trabajo considerados de tiempo completo. Referencias Ávalos, A. (2011), “Estimación del impacto económico de los Juegos Panamericanos Guadalajara 2011: análisis de insumo-producto”, Revista Econoquantum, Vol. 8 No. 1, pp. 35-60. aade, R. y Matheson, V. (2016), “Going for the gold: the economics of the Olympics”, Journal of Economic Perspectives, Vol. 30 No. 2, pp. 201-218. arajas, A., Salgado, J. y Sánchez, P. (2012), “Problemática de los estudios de impacto económico de eventos deportivos”, Estudios De Economía Aplicada, Vol. 30, pp. 441-462. arget, E. y Gouguet, J. (2008), “Un análisis de la copa del mundo de rugby 2007 en Francia”, RAE: Revista Asturiana De Economía, Vol. 42, pp. 103-127. DANE (2013), Metodología Para Calcular el Indicador de Importancia Económica Municipal, DANE, Bogotá. Devesa, M., Báez, A., Figueroa, V. y Herrero, L.C. (2012), “Repercusiones económicas y sociales de los festivales culturales. El caso del festival internacional de Cine de Valdivia”, EURE–Revista Latinoamericana de Estudios Urbano Regionales, Vol. 38 No. 115, pp. 95-115. Duque, H., Escobar, J., Aponte, E., Flórez, J. y Aguirre, A. (2013), Impacto Del TLC Con Estados Unidos en la producción y el empleo del Valle del Cauca. Gratton, C., Shibli, S. y Coleman, R. (2006), “The economic impact of major sports events: a review of ten events in the UK”, The Sociological Review, Vol. 54 No. s2, pp. 41-58. Levy, P. y Lameshow, S. (1991), Sampling of Populations: Methods and Applications, Wiley, New York, NY. Llopis-Goig, R. (2012), Megaeventos Deportivos: Perspectivas Científicas y Estudios de Caso, Editorial UOC, Barcelona. Matheson, V. (2006), “Economic impact analysis”, en Andreff, W. and Szymanski, S. (Edits), Handbook on the Economics of Sport, Edward Elgar, Cheltenham. Matheson, V. (2012), “Efectos de los principales megaeventos deportivos en las economías locales”, en Llopis-Goig, R. (Ed.), Megaeventos Deportivos. Perspectivas Científicas y Estudios de Caso, UOC, Barcelona, pp. 53-74. Porter, P. y Chin, D. (2012), “Economic impact of sports events”, en Maennig, W. and Zimbalist, R. (Eds), International Handbook on the Economics of Mega Sporting Events, Edward Elgar Publishing, pp. 246-268. 153 Radicchi, E. (2012), “Megaeventos deportivos y creación de valor para las economías anfitrionas”, en Llopis-Goig, R. (Ed.), Megaeventos deportivos. Perspectivas científicas y Estudios de Caso, UOC, Barcelona, pp. 25-51. Roche, M. (1994), “Mega-events and urban policy”, Annals of Tourism Research, Vol. 21 No. 21, pp. 1-19 Schuschny, A. (2005), Tópicos Sobre el Modelo de Insumo-producto: Teoría y Aplicaciones, Serie Estudios Estadísticos y Prospectivos No. 37. Referencias División de Estadística y Proyecciones Económicas de la CEPAL, Santiago de Chile. Siegfried, J. y Zimbalist, A. (2006), “The economic impact of sports facilities, teams and mega-events”, The Australian Economic Review, Vol. 39 No. 4, pp. 420-427. Zimbalist, A. (2010), “>Merece la pena?”, Revista Finanzas y Desarrollo, marzo, pp. 8-11. Zimbalist, A. (2015), “The illusory economic gains from hosting the Olympics and World Cup”, World Economics, Vol. 16 No. 1, pp. 35-42. Efectos de la realización de un megaevento deportivo For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com Corresponding author i A d b Luis F. Aguado can be contacted at: lfaguado@javerianacali.edu.co For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com
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https://europepmc.org/articles/pmc4857249?pdf=render
English
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Frozen elephant trunk reconstruction for right-sided aortic arch with aberrant left subclavian artery and aneurysm of the descending aorta: a case report
Journal of cardiothoracic surgery
2,016
cc-by
2,476
© 2016 Kokotsakis et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background A right-sided aortic arch is an uncommon congenital lesion occurring in 0.05–0.10 % of the population. It is often associ- ated with an aberrant left subclavian artery, and patients are particularly prone to developing aneurysm and dissection [1]. The optimal approach to this condition requires meticulous planning and depends on the extent of aneurysm formation. We report the successful single stage repair of this vascular anomaly using the frozen elephant trunk technique. Abstract Background: A 59-year old man being investigated for back pain was found to have aneurysmal dilatation of a right-sided aortic arch and descending thoracic aorta together with an aberrant left subclavian artery. Case Presentation: He underwent repair of this utilising the frozen elephant trunk technique, which dealt with all three pathologies in one-stage. He made an unremarkable recovery and was discharged home on the 8th post-operative day. Conclusions: This case report further demonstrates the flexibility and safety of the frozen elephant trunk in dealing with complex aortic pathology as a single-stage procedure. Keywords: Aortic arch, Endovascular procedures thoracic aorta (DTA, 6.8 cm at its largest point) located in the right hemi-thorax, which returned to normal size below the diaphragm. The root and ascending aorta were not aneurysmal. An aberrant left subclavian artery (ALSA) arose from a Kommerell’s diverticulum (3.5 cm) at the junction between the distal arch and the proximal DTA. The head and neck vessels arose individually from the arch in the following order: left common carotid artery (LCCA), right common carotid artery (RCCA), right sub- clavian artery (RSA) and finally the ALSA as mentioned above (Fig. 1). Coronary angiography, echocardiography and carotid doppler scan were unremarkable. After multi- disciplinary team discussion it was decided to proceed with the frozen elephant trunk technique. Frozen elephant trunk reconstruction for right-sided aortic arch with aberrant left subclavian artery and aneurysm of the descending aorta: a case report Frozen elephant trunk reconstruction for right-sided aortic arch with aberrant left subclavian artery and aneurysm of the descending aorta: a case report hn Kokotsakis1, Omar A. Jarral2*, Leanne Harling2, Panteleimon Tsipas1 and Thanos Athanasiou2 CASE REPORT Open Access Frozen elephant trunk reconstruction for right-sided aortic arch with aberrant left subclavian artery and aneurysm of the descending aorta: a case report John Kokotsakis1, Omar A. Jarral2*, Leanne Harling2, Panteleimon Tsipas1 and Thanos Athanasiou2 Abstract Background: A 59-year old man being investigated for back pain was found to have aneurysmal dilatation of a right-sided aortic arch and descending thoracic aorta together with an aberrant left subclavian artery. Case Presentation: He underwent repair of this utilising the frozen elephant trunk technique, which dealt with all three pathologies in one-stage. He made an unremarkable recovery and was discharged home on the 8th post-operative day. Conclusions: This case report further demonstrates the flexibility and safety of the frozen elephant trunk in dealing with complex aortic pathology as a single-stage procedure. Keywords: Aortic arch, Endovascular procedures Kokotsakis et al. Journal of Cardiothoracic Surgery (2016) 11:77 DOI 10.1186/s13019-016-0479-y CASE REPORT Open Access Frozen elephant trunk reconstruction for right-sided aortic arch with aberrant left subclavian artery and aneurysm of the descending aorta: a case report John Kokotsakis1, Omar A. Jarral2*, Leanne Harling2, Panteleimon Tsipas1 and Thanos Athanasiou2 Abstract Background: A 59-year old man being investigated for back pain was found to have aneurysmal dilatation of a right-sided aortic arch and descending thoracic aorta together with an aberrant left subclavian artery. Case Presentation: He underwent repair of this utilising the frozen elephant trunk technique, which dealt with all three pathologies in one-stage. He made an unremarkable recovery and was discharged home on the 8th post-operative day. Conclusions: This case report further demonstrates the flexibility and safety of the frozen elephant trunk in dealing with complex aortic pathology as a single-stage procedure. Keywords: Aortic arch, Endovascular procedures Kokotsakis et al. Journal of Cardiothoracic Surgery (2016) 11:77 DOI 10.1186/s13019-016-0479-y Kokotsakis et al. Journal of Cardiothoracic Surgery (2016) 11:77 DOI 10.1186/s13019-016-0479-y CASE REPORT Open Access Frozen elephant trunk reconstruction for right-sided aortic arch with aberrant left subclavian artery and aneurysm of the descending aorta: a case report John Kokotsakis1, Omar A. Jarral2*, Leanne Harling2, Panteleimon Tsipas1 and Thanos Athanasiou2 Abstract B k d A 59 ld b i i i d f b k i f d h l dil i f DOI 10.1186/s13019 016 0479 y CASE REPORT Open Access Frozen elephant trunk reconstruction for right-sided aortic arch with aberrant left subclavian artery and aneurysm of the descending aorta: a case report John Kokotsakis1, Omar A. Jarral2*, Leanne Harling2, Panteleimon Tsipas1 and Thanos Athanasiou2 * Correspondence: omarjarral@gmail.com 2Department of Surgery and Cancer, Imperial College London, London W2 1NY, UK Full list of author information is available at the end of the article Case presentation A 59-year old male was referred to our clinic with a six- month history of back pain. His medical history included hypertension and a 60-pack year history of smoking. Phys- ical examination including assessment of peripheral pulses was unremarkable. Chest radiograph revealed a laterally displaced trachea and a right-sided aortic arch. Computer- ized tomographic angiography (CTA) confirmed a right- sided aortic arch (4.1 cm) and an aneurysmal descending After induction of anaesthesia, arterial lines were placed in the left femoral and both radial arteries. A lumbar cerebrospinal fluid drain was placed in order to manipulate cord perfusion pressure. A soft guidewire was placed in the right femoral artery (RFA) and ad- vanced in to the arch. A median sternotomy was per- formed with left supraclavicular extension in order to expose the distal LSA. The right axillary artery (RAA) was exposed with an infraclavicular incision. After Page 2 of 4 Kokotsakis et al. Journal of Cardiothoracic Surgery (2016) 11:77 Fig. 1 Computer generated images of aorta Fig. 1 Computer generated images of aorta were transected 1 cm distal to their origin and the RSA was ligated at its origin from the aortic arch. Perfusion catheters were inserted in to the LCCA, the RCCA, and together with the RAA cannula already in place, continuous bilateral se- lective antegrade cerebral perfusion (SACP) was started. heparinization, a 8 mm graft was anastomosed to the RAA through this incision on the underside of the artery. The other end of the graft was tunnelled through the right pleural space in to the pericardial cavity and the arterial line (bifurcated) of the cardiopulmonary bypass (CPB) cir- cuit was connected to this end. Examination of the aorta revealed no evidence of dissection or rupture. At this point the soft guidewire in the RFA was exchanged for a stiff one. A hybrid stent-graft (E-vita OPEN PLUS 40 × 160 mm, Jotec Inc., Hechingen, Germany) was advanced in to the DTA in an antegrade fashion. The proximal landmark for stent deployment was just distal to the origin of the RSA. The incorpo- rated polyester graft was cut back and sutured to the transected distal arch with interrupted horizontal mattress sutures (3-0 Prolene, Ethicon, Somerville, NJ) reinforced with a strip of Teflon. A sterilized bronchoscope enabled the distal part of the stent graft to be checked under direct vision. Case presentation After going on to CPB a right superior pulmonary vein vent was placed and the patient was cooled to 25 °C. During cooling the head and neck vessels were dissected out, and upon fibrillation the ascending aorta was clamped and trans- ected just above the sinotubular junction. The heart was arrested using direct cold crystalloid cardioplegia. Subsequent shots were delivered retrogradely every 20 min. Once the target temperature was reached, CPB was arrested and the ascending aorta and part of the arch (up to 2 cm before the origin of the RSA) were resected. The LCCA and RCCA Page 3 of 4 Page 3 of 4 Kokotsakis et al. Journal of Cardiothoracic Surgery (2016) 11:77 Attention was then turned to the ascending aorta where a vascular prosthesis with four side branches (30 mm Tetrabranch, Jotec Inc.) was anastomosed distally to the arch cuff (comprising the native aorta and the E-vita pros- thesis/Teflon strip as described above) using a continuous suture (3-0 Prolene, Ethicon). Systemic perfusion was re- established by connecting the arterial line of the CPB circuit to the 4th side branch of the vascular prosthesis. The 3rd side branch (8 mm) was passed under the innom- inate vein and was anastomosed end-to-end to the RCCA (after removal of the perfusion catheter) with a continuous suture (5-0 Prolene, Ethicon). The proximal anastomosis of the main graft to the sinotubular junction was then per- formed using a continuous suture (3-0 Prolene, Ethicon) with an external strip of Teflon. Rewarming was com- menced; a de-airing drill completed and the cross-clamp was removed. The 1st side branch (10 mm) was passed under the innominate vein and anastomosed end-to-end to the LCCA (after removal of the perfusion catheter) with a continuous suture (5-0 Prolene, Ethicon). Arterial inflow to the RAA graft, which had been tunnelled to sit in the pericardial cavity, was then stopped and this end of the graft was anastomosed end-to-end to the 2nd side branch(8 mm) of the prosthesis using a continuous su- ture (5-0 Prolene, Ethicon). A separate 8 mm spiral graft was then anastomosed end-to-side to the ALSA in the left supraclavicular area and connected proximally end-to-side to the 1st branch of the main vascular pros- thesis (Fig. 2). The ALSA was ligated proximal to the origin of the left vertebral artery. After complete rewarming the patient was separated from CPB uneventfully. Fig. 2 Intraoperative photo References Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: Case presentation The bypass times were as follows: CPB (160 mins), SACP (90 mins) and, distal ischaemic time (50mins). Following haemostasis, the synthetic grafts were covered with a strip of pericardium to exclude them from the sternal wound prior to rou- tine closure. The patient was extubated 6 h following the operation and discharged on the 8th post-operative day at which point he was mobile and neurologically in- tact. Twelve-month follow-up with CTA confirmed sat- isfactory appearance of the reconstruction with absence of endoleak. Fig. 2 Intraoperative photo Fig. 2 Intraoperative photo Page 4 of 4 Kokotsakis et al. Journal of Cardiothoracic Surgery (2016) 11:77 Page 4 of 4 Received: 17 November 2015 Accepted: 2 May 2016 Received: 17 November 2015 Accepted: 2 May 2016 Received: 17 November 2015 Accepted: 2 May 2016 Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Authors’ contributions JK PT KV d TA f JK, PT, KV and TA performed the operation. TA and JK conceived the report. OJ and LH wrote the first draft with input from all authors. All authors read and approved the final manuscript. Acknowledgements None. Author details 1E l H 1Evaggelismos Hospital, Athens, Greece. 2Department of Surgery and Cancer, Imperial College London, London W2 1NY, UK. References 1. Cina CS, Althani H, Pasenau J, Abouzahr L. Kommerell’s diverticulum and right-sided aortic arch:a cohort study and review of the literature. J Vasc Surg. 2004;39(1):131–9. 1. Cina CS, Althani H, Pasenau J, Abouzahr L. Kommerell’s diverticulum and right-sided aortic arch:a cohort study and review of the literature. J Vasc Surg. 2004;39(1):131–9. 2. Easo J, Holzl P, Dikov V, Chavan A, Dapunt O. Frozen elephant trunk procedure in a dextropositioned arch and descending aorta. J Endovasc Ther. 2010;17(6):751–4. In this case we utilised the frozen elephant trunk technique in one-stage to deal with three pathologies: arch and descending aortic aneurysm together with an ALSA originating in a Kommerell’s diverticulum. To our knowledge, this is only the third such report in the literature [2, 3]. We consider it to be a safe and effective technique given the excellent outcome this patient achieved, especially as some of the above tech- niques are associated with very significant morbidity. There are now a number of other devices on the mar- ket (e.g., Thoraflex Hybrid), which would allow a simi- lar or more advanced outcome to be achieved. Whilst it could be suggested that TEVAR(after debranching) is a less invasive option, this was not considered due to the acute angle between the distal arch and the DTA, which may have led to difficulty in securing a good proximal landing. Proceeding with TEVAR in such a situation may have led to incomplete treatment of the arch aneurysm and perhaps unwanted compli- cations such as endoleak. A further disadvantage of this approach is that of embolic related complications in patients with extensive atheroma of the distal aorta. In retrospect, we could have simplified our operation further by establishing CPB via arch cannulation and reanastomosing the RSA to the new arch graft directly via the median sternotomy. 3. Munakata M, Itaya H, Fukui K, Ono Y. One-stage repair for aortic regurgitation and kommerell diverticulum with aneurysmal right aortic arch. J Thorac Cardiovasc Surg. 2007;133(3):798–9. Consent Right-sided aortic arch with ALSA is a rare congenital abnormality frequently associated with aneurysm and dissection. Whilst appropriate surgical indications are not yet clearly established, intervention in the asymp- tomatic is usually recommended for aneurysms over 3 cm [1]. Meticulous pre-operative imaging and plan- ning are essential. Current treatment options are tai- lored to the anatomical features (e.g., type I or II right- sided arch and the degree of aneurysm formation) of each case and include: right (or left) thoracotomy with replacement of the descending thoracic aorta and/or reconstruction of the ALSA, sternotomy and deb- ranching of the arch vessels with subsequent thoracic endovascular aneurysm repair (TEVAR), sternotomy and total arch replacement with insertion of elephant trunk and subsequent TEVAR, total arch replacement with subsequent thoracotomy for DTA replacement, arch replacement and DTA replacement through the clamshell approach, and left carotid to left subclavian artery bypass with ligation or coiling at the origin of the ALSA [1]. Written informed consent was obtained from the patient for publication of this case report and any accompanying images. Conclusion This case report demonstrates the flexibility and safety of the frozen elephant trunk in dealing with right-sided aortic arch aneurysms as a single-stage procedure.
https://openalex.org/W1606192030
https://revistas.pucp.edu.pe/index.php/boletindearqueologia/article/download/1854/1789, https://revistas.pucp.edu.pe/index.php/boletindearqueologia/article/download/1854/1789/
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El dominio del Inka, identidad local y complejidad social en las tierras altas del desierto de Atacama, Norte Grande de Chile (1450-1541 d.C.)
Boletín de arqueología PUCP
2,002
cc-by
13,200
301 DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... BOLETÍN EL DEDOMINIO ARQUEOLOGÍA PUCP, N.° 6, 2002, 301-336 EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN LAS TIERRAS ALTAS DEL DESIERTO DE ATACAMA, NORTE GRANDE DE CHILE (1450-1541 D.C.) 1 Mauricio Uribe,* Leonor Adán** y Carolina Agüero*** Resumen En este artículo se presenta una aproximación a la expansión del Tawantinsuyo en las tierras altas del desierto de Atacama (norte de Chile), proponiendo un enfoque alternativo a la tradicional concepción de una presencia débil o indirecta del Inka en este territorio. Por ello, se exponen aquellas evidencias y argumentos que parecen representar mejor la complejidad social, económica, política y étnica que se encontraba en pleno desarrollo cuando el imperio tomó contacto con las poblaciones de este desierto. En particular, los autores se centran en el caso de San Pedro de Atacama, porque aún cuando se tratán de una de las localidades que ha servido como punto de referencia para la comprensión de los grupos que habitaron sus tierras altas, todavía se conoce muy poco de aquellos momentos. Pero, sobre todo, porque el estudio de la convergencia de las poblaciones atacameñas e «incaicas» dentro de San Pedro, de acuerdo a experiencias en otras localidades de este territorio, permite vislumbrar en ese encuentro los elementos que particularizan la complejidad social del desarrollo local. Abstract THE INKA’S DOMINION, LOCAL IDENTITY AND SOCIAL COMPLEXITY IN THE HIGHLAND OF ATACAMA’S DESERT, CHILE’S NORTE GRANDE (1450-1541 AD) This paper presents our approach to the Tawantinsuyo expansionism in the highlands of Atacama Desert (Northern Chile), in which we suggest an alternative view to the traditional idea of a weak or indirect Inka presence in the Atacamenian territory. Therefore, we show the evidence and arguments that represent the politics, economic and ethnic complexity development when the empire had contact with the populations from the chilean desert. We concentrate in the case of San Pedro de Atacama, because it had been a point of reference to the archaeological understanding of groups that lived in its highlands, but the history is poorly known. In particular the convergence of the study of Atacamenian and Inca people in San Pedro, like in others parts of this space, allows to appreciate best the elements that characterize the social complexity of this local development. «La existencia de pucaras [...] hace suponer el deseo de defender un patrimonio territorial e indica también el establecimiento en esa zona del núcleo fuerte y posiblemente solitario de los atacameños, tomados como unidad étnica, política, religiosa y social» (Montandón 1950: 15). 1. Introducción Este trabajo sintetiza la aproximación de los autores acerca de la expansión del Tawantinsuyo en las tierras altas del desierto de Atacama, norte de Chile (Fig. 1). Sobre la base de las investigaciones * Universidad de Chile, Departamento de Antropología. E-mail: mur_cl@yahoo.com ** Universidad Austral de Valdivia, Museo Histórico Mauricio van de Maele. E-mail: ladan@uach.cl *** Universidad Católica del Norte, Instituto de Investigaciones Arqueológicas. E-mail: maguero@ucn.cl 302 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO realizadas por los autores en la zona desde 1994, se propone un enfoque alternativo a la tradicional concepción de una presencia débil o indirecta del Inka en este territorio (Llagostera 1976). Por el contrario, se perfila un conjunto de estrategias económicas, políticas y simbólicas implicadas en el proceso de anexión de su población al estado, sugeridas por el comportamiento y naturaleza de la cultura material de este periodo, distribuida entre el río Loa y San Pedro de Atacama (Uribe 19992000). Esto no concuerda con las interpretaciones que enfatizan el carácter marginal de estos territorios para los intereses del Tawantinsuyo, lo cual se ha discutido con datos arqueológicos, etnohistóricos y etnográficos, teniendo en cuenta las particularidades de la historia de la investigación sobre la temática y las actuales perspectivas de la arqueología regional (Uribe et al. 1999; Adán y Uribe e.p.). En este sentido, los antecedentes sugieren desde ya un dominio o intervención más directa del Estado Incaico en Atacama, el que habría privilegiado la interacción con grupos dirigentes locales, potenciando las contradicciones al interior de su sociedad, entre aquellos y sus bases (Uribe y Adán e.p.). Tales contradicciones, consecuentemente, comprometerían la identidad local como la del Inka. Elementos incaicos y locales fusionados, observados en segmentos específicos de la población atacameña (Ayala et al. 1999), representan a esos grupos dirigentes que asumieron el funcionamiento estatal en la región, explotando su propia fuerza de trabajo, sus excedentes agrícolas y riquezas minerales. La tutela del Inka, entonces, se expresaría materialmente pura en otros sectores, como los periféricos respecto al núcleo local, manifestándose con mayor claridad en la vialidad y los asentamientos asociados al capac ñam (Cornejo 1995). Estas ideas toman como referente trabajos previos (Castro y Cornejo 1990; Cornejo 1995; Gallardo et al. 1995) y se fundamentan en análisis tipológicos, tecnológicos y funcionales en cuanto a la arquitectura, alfarería, industria lítica, textilería y otros materiales de estos periodos, llevados a cabo por los autores. Los resultados obtenidos permiten, en esta oportunidad, acceder a la complejidad sociopolítica del desierto de Atacama y, en particular ,de San Pedro (Fig. 1), a través de una evaluación del substrato local frente al «cinturón» creado por más de una decena de instalaciones del Tawantinsuyo que se conocen a lo largo de la vertiente occidental circunpuneña de este segmento de los Andes centro-sur (v.g., Raffino 1981; Castro 1992; Lynch y Núñez 1994). 2. Poblaciones de Atacama en los siglos XVI y XVII Las poblaciones indígenas que habitaron el área de estudio durante los siglos XVI y XVII representan identidades locales distinguibles a través de fuentes etnohistóricas, ya sea por discursos externos recogidos por los hispanos, o por la propia documentación colonial local generada a través del contacto directo con las poblaciones originales de este territorio (v.g., Hidalgo 1983; Martínez 1985; Castro 1997; Manríquez 2002). Las informaciones para los siglos en cuestión mencionan, de manera general, a «indios», «gentes» o «naturales» de determinados lugares de Atacama «la Grande» o «la Chica» (San Pedro y el Loa Superior respectivamente), de sus ayllus o pueblos, diferenciando claramente entre éstos y los indios asentados en la costa. En el siglo XVI, Oviedo grafica a estos «naturales» como de «tierra alzada é de guerra, y la gente por los montes, fuera de sus casas é simientes, y puestos en montañas y sierras muy asperas» (Núñez 1992: 85), relacionando la actitud de los «naturales» con las determinadas características del entorno que habitaban, es decir, del desierto. EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 303 Fig. 1. Mapa de la región con las principales localidades y sitios mencionados en el texto (Cortesía: V. Castro). 304 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO Paralelamente, en la provincia de Atacama crónicas y documentos hispanos locales señalan la existencia de indígenas cuya designación coincide con la de la «provincia». Esta manera de nombrar subsiste durante el periodo colonial, en el cual la denominación otorgada a un espacio geográfico (valle), un hito (río), o una división jurídica territorial (provincia, partido, pueblo, etc.), comienzan a ser apelativos de ciertas poblaciones específicas (Manríquez 2002). En estos primeros siglos de ocupación hispana tales poblaciones son caracterizadas de manera genérica como «yndios», «gente que sirvió al inga» y «jente dispuesta y bien bestidos como los del Piru» (Vivar 1979 [1558]: 20), los cuales, desde ya, son adscritos al espacio territorial nombrados como indios de la provincia de Atacama. Y, en 1557, cuando se inicia el proceso de «pacificación» de la provincia, se los menciona como «yndios de Atacama que estaban de guerra».2 La segunda mitad del siglo XVI marca el inicio de la consolidación hispana en la zona. El virrey del Perú y el aparato administrativo colonial deciden tomar el control directo de la provincia de Atacama, por tratarse de uno de los lugares claves en la comunicación entre La Plata y Potosí, Tucumán y el reino de Chile (Hidalgo 1982). El control se concretó aún más con el traspaso de una parte de esta extensa encomienda a Juan Velázquez Altamirano, en porción de las distintas «provincias» que constituían la audiencia de Charcas en Bolivia (Manríquez 2002). Este hecho indica que los indígenas de Atacama y su territorio son integrados por los españoles dentro de dicha audiencia, idea que la arqueología y la etnohistoria también comparten al integrar a esta unidad como parte de la subárea circunpuneña (Lumbreras 1981). De acuerdo a ello, es muy probable que la administración europea estuviera utilizando, aunque con ciertos reacomodos, un ordenamiento territorial prehispánico, basado en dinámicas históricas particulares y en un profundo conocimiento del medioambiente local. Es entonces cuando estas poblaciones comienzan a ser denominadas en propiedad como «atacamas». Al respecto, la investigación etnohistórica no reconoce antecedente alguno que oriente hacia la mención de una autodenominación, puesto que, salvo por los hispanos, ningún indígena se define como «atacama» o «indio atacama». Por ello se reafirma la idea de una exodenominación que bien podría tener su origen en las categorizaciones realizadas por otras poblaciones originarias o por el Tawantinsuyo mismo (Manríquez 2002). El término «Atacamas», por lo tanto, denominaría genéricamente a indígenas adscritos a un determinado territorio, pero también distingue a los indígenas que siendo señalados como «originarios» o «naturales» de Atacama se desplazaban de manera permanente o por periodos variables de tiempo, de preferencia hacia lugares situados en el Noroeste argentino, Chichas, Tarapacá y el extremo norte del reino de Chile (v.g., Copiapó), considerados lugares al exterior de los límites de la provincia (Martínez 1998). Frente a esta representación externa, también se desarrollan otras identidades desde una escala local o «microscópica» (Manríquez 2002). Así, poblaciones de Atacama la Alta o San Pedro compartirían códigos de identidad cultural con aquellos que reconocen su origen en espacios y ayllus situados en Atacama la Baja o el río Loa, en tanto las mismas u otras reconocerían membresía en el Noroeste argentino, Chichas o Tarapacá (v.g., Odone 1995). Dentro de esta lógica, los indígenas parecieran relacionar su pertenencia con el origen y éste con el lugar donde se encuentran sus parientes. En toda la región y en cada uno de los ayllus serían las relaciones de parentesco y de un origen reconocido como común, piezas importantes en la articulación de sus relaciones sociales, económicas, políticas y religiosas. Se establece así un nexo significativo entre parentesco, territorio de origen e identidad que se define por el lugar donde se ubican los familiares muertos y sus expresiones sagradas (Manríquez 2002). Estas relaciones estarían documentadas ya en el siglo XVI por descripciones como las que realiza indirectamente Vivar (1979 [1558]), sobre el «pueblo o valle de Atacama» cuando se refiere a las «costumbres EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 305 de yndios» y describe que en un apartado de las casas, cerca de donde duermen: «...qu’ es el más prinçipal, está hecho de bobeda alta fasta el entresuelo y cuadrada. Aqueste es su enterramiento y sepulcro. Y allí dentro tienen a sus bisaguelos y aguelos y padres y toda su generaçion» (op. cit.: 20). Esta situación se advierte igualmente en prácticas religiosas del siglo XVII en las que cada especialista ofrendaba a un «ídolo», correspondiente a un determinado pueblo, por ejemplo, Qumaquma o Qumaquina de Chiuchiu, Socomba de Aiquina, Sintalacna de Caspana y otros (Castro 1997). Preliminarmente, por lo tanto, se desprende de estas descripciones y las inferencias hechas que, al momento del contacto, el territorio atacameño se encontraba constituido por distintas comunidades adscritas a un extenso espacio desértico, pero, al mismo tiempo, a los espacios de otros a los cuales podían acceder, de acuerdo a los nexos establecidos por el parentesco, los ancestros y las deidades propias como compartidas por cada una de esas unidades. 3. San Pedro de Atacama antes y durante el Inka De manera coherente con lo anterior, las investigaciones arqueológicas llevadas a cabo han permitido comenzar a definir en Atacama la existencia de un sistema social articulado por poblaciones segmentadas en el espacio, pero fuertemente vinculadas en el plano territorial por aspectos sagrados y, seguramente, parentales, alcanzando un importante grado de integración macroregional, al menos entre las poblaciones del río Loa y San Pedro. A continuación se presentan las evidencias y argumentos arqueológicos que soportan este planteamiento, particularmente aquellos de San Pedro de Atacama donde se centran actualmente los estudios de los autores. 3.1. Los asentamientos El estudio de la arquitectura de los periodos tardíos de San Pedro de Atacama señala un patrón temprano de construcción en barro representado por la ocupación de los Campos de Sólor (Fig. 2). La vigencia de esta técnica constructiva tradicional apunta a una cierta continuidad con los tiempos previos, particularmente con los periodos Formativo y Medio (Le Paige 1958), que se expresa a través de asentamientos-núcleos construidos de barro, articulando a los oasis con los principales cursos de agua del Salar como los ríos San Pedro y el Vilama (Núñez 1992; Llagostera 1996; Uribe et al. e.p.). La ocurrencia de estos asentamientos, fuertemente asociados al aprovechamiento económico de los recursos cruciales de la cuenca, los ríos San Pedro y Vilama, plantea la existencia de un sistema social y económico complejo, aunque, probablemente, poco competitivo en momentos tempranos del periodo (v.g., dispersión de los asentamientos, ausencia de fortalezas, etc.), pero con una creciente presión hacia el bien básico y crítico para la vida de las poblaciones atacameñas: el agua. Este sistema debió generar una dinámica social a lo largo del recurso hídrico, por la cual las poblaciones fueron acercándose al norte del salar y hacia las quebradas del oriente, resultando favorecidas aquellas comunidades que podían controlar las cabeceras de dichos cursos (cf. Núñez 1992). Esta situación podría haber generado una importante tensión social durante la evolución del sistema, lo que en algún momento se expresa en la construcción de pequeños sitios en lugares estratégicos como los pukara de Quítor y Vilama, en los sectores más septentrionales de la localidad (Fig. 3 a). Del mismo modo, se aprecia en la experimentación con técnicas agrícolas y de irrigación en sistemas estancieros de altura como los de Guatin (Fig. 3 b), inicialmente destinados al pastoreo (Serracino y Stehberg 1974). Por último, esta tensión también se identifica por la introducción de elementos de apariencia foránea, en especial altiplánica, correspondiente a las estructuras conoci- a b Fig. 2. Campos de Sólor. a. Planta de estructura habitacional de barro; b. Cántaro o tinaja usada para almacenamiento de líquidos y/o como urna funeraria. a b Fig. 3. a. Pucaras de Vilama. Reductos de cumbre asociados al río Vilama; b. Estancia de Guatin. Se observan campos o canchas de cultivo. 308 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO a b Fig. 4. a. Aldea de Zápar. Estructura tipo chullpa; b. Aldea de Peine. Estructura tipo chullpa. das como chullpa (Figs. 4 a, b), entre otras, que aparecen en poblados que se instalan a partir de aquellos pukara, como Quítor, o en otros completamente nuevos, por ejemplo, Zápar y Peine. Es casi un consenso, en consecuencia, que durante los primeros momentos de los Desarrollos Regionales se ocuparon pueblos «sin defensa», como Sólor y Guatin, para luego comenzar a gestarse los pukara típicos del periodo (Le Paige 1958; Bittman et al. 1978; Schiappacasse et al. 1989; Núñez 1992). Al comienzo del Periodo Intermedio Tardío se nota, por lo tanto, la existencia de un sistema de asentamiento centrado en la explotación de los recursos de los oasis regados por los principales ríos que drenan el Salar, articulándose los asentamientos-núcleos con otros de pisos más altos, con gran probabilidad, ocupados para la ganadería de camélidos. Esta arquitectura fue la que caracterizó a la región después del 950 d.C. hasta cerca del 1350 d.C. (Berenguer et al. 1986; Tarragó 1989; Uribe e.p.), surgiendo luego otro patrón constructivo más competitivo y directamente relacionado con el control de los recursos hídricos que se manifestó en la aparición de los pukara, sean o no amurallados, a partir de los cuales se constituyeron las comunidades donde justamente el Inka dejó rastros materiales notables. EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 309 Catarpe, Quítor, Zápar y Peine. El trabajo realizado se concentró en ocho sitios habitacionales de San Pedro, los que han sido registrados en su totalidad a través de la aplicación sistemática de una ficha arquitectónica (Castro et al. 1991). De éstos, fueron seleccionados los asentamientos Catarpe Oeste (W), Quítor, Zápar y Peine (Fig. 1), los cuales proveen de información significativa para entender la época del contacto entre el nuevo desarrollo atacameño y el Inka. En estos yacimientos (Tabla 1), los rasgos arquitectónicos más distintivos son el gran número de recintos (entre 141 y 210 estructuras), el trazado ortogonal de los poblados, su orientación cardinal O-E, con sus muros transversales 60º al Norte, así como la conformación de conjuntos aglutinados y densos constituyendo conglomerados que en algunos casos presentan una evidente distribución espacial en mitades o bipartita. Los sitios exhiben espacios domésticos con diferentes tipos de estructuras, algunos con notables concentraciones de molienda, los cuales comprometen cocinas-dormitorios, patios interiores, recintos sin techar a modo de ramadas y otros grandes a manera de plazas, corrales u otros espacios de uso comunal.3 Afuera, junto y dentro de ellos se disponen sepulturas y depósitos —silos o trojas—, formalmente correspondientes a «cistas» y estructuras tipo chullpa; del mismo modo que tumbas «...adosadas a bloque rocoso», «adosadas y bajo bloque rocoso» y «bajo bloque rocoso»4 (Fig. 5), acercándose a la composición constructiva de los asentamientos del Loa superior (Aldunate y Castro 1981; Castro et al. 1991; Adán 1996; Ayala 2000). Por más que el emplazamiento sea más o menos plano, los sitios se ubican siempre en quebradas o asociadas a éstas, lo que exigió la aplicación de técnicas constructivas de terrazas y muros de contención, hasta el día de hoy comunes en las quebradas (Fig. 6 a), lo que equivale a la arquitectura habitacional con los sistemas de cultivo (Adán 1999). Los muros son principalmente simples, rústicos y a plomo, pero también aparecen los dobles, sedimentarios y desaplomados. Las piedras con las que se edificaron las viviendas fueron unidas con argamasa de barro y, en su mayoría, se encuentran en estado natural, aunque con un alto porcentaje de piezas desbastadas y toscamente canteadas. Además de estar asociados a extensos campos de cultivo y elaborados sistemas de regadío en las quebradas o valles del río, otra característica relevante de estos poblados es su conexión con rutas de tránsito, seguramente de origen preincaico, que conectan los oasis con la región del Loa superior y, más lejos aún, con las regiones de Bolivia y Argentina, hacia el norte y oriente. Estas rutas son reforzadas luego por la política expansiva del Tawantinsuyo, como lo evidencian los tambos y camino incaicos que se ubican en ambos extremos del Salar, como Catarpe Este y Peine, los dos anexados a los poblados locales (Lynch 1977; Niemeyer y Rivera 1983; Lynch y Núñez 1994). En definitiva, la localización, el emplazamiento, la visibilidad y relación de los poblados con los recursos hídricos, agrícolas y viales sugieren una idea de control y defensa, razón por la que tradicionalmente estas construcciones han sido llamadas pukara. Algunos de ellos como Catarpe y Quítor, por su ubicación que aprovecha y controla las mejores aguas y tierras del Salar en el sector más septentrional de San Pedro, debieron generar notables relaciones de dependencia respecto a las comunidades o ayllus más meridionales. Sugerentemente, Latcham (1938: 96-98) propone que, adosado a uno de los riscos del Pukara de Quítor, se levantó el edificio «más grande y más perfecto» que, en opinión del autor, habría correspondido a un «cacique» o autoridad, lo cual sugiere la existencia de una población internamente diferenciada, muy poco igualitaria, que pudo estar jerarquizada de acuerdo a esta dependencia de los ayllus según su acceso al agua. A lo anterior se suma que la disposición de estos asentamientos en zonas de quebradas y la significativa ocurrencia de estructuras tipo chullpa en ellos, señalan la configuración de una tradición de «tierras altas» equivalente a la del curso superior del río Loa (Uribe y Adán 1995). Por lo mismo, 310 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO Catarpe E Catarpe W Quítor Zápar Peine 163 177 201 141 210 Superficie (m ) 12.000 10.080 17.400 8.400 16.000 Densidad (n.º rec./hec.) 135,83 163,89 115,52 167 131,25 Número de recintos 2 1a. Sitios estudiados, número de recintos, superficie total y densidad ocupacional Catarpe E Forma de recintos Circular Cuadrangular Elipsoidal Irregular Oval Rectangular Subcircular Subcuadrangular Subrectangular Subtriangular Trapezoidal n 0 16 0 13 0 56 3 10 52 0 2 % 0 9,82 0 7,98 0 34,36 1,84 6,13 31,90 0 1,23 Catarpe W Quítor % n % n % n % 7 3,48 2 1,42 1 0,48 18 8,96 10 7,09 8 3,81 3 1,49 0 0,00 1 0,48 45 22,39 46 32,62 57 27,14 6 2,99 3 2,13 13 6,19 17 8,46 18 12,77 30 14,29 24 11,94 13 9,22 28 13,33 1 0,50 3 2,13 8 3,81 53 26,37 32 22,70 48 22,86 5 2,49 1 0,71 8 3,81 0 0,00 1 0,71 0 0,00 12 8,51 210 3,81 141 100 100 100 n 1 13 0 39 1 31 8 9 54 0 1 0,56 7,34 0 22,03 0,56 17,51 4,52 5,08 30,51 0,00 0,56 No observable 11 6,75 20 11,30 22 10,95 Total 163 100 177 100 201 100 Zápar Peine 1b. Forma de planta de los recintos y su frecuencia en los sitios. Catarpe E Catarpe W Quítor Zápar Peine Rango de tamaño de los recintos (m 2) n % n % n % n % n % hasta 5 49 30,06 51 28,81 38 18,91 58 41,13 102 48,6 entre 5-10 40 24,54 33 18,64 25 12,44 23 16,31 33 15,7 entre 10-20 38 23,31 37 20,90 47 23,38 13 9,22 26 12,4 entre 20-40 21 12,88 34 19,21 54 26,87 14 9,93 22 10,5 entre 40-60 1 0,61 3 1,69 17 8,46 6 4,26 10 4,76 más de 60 8 4,91 7 3,95 7 3,48 19 13,48 7 3,33 Sin registro 6 3,68 12 6,78 13 6,47 8 5,67 10 4,76 Total 163 100 177 100 201 100 141 100 210 100 1c. Frecuencia del tamaño de los recintos y su frecuencia en los sitios. Tabla 1. Principales características arquitectónicas de sitios habitacionales del Periodo Tardío de San Pedro de Atacama EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 311 a b Fig. 5. a. Aldea de Zápar. Cistas funerarias asociadas a estructuras habitacionales; b. Pucara de Catarpe Oeste. Sepulturas adosadas y bajo bloque rocoso. la ocupación de estos espacios ecológicos, las quebradas, no sólo resalta por la mera incorporación de nuevos rasgos arquitectónicos, sino porque redunda en la adopción de un sistema de tecnologías, conocimientos y poder asociados que hacen habitables y productivos los territorios altos previamente desestimados o subutilizados por las poblaciones de los oasis (Adán y Uribe 1995). En este sentido, una singularidad en la ocupación «quebradeña» de San Pedro es la coexistencia de estructuras tipo chullpa con entierros en cistas al interior de las viviendas, lo que indica una combinación de elementos culturales propios del Salar, en tanto una ancestral tradición del desierto y otros rasgos de la llamada tradición Altiplánica (Schiappacasse et al. 1989). Tal situación expresaría una convivencia de tradiciones culturales que caracterizaría a las poblaciones del Salar a partir de esos momentos que, de acuerdo a los fechados absolutos con que se cuenta hoy día para San Pedro, se iniciaría hacia el 1340 d.C. y perdura hasta el contacto con el Inka (Uribe e.p.). Al mismo tiempo, estas clases de tumbas y estructuras, cistas y tipo chullpa, al ser aéreas, exhiben dos cualidades que las antiguas fosas y cementerios atacameños no poseían (cf. Costa y Llagostera 1992). En primer lugar son visibles, por lo tanto, opera una presencia física determinada 312 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO en parte por la percepción o contacto visual constante que puede establecerse entre vivos y muertos. Por otra parte, este tipo de sepulturas permiten una eventual manipulación de los restos mortales (Montt 2002). La inclusión de estas prácticas innovadoras es de suma relevancia social, porque los rituales relativos a la muerte ofrecen a los vivos la eficacia simbólica de percibir visualmente y movilizar a sus difuntos (Isbell 1997). Por consiguiente, los muertos debieron constituir un significante explícito, sobre todo porque se ha visto que en esos tiempos la posesión y usufructo de la tierra y el agua parece haber sido crucial, lo que se encontraría legitimado ante las fuerzas productivas que componen la población local y ante otros, a través de la noción de ancestro, cuyo valor pudo radicar en su origen altiplánico. Se integra así un segundo elemento de distinción entre las poblaciones de San Pedro, las cuales no sólo se diferencian por su ubicación respecto a los recursos críticos locales —y, de acuerdo a ello, a un territorio específico— sino también por su vínculo con lo externo, en especial, lo altiplánico (Uribe 1996; Ayala 2000). Frente a todo esto, Catarpe Este y los tambos de Licancábur, Peine y otros manifiestan la imposición de la arquitectura del Tawantinsuyo en la región de San Pedro de Atacama (Niemeyer y Schiappacasse 1988). Sin embargo, Catarpe Este es paradigmático en términos de las manifestaciones de un dominio e incorporación efectiva de las poblaciones del Salar al funcionamiento estatal. El asentamiento, si bien comparte varias características con el resto de los sitios habitacionales de la región (v.g., el trazado ortogonal), evidencia la presencia de una importante plaza doble en una ubicación central (Fig. 6 b), una frecuencia similar de unidades domésticas y de silos o collcas, así como una abundancia relativa de muros dobles. La arquitectura de Catarpe Este indica, por lo tanto, la presencia de abundantes actividades de almacenaje, la disposición de espacios habitacionales sin sepulturas y, sobre todo, la existencia de grandes espacios de utilidad para funciones administrativas como ceremoniales. Su construcción se remontaría según los fechados radiocarbónicos a 1510 d.C.5 (Uribe e.p.), indicando una lógica de ocupación que obviamente se aleja de la que se ha señalado para los poblados locales y que parece incidir en el funcionamiento de la estructura económica y social de sus habitantes. Cerámica. Cerca de 53.000 fragmentos de cerámica, recolectados sistemáticamente a partir de un muestreo dirigido del 20% de cada sitio considerado en la investigación (Tabla 2), permiten detectar variaciones tipológicas y funcionales en los recintos analizados. Estas diferencias tienen dos lecturas, una temporal y otra funcional, las que se articulan conformando un panorama complejo y dinámico de los periodos Intermedio Tardío y Tardío. A partir de un análisis tipológico y funcional de la cerámica, para lo cual se usó con fines comparativos y cuantitativos, la cantidad y el peso de los fragmentos provenientes de cada recinto y asentamiento, se pudo establecer una secuencia ocupacional de los yacimientos estudiados. De acuerdo a esto, Catarpe Oeste y Quítor aparecen relacionados por una tradición alfarera local (Figs. 7, 8), la cual se remonta a tiempos previos y a los antiguos espacios de los oasis del Salar como los campos de Sólor (v.g., a través de la presencia predominante de tipos cerámicos conocidos como Dupont y Aiquina, o de otros más esporádicos como el San Pedro rojo violáceo [Uribe 1997]). Sin embargo, estos mismos sitios presentarían un momento posterior, más «clásico» del desarrollo regional y, justamente, serían los que reciben el impacto incaico, elocuente —como se ha visto— en Catarpe Este. Lo anterior significa que no se reconocen los ejemplares más tempranos o éstos pierden relevancia frente a una diversidad tipológica mayor (Uribe 1996), en la cual es posible reconocer exponentes «exóticos» como la cerámica altiplánica Hedionda (Fig. 9). De este modo, los tipos foráneos se incrementan desde estos momentos y remiten a vínculos con el Altiplano meridional, la vertiente oriental circunpuneña e incluso los Valles Occidentales, expresándose especialmente con el Inka, el cual exhibe un carácter marcadamente trasandino (Fig. 10 d, e). Debido a que presentan esta misma situación y porque, en la práctica, no existen evidencias cerámicas más tempranas en ellos, se incluyen Zápar y Peine como asentamientos ocupados en forma contemporánea a Quítor y Catarpe Oeste. No obstante, éstos exhiben la introducción de una alfarería cuyas características se relacionarían con el mayor impacto del Tawantinsuyo en la zona, lo EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 313 a b Fig. 6. a. Pucara de Quítor. Aterrazamientos para construcción de estructuras habitacionales en ladera; b. Tambo de Catarpe E. Plaza rectangular doble en el centro del asentamiento (Cortesía: F. Maldonado). 314 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO Tipos cerámicos (%) Sitios Pre Intermedio Tardío Intermedio Tardío Tardío Foráneos Hispano-indígena Guatin 10,96 86,73 1,82 0,20 0,00 Sólor 10,45 88,78 0,31 0,22 0,00 Vilama Norte 13,69 82,89 2,69 0,00 0,00 Vilama Sur 2,33 95,35 1,55 0,00 0,00 Catarpe Este 0,93 93,39 4,23 1,32 0,00 Catarpe Oeste 0,64 96,62 1,27 0,80 0,56 Quítor 0,06 98,50 0,36 0,90 0,02 Zápar 1,19 86,71 11,12 0,49 0,22 Peine 1,97 62,47 34,29 0,52 0,30 n=26.692 fragmentos Tabla 2. Resumen de la frecuencia de tipos cerámicos de sitios habitacionales tardíos de San Pedro de Atacama de acuerdo a su adscripción cultural y cronológica. cual redundaría en la transformación de la cerámica local (Fig. 10 a). Además, aquí la ocupación de estos asentamientos se extendería hasta épocas coloniales tempranas, marcando un tercer momento de la secuencia regional coincidente con el Periodo Tardío. Ahora bien, la cerámica local que se vuelve significativa inmediatamente antes del Inka, indica, por una parte, el incremento de grandes contenedores para almacenamiento (Uribe y Carrasco 1999). En este sentido, destacan aquellos para líquidos que no sólo pudieron ser utilizados para el agua, sino también para chicha, lo cual es especialmente relevante en situaciones ceremoniales o festivas (Fig. 8). Por otro lado, aumentan los contenedores asociados a la manipulación y preparación de alimentos, incluida su cocción, lo que otra vez se vincula con actividades festivas que habrían involucrado la producción de alimentos, generalmente para muchas personas (Fig. 8 c). Un tercer aspecto interesante es la aparición de cerámica local revestida y roja de uso ceremonial en recintos habitacionales y basurales junto con el resto del material doméstico (Fig. 8 b). Lo anterior indica que esas prácticas ceremoniales y festivas tendrían lugar en áreas tanto públicas como en los espacios particulares de las unidades domésticas, generando correlatos materiales sumamente similares. En suma, el tipo de asentamiento inferido a partir de la anterior asignación funcional de sus construcciones indica que todos los sitios inmediatamente preincaicos como Catarpe, Quítor, Zápar y Peine tendrían un carácter eminentemente doméstico, con recintos habitacionales donde se preparan y sirven alimentos, siendo sumamente importante el almacenaje. No obstante, también ocurren actividades ceremoniales, las cuales difieren tanto en escala como en intensidad, pues en dichos momentos esta práctica pareciera incentivarse en cada uno de los yacimientos mencionados y realizarse de manera independiente de los otros. Tal situación es coherente con la inclusión en todos ellos de prácticas funerarias manifestadas en la arquitectura que se incorpora dentro, junto o cerca de los asentamientos, sugiriendo una dinámica de comunidad o ayllu para las sociedades que habitaron estos poblados. Así, dentro de la escala temporal se observa un primer momento del Periodo Intermedio Tardío caracterizado por sitios habitacionales en los que se encuentran representadas una amplia EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 315 Fig. 7. Tipos cerámicos locales de inicios del periodo Intermedio Tardío. a. Tipo Dupont (escudilla); b. tipo San Pedro rojo violáceo (cántaro doble cuerpo); c. Tipo Turi rojo burdo (cántaro o tinaja); d. Tipo Turi gris alisado (olla con protúberos). gama de actividades, aunque no todos los aspectos del ciclo de vida de las personas. Lo anterior se restringió a muy pocos lugares, los que actuarían como asentamiento-núcleo, tal cual se ha descrito más arriba para los Campos de Sólor. En cambio, un segundo momento, representado en los sitios Catarpe, Quítor, Zápar y Peine, corresponde a asentamientos mayores y aglutinados que integran todo un espectro de actividades: habitacionales, de almacenamiento, ceremoniales y mortuorias en particular, que los convierte en unidades independientes y autónomas, sin distinguirse uno o unos pocos núcleos como antes. Seguramente, este cambio se habría gestado en función de los trastornos económicos, sociales, políticos y demográficos experimentados por las poblaciones locales después del colapso tiwanaku, mostrando un énfasis en la segmentación social, extensión territorial, en las conductas de 316 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO Fig. 8. Tipos cerámicos locales clásicos del Periodo Intermedio Tardío. a. Tipo Aiquina (escudilla); b. Tipo Turi rojo revestido alisado (cántaro); c. Tipo Turi gris alisado (olla con asa); d. Turi rojo alisado (cántaro). almacenamiento, las relaciones con sus fronteras y un cambio en la naturaleza y escala de lo ceremonial y/o público (cf. Albarracín-Jordán 1996). En este ámbito, el desarrollo de una organización jerarquizada, la generación de excedentes y el rol de los líderes o autoridades en festividades redistributivas debieron jugar un papel protagónico al interior de los grupos de San Pedro de Atacama, muy ligado a la competencia sobre el acceso a los recursos de agua y tierra como a los contactos a larga distancia para la complementariedad de recursos. Material lítico. Siguiendo el mismo procedimiento de recuperación y la perspectiva analítica con que se abordó la cerámica, se estudiaron 4080 piezas líticas y 1216 artefactos de molienda, producto EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 317 Fig. 9. Tipos cerámicos altiplánicos del Periodo Intermedio Tardío. a, b. Tipo Hedionda negro sobre ante (escudilla y cántaro miniatura); c, d. Tipo Yavi-Chichas (cántaro y balde). de la recolección superficial en los yacimientos como del registro in situ de los mismos (Carrasco 2002). Considerando esta división en talla y molienda del material lítico, se llegó a establecer un comportamiento común o patrón dominado por desechos secundarios e implementos para moler (Tabla 3). En este contexto, la talla siempre se presenta más abundante en momentos tempranos y de manera muy escasa en los sitios tardíos, a pesar incluso de evidencias de loza y vidrio empleados en la elaboración de chaquiras en épocas indígena-coloniales. Se observa que los sitios con menor material tallado exhiben una alta proporción de instrumentos de molienda (Fig. 11 a), con la excepción de Sólor que habría sido un importante centro aglutinante de actividades en momentos tempranos como anteriores al desarrollo regional, tal cual lo 318 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO Fig. 10. Tipos cerámicos inkas locales y foráneos del Periodo Tardío. a. Tipo Lasana café-rojizo revestido pulido (aríbalo pasta con mica), b. Tipo Turi rojo revestido, exterior negro, alisado interior (jarro «aribaloide»), c. Tipo Turi rojo revestido pulido ambas caras (escudilla ornitomorfa); d, e. Tipo Yavi-La Paya (aríbalos). afirma la arquitectura y la cerámica. En la misma época, otros sitios, como Vilama, muestran una talla más expeditiva y relacionada con situaciones defensivas, quizás también aplicables a Quítor, vinculadas a la competencia por las cabeceras de los principales cursos que desaguan en el salar y que se manifiesta en concentraciones de piedras «huevillo» para usar como proyectiles (Fig. 11 b). EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... Categorías Guatin Sólor Vilama Vilama Catarpe Catarpe Quítor Norte Sur Este Oeste 319 Zápar Peine Total Adornos 0 18 0 0 2 0 2 3 14 39 Contenedores 0 1 0 0 0 0 0 0 0 0 Herramientas 30 30 2 0 2 6 4 2 9 5 Molienda 35 251 0 3 66 105 0 423 157 1.040 Talla 1.381 1.041 197 9 254 277 92 74 567 3.892 Polifuncionales 0 81 0 1 1 6 0 57 7 153 Indeterminados 3 13 0 0 0 4 0 9 0 29 Material temprano 6 44 0 0 1 0 1 0 5 57 Total 1.455 1.479 199 13 326 398 99 568 759 5215 Tabla 3: Resumen de las principales categorías líticas morfofuncionales de sitios habitacionales tardíos de San Pedro de Atacama. Junto con este carácter expeditivo adquirido por la tecnología lítica, es importante destacar que durante la parte tardía del periodo los productos líticos enfatizan la molienda, tanto agrícola como con otros fines (v.g., ceremoniales, especialmente ritos funerarios o minería), desplegando una gran diversidad morfológica y polifuncionalidad de los artefactos referidos a dicha actividad. De este modo, es posible apreciar que junto con lo funcional, el uso de los artefactos líticos o de la piedra en general comienzan a adquirir un carácter versátil y simbólico que se expresa en la challa de materiales molidos en tumbas y chullpas, así como también en la reutilización y polifuncionalidad de muchas herramientas asociadas a esos materiales, en especial la molienda. Así, se configura, una conducta común para el resto del área, es decir, en conjunto con el río Loa, donde es bastante equivalente la manera en que se producen los artefactos líticos, con un énfasis en la molienda y el procesamiento de minerales con connotaciones simbólicas, como la malaquita (Uribe y Carrasco 1999). De acuerdo con lo mencionado, esto es coherente con la concentración de estructuras tipo chullpa y sepulturas al interior de los poblados en Catarpe, Quítor y sobre todo en Zápar y Peine, lo cual es a su vez coincidente con las apreciaciones sobre el material cerámico y su énfasis en el almacenamiento y la actividad ceremonial. Al respecto, el material lítico confirma la necesidad de procesar y acopiar ciertos alimentos, los que seguramente se emplearon en las festividades comunales, pero, asimismo, en el mantenimiento de otras labores como una minería asociada a la extracción de recursos de valor simbólico (Núñez 1999). Dentro de esta lógica, resulta considerable la producción de cuentas de malaquita y calcedonia, aparte de las de concha, que se suman al ámbito de la lítica y que se registran de manera generalizada en todos los asentamientos estudiados, aunque en proporciones modestas. Esto, sin duda, potenciaría el intercambio y caravaneo característico de la región con el propósito de ampliar la complementariedad de recursos a través de contactos a larga distancia. Para ello, en esos momentos las autoridades de cada comunidad debieron liderar su promoción y organización, ya que debido a la segmentación territorial predominante cada unidad lo llevaría a cabo independientemente y no de forma tan centralizada en San Pedro, como parece haber ocurrido con actividades semejantes durante el Periodo Medio (Llagostera 1996). Restos de fauna y vegetales. Bajo la misma óptica con que se trabajaron los materiales cerámicos y líticos se recolectaron y analizaron restos óseos y vegetales de los asentamientos descritos (Tabla 4), los cuales fueron identificados por especies e inicialmente asignados a labores vinculadas con la preparación, manipulación y almacenamiento de alimentos, además de actividades funerarias 320 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO a b Fig. 11. a. Aldea de Zápar. Concentración de instrumentos de molienda asociados a estructuras habitacionales; b. Pucara de Vilama Norte. Concentraciones de piedras asociadas a muro perimetral, posiblemente usadas como proyectiles. (González 2002). No obstante, muchos también pudieron ser el resultado de otras actividades, como las pastoriles, y de esa manera haber ingresado al contexto arqueológico perdurando, más o menos, gracias a las condiciones de conservación específicas de cada uno de los yacimientos. De este modo, se recuperaron restos vegetales, entre los cuales predominan el chañar, maíz y algarrobo, aparte de ramas y maderas no consideradas dentro de las labores domésticas señaladas. Por su parte, el material óseo se encuentra constituido principalmente por astillas de hueso, siendo preponderantes los restos de camélidos (Lama sp.), además de fauna local, silvestre e introducida. Comparando cada uno de los sitios estudiados, desde Sólor a Peine, en todos se observa que la presencia de fauna se mantiene uniforme, mientras que el material vegetal adquiere un notorio aumento en Catarpe, Quítor, Zápar y Peine. Al respecto, comparando con la cerámica y el lítico, se sostiene que la segregación observada entre las proporciones de las evidencias óseas y vegetales refleja la existencia de dos modos de EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 321 Materiales orgánicos (%) Oseo Vegetal Sólor 34,9 0,40 Guatin 2,4 0,1 Vilama Norte 8,4 0,2 Vilama Sur 1,2 0,6 Catarpe Este 2,8 3,4 Catarpe Oeste 1,3 8,9 Quítor 4,9 13,8 Zápar 26,0 31,4 Peine 17,6 40,8 n=2275,5g n=2571g Sitios Tabla 4: Resumen de la frecuencia de materiales orgánicos de sitios habitacionales tardíos de San Pedro de Atacama. producción distintos en el tiempo. En los momentos más tempranos se trataría de una economía con énfasis en la ganadería, que sustentaba principalmente actividades de tráfico o caravaneo, como lo sugiere la clara configuración de un transecto pastoril entre Sólor, Vilama y Guatin, del mismo modo que la existencia de una industria para fabricación de cuentas en el primero, especialmente a partir de moluscos, e incluso de metales (cf. Llagostera 1996). Este sistema económico estaría apoyado en un sistema agrícola comparativamente poco relevante, quizás de autosubsistencia, con mayor énfasis en la recolección de productos de fácil manejo, si es que no silvestres, como son los frutos de chañar y algarrobo abundantes en todos los oasis de San Pedro, coherente con el patrón de asentamiento señalado entre 950 y 1350 d.C. En cambio, los sitios del segundo grupo, más tardíos, representarían una transformación de ese sistema ganadero y caravanero, el cual estaría apoyado en una producción agrícola con otra importancia, más estable y de mayor escala, capaz de sustentar una cantidad de población más grande y segmentada. Lo anterior confirma y es del todo coherente con las particularidades que adquieren los asentamientos y las actividades desarrolladas en ellos en términos de sus desechos cerámicos y líticos, coincidentes con una nueva realidad demográfica y cultural, evidentes en prácticas de almacenamiento y ceremoniales incluidas en los ámbitos habitacionales que constituyen unidades independientes, aunque relacionadas. Ajuares y ofrendas. Para acercarse a la población que habría protagonizado estos cambios, se estudiaron 90 cráneos de los contextos funerarios de Yaye, Sólor y Catarpe con el propósito de caracterizar biológicamente a los habitantes tardíos del Salar de Atacama, evaluándolos según su condición de salud (edad, sexo, patologías, etc.), a partir de atributos cráneo-faciales, mandibulares, dentales y deformación cefálica (Reyes 2002; Uribe et al. e.p.). De acuerdo a este estudio preliminar —y a pesar del grave problema de la muestra, debido al trabajo exclusivo con el esqueleto craneal, incluso incompleto a veces— el conjunto correspondería a una misma población, bastante relacionada con las tierras altas del río Loa (cf. Ayala et al. 1999), afectada toda ella por las mismas eventualidades durante su existencia (v.g., alta esperanza de vida, pero trabajo duro, muchas enfermedades dentales y a veces etapas de carencia de alimentos o de baja calidad nutricional que afectan bastante la salud, entre otros). Sobre esta misma base poblacional, sin embargo, se desarrollarían diferencias internas importantes, cronológicas o sociales que se manifiestan en ajuares y ofrendas. Pero, además, ciertos 322 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO rasgos óseos —como la deformación craneana que se registra en la mitad de la muestra de manera indistinta entre hombres y mujeres, bastante diversa, aunque casi siempre bajo el predominio de la deformación tabular erecta, seguida de la oblicua y las circulares— también podrían vincularse a la complejidad sociopolítica de este grupo (cf. Torres-Rouff 2002), aunque todavía se necesita más estudios para afirmarlo. Por lo mismo, se enfocará el comportamiento de los textiles y los objetos misceláneos de otras materias primas que componen sus ajuares y ofrendas. Material textil. Estudios textiles desarrollados con anterioridad distinguen, a partir del Periodo Intermedio Tardío, un comportamiento estilístico particular del territorio comprendido por el desierto de Atacama, el cual se desenvuelve en la configuración de una identidad que transforma los parámetros del periodo anterior e incorpora aquellos de sus zonas de frontera, fortaleciendo su propio carácter y, quizá con ello, su acceso a otros espacios y recursos (Agüero et al. 1997, 1999; Agüero 1998). Tomando en cuenta esos avances, el trabajo realizado consideró el análisis de tejidos funerarios de San Pedro de Atacama como de cementerios del río Loa y del Noroeste argentino (específicamente del río Doncellas), a través de un registro sistemático de sus atributos tecnológicos, formales y decorativos (Agüero 2000). De todo este territorio se estudió un total de 946 prendas de vestir, tocados y bolsas, distinguiéndose dos versiones textiles propias del Salar. Por un lado, aquella representada en los cementerios que continúan ocupados después del Periodo Medio (Fig. 13), los cuales comparten otros elementos mucho más populares en el río Loa e incluso de origen foráneo (v.g., Tarapacá). Y, paralela a la anterior, otra versión que consiste en tejidos estructuralmente idénticos, pero propios de cementerios sin ocupaciones previas y con una mayor continuidad hacia los momentos tardíos (Fig. 12). Esto significa que este segundo conjunto —formado por túnicas cuadradas o rectangulares tejidas en tramas múltiples, con bordados laterales en puntada satín, además de gorros tipo «corona», «saquitos-amuletos» y bolsas hechas en sprang o torzal oblicuo doble— coexiste con la textilería de mayor tradición en el oasis de San Pedro y que comienza a desarrollarse en el Loa y sus bordes, junto con aquella que se describe a continuación. Hacia el 1350 d.C. se aprecia en el Loa (Agüero 1998) y las tierras altas del Noroeste argentino (Boman 1908; Rolandi de Perrot 1973, 1979; Pelissero et al. 1997), la popularización de un grupo de prendas rectangulares correspondientes a túnicas y bolsas, que utilizan técnicas decorativas de urdimbres transpuestas con uso de tramas múltiples y tapicería dentada (dovetailed), los cuales reproducen motivos con forma de «V» o rombos en sucesión vertical (en el caso de la transposición de urdimbres), así como cuadrados y una característica figura zoomorfa de tres dedos (en el caso de la tapicería), todos ellos confeccionados con hilados de colores blanco, azul y rojo, al igual que en las numerosas bolsas elaboradas en faz de urdimbre (Fig. 14). Su distribución geográfica y la utilización de tramas múltiples permiten insertar estas prendas dentro del componente «atacameño» o, mejor dicho, circunpuneño. Al mismo tiempo, la amplitud geográfica que abarca este conjunto, sumada a la abundancia de bolsas, sugiere su asociación con la actividad caravanera, lo que abre la posibilidad de que puedan ser situadas hacia finales del Periodo Intermedio Tardío y que hayan tenido contacto con el Inka, extendiéndose su circulación hasta Calingasta por el sur (Michieli 1994) y por el norte hasta Camarones y Arica (Ulloa, comunicación personal 1998). Coincide con esta situación que ciertos sitios propiamente tardíos de San Pedro de Atacama exhiben algunas túnicas, bolsas y gorros tipo «corona» como los de dicho conjunto textil, en momentos que parecieran ingresar los atuendos incaicos pertenecientes a figurillas de santuarios de altura (CIADAM 1978; Agüero 2000). Por lo tanto, aunque el tamaño de la muestra es bastante exiguo, se ha logrado distinguir un nuevo conjunto textil —el incaico—, que presenta novedosas combinaciones de técnicas y colores (Fig. 15 a), aún cuando está reservado a un contexto sumamente específico, diferente al del resto de los textiles considerados. De acuerdo a ello, es evidente que el estudio de los tejidos tardíos se ha enfocado en prendas elitistas, lo cual impide tener información EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 323 Fig. 12. Textiles de Atacama del Periodo Intermedio Tardío, presentes en cementerios sin ocupaciones del periodo Medio. a. Gorros de piel tipo «corona»; b. «Saquitos-amuletos»; c. Bolsa en torzal oblicuo doble; d. Túnicas decoradas por bordados laterales en puntada satín. 324 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO Fig. 13. Túnicas de Atacama del Periodo Intermedio Tardío, presentes en cementerios con ocupaciones previas, así como en zonas de frontera, mostrando bordados en puntada satín y listas laterales que incluyen decoración por urdimbres flotantes y/o complementarias. EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 325 acerca de su comportamiento en otros contextos con los que eventualmente podrían ser comparadas las prendas locales de San Pedro (Rowe 1978, 1995-1996; Michieli 1990; Dransart 1995). Sin embargo, la situación anterior se ve compensada en parte por hallazgos en Peine (gorro tipo corona y bolsa), los cuales se vinculan con la textilería previa y local. En particular, una bolsa presenta urdimbres transpuestas, decoración y colores que la insertan dentro del estilo tardío propuesto para la cuenca del Loa y el Noroeste argentino (Agüero et al. 1997, 1999; Agüero 2000). Pero, al mismo tiempo, muestran una gran estandarización en el uso de los colores y en los patrones decorativos, en cierto sentido la misma de los tejidos de estatuillas y santuarios de altura, por lo que casi con certeza pueden ser atribuidos a la presencia del Inka en la zona. Esto, por otra parte, no sugiere una simple adscripción a los patrones estilísticos incaicos, porque de hecho estéticamente no parece haber sido así (no es el mismo estilo de los santuarios de altura), sino más bien se trataría de una consecuencia de la intervención de la industria local por parte del Tawantinsuyo. Esta intervención significaría una reorganización de la producción textil, cuyo propósito finalmente se vincularía con los cambios ocurridos por la integración a un aparato estatal. Al mismo tiempo, en el ámbito del intercambio caravanero implicaría un tráfico distinto, totalmente controlado por el Inka, perfilando una manufactura de bolsas bien evidenciada en el Noroeste argentino (Fig. 14 c), casi en serie para un movimiento de recuas a una escala mucho mayor, observándose a través de este estilo un indudable vínculo entre ambos territorios. Sin duda, el manejo de los líderes locales debió ser clave para la implementación de esta clase de producción en cada una de las comunidades comprometidas o reuniendo a sus propios grupos de especialistas. Tabletas, calabazas y otro objetos misceláneos. Siguiendo un estudio y comportamiento muy similar al de los tejidos, la observación de 3432 objetos misceláneos de contextos funerarios consistentes en piezas enteras permite distinguir como primera característica importante: la existencia de una sólida tradición o continuidad cultural desde los inicios del Periodo Intermedio Tardío en los oasis de San Pedro de Atacama. Lo anterior se observa principalmente en los tipos de tabletas, tubos, morteros y espátulas de madera y hueso que componen el característico complejo sicotrópico de la región. Junto a ellos, además, se encuentran calabazas con y sin decoración pirograbada, cucharas e instrumentos relacionados con el trabajo agrícola, textil, caravaneo y caza, entre otros. Dicha tradición habría perpetuado un estilo o, más bien, una «estética» respecto a las clases de objetos ofrendados, los modos de fabricación de los mismos y, con certeza, los criterios para la selección como adquisición de productos de intercambio (Catalán 2002). A inicios del periodo, en los contextos funerarios de San Pedro los ajuares y ofrendas tienden a mantener ciertas características de la época previa (Costa y Llagostera 1992), pero ya no tienen el despliegue iconográfico y estético de antes, apreciándose más bien una especie de «simplificación» iconográfica y formal de los objetos, que ha sido mal interpretada como un «empobrecimiento» de la sociedad atacameña (Le Paige 1964; Tarragó 1989). Al contrario, esto significa un mayor acercamiento a lo cotidiano y una potenciación de la producción de excedentes basado en una agricultura y ganadería intensivas (Adán y Uribe 1995). Esta característica se ve reforzada con la incorporación de implementos para labores cotidianas asociadas al trabajo agrícola, textil y la subsistencia en general, los cuales empiezan a ocupar un importante lugar en el rito mortuorio (cf. Ayala et al. 1999). Tal simplificación, entonces, radicaría posiblemente en la concepción más cercana de la convivencia entre los vivos y los muertos dentro de un espacio simbólico que se ha hecho explícito en la arquitectura ceremonial y para el almacenamiento de los asentamientos más clásicos del desarrollo regional. De este modo, los elementos del complejo sicotrópico que se habrían incrementado profusamente en esta región con la influencia tiwanaku persisten durante los periodos tardíos, pero no de forma masiva. Las tabletas, especialmente como las de Catarpe, se caracterizan por tener un recipiente rectangular y mango con extensión plana que puede o no llevar decoración, correspon- 326 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO diente a figuras antropomorfas y zoomorfas, en cuyo caso tienden a representar sólo la cabeza y el cuello del animal (Fig. 16 g, h). Por su parte, las cucharas son objetos que muestran una alta sensibilidad al cambio morfológico más que al decorativo, ya que en el Periodo Tardío no suelen llevar manifestaciones de esta clase, la que se restringe a motivos zoomorfos, al combinar trazos naturalistas con otros más esquemáticos o, bien, aserrados o «recortes» a lo largo de los mangos (Fig. 16 f). Pero el rasgo formal más diagnóstico es que la pala se ubica sobre el nivel del mango, el cual puede ser recto o levemente divergente hacia el extremo terminal y de pala oval (Figs. 16 e, f). Por último, las calabazas presentan una evolución clara con relación a sus temáticas iconográficas, distinguiéndose un primer momento en los cementerios de Yaye, donde se observa una decoración simple representada por triángulos dispuestos por fuera alrededor del borde que, al parecer, se trataría de un diseño local (Fig. 16 a). No obstante, luego se produciría una situación diferente vista en Catarpe, donde se distinguen motivos directamente importados desde el Noroeste argentino, representados por triángulos con espirales o volutas que se asemejan a los de la cerámica puneña de Humahuaca (Fig. 16 b, d). Todas estas evidencias materiales señalan contactos culturales con los cursos medio e inferior del río Loa, sobre todo a través de los objetos de madera (Ayala et al. 1999), al mismo tiempo que, como los textiles, aluden a una relación más profunda con el Noroeste argentino. Además, entre todas estas regiones se desarrollaron temáticas iconográficas similares, existiendo una tendencia hacia la humanización de las figuras en los objetos relacionados con el complejo del rapé (Fig. 15 b), donde los personajes muestran un aspecto humano pero sin los rasgos del felino que se observan en momentos de la influencia tiwanaku (cf. Torres 1984a, b). Tal situación sugiere que la organización social y el poder político no estarían basados en un conocimiento y manejo de lo sobrenatural, sino en el dominio estratégico de prácticas asociadas a las actividades cotidianas mencionadas. Consecuentemente se integraría al río Loa dentro de esta dinámica debido a lazos más estrechos que se establecen con su curso medio, en particular con las localidades de Lasana y Chiuchiu, ricas en tierras agrícolas y ganaderas, distinguiéndose motivos decorativos compartidos entre las tabletas y tubos, a los que se unen los de las calabazas. No obstante, también se perciben ciertas distancias estilísticas con el Loa superior (cf. Alliende 1981), mientras que en el Loa inferior se aprecian elementos que representan un espacio de contactos con las tradiciones de los Valles Occidentales de Arica y Tarapacá (cf. Cervellino y Téllez 1980). Por su parte, en estos mismos momentos del Periodo Intermedio Tardío se integraría una iconografía foránea y más abstracta que la observada en las calabazas y que incluso llegarían a ser más populares que el complejo sicotrópico. Estos, quizás, constituirían objetos de importación desde el Noroeste argentino donde son mucho más comunes (Durán 1976), siendo prácticamente idénticos los diseños y motivos que aparecen en ese territorio, particularmente los del río Doncellas (Gentile 1990). En suma, se confirma una situación cultural diferenciada en el tiempo, la cual se vincularía con la existencia de poblaciones que en una etapa inicial se mantienen dentro de las antiguas tradiciones materiales, frente a otras más tardías que remiten a elementos nuevos, especialmente compartidos con el Noroeste argentino y el Altiplano meridional de Bolivia, volviéndose predominantes. Esto implica la presencia de una importante interacción circunpuneña que podría extenderse, al menos, hasta Tarapacá. Sin duda, una importante complejidad social y política debió verse representada por esta situación, involucrando diversas negociaciones entre las autoridades que dirigían estas «empresas», dentro de una ardua competencia por el mejor posicionamiento de las distintas comunidades. Por lo mismo, las concepciones acerca del poder habrían cambiado como Fig. 14. (Desplegable en la página siguiente). Textiles de finales del Periodo Intermedio Tardío y del Periodo Tardío circunpuneño. a. Túnica con decoración por urdimbres transpuestas; b. Túnica con decoración por tapicería enlazada o dovetailed; c. Bolsas. EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 327 a b Fig. 15. a. Textil de estilo foráneo del Periodo Tardío; b. Tableta del complejo sicotrópico con mango de tallado volumétrico con figura antropomorfa de los periodos Intermedio Tardío y Tardío (Museo Caspana). parece expresarse en las nuevas manifestaciones iconográficas, en las cuales destacan las figuras humanas por sobre las imágenes de seres sobrenaturales. Esta complejidad «atacameña» debió implicar, por otro lado, un impulso de gran escala para el desarrollo experimentado en toda la región por la producción agrícola y caravanera (cf. Núñez 1992), igualmente explícito en los materiales funerarios, lo que el Inka supo anexar para su propio provecho, en particular empleando el vínculo con el Noroeste argentino. 4. Conclusiones: Atacama en tiempos del Inka A lo largo de este trabajo se han expuesto, en la medida de lo posible, aquellas evidencias y argumentos que parecen representar mejor la complejidad social, económica, política y étnica que se MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO 328 Fig. 16. Objetos funerarios misceláneos. a. Calabaza pirograba de inicios del periodo Intermedio Tardío; b, c, d. Calabazas pirograbadas de los periodo Intermedio Tardío y Tardío; e, f. Cucharas con pala sobre el mango de los periodos Intermedio Tardío y Tardío; g, h. Tabletas del complejo sicotrópico con mango plano y tallado volumétrico de los periodos Intermedio Tardío y Tardío. EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 329 encontraba en pleno desarrollo cuando el Inka tomó contacto con las poblaciones del desierto de Atacama. En particular, los autores se concentraron en el caso de San Pedro de Atacama, porque tratándose de una de las localidades que ha servido como punto de referencia para la comprensión arqueológica de los grupos que habitaron este desierto y sus tierras altas (v.g., Berenguer et al. 1986; Tarragó 1989; Núñez 1992), todavía se conoce muy poco de aquellos momentos. Es un hecho que la investigación se ha centrado en otros periodos de la prehistoria regional, en la que la esplendorosa época de relaciones con Tiwanaku ha cegado la mirada hacia los momentos posteriores y se ha asumido, sin mayor cuestionamiento, una continuidad cultural entre las poblaciones prehispánicas, coloniales y actuales, la cual casi no ha sido sometida a evaluación arqueológica (cf. Orellana 1963; Castro et al. 1979; Adán et al. 1995). Por último, se hace hincapié en el estudio de este momento de convergencia de las poblaciones atacameñas e «incaicas» dentro de San Pedro, porque la experiencia obtenida en otras localidades de este territorio —específicamente Caspana— ha permitido vislumbrar en ese encuentro los elementos que particularizaron a la complejidad social local durante el Periodo Intermedio Tardío. Retomando ese conocimiento (Uribe et al. 1999; Adán y Uribe e.p.; Uribe y Adán e.p.), se sabe que después de 1450 d.C., en el Loa superior las poblaciones locales y el Inka dialogan a través de la arquitectura, probablemente porque se trata de un dominio significativo para ambos actores sociales, demostrando cierta «tensión social» (Uribe y Adán e.p.). Esto, dado que, por un lado el Inka —o lo foráneo— construyen para dominar a través de su monumentalidad, estandarización y visibilidad, donde se exhibe y sobrerepresenta (Adán 1999), conectado por una notable red vial vinculada a recursos mineros y agrícolas, con importantes manifestaciones de arte rupestre. Sin duda, se trata de una jerarquía administrativa, funcional y productiva que, en este caso, relaciona sitios de distinta envergadura que se denominaron instalaciones, instaurando la soberanía de agentes externos. En este contexto, lo local, por su parte, está libre de la monumentalidad y notoriedad del dominio inkaico (ibid.); no obstante, las labores productivas muestran una importante intervención a través de una expresión inka local (cerámica) en todas las clases de contextos funcionales que abarcan ampliamente cada uno de los asentamientos como los estudiados —consumo de alimentos y líquidos, molienda, almacenamiento y elaboración de artesanías— al mismo tiempo que en ellos se incrementan los elementos foráneos y exóticos, en especial del Noroeste argentino (Uribe 1999; Uribe y Carrasco 1999). El Inka, con relación a la producción lítica, concentra en sus instalaciones las evidencias de la explotación de mineral y su procesamiento a través de martillos y cobre triturado como la malaquita, pero dentro de una práctica ya conocida localmente, implementada de manera ceremonial y, tal cual se vio, realizada en los mismos poblados. En su expansión, por lo tanto, el Tawantinsuyo habría impulsado el desarrollo de las fuerzas productivas a través de una fuerza de trabajo numerosa previa, apoyada en el crecimiento demográfico local y de sus medios de producción, sobre todo agrícolas, cuya mayor fuerza de trabajo requirió de una organización social más compleja. Eso mismo obligaría al control de esas fuerzas, a modo de una burocracia administrativa o un grupo encargado de dirigirla desde la actividad doméstica a la estatal, modificando las relaciones de propiedad objetiva y, consecuentemente, la división social del trabajo. Las poblaciones locales de Atacama, entonces, desarrollaron aún más sus fuerzas productivas y potenciaron la generación de excedentes impulsando una jerarquía social que conllevaba a un sistema políticamente cada vez menos igualitario (Uribe y Adán e.p.). De este modo, tal cual se aprecia en Catarpe Este, con el Inka son más claros los espacios públicos como expresión del aparato administrativo, que demuestran el control a través del cual se dirige la fuerza de trabajo hacia la producción estatal, distinguiéndose un grupo productor de bienes y alimentos, de otro especializado en las actividades político-ideológicas y administrativas expuestos en ciertos cementerios (Ayala et al. 1999), y las cuales tampoco faltan en el Salar (v.g., Hostería de 330 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO San Pedro). En definitiva, el Tawantinsuyo se apropia de la fuerza de trabajo y la producción de las unidades domésticas como un excedente que es manejado por los encargados de los segmentos ya diferenciados de la misma población local, que ahora se convierten en administradores, incluso políticos e ideológicos. Así, paralelamente, se genera un amplio control de la conciencia social a través de la participación ideológica en la formación económica-social del imperio (Uribe y Adán e.p.). De esta manera, lo inka local mantendría la cohesión y el dominio sin violencia real. Ello por medio de referentes sociales significativos como las fiestas de redistribución dirigidas por jefes locales, las que jugaron un papel crucial como lo evidencia el incremento de contenedores de líquidos y contextos de preparación de alimentos y almacenamiento que se venía dando de antes, convirtiéndose en la trama religiosa, social y estética que sustenta y reproduce al sistema inkaico. En suma, antes de la aparición del Inka, en el desierto de Atacama existían sociedades complejas, segmentadas e internamente jerarquizadas; con el Tawantinsuyo se agudiza la división social y la propiedad particular, dando como resultado sistemas sociales andinos originales, pero desiguales, que emplean prácticas sociales arraigadas como la reciprocidad y la redistribución, generando estructuras verticales al interior de las sociedades locales, entre éstas como las del Loa y San Pedro, y con las poblaciones del núcleo estatal más cercano como el Noroeste argentino. En este contexto, el carácter «sagrado» del aparato imperial acompañaría y encubriría las contradicciones anteriores, dejando que cada entidad actuara dentro de una aparente autonomía (segmentación), bajo la necesidad de complementariedad económica y en una armonía social, tal cual lo sugiere la presencia del Inka que articula el sistema en los bordes o periferias más que en los centros. En este marco, el Inka pareciera imponer un nuevo orden en la geografía y paisaje de las poblaciones locales, lo cual pareciera ser explícito en otras materialidades no abordadas en esta oportunidad, como la vialidad y el arte rupestre. Se sabe que instauran lugares especiales, quizás a modo de huacas, que al estar cerca de las áreas productivas como campos de cultivo, pastoreo y minas las incluyen dentro de su propiedad (Vilches y Uribe 1999; Sepúlveda 2002). Una clara evidencia material de este comportamiento lo constituye un ushnu entre el Loa y San Pedro —Cerro Verde—, que pareciera articular la unidad «atacameña» preexistente y consolidada por el Inka, la misma que en tiempos coloniales se arrogaría a una misma deidad y, en este sentido, a un solo orden: «...el idolo Llamado sotar condi a quien todos los yndios de estas Prouincias teníamos Por dios el qual idolo estaua bestido de cumbe con supillo y plumas en el de oro y Pajaro flamenco...» (Castro 1997: A-30, fol. 40r). A estas deidades los «...indios cassados y cassas de familias por uia de mita [daban] carneros de la tierra cuyes chicha plumas de pajaros flamencos cossas yerbas de olor Para saumerios del demonio y los que no eran cassados ni cabeças de cassas yban a asistir a los dichos sacrificios» (ibid.: A-30, fol. 39r). Agradecimientos Agradecemos en primer lugar a los doctores Peter Kaulicke, Ian Farrington y Gary Urton por permitirnos participar en el IV Simposio de Arqueología PUCP; al Fondo Nacional de Ciencia y Tecnología (FONDECYT) que financió esta investigación; al Instituto de Investigaciones Arqueológicas y al Museo R. P. Gustavo Le Paige S. J. de San Pedro de Atacama, por su apoyo constante y, asimismo, a las comunidades indígenas «atacameñas» de San Pedro, por su comprensión con este trabajo. Este trabajo no habría sido posible sin la colaboración del equipo conformado por amigos y colegas: Patricia Ayala, Carlos Carrasco, Bárbara Cases, Claudia del Fierro, Josefina González, Indira Montt, Viviana Manríquez, Sergio Morales, Omar Reyes y Lorena Sanhueza. Además, les damos gracias a todos aquellos que participaron responsable y desinteresadamente en las distintas campañas de campo. Asimismo, nuestro reconocimiento a Paulina Chávez, por sus excelentes ilustraciones. EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 331 Notas 1 Este artículo es resultado del Proyecto FONDECYT 1000148, denominado Historia cultural y materialidad en la arqueología de los periodos Intermedio Tardío y Tardío de San Pedro de Atacama y su relación con la cuenca del río Loa. 2 Archivo General de Indias (1557), Patronato, legajo 188, n.° 4. 3 Categorías funcionales para arquitectura tardía han sido propuestas por Adán (1995, 1996). 4 Tipología propuesta por Ayala (1998 y 2000). 5 UCTL-1432 (1590 ± 40 d.C.), UCTL-1433 (1540 ± 30 d.C.) y UCTL-1434 (1570 ± 40 d.C.). 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EL DOMINIO DEL INKA, IDENTIDAD LOCAL Y COMPLEJIDAD SOCIAL EN ATACAMA... 335 Rowe, A. P. 1978 Technical Features of Inca Tapestry Tunics, The Textile Museum Journal 17, 5-28, Washington, D.C. 1995 -1996 Inca Weaving and Costume, The Textile Museum Journal 34-35, 4-53, Washington, D.C. Schiappacasse, V., V. Castro y H. Niemeyer 1989 Los desarrollos regionales en el Norte Grande, en: J. Hidalgo, V. Schiapacasse, H. Niemeyer, C. Aldunate y I. Solimero (eds.), Culturas de Chile. Prehistoria. Desde sus orígenes hasta los albores de la Conquista, 181-220, Andrés Bello, Santiago. Sepúlveda, M. 2002 Imagen rupestre y espacialidad en el desierto de Atacama durante los periodos tardíos (950-1550 d.C.), memoria de licenciatura inédita, Departamento de Antropología, Universidad de Chile, Santiago. Serracino, G. y R. Stehberg 1974 Vida pastoril en la precordillera andina, Estudios Atacameños 3, 81-89, San Pedro de Atacama. Tarragó, M. 1989 Contribución al conocimiento arqueológico de las poblaciones de los oasis de San Pedro de Atacama en relación con los otros pueblos puñenos, en especial al sector septentrional del valle Calchaquí, tesis de doctorado inédita, Especialidad de Arqueología, Universidad Nacional de Rosario, Rosario. Torres, C. 1984a Tabletas para alucinógenos de San Pedro de Atacama: estilo e iconografía, en: Tesoros de San Pedro de Atacama, 23-36, Museo Chileno de Arte Precolombino, Santiago. 1984b Iconografía de las tabletas para inhalar substancias psicoactivas de la zona de San Pedro de Atacama, norte de Chile, Estudios Atacameños 7, 178-195, San Pedro de Atacama. Torres-Rouff, C. 2002 Cranial Vault Modification and Ethnicity in Middle Horizon San Pedro de Atacama, Chile, Current Anthropology 43, 163-171, Chicago. Uribe, M. 1996 Religión y poder en los Andes del Loa: una reflexión desde la alfarería (Periodo Intermedio Tardío), memoria de licenciatura inédita, Departamento de Antropología, Universidad de Chile, Santiago. 1997 La alfarería de Caspana y su relación con la prehistoria tardía del área circunpuneña, Estudios Atacameños 14, 243-262, San Pedro de Atacama. 1999 La alfarería inca de Caspana, Boletín de la Sociedad Chilena de Arqueología 27, 11-19, Santiago. 1999 -2000 La arqueología del Inka en Chile, Revista Chilena de Antropología 15, 63-97, Santiago. e.p. Sobre alfarería, cementerios, fases, procesos y la construcción de Atacama en la prehistoria tardía (800-1600 d.C.), Estudios Atacameños 23, San Pedro de Atacama. Uribe, M. y L. Adán 1995 Tiempo y espacio en Atacama: la mirada desde Caspana, Boletín de la Sociedad Chilena de Arqueología 21, 35-37, Santiago. e.p. Acerca del dominio Inka, sin miedo, sin vergüenza..., para publicarse en: Chungará, Arica. Uribe, M., L. Adán y C. Agüero e.p. Los periodos Intermedio Tardío y Tardío de San Pedro de Atacama y su relación con la cuenca del Loa, para publicarse en: Chungará, Arica. Uribe, M. y C. Carrasco 1999 Tiestos y piedras talladas: la producción cerámica y lítica en el Periodo Tardío del Loa superior, Estudios Atacameños 18, 55-72, San Pedro de Atacama. 336 MAURICIO URIBE, LEONOR ADÁN Y CAROLINA AGÜERO Uribe, M., V. Manríquez y L. Adán 1999 El poder del Inka en Chile: aproximaciones a partir de la arqueología de Caspana (río Loa, desierto de Atacama), en: Actas del Tercer Congreso Chileno de Antropología, tomo II, 706-722, Temuco. Varela, V., M. Uribe y L. Adán 1993 La cerámica arqueológica del sitio «Pukara» de Turi: 02-TU-001, Boletín del Museo Regional de la Araucanía 4, tomo II 107-122, Temuco. Vilches, F. y M. Uribe 1999 Grabado y pinturas del arte rupestre tardío de Caspana, Estudios Atacameños 18, 73-88, San Pedro de Atacama. Vivar, G. de 1979 Crónica y relación copiosa y verdadera de los reinos de Chile (edición de L. Sáez), Biblioteca Ibero[1558] Americana, Colloquium Verlag, Berlin.
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Direct serogrouping of Dichelobacter nodosus from Victorian farms using conventional multiplex polymerase chain reaction
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Best et al. BMC Res Notes (2018) 11:108 https://doi.org/10.1186/s13104-018-3229-5 Best et al. BMC Res Notes (2018) 11:108 https://doi.org/10.1186/s13104-018-3229-5 BMC Research Notes © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Objective:  Dichelobacter nodosus is the causative agent of footrot in sheep. Ovine footrot is a major problem in Australia that results in large economic losses and a represents a very significant animal welfare issue. D. nodosus is divided into 10 serogroups (A–I, M), based on sequence variation in the type IV fimbriae gene, fimA. Control of the bacteria is possible through use of serogroup-specific vaccination, however traditional identification of the sero- groups of D. nodosus on infected sheep is time-consuming and costly. With the aim of reducing time and cost, a PCR assay was used to identify serogroups of D. nodosus directly from foot swabs of infected sheep in Victoria. Results:  It was shown that serogroup B was most common (10 locations), followed by A, G and H (4 locations), I and C (2 locations), D, E and F (1 location). Infections with multiple serotypes were observed in 50% of farms, with the remaining 50% having only a single serogroup detected. The ability to identify serogroups quickly and cheaply direct from foot swabs will aid the understanding of the epidemiology of D. nodosus and support control programs. Keywords: Footrot Serogroup PCR Keywords:  Footrot, Serogroup, PCR from the host. Vaccination with serogroup-specific FimA antigen provides a protective response, however, cross protection between serogroups does not occur, and mul- tiple serogroup infections have been reported to be com- mon [4]. Understanding what D. nodosus serogroups are on a particular farm can support better epidemio- logical investigations, control and eradication programs [5]. Currently, the testing for serotype of D. nodosus is time-consuming and labour intensive. It requires bacte- rial isolation, culturing and slide agglutination testing to determine the serogroup. Slide agglutination requires the production antisera specific for each serogroup, which is beyond most diagnostic laboratories capacity. Direct serogrouping of Dichelobacter nodosus from Victorian farms using conventional multiplex polymerase chain reaction Nickala Best1, Jacek Gwozdz2, Robert Suter3, Grant Rawlin2 and Travis Beddoe1* *Correspondence: t.beddoe@latrobe.edu.au 1 Department of Animal, Plant and Soil Science and Centre for AgriBioscience (AgriBio), La Trobe University, Bundoora, Melbourne, VIC, Australia Full list of author information is available at the end of the article Introduction Ovine footrot is a bacterial infection in sheep, with viru- lent infections showing severe lesions in the hoof and resulting in lameness [1]. Footrot infections cost the sheep meat and wool industry $45 million per year in Australia [2]. The causative agent, Dichelobacter nodo- sus, is a genetically diverse, gram-negative anaerobe. D. nodosus is classified into different serogroups based on the sequence of the fimbrial A gene (fimA). In Australia, the bacteria are classed into 10 serogroups (A–I, M), with serogroups A–I being fully sequenced, while the Austral- ian serogroup M has been partially sequenced [3]. Each serogroup elicits a specific and strong immune response Recently, a multiplex PCR was developed to deter- mine the serogroup of D. nodosus from bacterial colo- nies grown on media [6]. This molecular assay has been shown to be highly specific and reduces time in identify- ing the serogroup of D. nodosus. The ability to determine D. nodosus serogroup directly from foot swabs would aid Best et al. BMC Res Notes (2018) 11:108 Page 2 of 6 particle handling system (Thermo Fisher Scientific, Fin- land), as per manufacturer’s instructions. epidemiological investigations of footrot in Australia. Here, we have identified the serogroups of D. nodosus from foot swabs collected on Victorian farms using this multiplex PCR assay. qPCR for detection of D. nodosus Detection of D. nodosus was performed by quantita- tive polymerase chain reaction (qPCR) as described by Stäuble et al. [7]. Briefly, the qPCR program consisted of an initial denaturation step of 2 min at 50 °C, a hold of 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C. The AgPath-IDTM One-Step RT-PCR Kit (Ambion, Austin, USA) was used as master mix accord- ing to manufacturer’s instructions, with final concentra- tions of 300  nM primers, 100  nM DnAprTM- vMGB, 250  nM DnAprTM-bMGB and 5  μL of template DNA used. Primers and probes were synthesised and supplied by Applied Biosystems (California, USA). Reactions were carried out and analysed using the 7500 Fast Real-Time PCR System (Life Technologies), with a set threshold of 0.05. Results are reported as cycling threshold (Ct) val- ues, the point at which the sample signal exceeds the threshold of 0.05. Materials and methods Field collectionh The clinical samples were obtained retrospectively from the Victorian Government Veterinary Diagnostic unit collection. The original samples were submitted by Victo- rian District Veterinary Officers, Animal Health Officers and private veterinary practitioners from the interdigital skin of lame sheep for routine diagnostic testing. Samples (n = 137) were collected from 16 farms across 15 Victo- rian locations. These farms were diagnosed as clinically virulent, benign or healthy during a disease surveillance survey from November 2014 to August 2015 (Fig. 1). The interdigital skin was swabbed and the cotton head of the swab transported at 4 °C in phosphate buffer saline (PBS) (8.1  mM ­Na2HPO4, 137  mM NaCl, 1.4  mM ­KH2HPO4 and 2.6  mM KCl) with an addition of 20  mM ethylen- ediaminetetraacetic acid (EDTA). Total nucleic acid was extracted using the MagMAX™ − 96 Viral RNA isolation kit (Ambion, Austin, USA) and Kingfisher-96 magnetic Results Foot swab samples were collected from farms that have been known previously to have clinical forms of footrot, or were clinically healthy yet had D. nodosus detected on animals. These samples and the collection method were used due to availability, as they were concurrently used for additional studies. Genomic DNA was extracted from all samples and the presence of D. nodosus in all 137 sheep were confirmed by qPCR as described by Stäuble et al. [7]. Initial experiments to determine the serotype of D. nodosus by PCR from DNA extracted from foot swabs was unsuccessful, however the addition of extra magnesium to the PCR reaction was able to achieve amplification of PCR products for the determination of serogroups present on different farms (Fig. 2). The repre- sentative PCR products were verified by DNA sequenc- ing as shown in Additional file 1: Figure S1. These results give a basic overview of the epidemiology of D. nodosus on farms throughout Victoria, which previ- ously has not been known. The ability to serotype directly from a swab greatly reduces the time and cost tradition- ally required to serotype field samples. This information and additional virulence testing may help in both under- standing and control of the disease in the future. i Overall the result revealed that 87 (63%) of the sheep tested resulted in a serogroup being determined by mul- tiplex PCR using DNA directly extracted from a foot swab sample (Table 1). From the animals that showed a serogroup band, 74% of the infected sheep had a single serogroup detected, while 26% had infections from mul- tiple serogroups, ranging from 2 to 4 serogroups detected (Table 1). Serogroup B was the most commonly detected serogroup as shown in Additional file 2: Figure S2), found in 48% of infections, on 56% of farms and in 60% of loca- tions in Victoria. Serogroup B was also involved in 91% of Discussion We have modified an existing protocol by increasing the concentration of ­MgCl2, for faster and more cost-effec- tive identification of D. nodosus serogroup determination by PCR, by analysing DNA extracted from foot swabs. This has allowed us to investigate the epidemiology of D. nodosus infection on farms throughout Victoria. It was found that on the sampled farms, there was a high level of detection of serogroup B. This is in agreement with a previous study that showed serogroup B was most com- monly detected [8]. Additionally, it was also found that serogroup B was most commonly found in multiple infections. Additional sampling across more locations is needed to gain an overall insight into the prevalence of differ- ent serogroups in Victoria, with this method providing a starting point for a higher throughput study. Currently, Victorian diagnostics laboratories are not equipped to isolate or grow D. nodosus or produce the antisera that is required for the traditional slide agglutination tests to determine serogroup. The further improvement of the methods here would allow for molecular diagnostics based laboratories to access this information. A more affordable and accessible method of serotyping would aid in epidemiological studies in Victoria and contribute to the improved control of the disease.h Serotyping by PCR The following amplification con- ditions consisted of an initial denaturation step of 4 min at 95 °C, followed by 5 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, followed by 25 cycles of 30 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C for 30 s and then a final elon- gation of 4 min at 72 °C. Amplicon size was checked by agarose electrophoresis migration by loading directly on 2% (w/v) ­MetaPhor® Agarose (Lonza, USA) gel, prepared as per manufacturer’s instructions, with the addition of 0.5 µL of ­Sybr® Safe DNA Gel Stain (Life Technologies). The gel was run at 100 V for 55 min and imaged using the GelDoc™ XR+ (BioRad) instrument and software. Sanger DNA sequencing of representative amplicons was per- formed at Australian Genomics Research Facility (AGRF) to verify amplified serogroups. the farms with multiple serogroup infections. Serogroups A, G and H were the next most common serogroups, found in 15, 25 and 12% of infections respectively. All were found in 4 locations in both single and multiple infections. Serogroup C and I were found in 2 farms in 2 separate locations, with D, E and F present in 1 location only. Serotyping by PCR Multiplex PCR for determination of serogroup was per- formed as described by Dhungyel et al. with the following Fig. 1  Victorian farms sampled. The locations of the 16 Victorian farms sampled and number of serogroups detected at each location. The figure was generated in Tableau 6.1 Fig. 1  Victorian farms sampled. The locations of the 16 Victorian farms sampled and number of serogroups detected at each location. The figure was generated in Tableau 6.1 Best et al. BMC Res Notes (2018) 11:108 Page 3 of 6 modifications [6]. ­GoTaq® Green Master Mix 2X (Pro- mega, Madison, USA) was used for amplification as per manufacturer’s instructions, with the addition of 1  µL 100  mM ­MgSO4 to increase the ­Mg+ concentration to 5.5 mM. Reactions were carried out in a final volume of 25  µL and amplified in a C-Master GT thermal cycler (Dynamica, Australia). The following amplification con- ditions consisted of an initial denaturation step of 4 min at 95 °C, followed by 5 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C, followed by 25 cycles of 30 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C for 30 s and then a final elon- gation of 4 min at 72 °C. Amplicon size was checked by agarose electrophoresis migration by loading directly on 2% (w/v) ­MetaPhor® Agarose (Lonza, USA) gel, prepared as per manufacturer’s instructions, with the addition of 0.5 µL of ­Sybr® Safe DNA Gel Stain (Life Technologies). The gel was run at 100 V for 55 min and imaged using the GelDoc™ XR+ (BioRad) instrument and software. Sanger DNA sequencing of representative amplicons was per- formed at Australian Genomics Research Facility (AGRF) to verify amplified serogroups. modifications [6]. ­GoTaq® Green Master Mix 2X (Pro- mega, Madison, USA) was used for amplification as per manufacturer’s instructions, with the addition of 1  µL 100  mM ­MgSO4 to increase the ­Mg+ concentration to 5.5 mM. Reactions were carried out in a final volume of 25  µL and amplified in a C-Master GT thermal cycler (Dynamica, Australia). Limitations It should be noted that not all samples tested returned a visible serogroup PCR band, and these will require further investigation. Experimentally, it may be possible bands were present yet were not detected using the current method. Additional detection methods were not inves- tigated. There is also the possibility that undetermined Best et al. BMC Res Notes (2018) 11:108 Page 4 of 6 l b l t M thi t i l d d i l ti lt i d lid l ti ti d t th l k Fig. 2  Identification of serogroups from foot swab samples by PCR. Samples from various farms were amplified in serogroups mixtures (ABC, DEF, and GHI) and analyzed on a 2% MetaPhor agarose gel. Banding is shown that corresponds to serogroup I (a and b), serogroups A, B, C and H (c, d and e), serogroups B, D and E (f, g and h) and serogroup F (i). The serogroup A and C positive control with no amplification seen in the negative, no template (NT), control Fig. 2  Identification of serogroups from foot swab samples by PCR. Samples from various farms were amplified in serogroups mixtures (ABC, DEF, and GHI) and analyzed on a 2% MetaPhor agarose gel. Banding is shown that corresponds to serogroup I (a and b), serogroups A, B, C and H (c, d and e), serogroups B, D and E (f, g and h) and serogroup F (i). The serogroup A and C positive control with no amplification seen in the negative, no template (NT), control isolation, culturing and slide agglutination due to the lack of suitable primers for identification of serogroup M by PCR. Therefore, it is imperative to develop primers suit- able for detection of serogroup M by PCR to understand the prevalence of serogroup M in Victorian sheep flocks. samples belong to serogroup M as this was not included in the PCR. There is limited information on the prevalence of serogroup M in Australian isolates, however, in two studies the numbers ranged from 5 to 40% of flocks [3, 9]. Currently, serogroup M identification requires bacterial Best et al. BMC Res Notes (2018) 11:108 Page 5 of 6 Table 1  Summary of serogroups found on farms across different locations in Victoria The animals tested were all confirmed to be D. Publisher’s Note Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Funding NB was responsible for the experimental work, data analysis, and writing of the manuscript. JG contributed to the writing of the manuscript and reviewed the drafts. RS, GR and TB coordinated the project, contributed to the experi- mental design, and reviewed the drafts. All authors read and approved the final manuscript. Funding The Victorian Sheep and Goat Compensation Fund provided funding for this project. The Victorian Sheep and Goat Compensation Fund provided funding for this project. Limitations nodosus positive via qPCR Location Farms Animals tested (n) Animals with serogroup detected (n) Serogroup (times detected) Animals with multiple serogroups (n) Avenel 1 10 6 G (6) 0 Bendoc 1 10 4 G (4), H (2) 2 Carboor 1 10 9 A (7), B (3), C (4), H (5) 8 Cavendish 1 10 9 B (1), G (8) 5 Giffard 1 1 1 C (1) 0 Hamilton 2 20 15 B (8), G (3), F (7) 3 Kernot 1 2 2 A (2), H (1) 1 Kialla East 1 10 5 I (5) 0 Kilmore East 1 10 4 A (1), B (2), H (2) 1 Lindenow South 1 10 1 I (1) 0 Murroon 1 9 6 B (1), D (4), E (4) 3 Newstead 1 10 8 A (3), B (5) 0 Rutherglen 1 9 6 B (6) 0 Strathbogie 1 6 1 B (1) 0 Tooborac 1 10 10 B (10) 0 Total 16 137 87 23 Table 1  Summary of serogroups found on farms across different locations in Victoria The animals tested were all confirmed to be D. nodosus positive via qPCR VIC, Australia. 3 Department of Economic Development, Jobs, Transport and Resources, Victorian Government, Attwood, Melbourne, VIC, Australia. Acknowledgements Additional file 1: Figure S1. PCR amplicons of different serogroups subjected to Sanger sequencing. Blastn alignment of serogroup G (panel A), I (panel B), B (panel C, E, F and G) and F (panel D) amplicon sequence obtained from Sanger sequencing from various farms. Accession number is shown in brackets. g We would like to thank the participating sheep owners for their time and assistance during sample collection, and the Victorian District Veterinary Offic- ers and Animal Health Officers for sample collection. Abbreviations PCR i i qPCR: quantitative polymerase chain reaction; EDTA: ethylenediaminetet- raacetic acid; DNA: deoxyribonucleic acid; PBS: phosphate buffer saline. Availability of data and materials The information supporting the conclusions of this article is included in the article. Competing interests p g The author(s) declared no potential competing interest with respect to the research, authorship, and/or publication of this article. Additional file 2: Figure S2. PCR amplicons of different serogroups subjected to Sanger sequencing. Blastn alignment of serogroup G (panel A), I (panel B), B (panel C, E, F and G) and F (panel D) amplicon sequence obtained from Sanger sequencing from various farms. Accession number is shown in brackets. 6. Dhungyel OP, Whittington RJ, Egerton JR. Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination. Mol Cell Probes. 2002;16(4):285–96. 7. Stauble A, Steiner A, Frey J, Kuhnert P. Simultaneous detection and discrimination of virulent and benign Dichelobacter nodosus in sheep of flocks affected by foot rot and in clinically healthy flocks by competitive real-time PCR. J Clin Microbiol. 2014;52(4):1228–31. 8. Claxton PD, Ribeiro LA, Egerton JR. Classification of Bacteroides nodosus by agglutination tests. Aust Vet J. 1983;60(11):331–4. 9. Chetwin DH, Whitehead LC, Thorley SE. The recognition and prevalence of Bacteroides nodosus serotype M in Australia and New Zealand. Aust Vet J. 1991;68(4):154–5. Author details 1 1 Department of Animal, Plant and Soil Science and Centre for AgriBioscience (AgriBio), La Trobe University, Bundoora, Melbourne, VIC, Australia. 2 Depart- ment of Economic Development, Jobs, Transport and Resources Centre for AgriBioscience (AgriBio), Victorian Government, Bundoora, Melbourne, Received: 8 December 2017 Accepted: 2 February 2018 Best et al. BMC Res Notes (2018) 11:108 Page 6 of 6 References 1. Kennan RM, Han X, Porter CJ, Rood JI. The pathogenesis of ovine footrot. Vet Microbiol. 2011;153(1–2):59–66. 2. Lane J, Jubb T, Shephard R, Webb-Ware J, Fordyce G: Final Report B.AHE.0010: Priority list of endemic diseases for the red meat industries. In: Edited by Australia. MaL; 2015. 3. Dhungyel O, Schiller N, Whittington R. Identification and characterization of serogroup M Dichelobacter nodosus from sheep with virulent footrot. Vet Microbiol. 2015;176(3–4):378–81. 4. Dhungyel OP, Lehmann DR, Whittington RJ. Pilot trials in Australia on eradication of footrot by flock specific vaccination. Vet Microbiol. 2008;132(3–4):364–71. 5. Dhungyel O, Hunter J, Whittington R. Footrot vaccines and vaccination. Vaccine. 2014;32(26):3139–46. 6. Dhungyel OP, Whittington RJ, Egerton JR. Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination. Mol Cell Probes. 2002;16(4):285–96. References References 1. Kennan RM, Han X, Porter CJ, Rood JI. The pathogenesis of ovine footrot. Vet Microbiol. 2011;153(1–2):59–66. 2. Lane J, Jubb T, Shephard R, Webb-Ware J, Fordyce G: Final Report B.AHE.0010: Priority list of endemic diseases for the red meat industries. In: Edited by Australia. MaL; 2015. 3. Dhungyel O, Schiller N, Whittington R. Identification and characterization of serogroup M Dichelobacter nodosus from sheep with virulent footrot. Vet Microbiol. 2015;176(3–4):378–81. 4. Dhungyel OP, Lehmann DR, Whittington RJ. Pilot trials in Australia on eradication of footrot by flock specific vaccination. Vet Microbiol. 2008;132(3–4):364–71. 5. Dhungyel O, Hunter J, Whittington R. Footrot vaccines and vaccination. Vaccine. 2014;32(26):3139–46. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: 7. Stauble A, Steiner A, Frey J, Kuhnert P. Simultaneous detection and discrimination of virulent and benign Dichelobacter nodosus in sheep of flocks affected by foot rot and in clinically healthy flocks by competitive real-time PCR. J Clin Microbiol. 2014;52(4):1228–31. y 9. Chetwin DH, Whitehead LC, Thorley SE. The recognition and prevalence of Bacteroides nodosus serotype M in Australia and New Zealand. Aust Vet J. 1991;68(4):154–5. 8. Claxton PD, Ribeiro LA, Egerton JR. Classification of Bacteroides nodosus by agglutination tests. Aust Vet J. 1983;60(11):331–4. 5. Dhungyel O, Hunter J, Whittington R. Footrot vaccines and vaccination. Vaccine. 2014;32(26):3139–46. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
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Trust and vaccine hesitancy during the COVID-19 pandemic: A cross-national analysis
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ERROR: type should be string, got "https://doi.org/10.1016/j.jvacx.2023.100299\n2590-1362/\u0001 2023 The Author(s). Published by Elsevier Ltd.\nThis is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ⇑Corresponding author.\nE-mail address: w.j.jennings@soton.ac.uk (W. Jennings). a r t i c l e\ni n f o Article history:\nReceived 21 November 2022\nReceived in revised form 31 March 2023\nAccepted 1 April 2023\nAvailable online 6 April 2023\nKeywords:\nCOVID-19\nVaccination\nTrust\nMisinformation Article history:\nReceived 21 November 2022\nReceived in revised form 31 March 2023\nAccepted 1 April 2023\nAvailable online 6 April 2023 Previous studies of vaccine hesitancy in the context of COVID-19 have reported mixed results in terms of\nthe role played by political and institutional trust. This study addresses this ambiguity with a global anal-\nysis of the relationship between trust and vaccine hesitancy, disentangling the effects of generalized trust\norientations, trust in specific institutions and conspiracy mentality. It first draws upon a cross-national\nsurvey of 113 countries to demonstrate that trust in government is a predictor of vaccine hesitancy across\nglobal regions. It further draws on original surveys fielded in seven countries (France, Germany, Spain,\nArgentina, Croatia, Brazil, India), which deploy a diverse range of measures, to disentangle the\nindividual-level predictors of vaccine hesitancy. Our findings confirm the robust effects of trust in gov-\nernment across countries, but when including other trust measures in the same models, the most robust\neffects are those of trust in health institutions and conspiracy mentality. Weaker associations are\nobserved for right-wing ideology and online political engagement, while the consumption of traditional\nmedia tends to predict the willingness of individuals to be vaccinated. Keywords:\nCOVID-19\nVaccination\nTrust\nMisinformation Keywords:\nCOVID-19\nVaccination\nTrust\nMisinformation \u0001 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license\n(http://creativecommons.org/licenses/by/4.0/). The global battle against COVID-19 has drawn attention to the\nperceived critical importance of political trust for public health\ninterventions designed to mitigate the spread of infectious disease\n[3,4,8,9]. This perception seems well-founded: citizens’ trust in\nauthorities and social institutions speaks to whether they believe\nthat they are actually looking out for their interests and has thus\nlong been understood as fundamental to cooperation between gov-\nernment and the public [8,13]. Indeed, trust has been found to be\nimportantly related to countries’ general success in combating\nthe pandemic on the aggregate level [3,30] and with citizens’ com-\npliance with public health guidelines on the individual level\n[12,29,27,29]. they report non-significant effects for trust in government in all\nbut one of their samples. On the other hand, Lazarus et al. Trust and vaccine hesitancy during the COVID-19 pandemic: A cross-\nnational analysis Will Jennings a,⇑, Viktor Valgarðsson a, Lawrence McKay a, Gerry Stoker a, Eduardo Mello b,\nHasan Muhammad Baniamin c a University of Southampton, UK\nb Fundação Getulio Vargas, Brazil\nc North South University, Bangladesh Vaccine: X 14 (2023) 100299 Vaccine: X 14 (2023) 100299 https://doi.org/10.1016/j.jvacx.2023.100299\n2590-1362/\u0001 2023 The Author(s). Published by Elsevier Ltd.\nThis is an open access article under the CC BY license (http://c Original survey data For the second part of the analysis, we use data from online\nnationally representative surveys in seven major countries –\nFrance, Germany, Spain, Croatia, Argentina, Brazil and India –\nwhich we conducted between September and December 2021. Each of our surveys apply quotas on multiple demographic factors,\nwhich include age, gender, and region for all surveys (and addition-\nally social class for NetQuest surveys and education and past vote\nfor YouGov surveys). Full details of the survey fieldwork, sample\nsize, survey company and mode/design are reported in Table 1. The dependent variable is vaccine willingness, the inverse of vac-\ncine hesitancy. At the time of fieldwork, the distribution of free\nvaccines was becoming a reality rather than (as with the data in\nour previous analysis) a theoretical future possibility, even in\ncountries where the rate of doses had been stalled by poor avail-\nability (such as in the case of India, which may be reflected in its\nlow level of first doses and full vaccinations received), so the mea-\nsures reflect actual reported prior behaviour, rather than reported\nvaccine intentions. In the survey, we asked respondents the follow-\ning pair of questions, coding as vaccine ‘willing’ (1) those respon-\ndents who answered that they had received a dose or that they\nwould if offered. Those who had declined the dose offered, or\nwould not get a future dose if offered, are coded as unwilling\n(0).2 The questions were formulated as follows: p\nOur empirical findings confirm that political trust is a consistent\npredictor of vaccine hesitancy across the world, one that is robust\nto the inclusion of multiple controls and holds in over 90 percent of\nthe more than 100 countries covered here. In our more detailed\nanalysis of the role of trust, using original survey data, we find that\ngeneralised distrust in government is positively related to vaccine\nhesitancy and generalised trust is negatively related to it, whereas\nthe effects of political mistrust and social trust are non-significant,\nbut conspiracy mentality has the strongest effect by some margin. When separating political trust into trust in different institutions,\nwe find, in line with previous studies, that trust in health institu-\ntions stands out as by far the most robust, but this is fairly strongly\nrelated to confidence in political institutions, which has a signifi-\ncant effect when the former is not included in the same model. Methods lumped together in statistical models, without great clarity about\nthe reasons behind the choice of variables, the correlations\nbetween them or how the measures overlap. In this study, we\nmove\nbeyond\nthese\nprevious\napproaches\nby\ndistinguishing\nbetween generalized trust orientations and more specific confi-\ndence in particular institutions, by distinguishing between political\ntrust, mistrust, distrust and conspiracy mentality as distinct types of\nattitudes within the ‘‘trust” family, and by explicitly interrogating\nthe inter-relationships between different types of trust and vaccine\nacceptance. Finally, our original surveys contain a broader range of\nrelevant measures of personal, attitudinal and behavioural factors\nthan most others, allowing us to compare the effects of trust with\nfactors such as media use, personality traits and demographics to a\ngreater extent than prior studies. Wellcome global monitor data For the global analysis, we use data from The Wellcome Global\nMonitor (WGM) included in the Gallup World Poll. Between Octo-\nber 2020 and January 2021 inclusive, telephone surveys were com-\npleted by nearly 120,000 respondents in 113 countries, which\ncollectively represent 90 % of the global population aged 15 +. The surveys concentrated on themes related to COVID-19, includ-\ning its economic impacts, and crucially featured questions on both\nvaccine uptake intentions and on trust. The measures used here\nasked respondents: (a) how much they trusted the national gov-\nernment in their country, and (b) whether they would agree to\nbe vaccinated if a vaccine to prevent coronavirus ‘‘was available\nright now at no cost”.1 Our approach in this paper thus seeks to advance our under-\nstanding of the role(s) of trust in vaccine acceptance by addressing\nthese limitations of prior studies. Firstly, we undertake a global\nanalysis of the degree to which trust predicts vaccine hesitancy,\nbased on high quality survey data from over 100 countries. This\nenables us to determine how general the link between trust and\nvaccine hesitancy is, covering a broader scope of countries than\nany prior study on the individual level. Secondly, we report results\nfrom our original surveys conducted in seven countries, which\nincluded an extensive set of measures which allow us to disentan-\ngle the relationship between different types of trust and vaccine\nacceptance and compare with the role of related factors such as\npersonality traits, cognition and political ideology. 1 The full wording for these questions is included in Online Appendix A.\n2 Those who give ‘prefer not to say’ responses at Q13, or respond ‘don’t know’ at\nQ14, are dropped from the analysis. Methods In terms of\ntrust, we include measures of generalized political trust, mistrust\nand distrust, reflecting insights from recent studies that suggest\nthat lack of trust may either denote a type of cautious scepticism\n(mistrust) or settled dissatisfaction (distrust) – and that the two\nmay have different implications for vaccine hesitancy, in that we\nwould expect distrust but not necessarily mistrust to drive it\n([2]; [28]; [12]). In addition, we include a scale measuring ‘conspir-\nacy mentality’ (developed by 5 and more conventional measures of\ntrust in representative institutions, health institutions and the\nmedia as well as a measure of social trust, separating these in our\nanalysis and comparing their relative effects. This offers a more\ncomprehensive test of the precise ways that trust influences beha-\nviour and its relative importance. The WGM also included demographic information on respon-\ndents’ age, sex, education, income and employment status. In\nterms of sample size, 98,166 respondents in 105 countries are valid\non the vaccine and trust measures, with 94,620 in 102 countries\nvalid once control variables are added in a regression model. a r t i c l e\ni n f o In this study, we\nmove\nbeyond\nthese\nprevious\napproaches\nby\ndistinguishing\nbetween generalized trust orientations and more specific confi-\ndence in particular institutions, by distinguishing between political\ntrust, mistrust, distrust and conspiracy mentality as distinct types of\nattitudes within the ‘‘trust” family, and by explicitly interrogating\nthe inter-relationships between different types of trust and vaccine\nacceptance. Finally, our original surveys contain a broader range of\nrelevant measures of personal, attitudinal and behavioural factors\nthan most others, allowing us to compare the effects of trust with\nfactors such as media use, personality traits and demographics to a\ngreater extent than prior studies. a r t i c l e\ni n f o [16] find\na significant effect for trust in government in their 19-country\nstudy and Lindholt et al. [17] find in their 8-country study that\ntrust in government has an effect in addition to trust in scientists\nand health authorities, although that effect is only marginally sig-\nnificant in their full model. Finally, Allington et al. [1] find in their\nstudy of the UK that trust in government had a significant effect\nbut a much smaller one than trust in scientists and medical profes-\nsionals. Other studies on the topic have been limited by their use of\nconvenience samples, although these tend to find significant\neffects for political trust [13,25,25]. However, when it comes to the particular topic of the role of\npolitical trust in shaping vaccine acceptance, studies have reported\nmixed results. There appears to be no doubt that some kind of trust\nmatters, but while Rozek et al. [21] report in their 17-country\nstudy that trust in health institutions matter, they report no effects\nfor trust in political leaders. Similarly, Kerr et al. [14] find in their\n25-sample study that trust in experts plays an important role, but In summary, of the four large, cross-national studies published\nto date on the topic, two report significant effects for political trust\non vaccine acceptance, whereas two report no significant effects. In\nthis study, we directly address this ambiguity in the existing liter-\nature, using secondary survey data from over 100 countries and\noriginal survey data from seven countries, with the latter explicitly\ndesigned to disentangle the role of different types of trust in seven\ncountries. The broad concept of political trust is typically not mea-\nsured in a systematic or theoretically driven way in prior studies;\ninstead, different measures of trust in various entities tend to be https://doi.org/10.1016/j.jvacx.2023.100299\n2590-1362/\u0001 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Vaccine: X 14 (2023) 100299 Vaccine: X 14 (2023) 100299 W. Jennings, V. Valgarðsson, L. McKay et al. lumped together in statistical models, without great clarity about\nthe reasons behind the choice of variables, the correlations\nbetween them or how the measures overlap. Original survey data Indeed, trust in health institutions, conspiracy mentality and age\nare the only predictors whose significant effects persist even after\nincluding all 29 variables in our analysis in the same model. Con-\nsuming news in traditional media is also a strong (negative) pre-\ndictor of hesitancy and those who identify on the right in politics\nare more likely to be hesitant, whereas the effects of other factors\nare weaker and less consistent. By combining breadth and depth of\nanalysis, we highlight the consistency of trust as a predictor of vac-\ncine hesitancy and shed light on the dynamic role that different\ntypes of trust play, with conspiracy mentality and trust in health\ninstitutions standing out as the most potent predictors. Q13. Which of these applies to you? (1) I have received one or two doses of a COVID-19 vaccine\n(2) I have been offered a vaccine against COVID-19, but declined the\noffer to be vaccinated\n(3) I have not yet been offered a vaccine against COVID-19\n(4) Prefer not to say\nQ14. To what extent, if at all, do you agree, or disagree, with the\nfollowing statements? If I was offered a vaccine for COVID-19, I would get it [ASK IF\nQ13 = 3]\n(1) Strongly agree\n(2) Tend to agree\n(3) Neither agree nor disagree\n(4) Tend to disagree (2) I have been offered a vaccine against COVID-19, but declined the\noffer to be vaccinated (3) Neither agree nor disagree (4) Tend to disagree 2 2 Vaccine: X 14 (2023) 100299 W. Jennings, V. Valgarðsson, L. McKay et al. Table 1\nOriginal survey details. Source: Our World In Data [18]. Country\nFieldwork\nSample\nCompany\nDesign\nVaccination rate*\nFrance\n23.09.2021 – 20.11.2021\n1,548\nYouGov\nOnline, nationally representative\n65 % (75 %)\nGermany\n14.10.2021 – 23.11.2021\n1,558\nYouGov\nOnline, nationally representative\n65 % (70 %)\nSpain\n23.09.2021 – 26.09.2021\n1,022\nYouGov\nOnline, nationally representative\n77 % (79 %)\nCroatia\n29.09.2021 – 06.10.2021\n1,017\nYouGov\nOnline, nationally representative\n40 % (46 %)\nArgentina\n29.09.2021 – 08.10.2021\n1,113\nYouGov\nOnline, nationally representative\n50 % (66 %)\nBrazil\n06.12.2021 – 28.12.2021\n2,123\nNetQuest\nOnline, nationally representative\n64 % (77 %)\nIndia\n22.09.2021 – 30.09.2021\n1,040\nNetQuest\nOnline, nationally representative (of the literate population)\n15 % (44 %)\nTotal\n22.09.2021 – 28.11.2021\n9,421\nNote: France includes a top-up sample of 498 respondents 19.11.2021–20.11.2021; Germany includes top-up sample of 534 respondents 19.11.2021–20.11.2021. 3 In Brazil, this item specifically referred to the publicly funded healthcare system,\n‘‘Sistema Único de Saúde (SUS)”. Global analysis: Wellcome Global Monitor data We begin with an analysis of the relationship between political\ntrust and vaccine hesitancy across the globe. Is this robust, and\ndoes it consistently emerge globally? To answer this, we turn to\nthe Wellcome Global Monitor included in the Gallup World Poll,\nwhich includes measures of trust in government and willingness\nto be vaccinated, along with demographic measures. Further infor-\nmation about this data source and the variables used is presented\nin the methods section below and in Online Appendix B. We first\npresent the country-level aggregate relationships between trust\nin\ngovernment\nand\nvaccine\nintention\non\nthe\ncountry-level\n(Fig. 1).4 The y-axis represents the percentage of respondents in each\ncountry who said that they would agree to be vaccinated against the\ncoronavirus if a vaccine was available at no cost,5 while the x-axis\nrepresents the percentage of respondents who said they trusted\nthe national government in their country ‘a lot’ or had ‘some’ trust\nin it.6 The aggregate correlation between these two variables is pos- Following previous work, we took the first three statements (1–\n3) to indicate trust, the next two (4–5) to reflect mistrust, and the\nlast three (6–8) to measure distrust. For each set of statements, we\ncreated scales for each respondent taking the mean level of agree-\nment with the statements (on the Likert scale) before rescaling the\nvalues to 0–1. Cronbach’s alpha scores vary between 0.60 (mis-\ntrust) to 0.84 (distrust) and 0.90 (trust), all of which pass the 0.6\nthreshold established as acceptable by Nunally and Bernstein [20]. Following previous work, we took the first three statements (1–\n3) to indicate trust, the next two (4–5) to reflect mistrust, and the\nlast three (6–8) to measure distrust. For each set of statements, we\ncreated scales for each respondent taking the mean level of agree-\nment with the statements (on the Likert scale) before rescaling the\nvalues to 0–1. Cronbach’s alpha scores vary between 0.60 (mis-\ntrust) to 0.84 (distrust) and 0.90 (trust), all of which pass the 0.6\nthreshold established as acceptable by Nunally and Bernstein [20]. Turning to our other independent variables, we provide more\ndetail in Online Appendix B, and only summarise our measures\nbriefly here. In terms of demographics, we include age, gender,\nand a dummy for university level education. Our models include\nleft–right ideological self-placement, measured on a 0–10 scale. (5) Strongly disagree\n(6) Don’t know Turning to our independent variables, the survey includes mul-\ntiple measures of trust. First, it includes conventional measures of\nconfidence in institutions, drawn from the World Values Survey,\nfrom which we derive three separate measures of institutional\ntrust: 1) trust in health institutions, which is calculated for each\nrespondent as the mean of trust in the World Health Organisation\n(WHO) and ‘‘the health service in your country”,3 2) trust in the\nmedia, which is the mean of trust in ‘‘the press” and ‘‘TV”, and 3)\ntrust in political institutions, which is calculated as the mean of trust\nin ‘‘parliament”, ‘‘political parties” and ‘‘the government in your\ncountry”. We also include a module developed in previous studies\nto measure generalized orientations of trust, mistrust and distrust\ntowards government [6,14]. The following set of questions was\nasked in all seven countries: To what extent do you agree, or disagree, with the following\nstatements? statements? (1) The government is honest and truthful\n(2) In general, the government usually does the right thing\n(3) The government usually has good intentions\n(4) I am usually cautious about trusting the government\n(5) I am unsure whether to believe the government\n(6) The government acts unfairly towards people like me\n(7) The government usually ignores my community\n(8) The government doesn’t respect people like me\n(1)\nStrongly agree\n(2)\nTend to agree\n(3)\nNeither agree nor disagree\n(4)\nTend to disagree\n(5)\nStrongly disagree\n(6)\nDon’t know 4 Readers should bear in mind the timing of data collection (October 2020 through\nJanuary 2021) and that the level of trust in each country may have increased or\ndecreased since (for instance in Myanmar – ‘MM’ in Fig. 1 – where trust may have\nfallen significantly since the 2021 coup d’etat).\n5 The response options were binary: ‘‘Yes, would agree” and ‘‘No, would not agree”.\nThose who responded ‘‘don‘t know” or refused to respond are coded as missing for the\npurposes of this analysis.\n6 The other response options were ‘‘not much” and ‘‘not at all”. Don’t knows and\nrefusals are once again treated as missing. Results Global analysis: Wellcome Global Monitor data Original survey data *Share of\npeople vaccinated against COVID-19 at start of survey fieldwork: complete initial vaccine protocol (partially vaccinated). Table 1\nOriginal survey details. Source: Our World In Data [18]. 4 Readers should bear in mind the timing of data collection (October 2020 through\nJanuary 2021) and that the level of trust in each country may have increased or\ndecreased since (for instance in Myanmar – ‘MM’ in Fig. 1 – where trust may have\nfallen significantly since the 2021 coup d’etat). Table 1 We further incorporate measures of the ‘Big Five’ personality traits,\nbased on the ‘Short 15-item Big Five Inventory (BFI-S)’ devised and\nvalidated by Lang et al. [15], creating scales for the mean (dis)\nagreement with the statements measuring each of the Big Five. We also include a scale of ‘conspiracy mentality’, designed for\nuse across cultures and languages, based on the 5-item scale by\nBruder et al. [5], albeit with a slightly modified question and\nresponse scale. We take the simple mean of these items as our\nmeasure of conspiracy mentality. Finally, we include a measure\nof cognitive reflection based on Frederick (2005; see also [25]),\nwhich principally measures the ability or disposition of respon-\ndents to reflect on a question and resist reporting the first response\nthat comes to mind (using classic games/experiments from beha-\nvioural economics). We take the sum of correct answers (0–3) to\nbe the respondent’s cognitive reflection score. Finally, we include\nmeasures of media consumption and activity: how much respon-\ndents follow news in different types of media and whether they\nhave recently ‘fact-checked’ articles or posted political content\nonline. All these scales and all other variables used in our analysis\nare rescaled to 0–1 to enable direct comparison of effects. After\nremoving all cases with missingness in the final model, 6,210\nrespondents are retained for the multivariate analysis. (5) Strongly disagree\n(6) Don’t know 6 The other response options were ‘‘not much” and ‘‘not at all”. Don’t knows and\nrefusals are once again treated as missing. 5 The response options were binary: ‘‘Yes, would agree” and ‘‘No, would not agree”.\nThose who responded ‘‘don‘t know” or refused to respond are coded as missing for the\npurposes of this analysis. Global analysis: Wellcome Global Monitor data Turning to our other independent variables, we provide more\ndetail in Online Appendix B, and only summarise our measures\nbriefly here. In terms of demographics, we include age, gender,\nand a dummy for university level education. Our models include\nleft–right ideological self-placement, measured on a 0–10 scale. 3 W. Jennings, V. Valgarðsson, L. McKay et al. Vaccine: X 14 (2023) 100299 Fig. 1. Trust in government vs self-reported vaccine intentions (aggregate-level), Wellcome Global Monitor. Countries denoted by Alpha-2 ISO codes, see: https://www. iban.com/country-codes. Fig. 1. Trust in government vs self-reported vaccine intentions (aggregate-level), Wellcome Global Monitor. Countries denoted by Alpha-2 ISO codes, see: https://www. iban com/country-codes Fig. 1. Trust in government vs self-reported vaccine intentions (aggregate-level), Wellcome Global Monitor. Countries denoted by Alpha-2 ISO codes, see: https://www. iban.com/country-codes. orted vaccine intentions (aggregate-level), Wellcome Global Monitor. Countries denoted by Alpha-2 ISO codes, see: https://www levels, which may drive both trust and vaccine intention). The full\nresults from these regression models are presented in Online Appen-\ndix, Table A1, and they show that moving through each stage from\nthe lowest to the highest level of trust in government is associated\nwith a significant increase in vaccine willingness, when controlling\nfor individual and contextual factors. Fig. 3 presents the marginal\neffects of each category of trust from these models, illustrating that\nthe global prediction (holding all control variables constant) for vac-\ncine intention is about 54 % for those who say they trust their\nnational government ‘‘not at all”, whereas it is about 71 % for those\nwho say they trust it ‘‘a lot”. Thus, the global relationship between\npolitical trust and vaccine intention appears to be significant and\nsubstantial even when accounting for demographic and contextual\nfactors. itive and moderate (0.49, p < 0.001). The simple bivariate regression\nline shows that at the lowest observed levels of trust (20 %), fewer\nthan 50 % are expected to take vaccines, while at the highest levels\n(near to 100 %), nearly 80 % would accept a no-cost vaccine. To explore how far this relationship is universal, we run bivari-\nate logistic regression models of vaccine intention on trust within\neach country on the individual level and present the odds ratios for\neach country in Fig. 2 (where trust in government is an ordinal\nvariable that takes the values 1 ‘not at all’, 2 ‘not much’, 3 ‘some’\nand 4 ‘a lot’). 7 Here, we include the full categorical variable with dummies for each category and\n‘‘not at all” as the baseline. Global analysis: Wellcome Global Monitor data While there is significant cross-national heterogene-\nity, it is striking that there is statistically significant (at the 95 per\ncent confidence level) positive relationship between trust in gov-\nernment and vaccine willingness in 75 out of 105 countries. In just\na single country (Bosnia Herzegovina), the relationship between\ntrust and vaccine willingness is significantly negative (i.e. confi-\ndence intervals of the estimated odds ratio do not cross at 1.0). In another 29 countries, the relationship was not statistically sig-\nnificant, interestingly including both high trust countries, such as\nNorway and Sweden, and low trust countries, such as Slovakia,\nSenegal and Serbia. The average odds ratio across countries is equal\nto 1.4, meaning that each one unit increase in the ordinal scale of\ntrust in government typically leads to a 40 % increase in the likeli-\nhood of being willing to be vaccinated. Detailed analysis: original surveys in seven countries While the Wellcome Global Monitor provides impressive global\nscope in terms of the total number of countries and proportion of\nthe global population covered, it is understandably limited in\nhow far it explores alternative measures of trust, as well as other\nrelevant values, beliefs and behaviours which may account for or\novershadow the relationship. We build upon this through fielding\nour own bespoke survey that specifically explored multiple dimen-\nsions of trust and a range of related factors in multiple countries. For our study, we are therefore able to draw on an array of mea-\nsures that distinguish between categories of generalized trust, mis-\ntrust and distrust in government, as well as more conventional\nmeasures of trust in specific institutions, where we differentiate\nbetween political institutions (parliament, political parties and\nthe national government), health institutions (the national health\nservice and WHO) and the media (the press and TV). We also\ninclude various measures of conspiratorial mentality, political ide-\nology, cognitive reflection, personality traits and traditional and\nonline media use, as well as demographic information. This\napproach enables us to explore the degree to which a much wider Turning to a more sophisticated analysis of this relationship, we\nnext run multi-level regression models on the entire dataset (with\ncountry as the second level). These again predict vaccine intent as a\nfunction of trust in government,7 and add several individual-level\ncontrols: age (in four categories), sex, education (a binary variable\nwhere those with university education are coded as equal to 1),\nemployment status and income on the individual level (in quintiles). At the country-level, we include the Human Development Index\n(HDI) score from the UN Human Development Reports (to partly\naccount for the potential confounding effect of societal welfare 4 Fig. 2. Bivariate regression of trust in government on self-reported vaccine willingness, odds ratios (individual-level, by country). Countries denoted by Alpha-3 ISO\nsee: https://www.iban.com/country-codes. W. Jennings, V. Valgarðsson, L. McKay et al. Vaccine: X 14 (2023) 1 W. Jennings, V. Valgarðsson, L. McKay et al. Vaccine: X 14 (2023) 100299 Fig. 2. Bivariate regression of trust in government on self-reported vaccine willingness, odds ratios (individual-level, by country). Countries denoted by Alpha-3 ISO codes,\nsee: https://www.iban.com/country-codes. Fig. 3. Marginal predictions of vaccine intention by levels of trust in government in the 2020 Wellcome Global Monitor survey, from multi-level regression models controlling\nfor individuals’ demographics and countries’ HDI. W. Jennings, V. Valgarðsson, L. Detailed analysis: original surveys in seven countries However, these measures are moderately correlated (the\npairwise Pearson’s r coefficients are between 0.45 and 0.5) and\nwhen the other institutional trust measures are included without\nthe others but with controls, both are significant predictors, so this\nshould not be interpreted to mean that trust in political or media\ninstitutions does not matter for vaccine willingness. In other\nwords, people’s general trust orientations towards government\nand their trust in political institutions matter for vaccine accep-\ntance, but when accounting for their trust in different specific state\ninstitutions, it turns out that health institutions are the most clo-\nsely relevant institution. We begin by considering the degree to which each of our mea-\nsures in isolation is associated with willingness to be vaccinated\nagainst COVID-19. Bivariate logistic regression models are calcu-\nlated for all predictors for the full, pooled seven-country sample,\nwith the odds ratios plotted in Fig. 4. In the figure, the dark circles\nindicate the coefficient value, while the whiskers indicate the 95\nper cent confidence intervals. The red line indicates the point at\nwhich there is no significant relationship between the variables if\nthose confidence intervals cross. Several measures of trust have statistically significant bivariate\nrelationships with vaccine willingness. The strongest positive rela-\ntionship\nis\nobserved\nfor\ntrust\nin\nhealth\ninstitutions\n(odds\nratio = 16.32, 95 % CI 12.40 to 21.48): in other words, those most\ntrusting were between 12 and 21 times more likely to accept the\nvaccine than those lowest in trust. Trust in the media (OR 4.96,\n95 % CI 3.70 to 6.64) and political institutions (OR 3.72, 95 % CI\n2.78 to 5.01) also have significant, but weaker, bivariate relation-\nships with vaccination willingness. p\ng\nAs expected, generalized trust orientations towards govern-\nment also have a positive effect on willingness (OR: 3.61, 95 % CI\n2.83 to 4.62), whereas distrusting and mistrusting orientations\nhave negative effects with mistrusters (OR: 0.49, 95 % CI 0.38 to\n0.62) and distrusters (OR: 0.52, 95 % CI 0.40 to 0.68) each about half\nas likely. Perhaps surprisingly, the difference between the effects of\ndistrust and mistrust are small, and social trust has weak and non-\nsignificant effects. Besides trust, we observe numerous other sig-\nnificant relationships. Detailed analysis: original surveys in seven countries McKay et al. Vaccine: X 14 (2023) 100299\n5 Fig. 2. Bivariate regression of trust in government on self-reported vaccine willingness, odds ratios (individual-level, by country). Countries denoted by Alpha-3 ISO codes,\nsee: https://www.iban.com/country-codes. Fig. 3. Marginal predictions of vaccine intention by levels of trust in government in the 2020 Wellcome Global Monitor survey, from multi-level regression models controlling\nfor individuals’ demographics and countries’ HDI. Fig. 3. Marginal predictions of vaccine intention by levels of trust in government in the 2020 Wellcome Global Monitor survey, from multi-level regression models controlling\nfor individuals’ demographics and countries’ HDI. 5 W. Jennings, V. Valgarðsson, L. McKay et al. Vaccine: X 14 (2023) 100299 set of measures of political attitudes are predictive of vaccine will-\ningness/hesitancy, and to situate different types of trust in that\ncontext. We provide further details on these data and measures\nin the methods section and the full questionnaire in Online Appen-\ndix A. age, and consumption of traditional media. However, we run all\nour block models on the same sample of respondents with non-\nmissing values on all variables (N = 6,235), to enable comparison\nof effects between models. The block models are displayed in Fig. 5, with effects again\nexpressed in terms of odds ratios. The first model includes gener-\nalized trust orientations, in both government and other people,\nand shows that generalized trust retains its positive effect\n(OR = 3.08, 95 % CI 2.08 to 4.56) on vaccine willingness and distrust\nits negative effect (OR = 0.58, 95 % CI 0.38 to 0.87), whereas mis-\ntrust becomes a non-significant predictor. Here, social trust\nbecomes a weakly negative predictor of willingness, albeit not sig-\nnificant at the 95 % level (p = 0.08), when accounting for political\ntrust orientations. In model 2, we include measures of institutional\ntrust, with intriguing results. Here, trust in political institutions\nbecomes non-significant and trust in the media only significant\nat the 90 % level (p = 0.08), but trust in health institutions remains\na strong predictor (OR = 15.37, 95 % CI 10.06 to 23.47) – suggesting\nagain that the latter is the most robust driver of vaccine willing-\nness. Detailed analysis: original surveys in seven countries Most notably, those with most ‘conspiracy\nmentality’ are about 10 % as likely as the least conspiratorial to\nbe willing to be vaccinated (OR = 0.10, 95 % CI 0.07 to 0.14) and\nthe oldest respondents are four times as likely as the youngest\n(OR = 3.98, 95 % CI 2.94 to 5.40). Engagement with different infor-\nmation sources also appears related to vaccine hesitancy: online\ninformation consumption, sharing and fact-checking material\nonline are associated with slightly lower willingness to get vacci-\nnated, though the positive association with consumption of tradi-\ntional media is slightly larger in magnitude. People who identify\nas on the right of the political spectrum are also somewhat less\nlikely to get vaccinated, as are those who score higher on some\nof the Big Five personality trait scales; especially on ‘openness’,\nwhereas cognitive reflection shows no association. Finally, as in\nother studies, university education is related to greater willingness\nto get vaccinated for COVID-19. Finally, the third model shows that conspiracy mentality is an\neven stronger negative predictor (OR = 0.07, 95 % CI 0.04 to 0.10)\nwhen including demographic controls. Because political trust and\nconspiracy mentality are closely related attitudes [10],8 we explore\nwhether trust is a confounder for the effects of conspiracy mentality\nor vice versa, by presenting the results of models including all of\nthese variables together in Online Appendix Fig. A2: these show that\nthe effects of conspiracy mentality survive controlling for all of the\ntrust measures, whereas the only trust measure that still has a sig-\nnificant effect is that of trust in health institutions, suggesting that\nboth shape vaccine willingness above and beyond political trust.9 Returning to other factors, we find that the effects of most per-\nsonality traits lose their significance in the block model (neuroti-\ncism is significant at the 90 % level, p = 0.06) but here, cognitive\nreflection\nshows\na\npositive\neffect\non\nvaccine\nwillingness\n(p < 0.01) and information engagement still has significant effects. In particular, the frequency of consuming traditional media (news-\npapers, television, radio) remains a strong predictor of vaccine\nwillingness (OR = 4.27, 95 % CI 2.89 to 6.32), while getting informa-\ntion from ‘people’ and online has negative effects, although rela-\ntively weak ones (and non-significant for the former). 8 The correlation between the conspiracy mentality scale and the generalized trust\nscales is between 0.22 and 0.28 Pearson’s r in the pooled dataset, with stronger\ncorrelations in France, Germany, and Spain.\n9 Generalized trust in government is still a significant predictor when including\nthose and conspiracy mentality together in a model, but its effects become\ninsignificant when controlling for trust in institutions as well. 8 The correlation between the conspiracy mentality scale and the generalized trust\nscales is between 0.22 and 0.28 Pearson’s r in the pooled dataset, with stronger\ncorrelations in France, Germany, and Spain. 9 Generalized trust in government is still a significant predictor when including\nthose and conspiracy mentality together in a model, but its effects become\ninsignificant when controlling for trust in institutions as well. Detailed analysis: original surveys in seven countries We find\nthat more ‘active’ internet users – those who post political content\n– are more vaccine hesitant (OR = 0.51, 95 % CI 0.37 to 0.69),\nalthough the effect of fact-checking becomes non-significant. We\nalso find that moving from left to right on a left/right scale is asso-\nciated with lower levels of vaccine willingness in all models. The\neffect of university education becomes non-significant in most\nmodels, but age retains a strong positive effect on willingness\n(while the effects of gender are still non-significant). The latter is While informative in a descriptive sense, these bivariate rela-\ntionships may well be confounded by various related demographic\nand attitudinal factors. To get closer to a causal relationship, we\nnext run a series of multivariate regression models, to isolate and\ndisentangle the effects of specific factors. We estimate six separate\n‘block’ models of related variables here, each retaining a selection\nof closely related variables. Each of the block models controls for\ndemographic factors (gender, age, education), left–right ideology\nand country dummies (because the dependent and independent\nvariables are likely to vary by country and the other control vari-\nables, in ways which might result in spurious effects reported in\nthe absence of these controls). In Online Appendix A, we also report\nresults of a single model including all 29 variables. We focus on\nblock models in this discussion, because including so many corre-\nlated variables in one model creates problems of multicollinearity,\nwide confidence intervals and very unintuitive interpretations of\nindividual coefficients 29. We see this model primarily as a conser-\nvative test of which predictors are the most robust of all: these\nturn out to be trust in health institutions, conspiracy mentality, 6 W. Jennings, V. Valgarðsson, L. McKay et al. Vaccine: X 14 (2023) 100299 Fig. 4. Bivariate logistic regression of vaccine willingness, odds ratios. Fig. 4. Bivariate logistic regression of vaccine willingness, odds ratios. traits interestingly appear to play a bigger role in those countries\nthan in the others. perhaps unsurprising given the sharp age gradient of the severity\nof COVID-19. Finally, we explore whether the determinants of vaccine hesi-\ntancy (or willingness) differ importantly between the countries\nin our dataset by conducting the bivariate and block models sepa-\nrately for each country. Coefficient plots from these models can be\nfound in the Online Appendix, Figs. A3-A23. Detailed analysis: original surveys in seven countries Most of the central\nfindings remain the same across countries, but generalized trust\norientations have by some margin the strongest relationship with\nvaccine hesitancy in France and Germany, whereas in Brazil polit-\nical trust negatively predicts vaccine willingness. This finding for\nBrazil is consistent with other studies suggesting that partisan cues\ncan reverse expected relationships between political trust and\nCOVID-related attitudes and behaviours (e.g., [12]). President Bol-\nsonaro was notoriously vaccine- and COVID-sceptic, so those\nrespondents expressing trust in his government were more likely\nto also hold anti-vax attitudes. Generally, most variables have\nweaker and often non-significant effects in India and particularly\nArgentina than in the other countries, but conversely, personality Discussion Our analysis here has served to demonstrate the critical link\nbetween political trust and vaccine hesitancy across the globe,\ndemonstrating how trust in government is predictive of willing-\nness to get vaccinated against COVID-19 for much of the world’s\npopulation. Generalized trust orientations towards government\nand trust in political institutions are consistent predictors of vac-\ncine hesitancy, although when including multiple interrelated\npolitical trust measures in the same models, trust in health institu-\ntions and conspiracy mentality stand out as the most robust pre-\ndictors. The design of this study has offered unprecedented\nglobal scope in its consideration of the degree to which trust in\ngovernment predicts vaccine hesitancy for COVID-19 (113 coun-\ntries) and systematic comparison of alternative measures of differ-\nent types of trust as well as other beliefs, opinions and traits, again\nin a substantial cross-national design (7 countries). This allows us 7 7 W. Jennings, V. Valgarðsson, L. McKay et al. Vaccine: X 14 (2023) 100299 onclude with increased confidence that trust is an essential\nmedia) are less willing. There is little evidence to support the clai\nFig. 5. Multivariate block models of vaccine willingness, odds ratios. Fig. 5. Multivariate block models of vaccine willingness, odds ratios. Fig. 5. Multivariate block models of vaccine willingness, odds ratios. to conclude with increased confidence that trust is an essential\nresource for societies in their fight against COVID-19, and that this\nis true both relative to other attitudes and controlling for the\ndemographics of individuals. Our findings confirm that willingness\nto be vaccinated against COVID-19 is positively linked to trust in\ngovernment, and negatively linked to distrust of government. Prior\ncross-national studies have reported mixed results in terms of the\neffects of political trust on vaccine hesitancy, but our findings sug-\ngest that this is likely because those that report non-significant\neffects of political trust do so via regression models which simulta-\nneously include various closely related measure of trust [14,24]. In\nterms of other factors, we find that consumers of traditional media\n(i.e. television, newspapers) are more willing to get vaccinated,\nwhereas the politically online (i.e. people who post on social media) are less willing. There is little evidence to support the claim\nthat personality traits are drivers of vaccine hesitancy, even in\nmodels that exclude strong predictors such as conspiracy beliefs\nand political trust. Discussion We find that cognitive reflection is associated\nwith greater vaccine willingness, but this is a small effect relative\nto other attitudinal and demographic predictors. Of the 29 predic-\ntors included in our regression models, political trust orientations\nand conspiracy mentality emerge as the strongest predictors of\nvaccine willingness, along with respondents’ age. to conclude with increased confidence that trust is an essential\nresource for societies in their fight against COVID-19, and that this\nis true both relative to other attitudes and controlling for the\ndemographics of individuals. Our findings confirm that willingness\nto be vaccinated against COVID-19 is positively linked to trust in\ngovernment, and negatively linked to distrust of government. Prior\ncross-national studies have reported mixed results in terms of the\neffects of political trust on vaccine hesitancy, but our findings sug-\ngest that this is likely because those that report non-significant\neffects of political trust do so via regression models which simulta-\nneously include various closely related measure of trust [14,24]. In\nterms of other factors, we find that consumers of traditional media\n(i.e. television, newspapers) are more willing to get vaccinated,\nwhereas the politically online (i.e. people who post on social We have here built on a still growing literature examining the\nlink between trust and vaccine hesitancy in the context of\nCOVID-19. Our study draws upon high quality survey data from\nthe Wellcome Global Monitor, enabling us to cover a substantially\ngreater number of countries than most empirical studies con- 8 W. Jennings, V. Valgarðsson, L. McKay et al. Vaccine: X 14 (2023) 100299 [7] Devine D, Gaskell J, Jennings W, Stoker G. Exploring Trust, Mistrust and\nDistrust (1st Digital Workshop of the ESRC ‘‘TrustGov” Project); 2020. https://\ntrustgov.net/s/TrustGov-Trust-mistrust-distrust-20042020.pdf. ducted during the pandemic, and through our own cross-national\nsurvey programme, allowing us to disentangle the roles of different\ntypes of trust and to directly compare their effects with other com-\nmonly cited factors. While the COVID-19 pandemic has eased in\nseverity by now, these results point to the importance of rebuilding\npolitical trust for the potential success of future mass vaccination\nprogrammes. [8] Devine D, Gaskell J, Jennings W, Stoker G. Trust and the Coronavirus Pandemic:\nWhat are the Consequences of and for Trust? An Early Review of the Literature. Polit\nStud\nRev\n2021;19(2):274–85. https://doi.org/10.1177/\n1478929920948684. [9] Devine D, Valgarðsson V, Smith J, Jennings W, di Vettimo MS, Bunting H,\nMcKay L. References [23] Nunnally JC, Bernstein IH. Psychometric Theory. McGraw-Hill; 1994. [24] Rozek LS, Jones P, Menon A, Hicken A, Apsley S, King EJ. Understanding Vaccine\nHesitancy in the Context of COVID-19: The Role of Trust and Confidence in a\nSeventeen-Country Survey. Int J Public Health 2021;66(May):. https://doi.org/\n10.3389/ijph.2021.636255636255. [1] Allington D, McAndrew S, Moxham-Hall V, Duffy B. Coronavirus conspiracy\nsuspicions, general vaccine attitudes, trust, and coronavirus information\nsource as predictors of vaccine hesitancy among UK residents during the\nCOVID-19\npandemic. Psychol\nMed\n2021. https://doi.org/10.1017/\nS0033291721001434. [25] Schernhammer E, Weitzer J, Laubichler MD, Birmann BM, Bertau M, Zenk L,\net al. Correlates of COVID-19 vaccine hesitancy in Austria: Trust and the\ngovernment. J Public Health 2021;44(1):E106–16. https://doi.org/10.1093/\npubmed/fdab122. [2] Bertsou E. Rethinking political distrust. Eur Polit Sci Rev 2019;11(2):213–30. https://doi.org/10.1017/S1755773919000080. [26] Schmeisser Y, Renström E, Bäck H. Who follows the rules during a crisis? Personality traits and trust as predictors of compliance with containment\nrecommendations during the COVID-19 pandemic in Sweden. Front. Polit. Sci. 2021;3(November):1–13. https://doi.org/10.3389/fpos.2021.739616. [3] Bollyky TJ, Hulland EN, Barber RM, Collins JK, Kiernan S, Moses M, et al. Pandemic preparedness and COVID-19: an exploratory analysis of infection\nand fatality rates, and contextual factors associated with preparedness in 177\ncountries, from Jan 1, 2020, to Sept 30, 2021. Lancet (London, England)\n2022;399(10334):1489–512. https://doi.org/10.1016/S0140-6736(22)00172-\n6. (\n)\np\n//\ng/\n/ p\n[27] Seyd B, Bu F. Perceived risk crowds out trust? Trust and public compliance\nwith coronavirus restrictions over the course of the pandemic. Eur Polit Sci Rev\n2022;14(2):155–70. https://doi.org/10.1017/S1755773922000078. [4] Brezzi M, Gonzalez S, Nguyen D, Prats M. An Updated OECD Framework on\nDrivers of Trust in Public Institutions to Meet Current (OECD Working Papers\non\nPublic\nGovernance,\nIssue\n48);\n2021. https://www.oecd-ilibrary. org/governance/an-updated-oecd-framework-on-drivers-of-trust-in-public-\ninstitutions-to-meet-current-and-future-challenges_b6c5478c-en. ( )\np\n//\ng/\n/\n[28] Stecula DA, Pickup M. Social Media, Cognitive Reflection, and Conspiracy\nBeliefs. Front. Polit. Sci. 2021;3(June):1–8. https://doi.org/10.3389/\nfpos.2021.647957. [29] Weinberg J. Can Political Trust Help to Explain Elite Policy Support and Public\nBehaviour in Times of Crisis? Evidence from the United Kingdom at the Height\nof the 2020 Coronavirus Pandemic. Polit. Stud. 2020. https://doi.org/10.1177/\n0032321720980900. [5] Bruder M, Haffke P, Neave N, Nouripanah N, Imhoff R. Measuring Individual\nDifferences in\nGeneric Beliefs\nin Conspiracy Theories Across Cultures:\nConspiracy Mentality Questionnaire. Front Psychol 2013;4(April). https://doi. org/10.3389/fpsyg.2013.00225. [30] Yuan H, Long Q, Huang G, Huang L, Luo S. Different roles of interpersonal trust\nand institutional trust in COVID-19 pandemic control. Ethical review [10] Easton D. A Re-Assessment of the Concept of Political Support. Br J Polit Sci\n1975;5(4):435–57. https://doi.org/10.1017/S0007123400008309. [11] Frederick S. Cognitive reflection and decision making. J Econ Perspect 2005;19\n(4):25–42. https://doi.org/10.1257/089533005775196732. This study was approved by the Faculty of Social Sciences\nResearch Ethics Committee at the University of Southampton\n(Ethics/ERGO Number: 61735). [12] Garry J, Ford R, Johns R. Coronavirus conspiracy beliefs, mistrust, and\ncompliance:\nTaking\nmeasurement\nseriously. Psychol\nMed\n2021;1–11. https://doi.org/10.1017/S0033291720005164. p\n//\ng/\n/\n[13] Hetherington MJ. Why Trust Matters: Declining Political Trust and the Demise\nof American Liberalism (Kindle Edi). Princeton University Press; 2005. Discussion Political trust in the first year of the COVID-19 pandemic: a meta-\nanalysis of 67 studies. J Eur Publ Policy 2023. https://doi.org/10.1080/\n13501763.2023.2169741. Declaration of Competing Interest 10.1007/s11135 017 0584 6. [16] Karic´ T, Mededovic´ J. Covid-19 conspiracy beliefs and containment-related\nbehaviour: The role of political trust. Pers Individ Differ 2021;175. https://doi. /10 1016/j\nid 2021 11069 /\n[16] Karic´ T, Mededovic´ J. Covid-19 conspiracy beliefs and containment-related\nbehaviour: The role of political trust. Pers Individ Differ 2021;175. https://doi. org/10.1016/j.paid.2021.110697. The authors declare the following financial interests/personal\nrelationships which may be considered as potential competing\ninterests: Will Jennings reports financial support was provided by\nthe UK Economic and Social Research Council (ESRC). Will Jennings\nreports financial support was provided by Research England QR\nGlobal Challenges Research Fund (GCRF). [17] Kerr JR, Schneider CR, Recchia G, Dryhurst S, Sahlin U, Dufouil C, et al. Correlates\nof\nintended\nCOVID-19\nvaccine\nacceptance\nacross\ntime\nand\ncountries: Results from a series of cross-sectional surveys. BMJ Open\n2021;11(8). https://doi.org/10.1136/bmjopen-2020-048025. [18] Lang FR, John D, Lüdtke O, Schupp J, Wagner GG. Short assessment of the Big\nFive: Robust across survey methods except telephone interviewing. Behav Res\nMethods 2011;43(2):548–67. https://doi.org/10.3758/s13428-011-0066-z. Acknowledgments [19] Lazarus JV, Ratzan SC, Palayew A, Gostin LO, Larson HJ, Rabin K, et al. A global\nsurvey of potential acceptance of a COVID-19 vaccine. Nat Med 2021;27\n(2):225–8. https://doi.org/10.1038/s41591-020-1124-9. Financial support was provided by the ESRC (ES/S009809/1) and\nResearch\nEngland\nQR\nGlobal\nChallenges\nResearch\nFund\n(519016146). [20] Lindholt MF, Jørgensen F, Bor A, Petersen MB. Public acceptance of COVID-19\nvaccines: Cross-national evidence on levels and individual-level predictors\nusing observational data. BMJ Open 2021;11(6):1–12. https://doi.org/10.1136/\nbmjopen-2020-048172. [21] Mathieu E, Ritchie H, Rodés-Guirao L, Appel C, Giattino C, Hasell J, et al. ‘‘Coronavirus Pandemic (COVID-19)”. Published online at OurWorldInData. org; 2022. Retrieved from: ’https://ourworldindata.org/coronavirus’ [Online\nResource]. Data will be made available on request. Data will be made available on request. [15] Johnston R, Jones K, Manley D. Confounding and collinearity in regression\nanalysis: a cautionary tale and an alternative procedure, illustrated by studies\nof British voting behaviour. Qual Quant 2018;52(4):1957–76. https://doi.org/\n10.1007/s11135-017-0584-6. Appendix A. Supplementary material Supplementary data to this article can be found online at\nhttps://doi.org/10.1016/j.jvacx.2023.100299. ]\n[22] McCarthy M, Murphy K, Sargeant E, Williamson H. Examining the relationship\nbetween conspiracy theories and COVID-19 vaccine hesitancy: A mediating\nrole for perceived health threats, trust, and anomie? Anal Soc Issues Public\nPolicy 2022;22(1):106–29. https://doi.org/10.1111/asap.12291. Data availability [14] Jennings W, Stoker G, Valgarðsson V, Devine D, Gaskell J. How trust, mistrust\nand distrust shape the governance of the COVID-19 crisis. J Eur Publ Policy\n2021;28(8):1174–96. https://doi.org/10.1080/13501763.2021.1942151. References Soc Sci Med 2022; 293\n(August 2021): 114677. doi:10.1016/j.socscimed.2021.114677. [6] Bunting H., Gaskell J., Stoker G. Trust, Mistrust and Distrust: A Gendered\nPerspective\non\nMeanings\nand\nMeasurements. Front. Polit. Sci. 2021:3\n(November):1-15. doi:10.3389/fpos.2021.642129. 9 9"
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Historicising Entanglements: Science, Technology and Socio-Ecological Change in the Postcolonial Anthropocene
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Document status and date: Published: 01/06/2022 Document status and date: Published: 01/06/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. p • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication Link to publication General rights C i ht d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study Y t f th di t ib t th t i l it f fit ki ti it i l i • You may not further distribute the material or use it for any profit-making activity o • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: Historicising Entanglements Citation for published version (APA): de Hoop, E., Sridhar, A., da Silva, C. M., & van der Vleuten, E. B. A. (2022). Historicising Entanglements: Science, Technology and Socio-Ecological Change in the Postcolonial Anthropocene . Global Environment, 15(2), 195-208. https://doi.org/10.3197/ge.2022.150201 Citation for published version (APA): de Hoop, E., Sridhar, A., da Silva, C. M., & van der Vleuten, E. B. A. (2022). Historicising Entanglements: Science, Technology and Socio-Ecological Change in the Postcolonial Anthropocene . Global Environment, 15(2), 195-208. https://doi.org/10.3197/ge.2022.150201 Citation for published version (APA): de Hoop, E., Sridhar, A., da Silva, C. M., & van der Vleuten, E. B. A. (2022). Historicising Entanglements: Science, Technology and Socio-Ecological Change in the Postcolonial Anthropocene . Global Environment, 15(2), 195-208. https://doi.org/10.3197/ge.2022.150201 DOI: 10.3197/ge.2022.150201 DOI: 10.3197/ge.2022.150201 Download date: 24. Oct. 2024 www.tue.nl/taverne Take down policy If you believe that this document breaches copyright please contact us at: openaccess@tue.nl providing details and we will investigate your claim. Download date: 24. Oct. 2024 Historicising Entanglements: Science, Technology and Socio- Ecological Change in the Postcolonial Anthropocene Evelien de Hoop, Aarthi Sridhar, Claiton Marcio da Silva and Erik van der Vleuten Evelien de Hoop, Aarthi Sridhar, Claiton Marcio da Silva and Erik van der Vleuten 1 P.J. Crutzen and E.F. Stoermer, ‘The “Anthropocene”’, IGPB Newsletter 41 (2000): 17–18; P.J. Crutzen, ‘Geology of mankind’, Nature 415 (51) (2002): 23. For a history, see H. Trischler, ‘The Anthropocene: A challenge for the history of science, technology, and the environment’, NTM Journal of the History of Science, Technology and Medicine 24 (3) (2016): 309–35. gy 2 J. Lorimer, ‘Multinatural geographies for the Anthropocene’, Progress in Hu- man Geography 36 (5): 593–612, at 606. Also, D. Chakrabarty, The Climate of History in a Planetary Age (Chicago and London: The University of Chicago Press, 2021); G. Caluya, ‘Fragments for a postcolonial critique of the Anthropocene: in- vasion biology and environmental security’, in J. Frawley and I. McCalman (eds), Rethinking Invasion Ecologies from the Environmental Humanities (London: Rout- ledge, 2014); A.S. Mathews, ‘Anthropology and the Anthropocene: criticisms, ex- periments, and collaborations’, Annual Review of Anthropology 49 (2020): 67–82; B. Latour, ‘Anthropology at the time of the Anthropocene: A personal view of what is to be studied’, in M. Brightman and J. Lewis (eds), The Anthropology of Sustainability (New York: Palgrave Macmillan, 2017), pp. 35–49; J. Lorimer, ‘The Anthropo-scene: A guide for the perplexed’, Social Studies of Science 47 (1) (2017):117–42. T.J. LeCain, ‘Against the Anthropocene: a Neo-materialist per- spective’, International Journal for History, Culture and Modernity 3 (2015): 1–28. Evelien de Hoop, Aarthi Sridhar, Claiton Marcio da Silva and Erik van der Vleuten his special issue focuses on connected histo- ries of science, technology and socio-ecolog- ical change in what we call the ‘postcolonial Anthropocene’. We used this term to guide the papers in this issue towards research questions that interrogate both human-na- ture relations and postcolonial relations, as entangled components of each inquiry. Of T course, both constituent terms ‘postcolonial’ and ‘Anthropocene’ are contentious and much-debated, with diverse connotations and perceived implications to diverse readerships; using both together can be a risky business. We nevertheless chose to do so, because we felt that, both in historiography and in wider academic discourses, Anthropocene and postcolonial research perspectives have too often remained poorly connected – despite the growing number of schol- ars arguing that Anthropocene research must include postcolonial perspectives and vice versa. In this introductory essay, we begin by discussing specific lines of (postcolonial) Anthropocene scholarship we chose to connect to, before we specify this issue’s overarching research questions and introduce the context and content of the in- dividual papers. GE 195 GE 195 The ‘Anthropocene’ and its critiques Caluya, ‘Fragments for a postcolonial critique of the Anthropocene: in- vasion biology and environmental security’, in J. Frawley and I. McCalman (eds), Rethinking Invasion Ecologies from the Environmental Humanities (London: Rout- ledge, 2014); A.S. Mathews, ‘Anthropology and the Anthropocene: criticisms, ex- periments, and collaborations’, Annual Review of Anthropology 49 (2020): 67–82; B. Latour, ‘Anthropology at the time of the Anthropocene: A personal view of what is to be studied’, in M. Brightman and J. Lewis (eds), The Anthropology of Sustainability (New York: Palgrave Macmillan, 2017), pp. 35–49; J. Lorimer, ‘The Anthropo-scene: A guide for the perplexed’, Social Studies of Science 47 (1) (2017):117–42. T.J. LeCain, ‘Against the Anthropocene: a Neo-materialist per- spective’, International Journal for History, Culture and Modernity 3 (2015): 1–28. The ‘Anthropocene’ and its critiques It has been over two decades since Paul J. Crutzen and Eugene F. Stoermer proposed the term ‘Anthropocene’ for the current geological epoch in the International Geosphere-Biosphere Programme IGBP newsletter (and soon after in a famous article in Nature). The term, informally in use since at least the 1980s, was intended to emphasise the ‘central role of mankind’ as ‘a powerful geological force’ changing the ‘systemic properties’ of the planet and the atmosphere.1 Today (at the time of writing) geologists are still debating whether to accept the Anthropocene as an official subdivision of geological time. Meanwhile humanities and social science scholars of many stripes have also mas- sively engaged with the Human Epoch as a geological as well as a cul- tural notion, based on their own (inter)disciplinary perspectives and concerns. Amongst those perspectives, we would like to highlight that many have lauded ‘the diagnosis of the Anthropocene’ as a decisive acknowledgement of ‘the public death of the modern understanding of Nature’, whereafter humans can no longer be regarded as operat- ing in isolation from the ecologies in which they live.2 This insight gy 2 J. Lorimer, ‘Multinatural geographies for the Anthropocene’, Progress in Hu- man Geography 36 (5): 593–612, at 606. Also, D. Chakrabarty, The Climate of History in a Planetary Age (Chicago and London: The University of Chicago Press, 2021); G. Caluya, ‘Fragments for a postcolonial critique of the Anthropocene: in- vasion biology and environmental security’, in J. Frawley and I. McCalman (eds), Rethinking Invasion Ecologies from the Environmental Humanities (London: Rout- ledge, 2014); A.S. Mathews, ‘Anthropology and the Anthropocene: criticisms, ex- periments, and collaborations’, Annual Review of Anthropology 49 (2020): 67–82; B. Latour, ‘Anthropology at the time of the Anthropocene: A personal view of what is to be studied’, in M. Brightman and J. Lewis (eds), The Anthropology of Sustainability (New York: Palgrave Macmillan, 2017), pp. 35–49; J. Lorimer, ‘The Anthropo-scene: A guide for the perplexed’, Social Studies of Science 47 (1) (2017):117–42. T.J. LeCain, ‘Against the Anthropocene: a Neo-materialist per- spective’, International Journal for History, Culture and Modernity 3 (2015): 1–28. gy 2 J. Lorimer, ‘Multinatural geographies for the Anthropocene’, Progress in Hu- man Geography 36 (5): 593–612, at 606. Also, D. Chakrabarty, The Climate of History in a Planetary Age (Chicago and London: The University of Chicago Press, 2021); G. 6 E.W. Krauss, ‘Anthropology in the Anthropocene: sustainable development, climate change and interdisciplinary research’, in H. Greschke and J. Tischler (eds), Grounding Global Climate Change (New York: Springer 2015), pp. 59–76; L. Ogden et al., ‘The politics of Earth stewardship in the uneven Anthropocene’, in R. Rozzi et al. (eds), Earth Stewardship. Linking Ecology and Ethics in Theory and Practice (New York: Springer 2015), pp. 137–57; J. Hope, ‘The anti‐politics of sustainable development: Environmental critique from assemblage thinking in Bolivia’, Transactions of the Institute of British Geographers 46 (1) (2021): 208–22. h p 4 R. Costanza et al., ‘Sustainability or collapse: what can we learn from in- tegrating the history of humans and the rest of nature?’, AMBIO: A Journal of the Human Environment 36 (7) (2007): 522–27; E. Russell, Evolutionary History: Uniting History and Biology to Understand Life on Earth (Cambridge: Cambridge University Press, 2011); J.L. Caradonna (ed.), Routledge Handbook of the History of Sustainability (New York, NY: Routledge, 2018); S. Sörlin, ‘Reform and respon- sibility – the climate of history in times of transformation’, Historisk tidsskrift 97 (1) (2018): 7–23.hh 5 C. Bonneuil and J.-B. Fressoz, The Shock of the Anthropocene. The Earth, His- tory and Us (London and New York: Verso, 2016), p. 13. 3 Trischler, ‘The Anthropocene’, 312. 7 J. Guldi and D. Armitage, The History Manifesto (Cambridge, Ma: Cam- bridge University Press, 2014), p. 69; Z.B. Simon, ‘History manifested: mak- ing sense of unprecedented change’, European Review of History 22 (5) (2015): 819–34; Z.B. Simon, The Epochal Event: Transformations in the Entangled Human, Technological, and Natural Worlds (London: Palgrave MacMillan, 2020). g g 8 D. Haraway et al., ‘Anthropologists are talking – about the Anthropocene’, Ethnos. Journal of Anthropology 81 (3) (2016): 535–64, p. 539. Also: D. Chandler, ‘The transvaluation of critique in the Anthropocene’, Global Society 33 (1): 26-44; Chakrabarty, The Climate of History in a Planetary Age; D. Haraway, ‘Anthropo- cene, Capitalocene, Plantationocene, Chthulucene: Making kin’, Environmental Humanities 6 (2015): 159–65; B. Latour, Down to Earth: Politics in the New Cli- matic Regime (Cambridge: Polity Press, 2018); A. Malm and A. Hornborg, ‘The geology of mankind? A critique of the Anthropocene narrative’, The Anthropocene Review 1 (1) (2014): 62–69; A. Blok and G.B. Jensen, ‘The Anthropocene event in social theory: On ways of problematizing nonhuman materiality differently’, The Sociological Review 67 (6) (2019): 1195–211. The classic is J. Ferguson, The Anti-politics Machine: ‘Development’, Depoliticiza- tion and Bureaucratic Power in Lesotho (Chicago: Chicago University Press, 1990).h INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN Ferguson, The Anti-politics Machine: ‘Development’, Depoliticiza- tion and Bureaucratic Power in Lesotho (Chicago: Chicago University Press, 1990).h g g 7 J. Guldi and D. Armitage, The History Manifesto (Cambridge, Ma: Cam- bridge University Press, 2014), p. 69; Z.B. Simon, ‘History manifested: mak- ing sense of unprecedented change’, European Review of History 22 (5) (2015): 819–34; Z.B. Simon, The Epochal Event: Transformations in the Entangled Human, Technological, and Natural Worlds (London: Palgrave MacMillan, 2020). INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN 196 inspired scholars to rethink interrelations between histories of science, technology and the environment.3 It also fostered, or resonated with, diverse initiatives towards collaboration across the natural sciences, social sciences and humanities to develop new knowledges informing the mitigation of the Anthropocene’s ecological challenges – including new historiographical knowledges.4 g p g However, and crucially, the proliferating use of the term An- thropocene has also met with substantial critique. We would like to emphasise two related critiques that are widely shared among histo- rians, anthropologists and geographers. First, concerning human re- lations, natural science-initiated accounts of the Anthropocene have been much criticised for depoliticising history by presenting ‘history as a contest between the human species as a whole and the planet, with societies as ignorant and passive masses who can only be guided by scientists and saved by green technologies’, as Bonneuil and Fres- soz bluntly phrased it.5 Research that universalises human agency tends to obscure differential human experiences, responsibilities and politics – some scholars therefore call such research an ‘anti-politics machine’.6 Second, concerning human-nature relations, the critique GE 197 G 197 G 197 is that omnipresent modernist uses of the term Anthropocene do not transcend the nature-society dualism at all, but instead enact, reproduce and amplify human exceptionalism. Implicitly and often quite explicitly, such modernist Anthropocene scholarship attributes to ‘humankind’ the position of primal Earth-changer and, as a con- sequence, Earth stewardship to repair environmental harm done in the ‘Human Epoch’. Such unabashed anthropocentrism also echoes in historiographical debate of the Anthropocene; for example, the History Manifesto endorsed calls for (long-term) histories to assists humans in their role as responsible Earth managers – and was fierce- ly criticised by others arguing that such human exceptionalist think- ing is part of the problem rather than the solution.7 Either way, the combination of over-emphasising and universalising human agency obscures how change on Earth emerges from complex, connected and situated interactions between a wide variety of human and non- human agents, and that such change may be distributed unequally across the earth. The world is not a ‘human species act’, as the term Anthropocene may portray it to be.8 h These critiques elicited many responses. One prominent line has been to develop a range of alternative terms to the notion of The classic is J. 9 J.W. Moore, Anthropocene or Capitalocene? Nature, History, and the Crisis of Capitalism (Oakland: PM Press, 2016). 10 D. Haraway, Staying with the Trouble: Making Kin in the Chthulucene (Dur- ham, NC: Duke University Press, 2016); M. Armiero, Wasteocene: Stories from the Global Dump (Cambridge: Cambridge University Press, 2021); C.M. da Silva and C. de Majo ‘Towards the soyacene: Narratives for an environmental history of soy in Latin America’s Southern Cone’, Historia Ambiental Latinoamericana y Caribeña 11 (1) (2021): 329–56. 11 Haraway, ‘Anthropocene, Capitalocene, Plantationocene, Chthulucene’; J. Davis et al., ‘Anthropocene, Capitalocene, … Plantationocene? A manifesto for ecological justice in an age of global crises’, Geography Compass 13 (5) (2019): e12438; B. Latour et al., ‘Anthropologists are talking – about capitalism, ecology, and apocalypse’, Ethnos 83 (3) (2018): 587–606. N. Myers, ‘From the Anthro- pocene to the Planthroposcene: Designing gardens for plant/people involution’, History and Anthropology 28 (3) (2017): 297–301. 9 J.W. Moore, Anthropocene or Capitalocene? Nature, History, and the Crisis of Capitalism (Oakland: PM Press, 2016). 10 D. Haraway, Staying with the Trouble: Making Kin in the Chthulucene (Dur- ham, NC: Duke University Press, 2016); M. Armiero, Wasteocene: Stories from the Global Dump (Cambridge: Cambridge University Press, 2021); C.M. da Silva and C. de Majo ‘Towards the soyacene: Narratives for an environmental history of soy in Latin America’s Southern Cone’, Historia Ambiental Latinoamericana y Caribeña 11 (1) (2021): 329–56. 11 Haraway, ‘Anthropocene, Capitalocene, Plantationocene, Chthulucene’; J. Davis et al., ‘Anthropocene, Capitalocene, … Plantationocene? A manifesto for ecological justice in an age of global crises’, Geography Compass 13 (5) (2019): e12438; B. Latour et al., ‘Anthropologists are talking – about capitalism, ecology, and apocalypse’, Ethnos 83 (3) (2018): 587–606. N. Myers, ‘From the Anthro- pocene to the Planthroposcene: Designing gardens for plant/people involution’, History and Anthropology 28 (3) (2017): 297–301. INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN 198 the Anthropocene, which explicitly invite scrutiny of human and multispecies differential politics, including postcolonial relations. To mention but a few: the notion of Capitalocene presents, in the words of Moore, the world as a ‘multi-species assemblage, a world- ecology of capital, power, and nature’.9 The term Plantationocene takes inspiration from the study of colonial plantation systems and refers to the radical simplification of previously diverse living sys- tems and their relocation elsewhere, and thereby producing life for value extraction. The Wasteocene concept invites historians to scru- tinise the embodied stratigraphy of power and toxicity with special attention to subaltern human and more-than-human communities; the notion of the Soyacene invites study of how soybean has medi- ated human and non-human lives especially in the last fifty years.10 Notions such as Chthulucene (derived from the Greek word chthon meaning earth) and Planthroposcene refer not to critical analysis of historical epochs, but to an ongoing attempt to deliberately shift the terms of encounter with Anthropocene-type-of-concerns for future modes of multispecies engagement.11 Note that these are only some of the alternative ‘Big Words’ that have been proposed recently to guide research on today’s major social and environmental challenges in more critical and sensitive directions. Still, as observed by Hara- way, the use of such terms comes with the risk of creating a Theory GE 199 GE 199 G 199 of Everything at the expense of empirical study that takes historical contingency, specificity and situatedness seriously.12 12 Haraway et al., ‘Anthropologists are talking – about the Anthropocene’, 550 and 651; C. Bos et al, ‘Steering with big words: Articulating ideographs in research programs’, Journal of Responsible Innovation 1 (2) (2014): 151–70.h p g f p 13 S. Harding (ed.),  The Postcolonial Science and Technology Studies Reader (Durham, NC: Duke University Press, 2011); J. Law and W. Y. Lin, ‘Provincial- izing STS: Postcoloniality, symmetry, and method’, East Asian Science, Technology and Society 11 (2) (2017): 211–27; A. Kumar et al. (eds), Dilemmas of Energy Transitions in the Global South (New York: Routledge, 2021); Ute Hasenörl, ‘His- tories of technology and the environment in post/colonial Africa: Reflections on the field’, Histories 1 (3)(2021): 122–44; J. van der Straeten, ‘The rhythms behind change. Historiography and the temporality of non-Western technological land- scapes’, Technikgeschichte 88 (2) (2021): 191–96. Understanding socio-ecological entanglements in the postcolonial Anthropocene This latter observation leads us to literatures highlighting and studying the historical, spatial, social and material situatedness of specific socio-ecological entanglements and changes, most notably historical and postcolonial Science and Technology Studies (STS).13 As such, this special issue approaches humans as diverse beings who are co-constituted by and act together with the (social, material, ecological) environments in which they live.14 This entanglement g f p 13 S. Harding (ed.),  The Postcolonial Science and Technology Studies Reader (Durham, NC: Duke University Press, 2011); J. Law and W. Y. Lin, ‘Provincial- izing STS: Postcoloniality, symmetry, and method’, East Asian Science, Technology and Society 11 (2) (2017): 211–27; A. Kumar et al. (eds), Dilemmas of Energy Transitions in the Global South (New York: Routledge, 2021); Ute Hasenörl, ‘His- tories of technology and the environment in post/colonial Africa: Reflections on the field’, Histories 1 (3)(2021): 122–44; J. van der Straeten, ‘The rhythms behind change. Historiography and the temporality of non-Western technological land- scapes’, Technikgeschichte 88 (2) (2021): 191–96. p g 14 Note also that in his ‘Postcolonial Studies and the Challenge of Climate Change’, Chakrabarty speaks of the postcolonial ‘view of the human as the same everywhere ... endowed with … anthropological difference’. This has received criti- cism from within postcolonial scholarship, and we follow the latter – work which recognises the emergence of difference without making the assumption of univer- sality being hidden behind difference (e.g. D. Boscov-Ellen, ‘Whose universalism? Dipesh Chakrabarty and the Anthropocene’, Capitalism Nature Socialism 31 (1): 70–83; G. Caluya, ‘Fragments for a postcolonial critique of the Anthropocene: inva- sion biology and environmental security’, in J. Frawley and I. McCalman (eds), Re- thinking Invasion Ecologies from the Environmental Humanities (London: Routledge, 2014); G. Jack, ‘Advancing postcolonial approaches in critical diversity studies’, in R. Bendi et al. (eds), The Oxford Handbook of Diversity in Organizations (Oxford: INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN 200 has implications not only for our understanding of humans, their agency and power imbalances in the Anthropocene, but also for our understanding of what constitutes these humans’ material and ecological ‘environments’. It means we do not approach such en- vironments as singular, not a ‘One-World world’ as geological and other natural sciences studying the Anthropocene would have it,15 Oxford University Press, 2015)). Postcolonial theorists’ conceptualisation of human beings and their agency as being co-constituted by the environments in which they live draws upon both (older) STS work, such as actor-network theory (B. Latour, Reassembling the Social: An Introduction to Actor-Network-Theory (New York: Oxford University Press, 2005)) and on more recent new materialist work which has been spurred in part by the challenge of the Anthropocene (A.F. Conty, ‘The Politics of nature: New materialist responses to the Anthropocene’, Theory, Culture and So- ciety 35 (7–8) (2018): 73–96). See, for example, the notion of trans-corporeality proposed by feminist critical theorist Alaimo, denoting that ‘the human is always intermeshed with the more-than-human world, ... ultimately inseparable from “the environment”’ (S. Alaimo, Bodily Natures: Science, Environment, and the Material Self (Bloomington, IN: Indiana University Press, 2010), p. 2). f g y p 15 M. De La Cadena and M. Blaser, A World of Many Worlds (Durham, NC: Duke University Press, 2018). This One-World world has been critiqued for creat- ing a colonial divide between ‘those who function within the OWW from those who insist on other ways of bringing a world into being, or “worlding”’ (A. Es- cobar, ‘Thinking-feeling with the Earth: Territorial struggles and the ontological dimension of the epistemologies of the South’, Revista de Antropología Iberoameri- cana 11 (1) (2016): 11–32, at 21). Indeed, scholarship on indigenous protests against extractivism, such as Australian Aboriginal resistance to drilling (D. Dan- owski and E. de Castro, The Ends of the World (Cambridge, UK and Malden, Ma: Polity Press, 2016)) or Meswaki and Sioux resistance to the Dakota Access Pipeline (G. Giuliani, Monsters, Catastrophes and the Anthropocene; A Postcolonial Critique (Abingdon, Oxon and New York, NY: Routledge, 2021)) has power- fully brought the power and relevance of a plurality of locally-situated and (often) non-anthropocentric cosmologies to the fore, and contrasted these with those en- acted through extractivist approaches. 16 A. Blunt and C. McEwan, Postcolonial Geographies (London and New York: Continuum, 2002). 17 R. Wenzlhuemer, Doing Global History: An Introduction in 6 Concepts (Lon- don and New York: Bloomsbury, 2020). On infrastructure, supply chain, and commodity circulation histories see e.g., A. Johnson, ‘Europe without borders: Environmental and global history in a world after continents’, Contemporary Eu- ropean History 31 (1) (2022): 129–41; L. Marques, ‘Commodity chains and the global environmental history of the colonial Americas’, Esboços: histórias em con- textos globais 28 (49) (2021): 640–97; P. Högselius et al., Europe’s Infrastructure Transition: Economy, War, Nature (London: Palgrave MacMillan, 2016). INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN Other ways of worlding have not only been identified in scholarship on resistance against extractivism, but of course also in, amongst others, work on Indigenous worldviews on ecology and American Indian cosmologies, and on non-modern philosophies of life such as Buen Vivier. See, amongst many others, J.M. Beijer, ‘Beyond hegemonic state(ment)s of Nature. Indigenous knowledge and non-state possibilities in international relations’, in C. Geeta and S. Nair (eds), Power, Postcolonialism and International Relations. Read- ing Race, Gender and Class (London: Routledge, 2002). GE 201 GE 201 G 201 G 201 but as multiple and possibly inequitable, arising together with the imaginations and practices of the humans who live in them. Indeed, and critically, understanding both humans and their environments as co-constituting each other helps to bring (post)colonial power re- lations and inequalities in the shaping of sited human-environment relationships to the fore. This resulted in the following question that guided the investigations of the papers published in this special is- sue: which human-environment constellations emerged at the site(s) studied, how did they relate to one another, and what inequalities were embedded therein? Second, we approach this process of co-constitution in (post) colonial worlds as transnationally entangled.16 In particular, the pa- pers in this special issue take the lead from two bodies of historical work in which such transnational entanglements feature particu- larly prominently, namely histories of relations and relational histo- ries. The former constitute historical investigations of connections and relations between different sites and the ways in which these relations and sited socio-ecological dynamics co-constituted each other.17 These histories of relations predominantly approach diverse sites’ histories from the perspective of a common logic (and, despite claims to the contrary, not seldom a European or global North-in- spired one), and their attentiveness to multi-sited historical diver- sity has therefore been limited. In contrast, relational and connected histories investigate how regional histories may be at once highly geographically distant, diverse, specific and situated, yet neverthe- INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN 18 See S. Subrahmaniam, Europe’s India. Words, People, Empires, 1500–1800 (Harvard: Harvard University Press, 2017). 19 A.L. Stoler, Along the Archival Grain: Epistemic Anxieties and Colonial Com- mon Sense (Princeton and Oxford: Princeton University Press, 2009). We consider this important, amongst others, in view of providing room to diverse ways of understanding and living in today’s world(s), cf. A. Escobar, Designs for the Pluriv- erse Radical Interdependence, Autonomy, and the Making of Worlds (Durham: Duke University Press, 2018).h INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN 202 less develop in mutual interaction.18 Such work may explicitly move against the archival grain, deconstructing the dominant narratives of archives and contemporary historical ‘sources’ that scholars use and contrasting these with the narratives of a wide diversity of voices.19 In both types of histories, relations are established through the trav- els of a wide variety of entities; in line with this issue’s grounding in postcolonial STS we particularly focus on (expert and lay) knowl- edges and technologies as phenomena that connect and co-construct geographically dispersed and locally situated socio-ecological histo- ries. This resulted in the second question that guided the investiga- tions of the papers published in this special issue: what roles did science and technology, which often travelled between diverse locations across the globe, play in the emergence of and inequalities embedded in the diverse human-environment constellations identified in response to the first question? 20 Crutzen and Stoermer, ‘The “Anthropocene”’, 18. 21 The initiative sprang from a workshop in Lisbon in Autumn 2019, organ- ised in the context of the Tensions of Europe programme Technology and Societal Challenges 1850–2050 and its ‘Technology, environment and resources’ working group. For the broader programme: E. van der Vleuten, ‘Technology, societal challenges, and global sustainability history’, Icon 24 (2018): 34–52; E. van der Vleuten, ‘History and technology in an age of grand challenges: Raising ques- tions’, Technology and Culture 61(1): 260–71. For the ‘Technology, environment and resources’ working group: M. Heymann et al. ‘Challenging Europe: Tech- nology, environment, and the quest for resource security’, Technology and Cul- ture 61 (1) (2020): 282–94; O. Sparenberg and M. Heymann (eds), ‘Resource challenges and constructions of scarcity in the 19th and 20th centuries’, Special Issue in European Review of History 27 (1–2) (2020): 243–369; C. Kehrt and J. Martin (eds), ‘Reconfiguring nature: Resource security and the limits of expert knowledge’, Global Environment 13 (3) (2020): 512–658; A. Åberg and F. Ve- raart (eds), ‘Creating, capturing and circulating commodities: The technology and politics of material resource flows, from the 19th century to the present’, Special section in The Extractive Industries and Society 7 (1) (2020): 1–67; J. Daheur (ed.), ‘Extractive Peripheries in Europe: Quest for Resources and Changing Environ- ments (Fifteenth-Twentieth Centuries)’, Special issue of Global Environment 15 (1) (2022): 7–147. Contributions to this special issue Crutzen and Stoermer offered the term ‘Anthropocene’ in order to inspire and empower the development of ‘a world-wide accepted strategy leading to sustainability of ecosystems against human in- duced stresses’; such ‘great future tasks of mankind’ in their words required intensive research efforts and the ‘wise application of the knowledge thus acquired’.20 Critiquing such calls and research for their universalisation of humanity and the obscuring of differential experiences, responsibilities and politics, this special issue has sought to study situated and diverse engagements with socio-ecological GE 203 GE 203 G 203 G 203 change, guided by the two questions raised in the foregoing para- graphs.21 This effort has resulted in the following papers. hf First, de Hoop and van der Vleuten study how human-environ- ment constellations were variously articulated in scientific knowl- edge on palm oil sustainability in Southeast Asia and Europe since 1980. They ask how such research enacted a postcolonial politics of difference between Southeast Asia and Europe with regard to defining ‘sustainability’ problems and solutions. They observe that palm oil sustainability research originating from Southeast Asia fore- grounded problems experienced and to be redressed within the re- gion itself. By contrast, diverse strands of research lead by scholars from Europe variously framed migrant, smallholder and large-scale palm oil farmers in Southeast Asia as primarily responsible for caus- ing and solving ‘global sustainability problems’, notably global de- forestation and climate change. This ‘global sustainability’ discourse by and large acquitted European actors and markets from such re- sponsibilities – even though these had long deforested their own ter- ritories, had played a major role in establishing palm oil cultivation, INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN 204 trade and use, and had constituted prime markets for most of the twentieth century. Next, da Silva and de Majo’s study of Southern Brazilian tradi- tional populations known as Caboclos shows how a relationship of mutual dependence between people and their forests had been built up over centuries. This ‘bio-anthropophagic’ relationship changed dramatically when a new wave of settlers advanced westward in the early twentieth century, creating new human-environment constel- lations based on governments’ and settlers’ ideas about both the for- est and its traditional populations. These ideas were prominently informed by the travels of both racial and eugenic theories and as- sociated sanitation policies. Thus followed a rearrangement of the exploitation of natural materials – such as yerba mate or herbal medicines – based on new hierarchies. Local populations survived through astute strategies of incorporating exogenous values, giving rise to deep inequalities with respect to the ability to both materially and ontologically define one’s one way of life. g yi y Also located in South America, França de Oliveira and Zarilli’s contribution analyses how the transformation of the Argentine pampas into an area of large-scale cattle farming went hand in hand with profound environmental, economic and demographic transfor- mations. This included the incorporation of over forty million hec- tares of pampas into market-based agriculture and cattle-farming; the introduction of new animal and plant species as well as a range of modern agricultural technologies; new economic models of farm- ing; and a substantial increase in the number of people living in the area. This paper demonstrates how these changes were mediated not only by the introduction of cattle and the early establishment of farms in the area in the sixteenth century, but also by transportation infrastructures whereby railroads connected the pampas to Argen- tina’s ports, reconfiguring the landscape. i Next, Abazeed and Hafez investigate how cultural discourse co- constructed the Nile as a ‘modern river’ through an analysis of imag- inaries and knowledges of rivers as represented in two different sets of writing: Egyptian renaissance writing by modernist Alī Mubārak, who hybridised a European education into his nationalist develop- GE 205 GE 205 ment visions, and a set of travelogue diaries of orientalist European travelers writing for European audiences. INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN Both cultural traditions harnessed modernist ideas, but did so in situated ways, using dif- ferent vantage points, to remake the free-flowing River Nile and its people into a modern ‘hybrid entity’ drawing in multiple cultural and natural attributes. Fotopoulos and Araposthatis highlight how explorations of hy- drocarbons, regional visions and governing practices were historical- ly co-constructed in the Eastern Mediterranean, with implications for who has access to Greece’s hydrocarbons. They argue that Euro- pean and North American expertise on hydrocarbon explorations as well as various foreign exploration technologies and infrastructures were critical to encoding these power constellations in regional hy- drocarbon development. p The final paper of the issue represents the tradition of investi- gating histories of relations and connections rather than relational histories: Veraart investigates how the development and travels of catalysis technologies, which enabled the production of margarine from a diversity of oils and fats, gave rise to a variety of new hu- man-environment constellations at different sites across the world, through putative supply chain relations forged in colonisation con- texts across Congo, the Dutch Indies and the Antarctic, between 1910 and 1940. Acknowledgements This special issue springs from the inspiring workshop on ‘Re- sources, Infrastructures and the Anthropocene: Dialogues between the Global-North and the Global-South’, organised at the Univer- sidade NOVA de Lisboa in 2019. We are particularly indebted to the workshop participants for vivid, critical and inspiring contribu- tions and debate. We also thank the organizers and funders of the workshop: the Centro Interuniversitário de História das Ciências e da Tecnologia, the Dutch research council NWO’s internationalising humanities program GREASE (nr. IG.18.036), the Danish Council INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN 206 for Independent Research’s research network programme EurReS, and the Foundation for the History of Technology SHT. for Independent Research’s research network programme EurReS, and the Foundation for the History of Technology SHT. Erik van der Vleuten teaches at Eindhoven University of Technology and researches the connected histories of technology and socioecological change in an infrastructured world. Earlier works include: Europe’s Infrastructure Transition: Economy, War, Nature (2015, with Per Högselius and Arne Kaijser) and Engineer- ing the Future, Understanding the Past: A Social History of Technology (2017, with Ruth Oldenziel and Mila Davids). His current research and education focus on diverse and contested sustainability histories and futures of distant-yet-connected regions across the globe. Email: e.b.a.v.d.vleuten@tue.nl INTRODUCTION / DE HOOP, SRIDHAR, DA SILVA, V. D. VLEUTEN for Independent Research’s research network programme EurReS, and the Foundation for the History of Technology SHT. Evelien de Hoop is based at the transdisciplinary Athena Institute (Vrije Universiteit Amsterdam, NL). With a background in Science and Technology Studies, she works with scholars from diverse disciplines and societal stakehold- ers to address contemporary health and sustainability challenges. Empirical foci include Indian farmers’ engagements with biofuels, sustainable Dutch country- sides, and inclusive food systems in deprived urban areas. She mobilises research on the historical and transnational entanglements of contemporary challenges, with a particular focus on knowledge politics, to render her contemporary work historically-sensitive and pluralise ways of thinking about the future. Aarthi Sridhar is one of the founder trustees of Dakshin Foundation and is pursuing a Ph.D. from the University of Amsterdam on a historical sociology of fisheries science in India. Her academic interests centre on historical and con- temporary socio-legal studies, Science and Technology Studies, and sociological approaches to knowledge and expertise. Her empirical focal areas are coastal and marine environments, resource politics, maritime infrastructures and practices of environmental norms and justice. She enjoys collaborative projects and has worked with diverse disciplinary teams on writing projects, documentary films, technical websites, field manuals and other learning material on the subject of marine environments and people. Email: aarthi77@gmail.com Email: aarthi77@gmail.com Claiton Marcio da Silva is Associate Professor at the Federal University of the Southern Frontier (UFFS), Santa Catarina, Brazil and CNPq Productiv- ity Researcher. He began studying the modernisation of Brazilian agriculture in 2002, completing his MA in history at the Federal University of Santa Catarina (UFSC) with a thesis on rural youth in Southern Brazil. At Casa de Oswaldo Cruz (COC/Fiocruz), his doctoral dissertation explored the role of Nelson Rockefeller’s American International Association for Economic and Social Development (AIA) in introducing the US extension service model in Latin America. Recently, he has been exploring the global environmental history of soybeans and its impacts for traditional populations in the Global South. Email: claiton@uffs.edu.br GE 207 GE 207 Erik van der Vleuten teaches at Eindhoven University of Technology and researches the connected histories of technology and socioecological change in an infrastructured world. Earlier works include: Europe’s Infrastructure Transition: Economy, War, Nature (2015, with Per Högselius and Arne Kaijser) and Engineer- ing the Future, Understanding the Past: A Social History of Technology (2017, with Ruth Oldenziel and Mila Davids). His current research and education focus on diverse and contested sustainability histories and futures of distant-yet-connected regions across the globe.
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Symmetry Extensions and Their Physical Reasons in the Kinetic and Hydrodynamic Plasma Models
Symmetry, integrability and geometry: methods and applications
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Symmetry Extensions and Their Physical Reasons in the Kinetic and Hydrodynamic Plasma Models⋆ Volodymyr B. TARANOV Institute for Nuclear Research, 47 Nauky Ave., 03028 Kyiv, Ukraine E-mail: wlatar@inet.ua URL: http://www.geocities.com/vebete/mypage.html p g yp g Received October 31, 2007, in final form January 14, 2008; Published online January 17, 2008 Original article is available at http://www.emis.de/journals/SIGMA/2008/006/ Received October 31, 2007, in final form January 14, 2008; Published online January 17 O igi al a ticle is a ailable at htt // i d /j l /SIGMA/2008/006/ Received October 31, 2007, in final form January 14, 2008; Published online January 17, 2008 Original article is available at http://www.emis.de/journals/SIGMA/2008/006/ Original article is available at http://www.emis.de/journals/SIGMA/2008/006/ Abstract. Characteristic examples of continuous symmetries in hydrodynamic plasma theory (partial differential equations) and in kinetic Vlasov–Maxwell models (integro- differential equations) are considered. Possible symmetry extensions conditional and ex- tended symmetries are discussed. Physical reasons for these symmetry extensions are clarified. Key words: symmetry; plasma; hydrodynamic; kinetic Key words: symmetry; plasma; hydrodynamic; kinetic 2000 Mathematics Subject Classification: 35A30; 35Q60; 45K05; 58J70; 22E70 2000 Mathematics Subject Classification: 35A30; 35Q60; 45K05; 58J70; 22E70 ⋆This paper is a contribution to the Proceedings of the Seventh International Conference “Symmetry in Nonlinear Mathematical Physics” (June 24–30, 2007, Kyiv, Ukraine). The full collection is available at http://www.emis.de/journals/SIGMA/symmetry2007.html Symmetry, Integrability and Geometry: Methods and Applications Symmetry, Integrability and Geometry: Methods and Applications SIGMA 4 (2008), 006, 7 pages 1 Introduction Symmetry considerations essentially help us to solve nonlinear problems of plasma physics. Among the recent publications, a survey of methods which allow us to find symmetries of integro- differential equations of kinetic plasma theory [1] and a review of applications of symmetry methods to the hydrodynamic plasma models based on the partial differential equations [2] can be cited. In the present paper, some characteristic examples of the symmetries are presented for var- ious plasma theory models: electron magnetohydrodynamics, collisionless electron plasma os- cillations, drift waves in plasma and multi-component collisionless plasma containing particles with equal charge to mass ratios. In Section 2, Lie point symmetries are obtained for the partial differential equations of the electron magnetohydrodynamics by means of the standard Maple 11 program. A simple invariant solution is presented, which is in fact a background solution of a perturbation theory formalism. An additional condition is underlined which can lead to the symmetry extension. In Section 3, continuous symmetries of the integro-differential kinetic equations of the colli- sionless electron plasma model are discussed. The symmetries were found in [3] by an indirect algorithm which allows us to obtain symmetries of the kinetic equations from the symmetries of an infinite set of partial differential equations for the moments of distribution functions. In Section 4, some conditional symmetries are presented for the Hasegawa–Mima hydrody- namic model describing drift waves in a plasma. Due to these symmetries, invariant solutions are possible describing the asymptotic structure of the nonlinear waves. In Section 5, additional symmetries for integro-differential equations of the kinetic theory of collisionless plasma containing particles with equal charge to mass ratio are considered. For example, alpha particles and deuterium ions participating in a thermonuclear reaction D+ + T+ →He++ + n + 17.6 MeV have close charge to mass ratios. By simple vector considerations V.B. Taranov 2 so called extended symmetry transformations are obtained which allow us to reduce the number of equations. so called extended symmetry transformations are obtained which allow us to reduce the number of equations. Conclusions are made in Section 6 Conclusions are made in Section 6. 2 Electron magnetohydrodynamics Let us consider the equations of the electron magnetohydrodynamics (EMHD) [4 ∂Ψ ∂t = ∇× (v × Ψ), Ψ = B −∆B, v = −∇× B, ∇· B = 0, where Ψ is the generalized vorticity, B is magnetic field strength and v is the hydrodynamic velocity of the electron plasma component. Nine functions, Ψ, v, B, of four independent variab- les t, x, y, z are present in this system of partial differential equations. Nevertheless, its symmetry has been obtained by the standard Maple 11 package program. y y p p g p g The infinitesimal operators of Lie point symmetries of the model are as follows: X1 = ∂ ∂t, X2 = ∂ ∂x, X3 = ∂ ∂y, X4 = ∂ ∂z , X5 = t ∂ ∂t −v ∂ ∂v −B ∂ ∂B −Ψ ∂ ∂Ψ, X6 = r × ∂ ∂r + v × ∂ ∂v + B × ∂ ∂B + Ψ × ∂ ∂Ψ. X1 = ∂ ∂t, X2 = ∂ ∂x, X3 = ∂ ∂y, X4 = ∂ ∂z , X5 = t ∂ ∂t −v ∂ ∂v −B ∂ ∂B −Ψ ∂ ∂Ψ, X6 = r × ∂ ∂r + v × ∂ ∂v + B × ∂ ∂B + Ψ × ∂ ∂Ψ. The Maple 11 program was tested previously on Korteweg–de Vries, nonlinear Schr¨odinger, Hasegawa–Mima and other nonlinear plasma theory models and reproduced well known sym- metries. So we can expect that operators X1–X6 form a full basis of EMHD Lie point symmetry algebra. A little doubt remains, however: maybe some additional symmetries could be obtained by the direct ‘manual’ use of the standard Lie algorithm. Due to time and space homogeneity (symmetries X1 to X4) the simplest exact solution exists corresponding to the constant external magnetic field which can be directed along the Oz axis without the loss of generality (because of the rotation symmetry X6). The field amplitude can be set as B0 = 1 due to the similarity transform generated by X5: (1) B = ez, v = 0. B = ez, v = 0. This solution can be chosen as a background for the perturbation theory. 3 Collision less electron plasma Let us consider electron collisionless plasma. In this case the Vlasov–Maxwell integro-differential system of equations holds: ∂f ∂t + v∂f ∂x −E ∂f ∂v = 0, (4) ∂E ∂x = 1 − Z ∞ −∞ fdv, ∂E ∂t = Z ∞ −∞ vfdv, (5) (4) (5) where f(t, x, v) is the distribution function of the electron component of the plasma, E(t, x) is the electric field strength. where f(t, x, v) is the distribution function of the electron component of the plasma, E(t, x) is the electric field strength. We cannot find symmetries of this integro-differential model by the traditional Lie method. This can be done, however, by the indirect algorithm [3]. [ ] First, let us introduce the moments: First, let us introduce the moments: First, let us introduce the moments: Mk(t, x) = Z ∞ −∞ vkfdv, k = 0, 1, 2, . . . (6) (6) and obtain an infinite set of partial differential equations for them, which is as follows: ∂Mk ∂t + ∂Mk+1 ∂x + EkMk−1 = 0, ∂E ∂x = 1 −M0, ∂E ∂t = M1. Then, we consider the reduced system involving only first N + 1 equations of this set, and we can obtain by the standard Lie procedure the symmetries of this system: Then, we consider the reduced system involving only first N + 1 equations of this set, and we can obtain by the standard Lie procedure the symmetries of this system: X1 = ∂ ∂t, X2 = ∂ ∂x, X3 = x ∂ ∂x + E ∂ ∂E + N+1 X k=1 kMk ∂ ∂Mk , X4 = cos (t)  ∂ ∂x + ∂ ∂E  −sin (t) N+1 X k=1 kMk−1 ∂ ∂Mk , (7) X5 = sin (t)  ∂ ∂x + ∂ ∂E  + cos (t) N+1 X k=1 kMk−1 ∂ ∂Mk , (7) (7) and an additional symmetry and an additional symmetry and an additional symmetry XFGN = F(t) ∂ ∂MN +  G(t) −x∂F(t) ∂t  ∂ ∂MN+1 , F(t) and G(t) being arbitrary functions of t. The standard Maple 11 program can help us to check these results for not very large N (depending on computer capacity). Only two highest moments MN and MN+1 are present in XFGN. So only moments with numbers greater than any previously chosen are involved in the transform generated by XFGN. 2 Electron magnetohydrodynamics After the substi- tution B →ez + B, Ψ →ez + Ψ the first EMHD equation becomes ∂Ψ ∂t = ∂v ∂z + ∇× (v × Ψ) (2) ∂Ψ ∂t = ∂v ∂z + ∇× (v × Ψ) (2) but other equations remain unchanged. In this way the system of equations describing helicon waves in a plasma is usually obtained. but other equations remain unchanged. In this way the system of equations describing helicon waves in a plasma is usually obtained. We can expect that EMHD model is much more symmetric. For example, nonlinear term in (2) vanishes under an additional condition: v × Ψ = ∇F, (3) (3) v × Ψ = ∇F, where F is arbitrary scalar function. In this way we obtain the set of linear partial differential equations and the nonlinear algebraic restriction (3). This property can be a source of rich additional symmetries. where F is arbitrary scalar function. In this way we obtain the set of linear partial differential equations and the nonlinear algebraic restriction (3). This property can be a source of rich additional symmetries. Symmetry Extensions in the Kinetic and Hydrodynamic Plasma Models 3 3 Collision less electron plasma In the limit N →∞the symmetry group is generated by X1–X5. In general, restoring kinetic symmetries from the symmetries of the corresponding set of the moments equations is a hard problem in pure mathematics. Nevertheless, for the relatively simple operators X1–X5 it was shown in [3] that in the case considered they correspond to the following infinitesimal operators of the kinetic theory: X1 = ∂ ∂t, X2 = ∂ ∂x, X3 = x ∂ ∂x + v ∂ ∂v + E ∂ ∂E −f ∂ ∂f , (8) (8) V.B. Taranov 4 X4 = cos (t)  ∂ ∂x + ∂ ∂E  −sin (t) ∂ ∂v, X5 = sin (t)  ∂ ∂x + ∂ ∂E  + cos (t) ∂ ∂v. (9) (9) Strictly speaking, from the definition (6) it can be readily seen that the transformations gen- erated by the operators (8), (9) in the space of variables t, x, v and f lead to the transformations generated by the infinitesimals (7) in the space of variables t, x and Mk, k = 0, 1, 2, . . . . Strictly speaking, from the definition (6) it can be readily seen that the transformations gen- erated by the operators (8), (9) in the space of variables t, x, v and f lead to the transformations generated by the infinitesimals (7) in the space of variables t, x and Mk, k = 0, 1, 2, . . . . The symmetries of the same electron collisionless plasma kinetic model (4), (5) were found in [5] by the direct algorithm, i.e. without using of an infinite set of moments equations. The same generators (8), (9) were obtained. 4 Hasegawa–Mima model Let us consider an inhomogeneous plasma slab in the external homogeneous magnetic field. Electrons, unlike ions, are magnetized, smoothing an electrostatic potential Φ along the magnetic field lines. In this case, Hasegawa–Mima model equations hold [6]: ∂Ψ ∂t + J(Φ, Ψ) = ∂Φ ∂y , Ψ = Φ −∆⊥Φ, ∂Ψ ∂t + J(Φ, Ψ) = ∂Φ ∂y , Ψ = Φ −∆⊥Φ, (10) Ψ = Φ −∆⊥Φ, (10) (10) where Ψ is the generalized vorticity, where Ψ is the generalized vorticity, J(F, G) ≡∂F ∂x ∂G ∂y −∂G ∂x ∂F ∂y , ∆⊥≡∂2 ∂x2 + ∂2 ∂y2 . J(F, G) ≡∂F ∂x ∂G ∂y −∂G ∂x ∂F ∂y , ∆⊥≡∂2 ∂x2 + ∂2 ∂y2 . The simultaneous presence of the Φ and ∆⊥Φ in the second equation of (10) leads to the symmetry reduction and, as a consequence, to the absence of self-similar solutions [7]. Nevertheless, self-similar solutions can exist as a consequence of conditional symmetries [8]. The notion of conditional symmetry is discussed and large bibliography is presented in a recent paper [9]. For example, if an additional condition ∂2Φ ∂x2 = 0 (11) ∂2Φ ∂x2 = 0 (11) ∂2Φ ∂x2 = 0 (11) is fulfilled, the term ∆⊥Φ is reduced to ∂2Φ ∂y2 , which leads to the symmetry extension. In this way we obtain more symmetric, but still nonlinear, set of equations, which admits the following similarity transform: is fulfilled, the term ∆⊥Φ is reduced to ∂2Φ ∂y2 , which leads to the symmetry extension. In this way we obtain more symmetric, but still nonlinear, set of equations, which admits the following similarity transform: X1 = x ∂ ∂x + Φ ∂ ∂Φ + Ψ ∂ ∂Ψ, which allows us to try solutions in the form: which allows us to try solutions in the form: Φ = xF(t, y), Ψ = xG(t, y). G = −1 is a trivial solution which is not interesting for physics. So only the simple nonlinear exact solution with G ̸= −1 important for physics as a nonlinear wave asymptotic, will be considered below. More complicated solutions can be presented and discussed in future papers. For the functions F(t, y) and G(t, y) we obtain the equations For the functions F(t, y) and G(t, y) we obtain the equations ∂(1/(G + 1)) ∂t + ∂(F/(G + 1)) ∂y = 0, G = F −∂2F ∂y2 . 4 Hasegawa–Mima model (12) ∂(1/(G + 1)) ∂t + ∂(F/(G + 1)) ∂y = 0, G = F −∂2F ∂y2 . (12) Equations (12) have a particular solution [8]: (13) F = α(1 + β cos((ω + δω)t + qy)), Symmetry Extensions in the Kinetic and Hydrodynamic Plasma Models 5 so that so that Φ = αx(1 + β cos((ω + δω)t + qy)), (14) αx(1 + β cos((ω + δω)t + qy)), (14) (14) Φ = αx(1 + β cos((ω + δω)t + qy)), where α is arbitrary constant amplitude, constant factor β determines the relative weight of the zonal flow and the monochromatic wave, the frequency is ω = q/(1+q2) and the frequency shift is δω = −αq3/(1 + q2). where α is arbitrary constant amplitude, constant factor β determines the relative weight of the zonal flow and the monochromatic wave, the frequency is ω = q/(1+q2) and the frequency shift is δω = −αq3/(1 + q2). Another class of conditionally symmetric invariant solutions became possible under the fol- lowing condition: ∂2Φ ∂y2 = 0. ∂2Φ ∂y2 = 0. In this case the following similarity transform appears as a conditional symmetry: In this case the following similarity transform appears as a conditional symmetry: X2 = t ∂ ∂t + y ∂ ∂y X2 = t ∂ ∂t + y ∂ ∂y X2 = t ∂ ∂t + y ∂ ∂y and another class of invariant solutions is possible Φ = F(x)y t + G(x), Ψ = (F(x) −F ′′(x))y t + G(x) −G′′(x). Pure self similar solutions G(x) = 0 are absent in this case, but there exist solutions corre- sponding to the arbitrary shear flow F(x) = 0, G(x) being an arbitrary function of x. 5 Multi-component collisionless plasma So the transformation [8] f′ 1 = f1 −e2F(f1, f2), f′ 2 = f2 + e1F(f1, f2), (19) where F(f1, f2) is an arbitrary function of its arguments, leaves the Vlasov–Maxwell equations invariant, at least if we perform in (17) the summation first and the integration later. The transformation (19) does not preserve, in general, the positiveness of the distribution functions. Moreover, they can lead to the divergence of the moments, which can be shown, for example, by choosing F = const. y g eless, we can choose F(f1, f2) = f1/e2 to obtain the simplifying transformation vertheless, we can choose F(f1, f2) = f1/e2 to obtain the simplifying transformation Nevertheless, we can choose F(f1, f2) = f1/e2 to obtain the simplifying transf e2f′ 2 = e1f1 + e2f2. (20) f′ 1 = 0, e2f′ 2 = e1f1 + e2f2. (20) The simplifying transform (20) can also be obtained from the symmetry deduced from the symmetry of moments’ equations, f′ 1 = f1 exp(a), f′ 2 = f2 + e1 e2 (1 −exp(a))f1, a is arbitrary constant, in the limit a →−∞. a is arbitrary constant, in the limit a →−∞. According to (20), we can choose the invariant (18) as a new distribution function and solve the system (15), (16) for e1f1 + e2f2, f3, . . . , fN e1f1 + e2f2, f3, . . . , fN and the fields E and B, so the number of equations is reduced by one. and the fields E and B, so the number of equations is reduced by one. 5 Multi-component collisionless plasma Let us consider N-component collisionless plasma. In this case the Vlasov–Maxwell integro- differential system of equations holds: ∂fα ∂t + v∂fα ∂r + eα mα  E + 1 c[v × B]  ∂fα ∂v = 0, (15) ∇× E + 1 c ∂B ∂t = 0, ∇· E = 4πρ, ∇× B = 1 c ∂E ∂t + 4π c j, ∇· B = 0, (16) (15) 1 c ∂B ∂t = 0, ∇· E = 4πρ, ∇× B = 1 c ∂E ∂t + 4π c j, ∇· B = 0, (16) (16) where fα(t, r, v) is the distribution function of the α−th component of the plasma, α = 1, . . . , N. Charge and mass of particles of the αth component are denoted by eα and mα, respectively. Charge and current densities have the form where fα(t, r, v) is the distribution function of the α−th component of the plasma, α = 1, . . . , N. Charge and mass of particles of the αth component are denoted by eα and mα, respectively. Charge and current densities have the form ρ = N X α=1 eα Z ∞ −∞ fαdv, j = N X α=1 eα Z ∞ −∞ vfαdv. (17) (17) Let α = 1 and α = 2 correspond to plasma components with equal charge to mass ratio of particles, for example, alpha particles and deuterium ion components, so we assume (and it is a good approximation: e1 m1 = e2 m2 . e1 m1 = e2 m2 . According to (17), these components enter the Maxwell equations only as the sum According to (17), these components enter the Maxwell equations only as the sum e1f1 + e2f2, (18) e1f1 + e2f2, (18) (18) e1f1 + e2f2, V.B. Taranov 6 V.B. Taranov V.B. Taranov which is in fact the distribution function of the charge density of the components α = 1 and α = 2. In addition, according to (15), the functions f1, f2 and e1f1 + e2f2 satisfy the same Vlasov equation. So the transformation [8] which is in fact the distribution function of the charge density of the components α = 1 and α = 2. In addition, according to (15), the functions f1, f2 and e1f1 + e2f2 satisfy the same Vlasov equation. 6 Conclusions Lie point symmetries of electron magnetohydrodynamics are obtained (Section 2), which include time and space homogeneity, self similarity and rotations. As a consequence of the symmetries, simple invariant solution (1) exists which is in fact the background solution which allows to explore nonlinear waves evolution by a perturbation formalism. Rich conditional symmetries are possible in this model due to additional conditions like (3). Lie point symmetries of the integro differential Vlasov–Maxwell model for collisionless elec- tron plasma obtained in [3] by the moments’ method, i.e. from the symmetry of an infinite set of partial differential equations for the moments of the distribution functions, are presented in Section 3. It is shown in Section 4 that hydrodynamic Hasegawa–Mima model for drift waves in magne- tized plasma, which, in general, is not self similar, has exact asymptotic self similar solutions (14). It is possible due to the conditional symmetry of the model as a consequence of an additional condition (11). The physical reason for this symmetry extension is that nonlinear waves have a simpler shape near their critical points – nodes, crests etc. In Section 5, by simple vector considerations, it is shown that multi-component collisionless plasma containing components with equal charge to mass ratio of particles is invariant under the transformation (19), if we perform the summation first and the integration later in the expressions for charge and current densities (17). This transformation, called the extended symmetry, does not preserve, in general, the global conditions of non negativity of distribution functions and existence of their moments. So, in contrast with the conditional symmetries, this symmetry extension is a consequence not of imposing additional conditions, but of neglecting Symmetry Extensions in the Kinetic and Hydrodynamic Plasma Models 7 some global constraints. Nevertheless, among the extended symmetries (19) we can find the simplifying transform (20), which allows us to reduce the number of equations by 1. The physical reason for the extended symmetry (19) is that particles with equal charge to mass ratios under the same initial conditions (coordinate and velocity) in a given electric and magnetic fields move along the same trajectories. The second physically important reason is that we consider very fast evolution of a plasma, faster than any collisions. References [1] Ibragimov N.H., Kovalev V.F., Pustovalov V.V., Symmetries of integro-differential equations: a survey of methods illustrated by the Benney equation, Nonlinear Dynam. 28 (2002), 135–165, math-ph/0109012. [2] Cicogna G., Ceccherini F., Pegoraro F., Applications of symmetry methods to the theory of plasma physics, SIGMA 2 (2006), 017, 17 pages, math-ph/0602008. [3] Taranov V.B., On the symmetry of one-dimensional high frequency motions of a collisionless plasma, Sov. J. Tech. Phys. 21 (1976), 720–726. [4] Gordeev A.V., Kingsep A.S., Rudakov L.I., Electron magnetohydrodynamics, Phys. Rep. 243 (1994), 215– 465. [5] Meleshko S.V., Application of group analysis in gas kinetics, in Proc. Joint ISAMM/FRD Inter-Disciplinary Workshop “Symmetry Analysis and Mathematical Modelling”, 1998, 45–60. [6] Horton W., Drift waves and transport, Rev. Modern Phys. 71 (1999), 735–778. [7] Taranov V.B., Drift and ion-acoustic waves in magnetized plasmas, symmetries and invari Ukrainian J. Phys. 49 (2004), 870–874. ] Taranov V.B., Symmetry extensions in kinetic and hydrodynamic plasma models, in Proceedin International Congress on Plasma Physics (2006, Kyiv), 2006, A041p, 4 pages. [9] Cicogna G., Laino M., On the notion of conditional symmetry of differential equations, Rev. Math. Phys. 18 (2006), 1–18, math-ph/0603021.
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REVISTA TEMPOS E ESPAÇOS EM EDUCAÇÃO AVALIADORES AD HOC 2017 Alice Copetti Dalmaso (UFSM) Amália Cristina Dias da Rocha Bezerra (UERJ) Ana Lucia Pereira (UEPG) André Luis Castro de Freitas (UFRN) Andreia Aparecida Guimarães Strohschoen (UNIVATES) Anne Alilma Silva Souza Ferrete (UFS) Antônio Amorim (UNEB) Antônio Pereira (UNEB) Antônio Roberto Xavier (UNILAB) Bréscia França Nonato (UFMG) Carlos Nogueira Fino (Universidade da Madeira – Portugal) Cassia Hack (UNIFAP) Cláudia Moraes (UFSC) Cláudio Pinto Nunes (UESB) Claudia Tavares Amaral (UFG) Célia Souza da Costa (UNIFAP) Cristiano Feronatto (UNIT) Cristiano Mezzaroba (UFS) Eduardo Galak (Universid Nacional de La Plata – Argentina) Eimard Gomes Antunes do Nascimento (Universidade de Aveiro – Portugal) Ezer Wellington Gomes Lima (UNIVASF) Fabiana Sena (UFPB) Gabriella Eldereti Machado (UFSM) Galdino Sousa (UFES) Gilberto Ferreira da Silva (UNILASALLE) Giovani Ferreira Bezerra (UFMS) Gislene de Araújo Alves (IFRN) Gustavo Roese Sanfelice (FEEVALE) Horácio Miguel Zapata (UNCAUS/UNNE/ISFDPAG) Jadson Justi (UFAM) Jean Carlos Miranda (UFF) Jenerton Arlan Schütz (UNIJUI) Joanice Conceição (UFF) Joaquim Tavares da Conceição (UFS) João Manuel de Oliveira (Instituto Universitário de Lisboa – Portugal) Jonathas Chaguri (Governo do Estado do Paraná) Lyana Virgínia Thédiga de Miranda (UFSC) Lucas Romário (UFPB) Lúcia Gracia Ferreira (UFRB) Lucken Bueno Lucas (UENP) Marcio Romeu Ribas de Oliveira (UFRN) Maria do Mar Ferreira (University of Warwick – Reino Unido) Maria Helena Santana Cruz (UFS) Maria Gouvêa Campesato (Prefeitura Municipal de Porto Alegre / RS) Mariana Mendonça Lisboa (UFSC) Marta Maria de Araújo (UFRN) Michelle Carreirão Gonçalves (UFRJ) Nilma Margarida de Castro Crusoé (UESB) Núbia Regina Moreira (UESB) Osmar Hélio Alves Araújo (UFC) Paulo Gerson Rodrigues Stefanello (UFSCar) Paulo Sergio Marchelli (UFS) Raphael Feitosa (UFC) Reginaldo Santos Pereira (UESB) Rita de Cássia Santos Souza (UFS) Rodrigo Duarte Ferrari (UFSC) Ronaldo Santos Santana (Secretaria Estadual de Educação de São Paulo/ SP) Rosana Carla Nascimento Givigi (UFS) Rosaria Nakashima (UFT) Sérgio Dorenski Dantas Ribeiro (UFS) Sílvia Ester Orrú (UNB) Simone Ferreira de Lucena (UFS) Tarcia Regina Silva (UFPE) Verônica Werle (UFSC) William Timóteo (UNIT) Wilma Amâncio da Silva Amâncio da Silva (Secretaria Municipal de Educação de Delmiro Gouveia/AL)
https://openalex.org/W2890832593
https://openresearch.surrey.ac.uk/view/delivery/44SUR_INST/12139765920002346/13140461640002346
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Metal Ion Interactions with Crude Oil Components: Specificity of Ca2+ Binding to Naphthenic Acid at an Oil/Water Interface
Colloids and interfaces
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Received: 17 August 2018; Accepted: 14 September 2018; Published: 18 September 2018 Abstract: On the basis of dynamic interfacial tension measurements, Ca2+ has been shown specifically to interact with naphthenic acid (NA) at the n-heptane/water interface, consistent with NA adsorption followed by interfacial complexation and formation of a more ordered interfacial film. Optimum concentrations of Ca2+ and NA have been found to yield lower, time-dependent interfacial tensions, not evident for Mg2+ and Sr2+ or for several alkali metal ions studied. The results reflect the specific hydration and coordination chemistry of Ca2+ seen in biology. Owing to the ubiquitous presence of Ca2+ in oilfield waters, this finding has potential relevance to the surface chemistry underlying crude oil recovery. For example, “locking” acidic components at water/oil interfaces may be important for crude oil emulsion stability, or in bonding bulk oil to mineral surfaces through an aqueous phase, potentially relevant for carbonate reservoirs. The relevance of the present results to low salinity waterflooding as an enhanced crude oil recovery technique is also discussed. Keywords: dynamic interfacial tension; interfacial complexation; low salinity waterflooding; metal ion interactions; naphthenic acid; oil recovery Article Metal Ion Interactions with Crude Oil Components: Specificity of Ca2+ Binding to Naphthenic Acid at an Oil/Water Interface Spencer E. Taylor * and Hiu Tung Chu Centre for Petroleum and Surface Chemistry, Department of Chemistry, University of Surrey, Guildford, Surrey GU2 5XH, UK; tungchi0906@gmail.com * Correspondence: s.taylor@surrey.ac.uk; Tel.: +44-1483-681999 Spencer E. Taylor * and Hiu Tung Chu Centre for Petroleum and Surface Chemistry, Department of Chemistry, University of Surrey, Guildford, Surrey GU2 5XH, UK; tungchi0906@gmail.com * Correspondence: s.taylor@surrey.ac.uk; Tel.: +44-1483-681999 Spencer E. Taylor * and Hiu Tung Chu Centre for Petroleum and Surface Chemistry, Department of Chemistry, University of Surrey, Guildford, Surrey GU2 5XH, UK; tungchi0906@gmail.com * Correspondence: s.taylor@surrey.ac.uk; Tel.: +44-1483-681999 Spencer E. Taylor * and Hiu Tung Chu   Received: 17 August 2018; Accepted: 14 September 2018; Published: 18 September 2018 colloids and interfaces colloids and interfaces colloids and interfaces colloids and interfaces www.mdpi.com/journal/colloids 1. Introduction Carboxylic acids are fundamental to the chemistry of all crude oils [1]. These are oil-soluble compounds with the general formula CnH2n+zO2 containing acyclic (z = 0) and alicyclic (z > 0) hydrocarbyl groups; in crude oils, they are collectively referred to as ‘naphthenic acid’ (NA), since the earliest examples identified contained saturated cyclopentyl and cyclohexyl groups [2,3]. Carboxylic acids are fundamental to the chemistry of all crude oils [1]. These are oil-soluble compounds with the general formula CnH2n+zO2 containing acyclic (z = 0) and alicyclic (z > 0) hydrocarbyl groups; in crude oils, they are collectively referred to as ‘naphthenic acid’ (NA), since the earliest examples identified contained saturated cyclopentyl and cyclohexyl groups [2,3]. As is the case for other polar species present in petroleum, organic acids are often implicated in various upstream and downstream operational problems. Thus, during crude oil production, the surface activity of NA can lead to alterations in reservoir wettability [4,5] and increased stability of water-in-oil (w/o) emulsions [6]. Additionally, under high pH conditions, ionized acid groups stabilize oil-in-water (o/w) emulsions [7] when dissociation of oil-soluble acids produces anionic surface-active species which strongly affect properties, such as interfacial tension and interfacial rheology [8]. During crude oil refining, the acidity of NA (pKa ~5 [9]) is responsible for corrosion in distillation units under the high temperature process conditions. The total acidity of the crude oil depends on its source, and oil-specific measures are often required to reduce potential damage when processing the highest acidity crudes (typically total acid number [TAN] values in excess of approximately 0.8 mg KOH/g) [10]. Naphthenic acids also exhibit specific interactions with calcium in certain oilfields, where Ca2+ coordination leads to precipitation and deposition of naphthenate-rich scale [11]. In this case, the naphthenates are tetrameric species, the original C80-tetra-acid member being identified as C80H142O8, molecular weight 1230 g/mol [12]. Following this discovery, other similar members Colloids Interfaces 2018, 2, 40; doi:10.3390/colloids2030040 www.mdpi.com/journal/colloids 2 of 21 2 of 21 Colloids Interfaces 2018, 2, 40 were identified as being responsible for naphthenate deposits in diverse locations [13,14]. Regarding the formation of these species, two points are noteworthy. First, if the tetra-acids are uncharged, it is likely that interactions must occur at an aqueous/oil interface. Second, from the composition of the deposits, there should be a specific affinity between the organic acid and aqueous Ca2+ ions, given the mixed cation composition of oilfield waters. 1. Introduction This is also confirmed using a range of techniques and a synthetic tetra-acid, for which the strongest affinity was found for Ca2+ [15,16]. There is evidence in the literature to show that acidic species contribute to the structure and properties of crude oil–water interfaces [17]. Dissociation of carboxylic acids, for example, explains the pH-dependent behavior of crude oil/water interfaces [18,19] leading to increased o/w emulsion stability at high pH. It is also evident that crude oil/water interfacial properties can be significantly influenced by the presence of divalent metal ions. Thus, Alvarado et al. [20] reported the increased stabilization of water-in-crude oil emulsions in the presence of Ca2+ ions compared with Na+ ions of the same ionic strength, suggesting that stronger interactions occur with Ca2+, although the authors suggested that further investigation is necessary [21]. Carboxylic acid groups at crude oil interfaces have also been implicated in improved recovery mechanisms during low salinity waterflooding (involving the so-called “low salinity effect”, LSE). One popular suggested LSE mechanism starts with the premise that oil is naturally bound to specific reservoir mineral surfaces via divalent metal ion bridging involving oil-based carboxylate groups [22–30]. It is considered that reducing the water salinity disrupts the bridges through ion exchange, resulting in reduced oil adhesion, thereby improving oil displacement and recovery. Other studies into the LSE have suggested that interfacial properties should be sensitive to complexation across oil/water interfaces [31,32]. Indeed, the specificity of interfacial complexation between metal ions and oil-soluble components is important elsewhere in liquid–liquid extraction processes [33]. Early work [34–36] identified that interfacial reactions involving metal ions and spread carboxylic acid monolayers at the air-water interface are relatively slow, with half-lives of several minutes. More recently, de Ruiter et al. studied the evolution of the interfacial tension upon placing aqueous solutions of metal ions in contact with a decane solution of stearic acid (HSt) [31]. When the solution pH > pKa, the acidity constant of stearic acid, distinct stages in the formation of metal stearate layers were noted, and interpreted as [31]: (i) deprotonation of adsorbed stearic acid (HSt), forming MSt2 complexes at the interface; (ii) nucleation of the MSt2 complexes, allowing further acid adsorption and complexation; (iii) partitioning of the three-dimensional MSt2 structures into the oil phase. 1. Introduction The present study is concerned with the influence of alkali and alkaline earth metal ions (as the respective chloride salts) on oil/water interfaces containing a commercial (technical-grade) sample of NA. Previous work by Brandal et al. investigated the effects of divalent metal ions (Mg2+, Ca2+, Sr2+ and Ba2+) on the interfacial tension behavior of different carboxylic acids under pH conditions where the acids were completely ionized [32]. However, we were keen to explore interactions between aqueous metal ions and the oil-soluble NA, initially in its unionized form as would be present in a native crude oil. 2.2.1. Synthesis of Calcium Naphthenate Calcium naphthenate was synthesized based on a method used by Pereira et al. to prepare calcium salts of long-chain carboxylates [38]. Thus, KOH (0.752 g, 13.0 mmol) was dissolved in ethanol (20 mL) and heated gently on a hot-plate until boiling. NA (3.270 g, ~3.3 mmol based on the above TAN value) was then added and boiling maintained. Separately, Ca(OAc)2·xH2O (1.062 g, ~6.5 mmol) was dissolved in the minimum quantity of water with sonication, after which this solution was carefully added dropwise to the boiling ethanolic KOH/NA. The resultant mixture was then allowed to cool and evaporate, whereupon an amber-colored gel separated, together with residual water (~2 mL). The water was decanted, and the gel product was washed with an approximately equal volume of deionized water, heated to 80 ◦C, and then allowed to cool. Again, the residual water was decanted. This washing process was repeated one further time. The drained product was then stirred with petroleum ether (40–60 ◦C boiling range) until dissolved (~40 mL required). This solution was filtered (Whatman #1 filter paper), which also served to remove residual water from the resultant clear amber filtrate. Solvent evaporation yielded viscous product (3.079 g, 87%). The stoichiometry of the product was determined by thermogravimetric analysis (TA Instruments TGA Q500 thermogravimetric analyzer, Elstree, Hertfordshire, UK). The sample was heated from ambient temperature to 900 ◦C at 10 ◦C/min in air to yield a residue (calcium oxide) corresponding to 7.14% Ca. Using the average NA molecular mass of 243 calculated above, the product has a composition consistent with the expected 1:2 Ca:NA stoichiometry. Figure 1 shows a comparison of the infrared spectra (Bruker Alpha spectrometer with Diamond ATR (Bruker UK Ltd., Coventry, UK), each sample applied in neat form) for the original naphthenic acid and its calcium salt, from which the shift of the carbonyl band from 1700 to 1543 cm−1 is characteristic of neutralized carboxylic acids [39,40]. The small band at 1709 cm−1 in the calcium naphthenate spectrum indicates a trace amount of unreacted naphthenic acid remains in the product. Loss of bands at 1265 and 943 cm−1 are also consistent with calcium salt formation [40]. Colloids Interfaces 2018, 2, x 4 of 21 Figure 1. Attenuated Total Reflection (ATR)-infrared spectra of naphthenic acid (red, dashed) and its calcium salt (blue, continuous). Figure 1. Attenuated Total Reflection (ATR)-infrared spectra of naphthenic acid (red, dashed) and its calcium salt (blue, continuous). 2.1. Materials Organic solvents (ethanol and n-heptane, both >98% purity) and the reagents naphthenic acid (technical grade), calcium chloride dihydrate (CaCl2·2H2O), strontium chloride hexahydrate (SrCl2·6H2O), magnesium chloride hexahydrate (MgCl2·6H2O), sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium hydroxide (KOH), calcium acetate (Ca(OAc)2·xH2O) were purchased from Sigma-Aldrich Co. Ltd. (Gillingham, Dorset, UK) and apart from n-heptane were used without further purification. The latter was passed through an alumina column to remove any surface-active impurities before use. According to the supplier, the commercial naphthenic acid is a technical mixture of alkylated cyclopentane carboxylic acids, with a total acid number (TAN) value of 230.5 mg KOH/g, 3 of 21 3 of 21 Colloids Interfaces 2018, 2, 40 which equates to an average molecular mass of 56/0.2305 = 243 g/mol with the reasonable assumption that the mixture only contains monoprotic acids. However, consistent with our own analysis, Hindle et al. reported that this product contains a high proportion of saturated fatty acids, with relatively low levels of true naphthenic components [37]. Notwithstanding the relatively low content of authentic naphthenic structures, its composition does provide a representative range of carbon chain lengths to investigate interactions which mainly involve the carboxylic acid function of crude oils. g g y y Deionized water (resistivity = 18.2 MΩ·cm) was from a Millipore Direct-Q system, with a pH of ~5.6 at room temperature (21 ◦C). 2.2.1. Synthesis of Calcium Naphthenate Figure 1. Attenuated Total Reflection (ATR)-infrared spectra of naphthenic acid (red, dashed) and its calcium salt (blue, continuous). Figure 1. Attenuated Total Reflection (ATR)-infrared spectra of naphthenic acid (red, dashed) and its calcium salt (blue, continuous). 4 of 21 Colloids Interfaces 2018, 2, 40 2.2.2. Dynamic Interfacial Tension Measurements 2.2.2. Dynamic Interfacial Tension Measurements The dynamic tension of water/n-heptane interfaces was determined using an in-house dynamic drop volume (DDV) method. The general basis of this approach has been detailed by van Hunsel and Joos [41] and is similar to the method used by Deshiikan et al. [42]. Briefly, aqueous drops are formed at the tip of a capillary using different (known) flow rates, f, and the corresponding drop formation times, td, measured. By knowing f and td allows corresponding drop volumes to be determined under dynamic conditions. From a comparison with the Ward-Tordai equation, the adsorption time is ~3/7td [43]. Strictly, drop volume (or weight) methods are used to determine equilibrium surface or interfacial tension for drops formed infinitely slowly, because at finite rates of drop formation hydrodynamic effects cause excess liquid to be incorporated into the detaching drop [41]. However, under dynamic conditions, the drop volume, V(td), is related to the drop formation time by the empirical relationship attributed to McGee (see ref. [41]): V(td) = V0 + St−0.75 d (1) (1) in which V0 is the drop volume at an infinitely slow formation time and S is the so-called McGee slope [41]. The latter is determined by measuring drop volumes at different flow rates for a series of pure liquids, under conditions of constant interfacial tension. Surface or interfacial tensions are then obtained using the established static pendant drop volume relationship: in which V0 is the drop volume at an infinitely slow formation time and S is the so-called McGee slope [41]. The latter is determined by measuring drop volumes at different flow rates for a series of pure liquids, under conditions of constant interfacial tension. Surface or interfacial tensions are then obtained using the established static pendant drop volume relationship: γ = V0∆ρg 2πrF (2) (2) in which r is the external tip radius, ∆ρ the density difference between the two phases, and F is a correction factor accounting for drop shape which was determined and tabulated as a function of r/V01/3 by Harkins and Brown [44] and extended by Wilkinson [45]. The more recent polynomial developed by Lee et al. [46] shown in Equation (3), which correlates F with r/V01/3 within the range 0 ≥r/V01/3 ≥1.2, was used in the present analysis. 2.2.2. Dynamic Interfacial Tension Measurements in which r is the external tip radius, ∆ρ the density difference between the two phases, and F is a correction factor accounting for drop shape which was determined and tabulated as a function of r/V01/3 by Harkins and Brown [44] and extended by Wilkinson [45]. The more recent polynomial developed by Lee et al. [46] shown in Equation (3), which correlates F with r/V01/3 within the range 0 ≥r/V01/3 ≥1.2, was used in the present analysis. F = 1 −0.9121(r/V01/3) −2.109(r/V01/3)2 + 13.38(r/V01/3)3 −27.29(r/V01/3)4 + 27.53(r/V01/3)5 −13.58(r/V01/3)6 + 2.593(r/V01/3)7 (3) (3) By using pure low viscosity liquids of constant surface tension over the range of drop formation times, hydrodynamic contributions to drop formation are effectively eliminated, which allows the relationship between the McGee slope and V0 to be defined. For our system, we found that the McGee slope is described well by S = 0.22V0 (4) (4) such that for a given drop time, Equation (2) becomes: such that for a given drop time, Equation (2) becomes: uch that for a given drop time, Equation (2) becomes: γ(td) = V(td)∆ρg 2πrF  1 + 0.22t−0.75 d  (5) (5) In the present method, water or aqueous salt solution drops were formed below the surface of the n-heptane phase from the tip of a stainless steel syringe needle, the end of which was cleaned and roughened with a fine grade sand paper before each measurement to ensure effective contact of the aqueous phase is made with the outer diameter (3.413 mm). Drop volumes were in the range ~30–120 mm3. The pH of the unadjusted aqueous phases was in the range 5.4–5.7 consistent with Robertson et al. [47]. Drops are then produced at up to ten known rates using a pre-calibrated syringe pump (B. Braun, Melsungen, Germany) at 21 ± 1 ◦C, and the drop times calculated by averaging the time taken for several drops to be produced. Drop counting was performed visually with an 5 of 21 Colloids Interfaces 2018, 2, 40 estimated mechanical (stopwatch) response time of ±0.02 s for a single drop; this limits the realistic rate of formation typically to ~1 drop per second. The procedure also requires the density difference between the liquid phases to be known under the same temperature conditions, and these were determined by pycnometry from the mass of a known volume of the respective liquids. 3. Results and Discussion In advance of discussing the results of this study, it is pertinent to consider the ionization behavior of carboxylic acids at oil/water interfaces. Thus, on electrostatic grounds it would be expected that ionization equilibria will be affected by the presence of an interface, which would then be reflected in the relevant dissociation constants. To illustrate this, if the acid dissociation constant of a carboxylic acid in bulk (aqueous) solution is pKa(∞), then the electrostatic image potential Ψ experienced by the acid group at a distance x from the interface is given by [49]: Ψ(x) = − Ce 2Dwx (6) (6) where C = 1/4πε0, e is the electronic charge (1.602 × 10−19 C), ε0 is the permittivity of a vacuum (8.854 × 10−12 F/m), and Dw is the dielectric constant of water (~80). By further considering the acid-base equilibrium as a function of distance from the interface, Dill and Bromberg [49] showed that the apparent pKa at a distance x from the interface is related to the bulk value by pKa(x) = pKa(∞) −0.4343eΨ(x) kBT = pKa(∞) + 0.4343Ce2 2xDwkBT (7) (7) in which kB is Boltzmann’s constant (1.381 × 10−23 J/K) and T is the absolute temperature. This simple model suggests that adsorbed acid groups within, say, 0.2–0.5 nm of an oil/water interface, would experience an increase in pKa of 0.3–0.8 units, purely on electrostatic grounds, without introducing specific ionic refinements. However, by using more sophisticated computational approaches, Andersson et al. found that the pKa of isolated acid molecules at an oil/water interface increased by an average of ~1 unit compared with the aqueous bulk solution value [50]. This reflects the greater affinity of the neutral molecule for the interface. The consequences of molecular confinement, at an interface, for example, were also probed using the same computational methods. In this situation, the possibility of deprotonated carboxylate groups stabilizing neighboring undissociated (neutral) carboxylic acid molecules arises through hydrogen bonding. Thus, the same workers also showed how the pKa shift produced in the first molecule, as described above, is almost exactly compensated by the stabilizing effect induced on the second molecule [50]. 2.2.2. Dynamic Interfacial Tension Measurements Thus, by measuring td at different flow rates allows the determination of γ(td) as a function of drop time using Equation (5). The combination of interfacial tension, drop volume, needle diameter and density difference translates for most of the measurements into values of the newly proposed Worthington number (Wo = ∆ρgVd/2πrγ) in the range ~0.7–0.9, for which a value of unity would be the ideal situation [48]. Additionally, replicate measurements using pure liquids with time-invariant interfacial tension, indicate a standard error of <3% of the quoted values, which is often better than ± 1 mN/m. 3.1. NA Adsorption at the n-Heptane/Water Interface Dynamic drop volume measurements betwee d f l d f 3.1. NA Adsorption at the n-Heptane/Water Interface Dynamic drop volume measurements betwee d f l d f Dynamic drop volume measurements between n-heptane containing NA and deionized water produce constant interfacial tensions over drop formation times in the approximate range ~0.3–500 s as shown in Figure 2a. The time-invariant values seen for different bulk NA concentrations (CNA) are due to the changes in the composition of the n-heptane phase, reducing slightly from the value for the n-heptane/water interface. The results also indicate that adsorption is rapid compared with the experimental timescale, being complete in <0.3 s. Using a microfluidic technique, adsorption at the hydrocarbon/water interface has recently been shown [55] to occur on the millisecond timescale, more rapidly than previously thought. roduce constant interfacial tensions over drop formation times in the approximate range ~0.3–500 s shown in Figure 2a. The time-invariant values seen for different bulk NA concentrations (CNA) ar ue to the changes in the composition of the n-heptane phase, reducing slightly from the value fo he n-heptane/water interface. The results also indicate that adsorption is rapid compared with th xperimental timescale, being complete in <0.3 s. Using a microfluidic technique, adsorption at th ydrocarbon/water interface has recently been shown [55] to occur on the millisecond timescale more rapidly than previously thought. Figure 2. (a) Examples of the dynamic interfacial tension of naphthenic acid solutions in n-heptane against deionized water. (b) Corresponding results against water containing 0.3 mol/L Ca2+. (c) Interfacial tension-log (naphthenic acid concentration) profiles for deionized water and the individual γi and γ∞ contributions for 0.3 mol/L Ca2+ (see Equation (8)). The inset shows the corresponding plot for n-heptane solutions of Ca(NA)2 against deionized water. All curves are fits to the Szyszkowski equation (Equation (9)). (d) The effect of Ca2+ concentration on the interfacial tension of the n-heptane/water interface. The line is based on the empirical equation γ = γ0 − Aγ0.5 + Figure 2. (a) Examples of the dynamic interfacial tension of naphthenic acid solutions in n-heptane against deionized water. (b) Corresponding results against water containing 0.3 mol/L Ca2+. (c) Interfacial tension-log (naphthenic acid concentration) profiles for deionized water and the individual γi and γ∞contributions for 0.3 mol/L Ca2+ (see Equation (8)). The inset shows the corresponding plot for n-heptane solutions of Ca(NA)2 against deionized water. All curves are fits to the Szyszkowski equation (Equation (9)). 3. Results and Discussion Several experimental studies have identified increased pKa values for acid groups present at surfaces and interfaces, including self-assembled monolayers [51], fatty acid micelles [52,53] and, more recently, at the air/water interface for fatty acid monolayers [54], the latter study also identifying the effect of chain length on the magnitude of the pKa change. In the context of the present paper, this will mean that adsorbed NA molecules will remain largely undissociated as the effective (interfacial) pKa is higher than the pH of the aqueous solutions. Colloids Interfaces 2018, 2, 40 ( ) p 1 NA Ad ti t th 6 of 21 6 of 21 3.1. NA Adsorption at the n-Heptane/Water Interface Dynamic drop volume measurements betwee d f l d f (d) The effect of Ca2+ concentration on the interfacial tension of the n-heptane/water interface. The line is based on the empirical equation γ = γ0 −Aγ0.5 + Bγ as described in the text. Figure 2. (a) Examples of the dynamic interfacial tension of naphthenic acid solutions in n-heptane gainst deionized water. (b) Corresponding results against water containing 0.3 mol/L Ca2+. (c) nterfacial tension-log (naphthenic acid concentration) profiles for deionized water and the ndividual γi and γ∞ contributions for 0.3 mol/L Ca2+ (see Equation (8)). The inset shows the orresponding plot for n-heptane solutions of Ca(NA)2 against deionized water. All curves are fits to he Szyszkowski equation (Equation (9)). (d) The effect of Ca2+ concentration on the interfacial ension of the n-heptane/water interface. The line is based on the empirical equation γ = γ0 − Aγ0.5 + Figure 2. (a) Examples of the dynamic interfacial tension of naphthenic acid solutions in n-heptane against deionized water. (b) Corresponding results against water containing 0.3 mol/L Ca2+. (c) Interfacial tension-log (naphthenic acid concentration) profiles for deionized water and the individual γi and γ∞contributions for 0.3 mol/L Ca2+ (see Equation (8)). The inset shows the corresponding plot for n-heptane solutions of Ca(NA)2 against deionized water. All curves are fits to the Szyszkowski equation (Equation (9)). (d) The effect of Ca2+ concentration on the interfacial tension of the n-heptane/water interface. The line is based on the empirical equation γ = γ0 −Aγ0.5 + Bγ as described in the text. Bγ as described in the text. Compared with the time-invariant interfacial tension behavior seen at the deionized water interface, the presence of 0.3 mol/L Ca2+ is accompanied by the appearance of time-dependent behavior, as shown in Figure 2b for equivalent naphthenic acid concentrations. Bγ as described in the text. Compared with the time-invariant interfacial tension behavior seen at the deionized water interface, the presence of 0.3 mol/L Ca2+ is accompanied by the appearance of time-dependent behavior, as shown in Figure 2b for equivalent naphthenic acid concentrations. These latter profiles suggest that the approach to equilibrium of a growing drop in the presence of Ca2+ occurs in (at least) two stages. 3.1. NA Adsorption at the n-Heptane/Water Interface Dynamic drop volume measurements betwee d f l d f The first stage involves a rapid γ decrease, as described above, as the interface re-equilibrates from γ0 ~48 mN/m to an “instantaneous” value (within the timescale Colloids Interfaces 2018, 2, 40 7 of 21 of the present technique), γi, which, for 5 mg/L ≤CNA ≤200 mg/L and 0.3 mol/L Ca2+, is lower than the corresponding values obtained for the deionized water interface. of the present technique), γi, which, for 5 mg/L ≤CNA ≤200 mg/L and 0.3 mol/L Ca2+, is lower than the corresponding values obtained for the deionized water interface. However, it is also evident in Figure 2b that within approximately the same CNA range given above, the interfacial tension behavior exhibits a slower second process which finally results in an equilibrium value, γ∞, i.e., γ0 →γi, followed by γi →γ∞. The interfacial tension curves for the slower process, as shown in Figure 2b, are satisfactorily represented by first-order kinetic curves constructed according to Equation (8), each of which being characterized by a first-order rate constant, k. γ = γ∞+ (γi −γ∞) exp(−kt) (8) (8) Since γ0 for each Ca2+ solution in the presence of NA is inaccessible using the present technique, we felt that it is reasonable to assume that it will be the same as the time-invariant value for the corresponding n-heptane/0.3 mol/L Ca2+ interface in the absence of NA. Thus, in Figure 2c are shown plots of γi and γ∞for a Ca2+ concentration, CCa of 0.3 mol/L as a function of CNA, together with the corresponding γ∞behavior at the deionized water interface, the latter being in good agreement with data published by Havre et al. [9] for a similar commercial NA sample. It is immediately apparent that the interfacial tension decreases in the presence of 0.3 mol/L Ca2+ in the aqueous phase for ~5 mg/L ≤CNA ≤200 mg/L, and increases once again above this concentration, towards the deionized water isotherm. In Figure 2c (inset), we also show interfacial tension data for the synthesized calcium salt, Ca(NA)2. In this case, only very small time-dependent effects were observed, the error bars reflecting the spread of results over the timescale of the measurements. However, it is evident that the calcium salt follows the same general behavior as NA, inasmuch as the interfacial tension is largely unaffected up to ~100 mg/L, but thereafter the data diverge. 3.1. NA Adsorption at the n-Heptane/Water Interface Dynamic drop volume measurements betwee d f l d f Within the NA concentration range considered, we find that the interfacial tension-log profiles in ure 2c are satisfactorily represented by the Szyszkowski equation [56]: γ = γ0 −RTΓm ln CNA a + 1  (9) (9) where CNA is the bulk NA concentration in the organic phase, Γm is the interfacial concentration of NA at monolayer adsorption (mol/m2) and a is a molecule-dependent constant (expressed as mol/L) related to the Gibbs energy of adsorption (∆G0) at infinite dilution by [57]: a = 55.3 exp ∆G0 RT  (10) (10) From Table 1 it is apparent that the “instantaneous” interfacial concentration of NA in the presence of Ca2+ is some ten times greater than in its equilibrium concentration. This translates into a much smaller apparent molecular area. The latter dimension is unreasonable, however, and most probably relates to a disordered interfacial accumulation of NA molecules in the initial stages, which would be consistent with the initial rapid response of the interfacial tension. The situation then transforms into a more structured interfacial film characterized by a more realistic molecular area, which is almost identical to the value at the deionized water interface. Moreover, the Gibbs energy of adsorption of NA is ~20 kJ/mol more favorable (i.e., lower) in the presence of Ca2+ ions, which presents an intriguing insight into the role played by Ca2+ ions in facilitating NA adsorption. Interestingly, this value is very close to the Gibbs energy for Ca2+ binding to hydrated carboxylate side chains of the protein β-lactoglobulin (−18 ± 1 kJ/mol) [58]. Whilst the interfacial packing of NA molecules appears to remain the same, the adsorption process itself is significantly more favorable, which suggests some degree of interaction occurs between NA and Ca2+ ions across the interface. 8 of 21 Colloids Interfaces 2018, 2, 40 Table 1. Summary of the adsorption parameters for NA in the presence and absence of Ca2+ ions based on the Szyszkowski equation. Table 1. Summary of the adsorption parameters for NA in the presence and absence of Ca2+ ions based on the Szyszkowski equation. 3.1. NA Adsorption at the n-Heptane/Water Interface Dynamic drop volume measurements betwee d f l d f Interface Species Γm (mol/m2) Molecular Area (nm2) ∆G0 (kJ/mol) NA 1.58 × 10−6 1.05 −27.9 NA + Ca2+ (i) 1.66 × 10−5 0.10 −27.5 NA + Ca2+ (∞) 1.60 × 10−6 1.04 −48.7 Ca(NA)2 3.34 × 10−6 0.50 −29.1 Finally, as a relevant aside, in Figure 2d are shown the effects of CCa on the n-heptane/water interfacial tension in the absence of NA. It is well-known that high electrolyte concentrations generally show an increase in surface and interfacial tension as a consequence of “negative adsorption”. However, relatively low electrolyte concentrations cause a small decrease in tension, a phenomenon known as the Jones-Ray effect, after the first researchers to report it, in 1937 [59]. While the original work focused on electrolyte effects on surface tension, some recent studies have considered the behavior at hydrocarbon/water interfaces [60], including the crude oil/water interface [61], because of their relevance to some of the applications mentioned earlier in the present paper. In each of these earlier studies, interfacial tension minima have been seen at low CCa, which is also evident in the present results. Thus, Figure 2d shows that the interfacial tension reduction for <0.1 mol/L Ca2+ is ~1.4 mN/m, similar to values obtained elsewhere [60]. Suggested reasons for this effect have been debated, and have included cation [60], anion [62–64] or impurity [65] adsorption, or electrostatic effects influencing water structure at the interface [66,67]. The curve fitted through the experimental data in Figure 2d is of the form γ = γ0 −Aγ0.5 + Bγ, where A and B are empirical constants (0.158 (mN/m)0.5 and 0.217, respectively) and γ0 is the interfacial tension of the n-heptane/water interface (48.4 mN/m). derived from Equation (13). 3 3 D d f I t f i l T i C d C 3.3. Dependence of Interfacial Tension on CNA and CCa q ( ) 3 3 D d f I f i l T i C d C 3.3. Dependence of Interfacial Tension on CNA and CCa 3.3. Dependence of Interfacial Tension on CNA and CCa The theoretical curves drawn through the data in Figure 3b were derived on the assumption that the interface can be described as a combination of two limiting situations, one based on pure water and the other containing a certain interfacial excess of Ca2+ (possibly representing a “saturated” interfacial film). The former condition is simply represented by deionized water data in Figure 2c, whilst the latter has to be obtained from the limiting interfacial tension data determined in the presence of different Ca2+ concentrations shown in Figure 3b (red line). As discussed above, both limiting conditions have been shown to be adequately described by the Szyszkowski equation (see Figure 2c). In the subsequent analysis, therefore, we have assumed that the overall adsorption conditions will be a combination of these two limiting cases, the extent to which each contributes being dependent on the (bulk) concentration ratio between NA and Ca2+. Thus, the interfacial tensions produced by NA adsorption at a water interface and at a saturated Ca2+-containing interface are given by Equations (9) and (11), respectively; it is unnecessary to define the bulk Ca2+ concentration responsible for the latter condition at this stage of the analysis. In Equation (11), Гm’ d ’ h i f i d l l f NA i h f h The theoretical curves drawn through the data in Figure 3b were derived on the assumption that the interface can be described as a combination of two limiting situations, one based on pure water and the other containing a certain interfacial excess of Ca2+ (possibly representing a “saturated” interfacial film). The former condition is simply represented by deionized water data in Figure 2c, whilst the latter has to be obtained from the limiting interfacial tension data determined in the presence of different Ca2+ concentrations shown in Figure 3b (red line). As discussed above, both limiting conditions have been shown to be adequately described by the Szyszkowski equation (see Figure 2c). In the subsequent analysis, therefore, we have assumed that the overall adsorption conditions will be a combination of these two limiting cases, the extent to which each contributes being dependent on the (bulk) concentration ratio between NA and Ca2+. 3.2. NA Adsorption at the n-Heptane/Water Interface in the Presence of Metal Ions With the exception of Ca2+, none of the other metal ions studied has been found to exhibit time-dependent interfacial tensions using the present experimental method. Thus, in Figure 3a, NA adsorption at the n-heptane/metal chloride solution (1.0 mol/L) interface is indistinguishable from the deionized water curve shown in Figure 2c. These data include the two other divalent metal ions studied, Mg2+ and Sr2+, which do not show the same interfacial tension behavior as a function of CNA as seen for Ca2+, i.e., interfacial tension minima that are dependent on CCa as highlighted in Figure 3b. As observed above, for NA concentrations beyond the minimum interfacial tension, the behavior returns to the curve defined by the other metal ion solutions and deionized water. This suggests a reduced influence of Ca2+ ions in lowering the Gibbs energy of adsorption of NA molecules (Table 1) as the interface becomes more saturated with NA. Crucially, however, it also suggests that the presence of specific calcium naphthenate species with higher surface activity may not be directly responsible for the increased interfacial activity of NA, but rather the Ca2+ ions promote NA adsorption, through specific interactions as will be discussed subsequently. 9 of 21 9 of 21 Colloids Interfaces 2018, 2, 40 Colloids Interfaces 2018, 2, x Figure 3. (a) Interfacial tension-log (naphthenic acid concentration) profiles at the n-heptane/water interface in the presence of the indicated cations (all chloride salts at 1 mol/L). (b) Corresponding profiles for different aqueous Ca2+ concentrations. The blue and red curves represent the respective limiting conditions for deionized water and a “Ca2+-saturated” interface, and the gray curves are derived from Equation (13). Figure 3. (a) Interfacial tension-log (naphthenic acid concentration) profiles at the n-heptane/water interface in the presence of the indicated cations (all chloride salts at 1 mol/L). (b) Corresponding profiles for different aqueous Ca2+ concentrations. The blue and red curves represent the respective limiting conditions for deionized water and a “Ca2+-saturated” interface, and the gray curves are derived from Equation (13). Figure 3. (a) Interfacial tension-log (naphthenic acid concentration) profiles at the n-heptane/water interface in the presence of the indicated cations (all chloride salts at 1 mol/L). (b) Corresponding profiles for different aqueous Ca2+ concentrations. The blue and red curves represent the respective limiting conditions for deionized water and a “Ca2+-saturated” interface, and the gray curves are derived from Equation (13) Figure 3. 3.2. NA Adsorption at the n-Heptane/Water Interface in the Presence of Metal Ions (a) Interfacial tension-log (naphthenic acid concentration) profiles at the n-heptane/water interface in the presence of the indicated cations (all chloride salts at 1 mol/L). (b) Corresponding profiles for different aqueous Ca2+ concentrations. The blue and red curves represent the respective limiting conditions for deionized water and a “Ca2+-saturated” interface, and the gray curves are derived from Equation (13). derived from Equation (13). 3 3 D d f I t f i l T i C d C 3.3. Dependence of Interfacial Tension on CNA and CCa The appearance of a plateau region in Figure 4 at CNA ~ 0.4 mol/L suggests that this bulk concentration corresponds to the interfacial “saturation” condition for Ca2+ ions, as alluded to earlier, and for which the maximum influence on NA adsorption is seen. 3 4 NA Ca2+ Interfacial Tension Kinetics derived from Equation (13). 3 3 D d f I t f i l T i C d C 3.3. Dependence of Interfacial Tension on CNA and CCa Thus, the general interfacial tension expression for different Ca2+ and NA concentrations is given by combining Equations (9) and (11), to give 𝛾= 𝛾0 −𝑅𝑇(𝑚(1 −exp (𝐶Ca 𝐶NA )) ln (𝐶NA 𝑎+ 1) + 𝑚′exp (𝐶Ca 𝐶NA ) ln (𝐶NA 𝑏+ 1)) (12) γ = γ0 −RT  Γm  1 −exp φCCa CNA  ln CNA a + 1  + Γm′ exp φCCa CNA  ln CNA b + 1  (12 Initial attempts at fitting the data shown in Figure 3b to Equation (12) indicated that, perha nsurprisingly, Гm ~ Гm’, allowing the simplified Equation (13) to be used to generate the fitt (12) erhaps Initial attempts at fitting the data shown in Figure 3b to Equation (12) indicated that, perhaps unsurprisingly, Γm ~Γm’, allowing the simplified Equation (13) to be used to generate the fitted curves shown in Figure 3b. For the purpose of this demonstration, the following values were used for all Ca2+ concentrations: γ0 (47.0 mN/m); Γm (1.60 × 10−6 mol/m2), a (0.03 mol/L) and b (145 mol/L), leaving the interaction term ϕ (mg/mol) as the only adjustable parameter. p g y g p q g curves shown in Figure 3b. For the purpose of this demonstration, the following values were used for all Ca2+ concentrations: γ0 (47.0 mN/m); Гm (1.60 × 10−6 mol/m2), a (0.03 mol/L) and b (145 mol/L) leaving the interaction term φ (mg/mol) as the only adjustable parameter. 𝛾= 𝛾0 −𝑅𝑇𝑚(1 −exp (𝐶Ca 𝐶 )) ln (𝐶NA 𝑎+ 1) + exp (𝐶Ca 𝐶 ) ln (𝐶NA 𝑏+ 1) (13) Initial attempts at fitting the data shown in Figure 3b to Equation (12) indicated that, perhaps unsurprisingly, Γm ~Γm’, allowing the simplified Equation (13) to be used to generate the fitted curves shown in Figure 3b. For the purpose of this demonstration, the following values were used for all Ca2+ concentrations: γ0 (47.0 mN/m); Γm (1.60 × 10−6 mol/m2), a (0.03 mol/L) and b (145 mol/L), leaving the interaction term ϕ (mg/mol) as the only adjustable parameter. p g y g p q g curves shown in Figure 3b. For the purpose of this demonstration, the following values were used for all Ca2+ concentrations: γ0 (47.0 mN/m); Гm (1.60 × 10−6 mol/m2), a (0.03 mol/L) and b (145 mol/L) leaving the interaction term φ (mg/mol) as the only adjustable parameter. derived from Equation (13). 3 3 D d f I t f i l T i C d C 3.3. Dependence of Interfacial Tension on CNA and CCa Thus, the interfacial tensions produced by NA adsorption at a water interface and at a saturated Ca2+-containing interface are given by Equations (9) and (11), respectively; it is unnecessary to define the bulk Ca2+ concentration responsible for the latter condition at this stage of the analysis. In Equation (11), Γm’ and a’ are the respective surface concentration and molecular constant of NA in the presence of the saturation concentration of Ca2+. Colloids Interfaces 2018, 2, 40 Colloids Interfaces 2018, 2, x 10 of 21 10 of 21 γ = γ0 −RTΓm′ ln CNA a′ + 1  (11) ases containing Ca2+ ions, and n-heptane phases containing NA, we nteraction at the water/n-heptane interface is a function of the (11) NA, we of the In the case of aqueous phases containing Ca2+ ions, and n-heptane phases containing NA, we assume that the extent of interaction at the water/n-heptane interface is a function of the concentration ratio CCa/CNA, such that the contributions arising from pure water and aqueous Ca2+ solution interfaces are [1 −exp(φCCa/CNA] and exp(φCCa/CNA), respectively, where the factor ϕ takes into account differences between concentration units as well as the relative effectiveness of Ca2+ and NA interactions at the interface. Thus, the general interfacial tension expression for different Ca2+ and NA concentrations is given by combining Equations (9) and (11), to give concentration ratio CCa/CNA, such that the contributions arising from pure water and aqueous Ca2 solution interfaces are [1 −exp(𝐶Ca/𝐶NA] and exp(𝐶Ca/𝐶NA), respectively, where the factor φ takes into account differences between concentration units as well as the relative effectiveness o Ca2+ and NA interactions at the interface. derived from Equation (13). 3 3 D d f I t f i l T i C d C 3.3. Dependence of Interfacial Tension on CNA and CCa 𝛾= 𝛾0 −𝑅𝑇𝑚(1 −exp (𝐶Ca 𝐶 )) ln (𝐶NA 𝑎+ 1) + exp (𝐶Ca 𝐶 ) ln (𝐶NA 𝑏+ 1) (13) γ = γ0 −RTΓm  1 −exp φCCa CNA  ln CNA a + 1  + exp φCCa CNA  ln CNA b + 1  (13) NA NA t is evident from Figure 3b that this simplistic analysis produces reasonable fits to the imental data and produces a sigmoidal variation of φ with the bulk Ca2+ concentration as (13) to the It is evident from Figure 3b that this simplistic analysis produces reasonable fits to the experimental data and produces a sigmoidal variation of ϕ with the bulk Ca2+ concentration as shown in Figure 4. The behavior seen in Figure 3b reflects the increasing concentration of Ca2+-NA interactions in the interface as the bulk Ca2+ concentration is raised. The appearance of a plateau region in Figure 4 at CNA ~0.4 mol/L suggests that this bulk concentration corresponds to the interfacial “saturation” condition for Ca2+ ions, as alluded to earlier, and for which the maximum influence on NA adsorption is seen. shown in Figure 4. The behavior seen in Figure 3b reflects the increasing concentration of Ca2+-NA interactions in the interface as the bulk Ca2+ concentration is raised. The appearance of a plateau region in Figure 4 at CNA ~ 0.4 mol/L suggests that this bulk concentration corresponds to the interfacial “saturation” condition for Ca2+ ions, as alluded to earlier, and for which the maximum influence on NA adsorption is seen. 3 4 NA Ca2+ Interfacial Tension Kinetics It is evident from Figure 3b that this simplistic analysis produces reasonable fits to the experimental data and produces a sigmoidal variation of ϕ with the bulk Ca2+ concentration as shown in Figure 4. The behavior seen in Figure 3b reflects the increasing concentration of Ca2+-NA interactions in the interface as the bulk Ca2+ concentration is raised. The appearance of a plateau region in Figure 4 at CNA ~0.4 mol/L suggests that this bulk concentration corresponds to the interfacial “saturation” condition for Ca2+ ions, as alluded to earlier, and for which the maximum influence on NA adsorption is seen. shown in Figure 4. The behavior seen in Figure 3b reflects the increasing concentration of Ca2+-NA interactions in the interface as the bulk Ca2+ concentration is raised. 3.4. NA-Ca2+ Interfacial Tension Kinetics f The additional decrease in inter A comparison with the data in Figure 3b shows that the maximum k corresponds to the minimum interfacial tension of ~15 mN/m Also for the low CNA region, the behavior of the second-order rate constant is shown as Also for the low CNA region, the behavior of the second-order rate constant is shown as a function of CCa in Figure 5b. Here, it is seen that a minimum rate region is apparent at CCa ~0.5–1.0 mol/L, consistent with the onset of the plateau region in Figure 4. g function of CCa in Figure 5b. Here, it is seen that a minimum rate region is apparent at CCa ~ 0.5–1.0 mol/L, consistent with the onset of the plateau region in Figure 4. We speculate that the cause of the slower reduction of interfacial tension shown in Figure 2b i We speculate that the cause of the slower reduction of interfacial tension shown in Figure 2b is due to complex formation involving Ca2+ and NA, and reorientation at the interface. This is evident only in the case of Ca2+, but not for any of the other studied cations. With reference to the adsorption parameters in Table 1, it appears probable that the hydrophobic side of the interfacial region rapidly becomes saturated with randomly-distributed NA molecules upon contacting water containing Ca2+ ions. The increased local NA concentration then reconfigures into a more ordered adsorbed layer with the same adsorbed area/molecule as for NA at the n-heptane/deionized water interface. With respect to the adsorption data for pre-formed Ca(NA)2 in Table 1, this finding suggests that complete complexation between NA and Ca2+ is unlikely to have taken place. The question then remains as to the role of Ca2+ ions in the adsorption process. To try to answer this, consideration needs to be given to the behavior of metal ions in aqueous solution, and especially with regard to interactions with carboxylates. p g due to complex formation involving Ca2+ and NA, and reorientation at the interface. This is eviden only in the case of Ca2+, but not for any of the other studied cations. With reference to the adsorption parameters in Table 1, it appears probable that the hydrophobic side of the interfacial region rapidly becomes saturated with randomly-distributed NA molecules upon contacting water containing Ca2 ions. 3.4. NA-Ca2+ Interfacial Tension Kinetics f The additional decrease in inter The additional decrease in interfacial tension of NA at the n-heptane/water interface in the presence of Ca2+ ions shown in Figure 2b occurs at rates that are measurable using the present technique. Kinetic data associated with these changes are given in Figure 5 in the form of two plots. The first shows the effects of CNA on the first-order rate constant k at constant CCa (0.3 mol/L; Figure 5a), and the second shows the variation in the second-order rate constant (=k/CNA; for CNA = 5 and 10 mg/L; Figure 5b) as a function of CCa. presence of Ca2+ ions shown in Figure 2b occurs at rates that are measurable using the present technique. Kinetic data associated with these changes are given in Figure 5 in the form of two plots The first shows the effects of CNA on the first-order rate constant k at constant CCa (0.3 mol/L; Figure 5a), and the second shows the variation in the second-order rate constant (=k/CNA; for CNA = 5 and 10 mg/L; Figure 5b) as a function of CCa. Figure 4. The effect of Ca2+ concentration on the interaction factor φ used in the construction of the fitted curves in Figure 3b. Figure 4. The effect of Ca2+ concentration on the interaction factor ϕ used in the construction of the fitted curves in Figure 3b. Figure 4. The effect of Ca2+ concentration on the interaction factor φ used in the construction of the fitted curves in Figure 3b. Figure 4. The effect of Ca2+ concentration on the interaction factor ϕ used in the construction of the fitted curves in Figure 3b. 11 of 21 11 of 2 Colloids Interfaces 2018, 2, 40 Colloids Interfaces 2018, 2, x 11 of 21 11 of 2 Figure 5a illustrates the NA concentration range over which dynamic interfacial tension effects are observed for a Ca2+ concentration of 0.3 mol/L. This indicates that there is a CNA “window”, spanning almost three orders of magnitude, in which time-dependent interfacial processes are occurring. Moreover, for CNA < 50 mg/L at this particular Ca2+ concentration, k is proportional to the NA concentration, the slope of the plot shown in the inset to Figure 5a representing the second-order rate constant for the process. 3.4. NA-Ca2+ Interfacial Tension Kinetics f The additional decrease in inter As CNA increases, however, not only is a maximum rate constant apparent at ~100 mg/L, but the uncertainties in the data are also seen to increase, as a result of the changes in the i t f i l t i b i ll i thi i A i ith th d t i Fi 3b h g g y are observed for a Ca2+ concentration of 0.3 mol/L. This indicates that there is a CNA “window” spanning almost three orders of magnitude, in which time-dependent interfacial processes ar occurring. Moreover, for CNA < 50 mg/L at this particular Ca2+ concentration, k is proportional to th NA concentration, the slope of the plot shown in the inset to Figure 5a representing the second-orde rate constant for the process. As CNA increases, however, not only is a maximum rate constan apparent at ~100 mg/L, but the uncertainties in the data are also seen to increase, as a result of th changes in the interfacial tension becoming smaller in this region. A comparison with the data in Fi 3b h h h i k d h i i i f i l i f 15 N/ p NA , , y pp ~100 mg/L, but the uncertainties in the data are also seen to increase, as a result of the changes in the interfacial tension becoming smaller in this region. A comparison with the data in Figure 3b shows that the maximum k corresponds to the minimum interfacial tension of ~15 mN/m. apparent at 100 mg/L, but the uncertainties in the data are also seen to increase, as a result of th changes in the interfacial tension becoming smaller in this region. A comparison with the data in Figure 3b shows that the maximum k corresponds to the minimum interfacial tension of ~15 mN/m Also for the low CNA region, the behavior of the second-order rate constant is shown as ~100 mg/L, but the uncertainties in the data are also seen to increase, as a result of the changes in the interfacial tension becoming smaller in this region. A comparison with the data in Figure 3b shows that the maximum k corresponds to the minimum interfacial tension of ~15 mN/m. pp g changes in the interfacial tension becoming smaller in this region. 3.5. Interfacial Behavior of Ions and Metal Ion–Carboxylate Bonding 3.5. Interfacial Behavior of Ions and Metal Ion–Carboxylate Bonding In order to provide a plausible explanation for the observed behavior described above, three particular factors need to be considered: (i) the behavior of hydrated cations at interfaces; (ii) exchange of ligated water in the inner coordination sphere; and (iii) specific interactions between cations and carboxylate groups. It has long been known that aqueous ions influence interfacial behavior [68]. Typically, aqueous salts are considered to “negatively adsorb” at air/water or oil/water interfaces, producing an increase in the respective surface or interfacial tensions in accord with the Gibbs adsorption theory, and to an extent that is specific to each ion [69]. Ion theories have been developed over the years, with increasingly sophisticated models proposed to predict their behavior at interfaces [70,71]. Herein, we have considered several different monovalent and divalent cations and found that, of these, only Ca2+ affects the n-heptane/water interfacial tension in the presence of NA. All cations have a tendency to be hydrated, owing to their ionic charge [72], and as a consequence will be excluded from the interfacial region [73,74]. Therefore, we have to examine why Ca2+ behaves differently from the rest of the studied cations. Ikeda et al. compared Ca2+ and Mg2+ ions in solution using constrained molecular dynamics, and found that the hydrated structure of Ca2+ is highly variable compared with that of Mg2+ [75]. Using density functional theory, Pavlov et al. identified that Ca2+ has a lower water binding energy compared with Mg2+ (as well as Be2+ and Zn2+), notwithstanding the different water structures around the respective ions [76]. This implies that Ca2+ will show potentially greater lability of its inner-shell coordinated water molecules, which is of importance for adsorption as well as coordination to ligands. Specific ion effects were originally classified based on the different abilities of salts to precipitate proteins from aqueous solution, a phenomenon which is accredited to Hofmeister in 1888 [77,78]. The “Hofmeister effect”, as it came to be known, now refers more generally to the relative effects of either cations or anions on colloidal, surface or biological processes. The commonly-held view that has been developed, discussed and reviewed by many authors over the years is that ion hydration is fundamental to the Hofmeister effect [73,74,79]; this includes the behavior of ions at liquid interfaces [70]. 3.4. NA-Ca2+ Interfacial Tension Kinetics f The additional decrease in inter The increased local NA concentration then reconfigures into a more ordered adsorbed laye with the same adsorbed area/molecule as for NA at the n-heptane/deionized water interface. With respect to the adsorption data for pre-formed Ca(NA)2 in Table 1, this finding suggests that complete complexation between NA and Ca2+ is unlikely to have taken place. The question then remains as to the role of Ca2+ ions in the adsorption process. To try to answer this, consideration needs to be given to the behavior of metal ions in aqueous solution, and especially with regard to interactions with carboxylates. Figure 5. (a) The effect of NA concentration on the first-order kinetic rate constant for equilibration of the n-heptane/water interfacial tension in the presence of 0.3 mol/L Ca2+ ions. The inset shows the second-order plot for low NA concentrations. (b) The effect of Ca2+ concentration on the second-order rate constant in the low NA concentration range (red and blue symbols are for CNA = 5 and 10 mg/L, respectively). Figure 5. (a) The effect of NA concentration on the first-order kinetic rate constant for equilibration of the n-heptane/water interfacial tension in the presence of 0.3 mol/L Ca2+ ions. The inset shows the second-order plot for low NA concentrations. (b) The effect of Ca2+ concentration on the second-order rate constant in the low NA concentration range (red and blue symbols are for CNA = 5 and 10 mg/L, respectively). Figure 5. (a) The effect of NA concentration on the first-order kinetic rate constant for equilibration of the n-heptane/water interfacial tension in the presence of 0.3 mol/L Ca2+ ions. The inset shows the second-order plot for low NA concentrations. (b) The effect of Ca2+ concentration on the second-order rate constant in the low NA concentration range (red and blue symbols are for CNA = 5 and 10 mg/L, respectively). Figure 5. (a) The effect of NA concentration on the first-order kinetic rate constant for equilibration of the n-heptane/water interfacial tension in the presence of 0.3 mol/L Ca2+ ions. The inset shows the second-order plot for low NA concentrations. (b) The effect of Ca2+ concentration on the second-order rate constant in the low NA concentration range (red and blue symbols are for CNA = 5 and 10 mg/L, respectively). 12 of 21 Colloids Interfaces 2018, 2, 40 12 of 21 3.5. Interfacial Behavior of Ions and Metal Ion–Carboxylate Bonding The size of the ion and its nuclear charge control its hydration properties in aqueous solution and serve to define kosmotropic (strongly hydrated) and chaotropic (weakly hydrated) ions. Thus, kosmotropes are likely to possess high charge densities and their consequent strong hydration contributes to repulsion from an interface [73,74]. Adsorption of these ions would be accompanied by liberation of the strongly-bound hydration water, requiring large positive enthalpic contributions to the Gibbs energy which would not necessarily be outweighed by the corresponding entropy changes, i.e., ∆Hads −T∆Sads > 0. On the other hand, water molecules bound to chaotropic ions are more easily lost on approaching an interface [73,74], making adsorption more favorable, i.e., ∆Hads −T∆Sads < 0. Considering the second factor mentioned above, that of the lability of the coordinated inner-shell water molecules, consistent with the theoretical investigations [75,76], it was found that water exchange rates from the innermost hydration shells are almost four orders of magnitude faster for Ca2+ than for Mg2+ ions [80]. This trend reflects the relative sizes of the anhydrous cations, and the greater charge density of Mg2+. Using the same arguments, this would suggest that Sr2+ should behave in a similar fashion to Ca2+, but we have not found this to be the case. We therefore turn to the third factor mentioned above, that of specific effects involving the coordination chemistry of Ca2+ and the carboxylate group. In a recent example, Kherb et al. studied the strength of association of monovalent and divalent cations with carboxylate sites on a polypeptide biopolymer, and identified the divalent ion sequence Zn2+ > Ca2+ > Ba2+ > Sr2+ > Mg2+, whilst monovalent cations showed weaker binding [81]. The inference made from this was that the most hydrated cations bind more strongly to carboxylate [81]. The structures of metal carboxylate complexes have been extensively studied over the years and have allowed the detailed examination of X-ray crystallographic data by Einspahr and Bugg [82] 13 of 21 13 of 21 Colloids Interfaces 2018, 2, 40 Colloids I terfaces 2018 2 and Carrell et al. [83], which, together with other investigations [47,84–86] has culminated in the principal binding modes shown in Figure 6 being identified. Thus, specifically for Ca2+, monodentate coordination with one carboxylate O atom and hydrogen bonded water, bidentate coordination, with both carboxylate O atoms, or bridging (see Figure 6a–c) are notable [82]. 3.5. Interfacial Behavior of Ions and Metal Ion–Carboxylate Bonding On the other hand, a major difference in bonding mode with carboxylic acids in the case of magnesium is the formation of uncoordinated ionic species of the form shown in Figure 6d [87]. The crystal structure of the magnesium complex of p-anisic acid (4-methoxybenzoic acid) contains a layer structure of alternating [Mg(H2O)6]2+ cations and p-anisate anions with the lattice water functioning as a link between the layers. On the other hand, the monohydrated Ca(II) salt is a two-dimensional coordination polymer [87]. principal binding modes shown in Figure 6 being identified. Thus, specifically for Ca2+, monodentate coordination with one carboxylate O atom and hydrogen bonded water, bidentate coordination, with both carboxylate O atoms, or bridging (see Figure 6a–c) are notable [82]. On the other hand, a major difference in bonding mode with carboxylic acids in the case of magnesium is the formation of uncoordinated ionic species of the form shown in Figure 6d [87]. The crystal structure of the magnesium complex of p-anisic acid (4-methoxybenzoic acid) contains a layer structure of alternating [Mg(H2O)6]2+ cations and p-anisate anions with the lattice water functioning as a link between the layers. On the other hand, the monohydrated Ca(II) salt is a two-dimensional coordination polymer [87]. Figure 6. Bonding modes identified for divalent ion interactions with carboxylates [47,84–86]. 1:1 coordination is shown in binding modes (a) and (b), with bridging exhibited in (c). (d) shows ionic bonding. Figure 6. Bonding modes identified for divalent ion interactions with carboxylates [47,84–86]. 1:1 coordination is shown in binding modes (a) and (b), with bridging exhibited in (c). (d) shows ionic bonding. Figure 6. Bonding modes identified for divalent ion interactions with carboxylates [47,84–86]. 1:1 coordination is shown in binding modes (a) and (b), with bridging exhibited in (c). (d) shows ionic bonding. Figure 6. Bonding modes identified for divalent ion interactions with carboxylates [47,84–86]. 1:1 coordination is shown in binding modes (a) and (b), with bridging exhibited in (c). (d) shows ionic bonding. The comparative behavior between Ca2+ and Mg2+ interactions at carboxylate monolayers has also been studied by Tang et al. using vibrational sum frequency generation spectroscopy [88,89]. 3.5. Interfacial Behavior of Ions and Metal Ion–Carboxylate Bonding Thus, binding of these ions to palmitic acid monolayers indicated different concentration-dependent metal ion binding modes [88], concluding that as the Ca2+ concentration increases (to 0.3 mol/L), complexation favors the 2:1 bridging configuration shown in Figure 6c, whereas Mg2+ adopts the ionic complex in Figure 6d consistent with solution and crystallographic results. Moreover, the stronger Ca2+ binding was shown to have a much greater impact on the underlying hydrogen bonding below the carboxylic acid monolayer [89], which is arguably consistent with the differences seen between Ca2+ and Mg2+ ions in the present study at the oil/water interface The comparative behavior between Ca2+ and Mg2+ interactions at carboxylate monolayers has also been studied by Tang et al. using vibrational sum frequency generation spectroscopy [88,89]. Thus, binding of these ions to palmitic acid monolayers indicated different concentration-dependent metal ion binding modes [88], concluding that as the Ca2+ concentration increases (to 0.3 mol/L), complexation favors the 2:1 bridging configuration shown in Figure 6c, whereas Mg2+ adopts the ionic complex in Figure 6d consistent with solution and crystallographic results. Moreover, the stronger Ca2+ binding was shown to have a much greater impact on the underlying hydrogen bonding below the carboxylic acid monolayer [89], which is arguably consistent with the differences seen between Ca2+ and Mg2+ ions in the present study at the oil/water interface. seen between Ca and Mg ions in the present study at the oil/water interface. We have also looked to biological systems to provide an understanding of the differences between divalent ion coordination modes, especially with respect to carboxylate ligands found in a wide range of protein environments [90–94]. Williams provides a clear indication that the most significant factors responsible for ion binding in biological systems are, inter alia, ion charge, ion size, ligand donor atom, and preferred coordination geometry [95]. Thus, in connection with biological systems, binding of ions such as those considered in the present study is restricted to oxygen donor ligands, and specificity of Ca2+ binding (e.g., relative to Mg2+) is due to its size and less-restricted steric preferences compared with other ions. Ca2+ shows a greater tendency for bidentate binding to carboxylate, compared with Mg2+ [91], and a lower affinity for water [92,94] We have also looked to biological systems to provide an understanding of the differences between divalent ion coordination modes, especially with respect to carboxylate ligands found in a wide range of protein environments [90–94]. 3.6. Implications for Crude Oil Surface Chemistry 3.6. Implications for Crude Oil Surface Chemistry Over the past two decades in particular, the importance of injection water composition on crude oil reservoir chemistry has been established through various laboratory coreflood investigations and field evaluations. Studies have shown that low salinity conditions contribute to improvements in oil recovery, but although various mechanisms have been proposed, a full understanding of the low salinity effect is still lacking. However, a common factor throughout some proposed mechanisms is the role of the ionic composition of the injection and formation waters [98] and especially their effects on reservoir wettability and the corresponding affinity of the oil for the rock surface [99]. Based on our present findings the specific role of Ca2+ is considered to be significant. From the data given in Figure 3b, for example, it is evident that the minimum interfacial tension of the present NA system is dependent on the Ca2+ concentration, as shown in Figure 7a. This representation of the data assumes that there is an excess of NA in the oil phase and that the bulk Ca2+ concentration governs the interfacial tension. Over the past two decades in particular, the importance of injection water composition on crude oil reservoir chemistry has been established through various laboratory coreflood investigations and field evaluations. Studies have shown that low salinity conditions contribute to improvements in oil recovery, but although various mechanisms have been proposed, a full understanding of the low salinity effect is still lacking. However, a common factor throughout some proposed mechanisms is the role of the ionic composition of the injection and formation waters [98] and especially their effects on reservoir wettability and the corresponding affinity of the oil for the rock surface [99]. Based on our present findings the specific role of Ca2+ is considered to be significant. From the data given in Figure 3b, for example, it is evident that the minimum interfacial tension of the present NA system is dependent on the Ca2+ concentration, as shown in Figure 7a. This representation of the data assumes that there is an excess of NA in the oil phase and that the bulk Ca2+ concentration governs the interfacial tension. Figure 7. (a) The effect of Ca2+ ion concentration (expressed here in mg/L) on the minimum interfacial tension of the n-heptane/water interface when NA is in excess. 3.5. Interfacial Behavior of Ions and Metal Ion–Carboxylate Bonding Williams provides a clear indication that the most significant factors responsible for ion binding in biological systems are, inter alia, ion charge, ion size, ligand donor atom, and preferred coordination geometry [95]. Thus, in connection with biological systems, binding of ions such as those considered in the present study is restricted to oxygen donor ligands, and specificity of Ca2+ binding (e.g., relative to Mg2+) is due to its size and less-restricted steric preferences compared with other ions. Ca2+ shows a greater tendency for bidentate binding to carboxylate, compared with Mg2+ [91], and a lower affinity for water [92,94]. carboxylate, compared with Mg [91], and a lower affinity for water [92,94]. Ca2+ binding to carboxylate is also considered to be advantageous based on Collins’ “matching water affinity” approach, according to which specific pairing interactions between ions, resulting in the formation of ion pairs, is most favorable for ions of comparable hydration energy [96]. The latter is quantified [97] by the apparent dynamic hydration number (ADHN) which, for Ca2+ and carboxylate are 2.1 and 2.0, respectively, which therefore match up surprisingly well (cf. 5.7 for Mg2+). Kiriukhin and Collins consider that interaction between these two species will result in dissipation of the hydration shell, thereby benefitting coordination [97], which we suggest may also feature in specific Ca2+-naphthenic acid interactions across an interface. Such interactions would be likely to require water to be displaced from the first coordination shell Ca2+ binding to carboxylate is also considered to be advantageous based on Collins’ “matching water affinity” approach, according to which specific pairing interactions between ions, resulting in the formation of ion pairs, is most favorable for ions of comparable hydration energy [96]. The latter is quantified [97] by the apparent dynamic hydration number (ADHN) which, for Ca2+ and carboxylate are 2.1 and 2.0, respectively, which therefore match up surprisingly well (cf. 5.7 for Mg2+). Kiriukhin and Collins consider that interaction between these two species will result in dissipation of the hydration shell, thereby benefitting coordination [97], which we suggest may also feature in specific Ca2+-naphthenic acid interactions across an interface. Such interactions would be likely to require water to be displaced from the first coordination shell. 14 of 21 14 of 21 Colloids Interfaces 2018, 2, 40 In summary, there is compelling evidence for specific Ca2+ interactions with carboxylic acids from biology. 3.5. Interfacial Behavior of Ions and Metal Ion–Carboxylate Bonding Therefore, a combination of evidence from biological and coordination chemistry appears to offer an interpretation of the present surface chemical findings, which may be of relevance in other practical and technological areas, such as our current interest in crude oil surface chemistry. Colloids Interfaces 2018, 2, x 14 of 21 appears to offer an interpretation of the present surface chemical findings, which may be of relevance in other practical and technological areas, such as our current interest in crude oil surface chemistry. 3.6. Implications for Crude Oil Surface Chemistry 3.6. Implications for Crude Oil Surface Chemistry Young’s equation is derived from the balance of forces acting on the contact line of a drop on a ace (Figure 7b), i.e., γWS=γOS + γWO cos θ (14) (14) where the γ terms are the respective Gibbs energies of the water/solid (WS), oil/solid (OS) and oil/water (OW) interfaces and θ is the contact angle made by oil on the solid surface. We can therefore consider two hypothetical situations by way of illustrating the potential role of Ca2+ ions in oil recovery, based on an initial oil/rock system in equilibrium with formation water, and a final waterflooded system with reduced salinity, and a concomitant lower Ca2+ concentration. We assume that Equation (14) can be applied to each situation, such that the change in Ca2+ concentration causes a resultant change in the water/oil interfacial tension, γWO. For this elementary analysis, we also assume that γWS and γOS remain relatively unaffected by the change in Ca2+ concentration. However, although we recognize that, in practice it is likely that wetting films comprising, for example, hydrophobic species [101] or hydration layers [102], can modify water/solid and oil/solid interfaces and their respective interfacial energies, predicting the effects on the contact angle, for example, is not easy. Therefore, we consider, as an example, the simple analysis involving the initial and final conditions as introduced above, for which it is apparent that (15) γi OW cos θi = γ f OW cos θ f (15) where i and f refer to initial and final states, respectively. As shown in Table 2, formation waters are generally more saline than sea water, the ionic composition being source-dependent but also dependent on the geologic period [103]. Thus, it is possible that during geologic time the oil/rock system will have been “conditioned” with a relatively highly saline brine containing a high Ca2+ concentration, say ~20,000 mg/L as an illustrative value (Table 2). Therefore, with reference to Figure 7a, dilution of the formation brine to ~5000 mg/L Ca2+ would approximately double the interfacial tension, such that (16) cos θ f = (γi OW/γ f OW) cos θi ≈0.5 cos θi (16) cos θ f = (γi OW/γ f OW) cos θi ≈0.5 cos θi (16) Table 2. Na+ and Ca2+ compositions of formation waters from different geologic periods. For comparison, sea water contains ~10,500 mg/L Na+ and ~400 mg/L Ca2+. Data compiled from Collins [103]. Table 2. 3.6. Implications for Crude Oil Surface Chemistry 3.6. Implications for Crude Oil Surface Chemistry (b) Schematic of an oil drop adhering to a rock surface in water showing the three-phase contact point defining the contact angle. (c) Demonstrating the effect of a decrease in Ca2+ concentration from 20,000 mg/L to 5000 mg/L on the final contact angle for different initial contact angles, and a simulated visual result on a sand surface to illustrate. Figure 7. (a) The effect of Ca2+ ion concentration (expressed here in mg/L) on the minimum interfacial tension of the n-heptane/water interface when NA is in excess. (b) Schematic of an oil drop adhering to a rock surface in water showing the three-phase contact point defining the contact angle. (c) Demonstrating the effect of a decrease in Ca2+ concentration from 20,000 mg/L to 5000 mg/L on the final contact angle for different initial contact angles, and a simulated visual result on a sand surface to illustrate. Figure 7. (a) The effect of Ca2+ ion concentration (expressed here in mg/L) on the minimum interfacial tension of the n-heptane/water interface when NA is in excess. (b) Schematic of an oil drop adhering to a rock surface in water showing the three-phase contact point defining the contact angle. (c) Demonstrating the effect of a decrease in Ca2+ concentration from 20,000 mg/L to 5000 mg/L on the final contact angle for different initial contact angles, and a simulated visual result on a sand surface ill Figure 7. (a) The effect of Ca2+ ion concentration (expressed here in mg/L) on the minimum interfacial tension of the n-heptane/water interface when NA is in excess. (b) Schematic of an oil drop adhering to a rock surface in water showing the three-phase contact point defining the contact angle. (c) Demonstrating the effect of a decrease in Ca2+ concentration from 20,000 mg/L to 5000 mg/L on the final contact angle for different initial contact angles, and a simulated visual result on a sand surface to illustrate. Colloids Interfaces 2018, 2, 40 15 of 21 With reference to Figure 7b, showing a schematic of oil coating a solid (rock) surface, two equilibrium conditions can be envisaged during oil recovery. The first is the original condition within the reservoir, in which the crude oil and rock are in equilibrium with the formation brine, and the second is during waterflooding conditions where sea water, softened water or low salinity water will change the aqueous phase composition [100]. 3.6. Implications for Crude Oil Surface Chemistry 3.6. Implications for Crude Oil Surface Chemistry Na+ and Ca2+ compositions of formation waters from different geologic periods. For comparison, sea water contains ~10,500 mg/L Na+ and ~400 mg/L Ca2+. Data compiled from Collins [103]. Geologic Period Average (Maximum) Metal Ion Concentration (mg/L) Na+ Ca2+ Oligocene/Paleogene 39,000 (103,000) 2530 (38,800) Cretaceous 31,000 (88,600) 7000 (37,400) Jurassic 57,300 (120,000) 25,800 (56,300) Permian 47,000 (109,000) 8600 (22,800) Carboniferous (Pennsylvanian) 43,000 (101,000) 9100 (205,000) Carboniferous (Mississippian) 41,500 (115,800) 8900 (37,800) Devonian 48,000 (101,000) 18,000 (129,000) Thus, Equation (16) enables the plot shown in Figure 7c to be constructed, illustrating how the contact angle between the oil and rock surface is affected by the Ca2+ concentration change in the example given, from 20,000 mg/L to 5000 mg/L during waterflooding. Here, it is evident that the lowest initial contact angles, indicating high oil affinity with the surface, are influenced most by the 16 of 21 Colloids Interfaces 2018, 2, 40 16 of 21 change in interfacial tension. In this region, contact angles increase by as much as 50◦, potentially improving oil recovery. Such a scenario is depicted in the inset images in Figure 7c, added for illustrative purposes only, but is consistent with the results of the study by Shehata and Nasr-El-Din regarding the dominant role of formation water salinity and composition in improving oil recovery during low salinity waterflooding [98]. Thus, the findings presented herein may provide further insight into potential mechanisms for the low salinity effect. In addition, it is apparent that the ability to predict the effect of water chemistry on oil recovery reservoir requires not only knowledge of the interfacial chemistry of inorganic ions, but also the range of surface-active species present in the crude oil; here, we have only examined the effects of naphthenic acids. For real crude oils, however, the increased complexity and competition resulting from the presence of other interfacially active species, such as asphaltenes and resin components, would be expected to produce a different interfacial behavior [104]. This has been borne out in studies on crude oil fractions to be reported elsewhere. 4. Conclusions It seems to be a recurring theme that Ca2+ plays significant roles in the aqueous colloid and interfacial chemistry of crude oil and, in this respect, is more important than other metal ions. As discussed in the present paper, the characteristics of this ion make it favorable to undergo specific interactions with carboxylic components found in crude oil, to some extent mimicking its unique position in biology [95]. Of the metal ions studied herein, both monovalent and divalent, only Ca2+ has been shown to exhibit interfacial coordination with carboxylate groups of a commercial naphthenic acid sample. The present findings have indicated that, on their own, naphthenic acids (NAs) are only weakly active at the n-heptane/water interface at concentrations below ~200 mg/L as determined using the dynamic drop volume technique. However, this behavior is modified in the specific presence of Ca2+, whereas Mg2+ and Sr2+, as well as several alkali metal cations, exhibit no comparable effect. We have found that, for a given aqueous Ca2+ concentration, there exists an “optimum” NA concentration in the n-heptane phase at which a minimum interfacial tension is attained. As shown in Figure 7, the lowest interfacial tension is ~10–15 mN/m, to be compared with a value of ~45 mN/m for the other cations and deionized water. The interaction is not stoichiometric with respect to the respective bulk concentrations, in that the minimum interfacial tension occurs at a bulk Ca2+:NA molar concentration ratio of ~1000:1. The interfacial stoichiometry is unclear at present, but probably relates to the bidentate preference of Ca2+, but further interfacial studies will be necessary in order to provide more definitive evidence. In fact, there may be no single factor responsible for the results presented in this study. Rather, for Ca2+, the combination of its ionic hydration structure and its thermodynamic and kinetic properties as well as its coordination preferences, particularly relating to carboxylate, may contribute to its behavior at the oil/water interface. Conclusions from studies undertaken some 80 years ago by Alexander et al. also highlighted the specific relevance of Ca2+ ions (compared with Na+, K+ and Mg2+) to the chemistry of biological films, such as cephalin (kephalin) at the benzene/water interface [105]. Author Contributions: S.E.T. conceptualized and supervised the research and conducted some of the experiments. 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What Explains Vietnam’s Exceptional Performance in Education Relative to Other Countries? Analysis of the 2012 and 2015 PISA Data
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Abstract On the 2012 and 2015 PISA assessments, Vietnam’s performance ranks much higher than that of all other developing countries, and even ahead of richer countries such as the U.S. and the U.K. This is especially remarkable since Vietnam was the poorest or second-poorest PISA participant. We provide the first rigorous investigation of Vietnam’s strong performance. Despite various corrections for potentially non-representative PISA samples, including bias due to Vietnam’s large out-of-school population, Vietnam remains a large positive outlier conditional on its income. Possible higher motivation of Vietnamese students at most only partly explains Vietnam’s performance. We also find that household- and school-level variables explain little of Vietnam’s exceptional performance. Finally, Blinder-Oaxaca decompositions indicate that the gap in average test scores between Vietnam and the other participating countries is due not to differences in students’ and schools’ observable characteristics, but is mostly caused by the greater “productivity” of those characteristics in Vietnam. JEL Classifications: H0, I2, O1, P3 JEL Classifications: H0, I2, O1, P3 JEL Classifications: H0, I2, O1, P3 Keywords: education, student learning, test scores, enrolment, PISA, Vietnam Keywords: education, student learning, test scores, enrolment, PISA, Vietnam What Explains Vietnam’s Exceptional Performance in Education Relative to Other Countries? Analysis of the 2012 and 2015 PISA Data Hai-Anh Dang World Bank Acknowledgements: Acknowledgements: We thank Francesco Avvisati, Luis Felipe Saenz, and Nic Spaull, as well as seminar participants at Columbia University (Teachers College), the City University of New York, the University of Copenhagen, the 2017 LACEA conference in Buenos Aires, the 2017 RISE conference, the University of Hawaii, and the OECD, for helpful comments. This is one of a series of working papers from “RISE”—the large-scale education systems research programme supported by funding from the United Kingdom’s Department for International Development (DFID), the Australian Government’s Department of Foreign Affairs and Trade (DFAT), and the Bill and Melinda Gates Foundation. The Programme is managed and implemented through a partnership between Oxford Policy Management and the Blavatnik School of Government at the University of Oxford. Please cite this paper as: Dang, H., Glewwe, P., Lee, J., and Vu, K. (2020). What Explains Vietnam’s Exceptional Performance in Education Relative to Other Countries? Analysis of the 2012 and 2015 PISA Data. RISE Working Paper Series. 20/036. https://doi.org/10.35489/BSG-RISE-WP_2020/036 Dang, H., Glewwe, P., Lee, J., and Vu, K. (2020). What Explains Vietnam’s Exceptional Performance in Education Relative to Other Countries? Analysis of the 2012 and 2015 PISA Data. RISE Working Paper Series. 20/036. https://doi.org/10.35489/BSG-RISE-WP_2020/036 Use and dissemination of this working paper is encouraged; however, reproduced copies may not be used for commercial purposes. Further usage is permitted under the terms of the Creative Commons License. The findings, interpretations, and conclusions expressed in RISE Working Papers are entirely those of the author(s) and do not necessarily represent those of the RISE Programme, our funders, or the authors’ respective organisations. Copyright for RISE Working Papers remains with the author(s). Research on Improving Systems of Education (RISE) www.riseprogramme.org information@riseprogramme.org Research on Improving Systems of Education (RISE) www.riseprogramme.org information@riseprogramme.org The lower secondary rate is from Dang and Glewwe (2018), while the primary completio he authors using the 2014 VHLSS data; the VHLSS data are described further below. y , w p v v , w g g, aiwan as countries, although the first two are Chinese territories, and Taiwan’s status is disputed. er considers only countries, and thus excludes Shanghai, which is obviously not representative of C nd the territory of Perm, which not representative of Russia. For convenience, we refer to Hong Ko ower secondary rate is from Dang and Glewwe (2018), while the primary complet hors using the 2014 VHLSS data; the VHLSS data are described further below. g 2 This paper considers only countries, and thus excludes Shanghai, which is obviously not representative of China as a whole, and the territory of Perm, which not representative of Russia. For convenience, we refer to Hong Kong, 3 The GDP per capita for Indonesia was $US 3,347 in 2015, which is about 50 percent higher than that of Vietnam ($US 2,110) in the same year (World Bank, 2017). g 2 This paper considers only countries, and thus excludes Shanghai, which is obviously not representative of China as a whole, and the territory of Perm, which not representative of Russia. For convenience, we refer to Hong Kong, Macao and Taiwan as countries, although the first two are Chinese territories, and Taiwan’s status is disputed. 1 The lower secondary rate is from Dang and Glewwe (2018), while the primary completion rate was calculated by g ; er considers only countries, and thus excludes Shanghai, which is obviously not representative of C The lower secondary rate is from Dang and Glewwe (2018), while the primary completion rate wa he authors using the 2014 VHLSS data; the VHLSS data are described further below the authors using the 2014 VHLSS data; the VHLSS data are described further below. 2 This paper considers only countries, and thus excludes Shanghai, which is obviously not representative of China as a whole, and the territory of Perm, which not representative of Russia. For convenience, we refer to Hong Kong, Macao and Taiwan as countries, although the first two are Chinese territories, and Taiwan’s status is disputed. 3 The GDP per capita for Indonesia was $US 3,347 in 2015, which is about 50 percent higher than that of Vietnam ($US 2 110) in the same year (World Bank 2017) 1 The lower secondary rate is from Dang and Glewwe (2018), while the primary completion rate was calculated by the authors using the 2014 VHLSS data; the VHLSS data are described further below. 2 This paper considers only countries, and thus excludes Shanghai, which is obviously not representative of China as a whole, and the territory of Perm, which not representative of Russia. For convenience, we refer to Hong Kong, Macao and Taiwan as countries, although the first two are Chinese territories, and Taiwan’s status is disputed. 3 The GDP per capita for Indonesia was $US 3,347 in 2015, which is about 50 percent higher than that of Vietnam ($US 2,110) in the same year (World Bank, 2017). 1 The lower secondary rate is from Dang and Glewwe (2018), while the primary completion rate was calculated by the authors using the 2014 VHLSS data; the VHLSS data are described further below. a whole, and the territory of Perm, which not representative of Russia. For convenience, we refer to Hong Kong, Macao and Taiwan as countries, although the first two are Chinese territories, and Taiwan’s status is disputed. 3 The GDP per capita for Indonesia was $US 3,347 in 2015, which is about 50 percent higher than that of Vietnam ($US 2,110) in the same year (World Bank, 2017). I. Introduction Vietnam’s rapid economic growth in the last 30 years has transformed it from one of the world’s poorest countries to a middle-income country (World Bank, 2013). While Vietnam’s economic achievements have attracted much attention, in more recent years its accomplishments in education have also generated a great deal of international interest. Vietnam’s high performance in the “quantity” of education is exemplified by its high primary school completion rate of 97%, and its high lower secondary enrollment rate of 95%.1 More striking still are the results of the 2012 PISA assessment: Vietnam’s performance ranked 16th in math and 18th in reading out of 63 countries and territories,2 ahead of both the US and the UK and much higher than that of any other developing country (OECD, 2014a). Its 2012 PISA mathematics and readings scores (at 511 and 508), for example, were more than one standard deviation higher than those of Indonesia (375 and 396), a nearby country whose GDP per capita is most similar to that of Vietnam among all countries that participated in the 2012 PISA.3 A visual depiction of Vietnam’s performance on the PISA in 2012, given its income, is shown in Figures 1 and 2, which plot PISA scores in math and reading by the log of per capita GDP for all 63 countries. Vietnam is in the upper left in both figures, higher than any other country above the line that shows the expected test score given per capita GDP. Vietnam is also the largest positive outlier when PPP (purchasing power parity) GDP is used (see Figures B1 and B2 in Appendix B). More recently, Vietnam was again the largest positive outlier in the 2015 PISA assessment, as seen in Figures 3 and 4. Finally, if the sample is limited to the nine East 1 1 Asian and Southeast Asian participants in the 2012 PISA, Vietnam is still the largest positive outlier (see Figures B3 and B4 in Appendix B). Asian and Southeast Asian participants in the 2012 PISA, Vietnam is still the largest positive outlier (see Figures B3 and B4 in Appendix B). This paper uses the 2012 and 2015 PISA data to try to understand Vietnam’s unusually high performance on these assessments of student learning. More specifically, it has three objectives. I. Introduction First, it examines whether Vietnam’s impressive performance on these PISA assessments may be exaggerated because: i) the 15-year-old Vietnamese students who participated in these assessments are not representative of 15-year-old students in Vietnam; ii) the enrollment rate of 15-year-olds in Vietnam is much lower than those rates in other PISA countries; or iii) Vietnamese students put more effort into those assessments than other students. Second, it uses regression methods to investigate whether family, teacher or school characteristics in the PISA data can explain Vietnamese students’ high performance. Third, it applies the Oaxaca-Blinder decomposition to disaggregate the difference in average test scores between Vietnamese students and students in the other countries that participated in the 2012 and 2015 PISA assessments. This paper’s first finding is that Vietnam’s striking performance on the 2012 and 2015 PISA assessments is at most only partially reduced by adjustments to reduce the possible sources of upward bias discussed above. In particular, adjusting Vietnam’s PISA scores to control for possible oversampling of wealthier, more urban students reduces its scores by only 20-24 (14-15) points in 2012 (2015). Moreover, three different methods to adjust for Vietnam’s low coverage (enrollment) rates (after correcting the OECD coverage rates for Vietnam) have little effect on its outlier status. Finally, back-of-the-envelope calculations to account for possible higher motivation of Vietnamese students on the PISA account for less than half of Vietnam’s outlier status. 2 The second finding it that accounting for household- and school-level variables in the PISA data explains little of Vietnam’s high performance on the 2012 and 2015 PISA assessments relative to its income level. A third finding is that Oaxaca-Blinder decompositions show that the gap in average test scores between Vietnam and the other countries in the 2012 and 2015 PISA is not due to differences in observable child, household and school characteristics; it is mostly due to Vietnam’s higher “productivity” of those characteristics relative to the other PISA countries. This paper focuses on Vietnam, and is most relevant to education policies in that country. I. Introduction But to the best of our knowledge, it also the first study to offer a rigorous and detailed analysis of the performance of one PISA country participant in comparison with the others.4 As such, the framework of analysis used here may be useful for studies of other countries’ performance in a similar context, not only for the PISA but also other skill assessments of multiple countries. This paper consists of five sections. It examines the possible mechanisms that could exaggerate Vietnam’s performance in the next section, before employing regression methods to investigate the role of family, teacher and school characteristics in explaining this performance in Section III. It then provides in Section IV Oaxaca-Blinder decompositions of the test score gap between Vietnam and the other PISA participants, focusing on child, household and school characteristics. Conclusions are offered in Section V. ent investigation of the qualitative aspects of the PISA, see Waldow and Steiner-Khamsi (2019). See, for example, the comments by Deputy Minister of Education Nguyen Vinh Hien in Thanh Ni 4 For a recent investigation of the qualitative aspects of the PISA, see Waldow and Steiner-Kh g q p , ( ) e, the comments by Deputy Minister of Education Nguyen Vinh Hien in Thanh Nien News (2013). 6 Most PISA countries, including Vietnam, conducted testing on April 12-14 of 2012. Thus children born in 1996 would be from 15 years and 3 (completed) months of age (born in December of 1996) and 16 years to 2 (completed) months (born in January of 1996). The target population was defined as “all students aged from 15 years and 3 com- pleted months to 16 years and 2 completed months at the beginning of the assessment period” (OECD, 2014b, p.66). 7 PISA’s urban classification refers to schools, not students. Some students may live in rural areas and attend urban schools; they would be classified as urban in the PISA but as rural in the VHLSS and so may explain some of the urban/rural difference in the two samples. Yet this cannot explain the other differences in these samples in Table 1. 6 Most PISA countries, including Vietnam, conducted testing on April 12-14 of 2012. Thus children born in 1996 would be from 15 years and 3 (completed) months of age (born in December of 1996) and 16 years to 2 (completed) months (born in January of 1996). The target population was defined as “all students aged from 15 years and 3 com- pleted months to 16 years and 2 completed months at the beginning of the assessment period” (OECD, 2014b, p.66). 7 PISA’s urban classification refers to schools not students Some students may live in rural areas and attend urban p y p g g p ( , , p ) 7 PISA’s urban classification refers to schools, not students. Some students may live in rural areas and attend urban schools; they would be classified as urban in the PISA but as rural in the VHLSS and so may explain some of the urban/rural difference in the two samples. Yet this cannot explain the other differences in these samples in Table 1. II. Is Vietnam’s Performance on the 2012 and 2015 PISA Assessments Exaggerated? Some observers, both Vietnamese and international, of Vietnam’s high performance on the 2012 and 2015 PISA assessments have expressed surprise that Vietnam could perform so well.5 This section investigates three possible mechanisms that could exaggerate Vietnam’s 3 performance: 1. The 15-year-old Vietnamese PISA participants in 2012 and 2015 were not representative of 15-year-old Vietnamese students in those years; 2. Vietnam’s relatively low enrollment rate for 15-year-olds selects higher-performing PISA participants; and 3. Vietnamese students exert more effort when participating in the PISA assessments. A. Were Vietnam’s PISA Participants “Better than Average” 15-year-old Students? Were the Vietnam’s PISA participants in 2012 and 2015 representative of the students that those assessments were intended to sample? Consider the 2012 PISA. In each participating country, the 2012 PISA participants were to be a random sample of all children born in 1996 (and thus were 15 years old in January of 2012) who were enrolled in school in 2012 (OECD, 2014b).6 Whether the students who participated in the 2012 PISA in Vietnam are a representative sample of individuals born in 1996 who were students in 2012 can be considered using data from the 2012 Vietnam Household Living Standards Survey (VHLSS), which Vietnam’s General Statistical Office conducts every two years on a sample of about 9,000 households in Vietnam. The 2012 VHLSS can be used to compare Vietnamese children in that survey who were born in 1996 and were students in 2012 with the Vietnamese students who participated in the 2012 PISA. Table 1 uses data from the 2012 PISA assessment and the 2012 VHLSS to assess the representativeness of Vietnam’s PISA participants in 2012. These two data sources have several discrepancies. Compared to the VHLSS data, the students who participated in the 2012 PISA are more likely to be from urban areas (50% vs. 26%),7 are slightly more likely to be in grade 10 4 (86% vs. 84%) and less likely to be in grade 9 (10% vs. 14%), have more educated fathers (9.0 vs. 7.2 years of schooling) and mothers (8.3 vs. 6.8 years), and are more likely to live in homes with air conditioners, cars, computers and televisions, and so are from wealthier families. 5-year-old students interviewed in the first two rounds of the 2012 VHLSS, about half were inter- h il d b h lf i i d i l 5 i i d i d i 8 Of the 236 15-year-old students interviewed in the first two rounds of the 2012 VHLSS, about half were inter- viewed in March or April, and about half were interviewed in June. Only 5 were interviewed in May, and 4 in July. or April, and about half were interviewed in June. Only 5 were interviewed in May, and 4 in July. II. Is Vietnam’s Performance on the 2012 and 2015 PISA Assessments Exaggerated? The discrepancy regarding the likelihood of being in grades 9 and 10 is larger if one notes that the 2012 PISA assessment was administered in Vietnam in April of 2012, which is a time when 22% of the children born in 1996 are still in grade 9, as seen in the third column of Table 1. More specifically, of the children born in 1996 who were still in school and were interviewed between March and July in the 2012 VHLSS (and thus had not yet reached the next grade of schooling in September of 2012),8 76% were in grade 10, while 22% were in grade 9; in contrast, of PISA participants in April of 2012, 86% were in grade 10 and only 10% were in grade 9. The distinction between grades 9 and 10 is important in Vietnam, because almost all children complete grade 9, but in many provinces, students must pass provincial entrance exams to enroll in grade 10. Thus 86% of the students in the PISA sample are students who have passed an exam that selects better performing students for upper secondary school, but the VHLSS data indicate that only about 76% of children in Vietnam who were eligible to participate in the PISA exam when it was administered (in April of 2012) were in grade 10 and thus had passed that exam. Similar patterns are seen in the last four columns of Table 1, which compare the students in the 2015 PISA (who were 15 years old when they took that test, and so should be a random sample of students born in 1999) to an average of students who were 15 years old in 2014 (more precisely, born in 1998) in the 2014 VHLSS data and students who were 15 years old in 2016 (more precisely, born in 2000) in the 2016 VHLSS data (this average is used because there is no 5 VHLSS for 2015). Relative to the averaged VHLSS data (focusing on those interviewed between March and July), the 2015 PISA participants are more likely to be in urban areas (50% vs. 29%), have more educated mothers (8.4 vs. 6.9 years of schooling) and fathers (7.9 vs. 6.4 years), and are more likely to live in homes with air conditioners, cars, computers and televisions. II. Is Vietnam’s Performance on the 2012 and 2015 PISA Assessments Exaggerated? In contrast to the findings for 2012, the students in the 2015 PISA assessment are only slightly more likely to be in grade 10 (85.5%) relative to their counterparts in the averaged VHLSS data (84.3%). The differences in Table 1 between the PISA and the VHLSS data raise a question: How would Vietnam’s students have scored on the PISA if the PISA sample had had the same student characteristics as the VHLSS sample? This can be assessed by using the PISA data for Vietnam to predict Vietnamese students’ performance on the PISA, assuming that this predictive power of the student-level characteristics is valid for the same characteristics as measured by the VHLSS. More specifically, consider an ordinary least squares (OLS) regression that uses the PISA data for Vietnam to predict students’ scores on that assessment based on the variables in Table 1: PISAscorei = βʹXi + ui (1) (1) where Xi is a vector, for student i, of the student characteristics in Table 1. The regressions for the 2012 and 2015 PISA data, using Equation (1), are shown in Tables B1 and B2, Appendix B.9 A convenient property of OLS regressions is that the mean values of the explanatory variables perfectly predict the mean value of the dependent variable. That is: PISAscore ̅̅̅̅̅̅̅̅̅̅̅̅̅ = 𝛃̂OLSʹ𝐗̅PISA (2) A convenient property of OLS regressions is that the mean values of the variables perfectly predict the mean value of the dependent variable. That is: PISAscore ̅̅̅̅̅̅̅̅̅̅̅̅̅ = 𝛃̂OLSʹ𝐗̅PISA (2) (2) PISAscore ̅̅̅̅̅̅̅̅̅̅̅̅̅ = 𝛃̂OLSʹ𝐗̅PISA where the horizontal bars indicate mean values and 𝛃̂OLS is the OLS estimate of β. This is shown in the top halves of Tables 2 (math) and 3 (reading); the first columns depict 𝐗̅ from the 2012 PISA data in Table 1, the fourth column shows the β coefficients (from Table B1, Appendix B), 9These regressions have high predictive power; for example, the R2 is 0.341 for reading and 0.310 for math for the 2012 PISA data. Most variables are highly significant, and almost all of the signs are in the expected direction. 6 and the fifth column shows the product of each variable with its respective coefficient. Summing the fifth column produces the actual PISA scores, 512.7 for math and 509.8 for reading. and the fifth column shows the product of each variable with its respective coefficient. II. Is Vietnam’s Performance on the 2012 and 2015 PISA Assessments Exaggerated? Summing the fifth column produces the actual PISA scores, 512.7 for math and 509.8 for reading. These regression coefficients can also be used to predict what the 2012 PISA score would have been if 𝐗̅ had been the means in the 2012 VHLSS data. The 2012 VHLSS means for the interviews conducted from March to July of 2012 (since the PISA was administered in April of 2012), from the third column of Table 1, are shown in the second column of Tables 2 and 3, the products of these variables and their coefficients are in the sixth column, and the predicted 2012 PISA scores are at the bottom of that column. When the 2012 VHLSS means are used to predict the PISA scores, the math score by about 24 points, to 489.0, and the reading score declines by about 20 points, to 489.5. Almost half of the difference between the 2012 PISA score and the predicted score that adjusts for the potential non-representative sample is due to the larger percentage of grade 10 students in the PISA sample, as seen in the last columns of Tables 2 and 3 10 Albania’s coverage rate, which is much higher for both the 2009 and 2015 PISA assessments, could be an error. We thank Francesco Avvisati for pointing this out. 3. A similar analysis based on equation (2) for the 2015 PISA data and the average of the 2014 and 2016 VHLSS data is shown in the bottom of Tables 2 and 3. Using the means 𝐗̅ from the averaged VHLSS data (the households interviewed from March to July), instead of the 2015 PISA data, Table 2 shows that the math score decreases by 13.6 points, and Table 3 shows that the reading score decreases by 14.7 points. These are smaller than the drops for the 2012 data. The overall message from this exercise is that the differences in child, parent and house- hold characteristics seen in Table 1 between the 2012 PISA sample and the 2012 VHLSS sample imply a drop of only about 20-24 points (or 0.20-0.24 standard deviations) of Vietnam’s performance on the 2012 PISA. Yet a quick glance at Figures 1 and 2 shows that Vietnam is still an outlier even after doing this adjustment. A similar adjustment comparing the 2015 PISA with 7 7 the 2014 and 2016 VHLSS has an even smaller effect on Vietnam’s outlier status in Figures 3 and 4. Correcting, and Three Methods to Adjust for, Vietnam’s Low Enrollment Rate B. Correcting, and Three Methods to Adjust for, Vietnam’s Low Enrollment Rate. Another possible explanation for Vietnam’s strong PISA performance is that many Vietnamese 15-year-olds are not enrolled in school, and those who are not in school are likely to have lower academic skills than those who are. Thus, one possible explanation for Vietnam’s strong performance on the PISA assessments is that, relative to other PISA countries, a larger proportion of Vietnam’s less academically inclined 15-year-olds did not participate in the PISA assessment, which includes only 15-year-olds enrolled in school. Indeed, Vietnam’s “coverage index” indicates that only 55.7% of its 15-year-olds participated in the 2012 PISA, primarily because of this age group’s low enrollment rate (OECD, 2014a, Table A2.1). This is the third lowest coverage rate of the 63 countries that participated in the 2012 PISA assessment; only Albania (55.2%) and Costa Rica (49.6%) had lower rates.10 Vietnam’s lower coverage rate is even more extreme in the 2015 PISA assessment; of the 66 participating countries, Vietnam’s coverage rate was the lowest, at only 49% (OECD, 2016, Table I.6.1). The next lowest country, Mexico, had a much higher rate of 62%. This subsection first corrects the Vietnam coverage rates reported by the OECD, and then presents three different methods to adjust for Vietnam’s (corrected) lower coverage rate. Even after this correction and these adjustments, Vietnam still remains a positive outlier, given its low income, in its performance on the 2012 and 2015 PISA assessments. 1. Correcting PISA Coverage Rates. Analysis of the PISA data demonstrates that Vietnam’s coverage rates were incorrectly calculated, and correcting these errors leads to 8 sizeable increases in those rates. As explained in Appendix A, census data from Vietnam were incorrectly used to calculate the number of 15-year-olds in Vietnam in 2012 and 2015. Correctly applying the census data to the school enrollment data in the OECD reports shows that the correct PISA coverage rates for 2012 and 2015 are, respectively, 65.9% and 65.6%. Yet even after these corrections, 34.1% of 15-year-olds in Vietnam did not participate in the 2012 PISA assessment, and 34.4% did not participate in the 2015 PISA assessment. These individuals were likely weak students before leaving school, since most of the PISA participants were in grade 10 and, unlike grade 9, grade 10 students are a selected group, as explained above. 11 These differ slightly from the numbers in OECD (2014a) because sample weights were not used, for comparabil- ity with columns 3 and 4, which cannot use sampling weights to exclude 15-year-olds who did not participate. Correcting, and Three Methods to Adjust for, Vietnam’s Low Enrollment Rate The rest of this subsection applies three different methods to adjust PISA scores for differences in countries’ coverage rates. Method 1: Focus on Top 50%. One way to adjust each country’s performance to account for differential participation in the PISA is to focus on the “top 50%” of 15-year-olds. This can be done by assuming that non-participating 15-year-olds would have scored in the lowest 50% of the distribution of test scores among all 15-year-olds in their respective countries if they had participated in the assessment, and then excluding the bottom 50% of 15-year-olds for all countries. In fact, for countries with a lower coverage rate, such as Vietnam, this assumption leads to underestimation of the performance of the top 50% of students since, for these countries, it is more likely that some not in school would be in the top 50% if they were in school, which means that some 15-year-olds classified as in the top 50% for these countries were in fact in the bottom 50%. The results of doing so for the 2012 PISA assessment are shown in Table 4A. The first two columns of Table 4A show the widely reported scores in the PISA reports, which include all test participants (and, of course, exclude nonparticipants).11 Vietnam ranks 16 11 These differ slightly from the numbers in OECD (2014a) because sample weights were not used, for comparabil- ity with columns 3 and 4, which cannot use sampling weights to exclude 15-year-olds who did not participate. 9 out of 63 in math and 18 out of 63 in reading. However, when 15-year-olds in the bottom 50% of the population of all 15-year-olds are excluded, using the method described above, 12 the performance of Vietnam’s “top 50%” of 15-year-olds is less impressive, ranking only 34 out of 63 in math and 39 out of 63 in reading (see the third and fourth columns of Table 4A). The same exercise using the 2015 PISA results yields a similar conclusion. As seen in Table 4B, Vietnam ranks 24 out of 66 countries in math and 28 out of 66 in reading. While these rankings are not as high as the 2012 PISA rankings, this is still a very strong performance given that Vietnam is the second poorest of all participating countries (see Figures 3 and 4). 12 Table 4A shows the mean scores of the top 50% of 15-year-olds under the assumption that those who did not par- ticipate would not have scored in the top 50% had they participated. Mathematically, denote the coverage rate by c, which is ≥ 50 for all countries except Costa Rica (its 49.6% rate is set to 50%). The goal is to drop the d% of the test participants who were not in the top 50%, thus d = c - 50. Thus one must drop (d/c)×100%, i.e. ((c-50)/c)×100%, of test participants. For each country the bottom ((c-50)/c)×100% of test takers were dropped, separately for each test. Correcting, and Three Methods to Adjust for, Vietnam’s Low Enrollment Rate However, when 15-year-olds in the bottom 50% of Vietnam’s 15-year-olds are excluded, as shown in the third and fourth columns of Table 4B, the performance of Vietnam’s “top 50%” of 15-year-olds is less impressive, ranking only 37 out of 66 in math and 41 out of 66 in reading. Yet these lower rankings are still impressive when one recalls Vietnam’s relatively low income. First, it still outperforms almost all other developing countries in the PISA, the sole exception being that Chile’s top 50% of 15-year-olds outperformed Vietnam’s top 50% on the 2015 reading assessment (and note that Chile is much wealthier than Vietnam). Second, as Figures 5 and 6 show, given that Vietnam was the poorest of the 63 countries in the 2012 PISA it is still by far the largest positive outlier when the scores of the “top 50% of all 15-year-olds” are plotted against log of per capita GDP. Although Vietnam’s “top 50%” scores in mathematics and reading are not much higher than their “unadjusted” scores, and the increase in test scores of the “top 50% of 15-year-olds” was much higher for other countries, the increases were highest in 10 the wealthier countries, which usually have high PISA participation rates. This increases the slope of the lines in Figures 5 and 6, relative to Figures 1 and 2, and since Vietnam is at the far left in these figures the higher slope makes it more of an outlier. This is also the case for the 2015 PISA assessment, as shown in Figures 7 and 8. Method 2: Bounds Analysis – Inferring Full Distribution Mean from Truncated Distribution Mean. A second method to correct for Vietnam’s relatively low enrollment rate is to build on the intuition of Method 1 that individuals not in school have lower academic skills in order to estimate bounds on the average test score of all 15-year-olds for the countries that participated in the PISA. To begin, assume that the PISA test scores follow a normal distribution when the entire population of 15-year-olds is included. Figure B5 in Appendix B shows that this assumption is reasonable for four 2015 PISA countries from different regions of the world that had enrollment (coverage) rates above 90%: Australia, Germany, South Korea and Tunisia. Correcting, and Three Methods to Adjust for, Vietnam’s Low Enrollment Rate One can derive a lower bound for the mean of the distribution of the test scores of all 15-year-olds (students and non-students) by making a second assumption: that the test scores of all children not in school, if they had participated in the PISA, would be lower than those of all children in school. This assumption is illustrated in Figure B6. Under these two assumptions, PISA participants constitute a normal distribution that is truncated from below (as in Figure B6). However, assuming that the test score of each 15-year-old PISA participant is higher than the test scores of all 15-year-old non-participants is quite extreme. It is almost certain that some PISA non-participants would have scored higher on that assessment than some participants. If so, the adjustment described below would underestimate (and so provide a lower bound for) the true mean (for all 15-year-olds). To see the intuition, consider Figure B6. Suppose that some 15-year-olds to the right of the truncation point did not participate in the PISA, and that some 15- 11 11 year-olds to the left of that point did participate. This would reduce the extent to which the mean of the distribution of the PISA participants overestimates the mean of the distribution for all 15- year-olds; applying the truncation formula that is proved below in Proposition 1 overcorrects and so provides a lower bound of that mean. While standard formulas for the mean of a truncated normally distributed variable obtain that mean using the mean of the overall (untruncated) distribution, our goal is to go in the other direction; we want to obtain the mean of the overall distribution using the mean of the truncated distribution. Proposition 1 below provides the formulas for doing this. Proposition 1: Estimating lower bounds and upper bounds of test scores Assume that the test scores of the entire population of 15-year-olds follow a normal distribution with mean µ and standard deviation 𝜎. The truncated mean of this distribution is given by the sample mean test scores 𝑇̅k, where k indexes truncation from above (a) or below (b), with 𝜏 being the truncation point. Define 𝛼 as 𝜏−µ 𝜎, and let r represent the given school enrollment rate. 1.1. If the PISA’s tested samples capture only academically better-performing children (as in Figure B6) the true mean test scores, denoted by µ lt (lt denotes lower truncation), is given by: ̅ 𝜇𝑙𝑡 = 𝑇̅𝑏− 𝜆𝑏(𝛼) 𝑇̅𝑏−𝑇𝑚𝑖𝑛 𝜆𝑏(𝛼)−𝛼 (1) where 𝑇̅𝑏= 𝐸(𝑇|𝑇> 𝜏) , 𝛼= Φ−1(1 −𝑟), 𝜆𝑏(𝛼) = 𝜙(𝛼) 1−Φ(𝛼) , and the truncation point 𝜏 is given by Tmin, the lowest observed test score in the data. (1) 1.2. If the PISA’s tested samples capture only the academically worse-performing children, the true mean test scores, denoted by µ ut (ut denotes upper truncation) is given by ̅ 1.2. If the PISA’s tested samples capture only the academically worse-performing children, the true mean test scores, denoted by µ ut (ut denotes upper truncation) is given by 𝜇𝑢𝑡 = 𝑇̅𝑎+ 𝜆𝑎(𝛼) 𝑇𝑚𝑎𝑥−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 (2) 𝜇𝑢𝑡 = 𝑇̅𝑎+ 𝜆𝑎(𝛼) 𝑇𝑚𝑎𝑥−𝑇𝑎 𝜆𝑎(𝛼)+𝛼 (2) where 𝑇̅ = 𝐸(𝑇|𝑇< 𝜏), 𝛼= Φ−1(𝑟), 𝜆𝑎(𝛼) = 𝜙(𝛼) Φ(α) , and the truncation point 𝜏 is given by Tmax, the highest observed test score in the data. (2) 𝜇𝑢𝑡 = 𝑇̅𝑎+ 𝜆𝑎(𝛼) 𝑇𝑚𝑎𝑥−𝑇𝑎 𝜆𝑎(𝛼)+𝛼 (2) where 𝑇̅ = 𝐸(𝑇|𝑇< 𝜏), 𝛼= Φ−1(𝑟), 𝜆𝑎(𝛼) = 𝜙(𝛼) Φ(α) , and the truncation point 𝜏 is given by Tmax, the highest observed test score in the data. The proof is given in Appendix A. Yet, since the assumptions that the PISA’s tested samples capture only either the academically better-performing or the academically worse-performing children in Proposition 1 are rather extreme, Equations (1) and (2) would provide, respectively, 12 lower bound and upper bound estimates of the true mean test scores. Equation (1) is the derivation of interest given that the mean test score of the PISA participants is almost certainly higher than the mean of the PISA non-participants (this is also shown below for the Young Lives data from Vietnam). We show Equation (2) only for mathematical completeness. 13 For more information on the Young Lives data, consult its website: www.younglives.org.uk g , y g g 14 The only other PISA country for which there are Young Lives data is Peru. 15 Another possibility is that Vietnamese teachers provided answers to students, along the lines that Jacob and Levitt (2003) found in Chicago public schools. Unfortunately, we cannot apply most of the methods that that paper used to check for such cheating because we do not have panel data. Also, students taking the PISA exam are given many different versions of the test so that any given student does not have the identical questions as the students sitting nearby. Multiple versions of the test also make it much harder for teachers to provide students the correct answers. Proposition 1: Estimating lower bounds and upper bounds of test scores Applying Equation (1) of Proposition 1 to the PISA math test, Figure 9 shows these estimates, as well as the sample means of the scores of the observed (truncated) distributions of 2012 PISA participants, which one can view as upper bounds, as explained above. Figure 10 shows the same for the 2015 PISA assessments. The gap between these two bounds is a decreasing function of the enrollment (coverage) rate, and will equal zero when the enrollment rate is 100%. The bounds in Figures 9 and 10 for Vietnam, and for some other countries, are rather wide. A “natural” approximation of the test score means that would be observed if all 15- year-olds in each country were tested is the mid-point between the lower bound derived in Proposition 1 and the upper bound given by the observed mean for the truncated sample, that is the midpoints of the gaps in Figure 9. These mid-point values are shown in Figures 11-14, where the countries are again plotted against their log of per capita GDP. Vietnam still stands out as an outlier, especially for math. Method 3: Adjustment with Auxiliary Data. A third way to adjust the mean of the test scores for Vietnamese students to include the scores of the PISA non-participants is to use the Young Lives data.13 This is done only for Vietnam, because only one other PISA country has such data;14 yet doing this adjustment only for Vietnam will be biased against Vietnam being an outlier. The younger cohort in the Young Lives Study were 15 years old in Round 5 of that study. This round, which took place in 2016, included administration of math and reading 13 comprehension tests to all 15-year-olds in that sample, about 1,940 15-year-olds, both those in, and those not in, school; as expected, those not in school had lower average math (9.4 out of 21) and reading (10.9 out of 25) scores than those in school (15.5 and 14.8). Assuming that the Young Lives reading and math scores rank 15-year-olds in a way that is similar to the PISA test rankings, one can adjust the observed PISA test scores to include 15-year-olds who are not in school; see Appendix A for the details of this method. Proposition 1: Estimating lower bounds and upper bounds of test scores Making this adjustment to the 2012 PISA scores reduces the mean math score for Vietnam by about 13 points, from 511.2 to 498.4, and the mean reading score by about 11 points, from 508.2 to 496.9. The same adjustment for the 2015 PISA has similar reductions, about 12 points for math and 11 for reading. These relatively small changes do not change the overall finding that Vietnam’s PISA performance was exceptional. C. Were Vietnamese Students More Motivated and Better Prepared for the PISA? A final possible explanation for Vietnam’s performance in the PISA assessments is that Vietnamese students really did outperform those of most other countries, but not due to higher skills; rather, they were highly motivated when they took the PISA tests, and they received extensive preparation for those tests.15 No studies have examined the motivation of Vietnamese students when taking international tests, but there are many anecdotes that Vietnamese students (and their teachers) are very competitive test takers. In contrast, there is evidence that students in developed countries exert little effort on tests for which there are no consequences. Gneezy et al. (2019) administered tests based on questions from previous PISA math t t t Chi t d t d U S t d t Th Chi t d t d h hi h th U S Gneezy et al. (2019) administered tests based on questions from previous PISA math tests to Chinese students and U.S. students. The Chinese students scored much higher than U.S. Gneezy et al. (2019) administered tests based on questions from previous PISA math tests to Chinese students and U.S. students. The Chinese students scored much higher than U.S. 14 students under standard conditions. However, randomly selected U.S. students who were offered financial incentives for high scores on the exam performed much better (22-24 points higher), while Chinese students performed no differently. The lack of an effect for Chinese students suggests that they are highly motivated to take tests despite no direct benefits. Vietnamese culture has many similarities to Chinese culture, and thus it is possible that Vietnamese students’ intrinsic motivation to do well on tests increased their PISA scores by 22-24 points relative to U.S., and perhaps other, students. There is also evidence that teachers and schools prepared Vietnamese students to take the 2012 and 2015 PISA tests.16 Studies in the U.S. and elsewhere have shown that preparation sessions for academic tests can greatly increase students’ scores. For example, Bangert-Drowns et al. (1983), summarizing a large number of studies, found that programs involving coaching sessions of more than nine hours total duration increased average test scores by 0.39 standard deviations (of the distribution of test scores, which for the PISA is equivalent to 39 points). A rough estimate of the combined impact of intrinsic motivation and preparation for the PISA exam would be 62 points (23 from being more motivated, and 39 from exam preparation). 16 When Vietnamese students took a draft version of the PISA exam in 2011 in preparation for the 2012 PISA, their performance was lower than expected, and Vietnam’s Ministry of Education and Training took several steps to increase their performance. This does not violate rules of the PISA assessment; schools can have students practice, using old exams, to become “accustomed” to PISA exams. In each country, the schools that participate in the PISA exam are selected several months before the exam, and the students who participate are selected 3-4 weeks before the exams. The selected Vietnamese students were told that a strong performance would bring honor to Vietnam, and were given special t-shirts indicating that they were PISA participants. The information on the implementation of the PISA in Vietnam is based on emails and discussions with Francesco Avvisati, who works on the PISA for the OECD. 17 For example, when presenting an earlier version of this paper in Colombia, we were told that Colombia has made similar efforts to increase its students’ performance on the PISA, but their efforts were not particularly effective. C. Were Vietnamese Students More Motivated and Better Prepared for the PISA? This would explain about half of Vietnam’s exceptional performance in terms of the positive residuals discussed below (Table 5, columns 1 and 2), assuming that all other countries that participated in the PISA took no steps to increase their students’ test scores and had students as unmotivated as U.S. students. Yet this assumption is rather extreme, and anecdotal evidence 15 suggests that other countries also try to increase their students’ performance on the PISA.17 Thus these two factors together likely explain less than half of Vietnam’s exceptional performance. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? The evidence in Section II shows that 15-year-old students in Vietnam scored unusually high on the 2012 and 2015 PISA assessments given Vietnam’s low GDP per capita, even after adjusting for the possibility that the PISA sample was not representative of the 15-year-olds enrolled in school and for the low enrollment rate of 15-year-olds in Vietnam. Presumably there is some reason why Vietnamese students outperform those in other countries conditional on per capita GDP. This section uses the PISA data to investigate Vietnam’s performance on the PISA. A. From Country Level to Student Level Regressions. Figures 1-4 in Section II are based on the following simple linear regression equation: Test Score = β0 + βgdp×Log(GDP/capita) + u Test Score = β0 + βgdp×Log(GDP/capita) + u (3) In these figures, the gap between any country’s actual performance on the test and its predicted performance given its (log) GDP per capita is given by u in equation (3). These figures show that Vietnam has a very high value of u. The regressions that generated these figures have one observation per country, yet analogous regressions with one observation per student for each country participating in the PISA assessments yield the same finding. Such regressions, which regress the student-level PISA test score data on a constant term and the log of per capita GDP, are shown for the 2012 PISA data in the first two columns of the top half of Table 5. As expected, the coefficient on GDP per capita is positive: countries with a higher GDP tend to have higher scores. However, Vietnam’s test scores in the 2012 PISA are much higher than those (3) 16 predicted by this regression. In particular, for the math regression Vietnam’s average value of u (the residual, shown in bold in the fifth row of Table 5) is 135.8, and for the reading regression it is 119.0. These are the highest values among all the countries included in the regression, as indicated by the “Residual Rank” row in Table 5, just as Vietnam is the largest positive outlier in the country-level regressions that generated Figures 1-4. The question is: Why is Vietnam’s residual so high? In particular, would adding more variables to the regression result in a “better fit” in which the (average) residual for Vietnam would not be so high? oner could not be used since it was collected for some countries (including Vietnam) but not others. 18 Air conditioner could not be used since it was collected for some countries (including Vietnam) but not others. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? This question is addressed in the rest of this section, first by adding household and student level characteristics, and then adding by school characteristics, using information from the 2012 and 2015 PISA data, which not only administered reading and mathematics tests but also collected data from students, parents and schools. The remaining columns in Table 5 explore the simple relationship between student test scores and national and household level income and wealth. One disadvantage of the regressions in the first two columns of that table is that the variable for the log of GDP per capita does not vary over students in the same country; ideally, it would be useful to have a wealth or income variable that varies within countries, which should provide more explanatory power in student- level regressions. A wealth variable can be generated from the PISA data by using information on students’ households from the student questionnaire. This was done by applying principle components analysis to the following household level variables in the PISA: internet connection, dishwasher, DVD, number of cell phones, number of televisions, number of computers, and number of cars.18 The first estimated principle component is used as a wealth variable in the analysis of this section. The third and fourth columns of Table 5 show that, for the 2012 PISA, 17 when this variable is used instead of the log of GDP per capita, Vietnam is still the largest outlier in the math regression, though it is only the second largest outlier in the reading regression, after Hong Kong. The rest of the analysis of this paper will use this wealth variable instead of log of GDP per capita because the former varies across students within each country in the PISA data. Before adding other variables to equation (3), which is the focus of this section, the last four columns of Table 5 explore two aspects of the wealth variable that was generated by principal components analysis. First, the third and fourth columns in Table 5 use country averages of the wealth variable, for comparability with the first two columns in that table, which are based on the log of GDP per capita. In contrast, the fifth and sixth columns allow each student to have his or her own household-specific value of wealth, instead of the national average. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? This allows the wealth variable to explain not only the differences in test scores across countries but also within countries. This reduces the coefficients on the wealth variable somewhat, but it is still highly significant. More interesting is that Vietnam falls slightly in terms of its outlier status. For math it is now the fourth highest outlier, while for reading it is the second highest. The main reason for this is that the predictive power of the wealth variable falls by about one fifth when it varies within countries, which indicates that it has a stronger role to play when explaining differences between countries than within them. This drop in the coefficient in effect leads to a less steep slope in the fitted lines in Figures 1-4, reducing the size of Vietnam’s residual and increasing the size of the residuals for the wealthiest top performers, such as Hong Kong, Singapore and South Korea. Yet Vietnam is still a large outlier, and much poorer than these other outlier countries. Second, the last two columns of Table 5 add country fixed effects, which again reduces the impact of wealth somewhat. The reported residuals in those two columns are simply the 18 estimated country fixed effects. Again, Vietnam is still an outlier, although slightly less of an outlier in that it has the fifth highest fixed effect for math and the third highest for reading. A similar analysis is done for the 2015 PISA data in the bottom half of Table 5. The overall pattern is the same. The average residual for Vietnam slightly decreases when the average wealth variable is used instead of GDP per capita, and decreases slightly more when the wealth variable is allowed to vary at the student level, but Vietnam is still one of the largest, if not the largest, outlier. This is also the case when country fixed effects are used. Again, the countries that occasionally are larger outliers than Vietnam are much wealthier than Vietnam. B. Adding Other Variables to Explain Vietnam’s Performance. The student-level regressions with country fixed effects in the last two columns of Table 5 are a useful starting point for a more systematic analysis to find characteristics of Vietnamese students, households, teachers and schools that explain Vietnam’s outlier status in the 2012 and 2015 PISA assessments. To begin, assume that the underlying skill (e.g. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? math) measured by the PISA test score of student i in country c, denoted by Sic, is a linear function of the characteristics of that student, of his or her household, the teachers he or she has had, and the school(s) he or she has attended: assessments. To begin, assume that the underlying skill (e.g. math) measured by the PISA test score of student i in country c, denoted by Sic, is a linear function of the characteristics of that student, of his or her household, the teachers he or she has had, and the school(s) he or she has attended: Sic = βʹxic + εic (4) Sic = βʹxic + εic (4) (4) (4) where the xic variables are all the student, household, teacher and school characteristics that affect students’ underlying skills, β measures the causal impacts of those characteristics on that skill, and εic is measurement error in the PISA test. The linearity assumption is not very restrictive since xic could include higher order and interaction terms. An important distinction to make is between the observed and unobserved xic variables: 5 An important distinction to make is between the observed and unobserved xic variables: An important distinction to make is between the observed and unobserved xic variables: Sic = βoʹxico + βuʹxicu + εic (5) important distinction to make is between the observed and unobserved xic variables Sic = βoʹxico + βuʹxicu + εic (5) (5) Sic = βoʹxico + βuʹxicu + εic ( Sic = βoʹxico + βuʹxicu + εic 19 = βoʹxico + βuʹ𝐱̅cu + βuʹxicu,d + εic = βoʹxico + βuʹ𝐱̅cu + βuʹxicu,d + εic = βoʹxico + βuʹ𝐱̅cu + βuʹxicu,d + εic where the superscripts o and u indicate observed and unobserved, respectively. The second line of equation (5) disaggregates xicu into its country specific mean, 𝐱̅cu, and the within-country deviation from that mean for student i, xicu,d, where the superscript d indicates that deviation. This disaggregation implies that the within-country mean of xicu,d equals zero for all countries. where the superscripts o and u indicate observed and unobserved, respectively. The second line of equation (5) disaggregates xicu into its country specific mean, 𝐱̅cu, and the within-country deviation from that mean for student i, xicu,d, where the superscript d indicates that deviation. This disaggregation implies that the within-country mean of xicu,d equals zero for all countries. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? In a regression with country fixed effects, the fixed effect for country c would be βuʹ𝐱̅cu, and the error term would be βuʹxicu,d + εic. The last two regressions in Table 5 have only one observed variable, the wealth indicator. The goal of the rest of this section is to add additional variables to equation (5), which in effect moves those variables out of xicu and into xico in that equation, to see whether Vietnam’s outlier status can be explained by observed variables in the PISA data. This approach was used by Fryer and Levitt (2004) to investigate the gap in test scores between black and white students in the U.S., and by Singh (2019) to explain differences in test scores of primary and secondary school age children across Ethiopia, India (Andhra Pradesh), Peru and Vietnam. If the PISA data contain the key factors that explain Vietnamese students’ success, then adding them as regressors will yield small and statistically insignificant country fixed effect for Vietnam by removing the variables that contribute to the βuʹ𝐱̅cu term in the second line of Equation (5). If all variables are included that explain the performance of all the countries in the PISA data set, then all country fixed effects will become insignificant and the error term will become the (within-country) variation in the measurement error, εic. Even if the PISA data lack some of the key variables that explain Vietnam’s success, and more generally explain student learning in all the countries that participated in the 2012 and 2015 PISA assessments, it may be that the country fixed effects, while statistically significant, are greatly reduced and thus at least part of the reasons for Vietnam’s success would be explained by 20 20 the PISA data. Even if Vietnam is still one of the largest outliers, it may be a much smaller outlier – relative to the overall variation in the PISA test score data – after adding the variables available in the PISA data. In contrast, if the student, household, teacher and school variables that explain Vietnam’s success are for the most part not in the PISA data, then Vietnam will continue to be a large, positive outlier and the reason(s) for its outlier status will be due to factors that are not measured, or at least not well measured, in the PISA data. the PISA data. 19 Missing values were particularly common for the sibling index. To avoid losing observations due to that variable being missing, missing values were assigned its average value and a variable was added indicating that it is missing. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? Even if Vietnam is still one of the largest outliers, it may be a much smaller outlier – relative to the overall variation in the PISA test score data – after adding the variables available in the PISA data. In contrast, if the student, household, teacher and school variables that explain Vietnam’s success are for the most part not in the PISA data, then Vietnam will continue to be a large, positive outlier and the reason(s) for its outlier status will be due to factors that are not measured, or at least not well measured, in the PISA data. To begin, student and household level variables from the 2012 PISA assessment are added to the regression equation in Table 6A. The first two columns of that table show regressions identical to those in the last two columns of Table 5, except that the sample size is reduced so that the sample is identical to that in the third and fourth columns of Table 6A, which add four additional household variables. The estimates in the first two columns of Table 6A are very similar to those in the last two columns of Table 5; the rank of Vietnam’s estimated country fixed effects is the same, and the countries with larger fixed effects are also the same. The third and fourth columns of Table 6A add four additional household characteristics that are “pre-determined” and may also explain students’ test performance: a dummy variable for girl students, an index of the number of siblings in the home (0 = none, 1 = brothers but no sisters, or sisters but no brothers, and 2 = sisters and brothers); and mother’s and father’s years of schooling. Each of these household variables has some missing values, which reduces the sample size to 401,489, compared to 455,971 in the last two columns of Table 6.19 The key question for Table 6A is whether adding these additional household-level variables “explains” much of the very large country fixed effect found for Vietnam when house- hold wealth is the only regressor. The third and fourth columns in Table 6A shows that adding 21 these four variables to the regression reduces the explanatory power of the wealth index variable by about one third (although it is still highly significant) but it has very little impact on the Vietnam country fixed. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? Indeed, these fixed effects increase slightly, from 78.2 to 80.6 for the math test and from 68.3 to 70.7 for the reading test. Vietnam’s outlier status is also largely unchanged; its fixed effect in the math regression drops from fifth place to sixth place, but increases from third place to second place in the reading regression. Thus, these four household-level variables in the PISA data do not explain Vietnam’s strong performance on the 2012 PISA assessment. This is not surprising when the means of these variables are compared for Vietnam and these other countries. In particular, Table 8 shows that the average of Vietnam’s sibling index is almost identical to that of the other PISA countries combined (1.048 vs. 1.086, respectively), and that Vietnamese parents have, on average, fewer years of schooling (8.3 for mothers and 8.9 for fathers) than do parents in the other PISA countries (11.0 for mothers and 11.1 for fathers), so these variables cannot explain why Vietnam outperforms other countries; indeed, its lower parental education levels make its performance all the more remarkable. The 2012 PISA data contain several variables that are directly related to students’ education, such as the grade they are in, years of preschool, several educational inputs, days of school attendance (in the past two weeks), books in the home not related to the child’s schooling, and hours per week in tutoring classes. These variables are likely to be endogenous (parents may provide more educational inputs to children not doing well at school, or perhaps to their most promising children), so adding them to the regression analysis likely produces biased estimates of the causal impacts of these variables. Despite this possible bias, these variables may provide informative explanatory power that may shed light on why Vietnamese students perform so well 22 on the PISA. For example, Table 8 shows that, on average, Vietnamese students spend more hours per week in tutoring classes (1.3 for reading, 2.7 for math) than do students in other PISA on the PISA. 0 Unfortunately, data on tutoring classes and on hours studying at home were not collected for Viet y, g y g PISA, so those variables can be used only for the 2012 analysis. y y 21 The education input index is the first principal component of the following variables: quiet place to study, desk, educational software, classical literature books, poetry books, educational books, technical books, and a dictionary. 20 Unfortunately, data on tutoring classes and on hours studying at home were not collected for Vietnam for the 2015 PISA so those variables can be used only for the 2012 analysis data o tuto g c asses a d o ou s study g at o e we e ot co ected o V et a o t e 0 5 ariables can be used only for the 2012 analysis. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? For example, Table 8 shows that, on average, Vietnamese students spend more hours per week in tutoring classes (1.3 for reading, 2.7 for math) than do students in other PISA countries (0.9 for reading, 1.3 for math), so even if one cannot estimate the causal impact of these classes on student test scores, even biased estimates may reduce Vietnam’s outlier status.20 The last two columns of Table 6A add these more education-focused child and household variables to the regression that had only gender, parental education and the sibling index variable (this reduces the sample size, so the fifth and sixth columns show the results with only household wealth but the same samples as in the last two columns). Adding these variables further reduces the coefficient on household wealth, and reduces Vietnam’s estimated country fixed effect (from 79.1 to 65.0 for math, and from 68.9 to 55.1 for reading), but it does not reduce Vietnam’s outlier status: it remains the fifth highest outlier for math and the third largest for reading. Again, the reason for this is that, for some education variables added to the regression, Vietnamese students have lower average values than do the students in the other PISA countries. For example, Vietnamese students have fewer educational inputs21 and fewer books at home (see Table 8). Vietnamese students have fewer educational inputs21 and fewer books at home (see Table 8). A similar analysis for the 2015 PISA data is presented in Table 6B. The overall results are similar. The country fixed effect for Vietnam changes very little when additional household- level variables are added to the regression, and Vietnam is always one of the top five positive outliers. Also, all of the other top five outliers are much wealthier than Vietnam. Since child and household variables in the PISA data do little to explain Vietnam’s exceptional performance (outlier status) in education, perhaps that performance is due to better schools and teachers. This is examined in Tables 7A and 7B, which add school and teacher 23 characteristics to the regressions. As before, the first two columns show, for comparison purposes, regressions that include only the wealth variable, but have the same samples as the regressions that include the school and teacher variables. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? The third and fourth columns of Tables 7A and 7B show regression results that add not only child and household variables (which are not shown to reduce clutter) but also school and teacher variables. The school and teacher variables are: class size; the proportion of teachers who have the required qualifications; computers per student; a variable indicating whether student performance is used to assess teachers’ performance (a higher value indicates a “no” response); an indicator of teacher absenteeism; an index of whether parents put pressure on teachers (2012 only); two variables indicating whether school principals and outside inspectors, respectively, observe teachers in the classroom; an indicator of the extent to which student performance determines teacher pay (2012 only); and an index that measures teacher mentoring. Most of these school characteristics have the expected signs, but the key question is whether they can “explain” at least part of Vietnam’s outlier status as measured by its country fixed effect. The results for the 2012 PISA in Table 7A show that adding school and teacher variables reduces Vietnam’s outlier status in the sense that Vietnam’s estimated fixed effects are reduced by nearly one fourth (from 76.7 to 58.1) for math and almost one third (from 66.1 to 44.7) for reading. Yet little has changed in the sense that adding these variables to the math test yields only a small reduction in the rank of Vietnam’s estimated fixed effect (from five to eight), and its rank for the reading test (four) is unchanged. The same analysis using the 2015 PISA data is shown in Table 7B, and the results are similar. In particular, adding school and teacher variables reduces Vietnam’s country fixed 24 effects by about one fifth for math (from 71.4 to 59.2) and for reading (from 59.2 to 46.5), but its relative rank declines by only one for math and five for reading. effects by about one fifth for math (from 71.4 to 59.2) and for reading (from 59.2 to 46.5), but its relative rank declines by only one for math and five for reading. To summarize, this section shows that the observed child, household, school and teacher variables in the PISA data explain very little of Vietnam’s impressive performance on the 2012 and 2015 PISA assessments relative to its income level. III. What Observed Variables in PISA Explain the Gaps Conditional on Income? At most, adding these variables explains one fourth of Vietnam’s exceptional performance in math and one third of its exceptional performance in reading. Thus, most of the explanation for that performance must be found elsewhere. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? The analysis in the previous section assumed that the impact of each variable on test scores is the same for all 63 countries in the analysis. But perhaps Vietnam’s exceptional performance is partly due to it being “more effective” in using various “inputs”. For example, it may be that each year of Vietnamese parents’ years of schooling represents a higher level of cognitive skills than does the average year of parental schooling in the other PISA countries. To explore this possibility, a Oaxaca-Blinder decomposition (Blinder, 1973; Oaxaca, 1973) is applied to differences in test scores between Vietnam and all other countries. Test scores (S) are assumed to be linear functions of the variables used in the last two columns of Tables 7A and 7B, again denoted by x. The impacts of these variables on test scores, denoted by β, are allowed to differ between Vietnam and the other countries in the PISA assessment. This yields the following regression equations (omitting the i subscript to reduce clutter): Svn = βvnʹxvn + uvn (Vietnam) (6) So = βoʹxo + uo (Other countries) (7) Svn = βvnʹxvn + uvn (Vietnam) (6) So = βoʹxo + uo (Other countries) (7) 25 The constant term in both of these equations can be normalized so that the means of the residuals equal 0. Taking the mean of both sides of each regression equation gives the following The constant term in both of these equations can be normalized so that the means of the residuals equal 0. 22 Note that this decomposition holds algebraically even when 𝛃̅ is replaced by any vector β of the same dimension; the arithmetic average is used here for its intuitive appeal. Other β’s have been suggested in the literature. See Fortin et al. (2011) and Jann (2008) for further discussion of this decomposition. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? Taking the mean of both sides of each regression equation gives the following i f th t t i Vi t S̅ d i th th 62 PISA t i S̅ expressions for the average test scores in Vietnam, S̅vn, and in the other 62 PISA countries, S̅o: expressions for the average test scores in Vietnam, S̅vn, and in the other 62 PISA countries, S̅o: S̅vn = βvnʹ𝐱̅vn (8) S̅o = βoʹ𝐱̅o (9) S̅o = βoʹ𝐱̅o (9) (9) The standard Oaxaca-Blinder decomposition uses equations (8) and (9) to express the difference in the mean test scores between Vietnam and the other PISA countries as follows: S̅vn – S̅o = βvnʹ𝐱̅vn – βoʹ𝐱̅o (10) = βvnʹ𝐱̅vn – βoʹ𝐱̅o + βoʹ𝐱̅vn – βoʹ𝐱̅vn = βoʹ(𝐱̅vn –𝐱̅o) + (βvn – βo)ʹ𝐱̅vn (10) = βoʹ(𝐱̅vn –𝐱̅o) + (βvn – βo)ʹ𝐱̅vn Thus, the difference in the average test scores in Vietnam and the average scores in the other countries has two components. The first is the difference in the means of the x variables between Vietnam and the other countries, multiplied by the β for the other countries (denoted by βo). The second is the difference between Vietnam and the other countries in the “effectiveness” of the x variables, βvn – βo, multiplied by the means of Vietnam’s x variables (denoted by 𝐱̅vn). Thus, the difference in the average test scores in Vietnam and the average scores in the other One criticism of equation (10) is that the differences in the means of the explanatory variables (𝐱̅vn –𝐱̅o) are “weighted” by the coefficient for the other 62 countries (βo) while the differences in the coefficients (βvn + βo) are weighted by the means of the explanatory variables for Vietnam (𝐱̅vn). Intuitively, it seems unbalanced that these weights are all from one group or the other group; it may be better for the weights to be weighted averages of the β’s and the 𝐱̅’s of both groups. 23 These means are for the sample for which the Oaxaca-Blinder composition is implemented. Observations with missing values for the x variables are dropped, and so the means are slightly different from those in previous tables. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? One way to do this is to take the average of the two vectors of β coefficients and use that β as the weight for the differences in the means; this yields the following decomposition: (11) S̅vn – S̅o = βvnʹ𝐱̅vn – βoʹ𝐱̅o 26 = 𝛃̅ʹ(𝐱̅vn –𝐱̅o) + [(βvn – 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo)ʹ𝐱̅o] where 𝛃̅ = (βvn + βo)/2.22 The first term, 𝛃̅ʹ(𝐱̅vn –𝐱̅o), weights the influence of the differences in the x variables by the simple average of the two β coefficients. The second term, (βvn – 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo)ʹ𝐱̅o, accounts for the influence of the differences in the βvn and βo coefficients by splitting that difference into two parts, the difference between βvn and 𝛃̅, weighted by 𝐱̅vn, and the differ- ence between 𝛃̅ and βo, weighted by 𝐱̅o. As in the original Oaxaca-Blinder decomposition, the first term “explains” how much of the difference in the mean test scores between Vietnam and the other countries is due to Vietnamese students having different characteristics than the other students, and the second term “explains” how much of the difference stems from the differences in the impacts of the various x variables, as measured by the difference between βvn and βo. In addition to decomposing the differences in the mean test scores, S̅vn – S̅o, into the above two components (the “explained” part due to differences in the x’s and the “unexplained” part due to differences in the β’s), both components can be further decomposed into the contributions of the individual variables, which sum up to equal the overall component. For example, one variable used below measures hours per week that children receive math tutoring, which for the 2012 PISA is much higher in Vietnam (2.7) than the average for the other PISA countries (1.3), as seen in Table 8. This tutoring could explain Vietnam’s strong performance by contributing to the first component, 𝛃̅ʹ(𝐱̅vn –𝐱̅o). That is, part of this component is β̅t(x̅vn,t – x̅o,t), where the t subscript indicates that this is the tutoring variable. Similarly, the impact of tutoring could also contribute to the second component via the difference in the βt coefficients; its contribution to the second component is [(βvn,t – β̅t)ʹx̅vn,t + (β̅t – βo,t)ʹx̅o,t]. 27 However, there are some potential problems with determining the roles played by specific variables in these decompositions. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? First, sets of categorical variables, such as region or ethnic group codes, require an omitted (base) category, and different base categories can produce different results when assessing the impacts of the differences in the mean values of the x However, there are some potential problems with determining the roles played by specific variables in these decompositions. First, sets of categorical variables, such as region or ethnic group codes, require an omitted (base) category, and different base categories can produce different results when assessing the impacts of the differences in the mean values of the x variables across the two groups. Fortunately, this is not a problem here because this paper does not use any sets of categorical variables. Second, variables that do not have “natural” zero points can yield different results when assessing the impacts of the differences in the values of the β terms across the two groups; adding an arbitrary constant to such variables will change the contribution of the difference in the β terms because that difference is multiplied by the mean of that variable, and the mean has changed. While almost all variables in the regressions have natural “zeros”, as explained above the wealth index and the education inputs index were constructed by using principal components analysis, and the first principal components for both of these variables take both positive and negative numbers. Both of these variables are “re- centered” by adding a constant that ensures that their minimum values are close to zero. Table 8 shows the means of the x variables separately for Vietnam and for the other PISA countries for both PISA assessments. The 2012 means are also shown in the second and fifth columns of Table 9A (and Table 10A). The bottom of the Table 9A shows the mean math test score for Vietnam, 516.5 (in the third column), which is S̅vn, and the mean math test score for the other 62 countries, 462.8 (the sixth column), which is S̅o.23 The gap between these two means is 53.7. Similarly, Table 10A shows that the gap between the two mean reading scores is 40.3. 24 The impact of this variable is similar in both Vietnam (6.77) and in the other 62 countries (7.72), although it is statistically significant only for the 62 countries, which reflects the much larger sample size for that estimate. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? These gaps are smaller than the average residuals for Vietnam in Table 5 because those residuals effectively compared Vietnam to a hypothetical “typical” other country that had the same level These gaps are smaller than the average residuals for Vietnam in Table 5 because those residuals effectively compared Vietnam to a hypothetical “typical” other country that had the same level 28 of wealth as Vietnam, while the gaps in Tables 9A and 10A compare Vietnam, which has a relatively low wealth of 4.14, with the other 62 countries, that have a higher wealth of 6.10. Returning to Table 9A, the x variables that have higher means in Vietnam than in the other 62 countries, and for which the corresponding 𝛃̅ coefficients are positive, can potentially explain part of the gap between the mean test scores in Vietnam and the other 62 countries in the 2012 PISA assessment. That is, the contribution of such variables to the 𝛃̅ʹ(𝐱̅vn – 𝐱̅o) component in equation (10) is positive. The contribution is also positive when the mean for Vietnam is lower than for the other 62 countries and the corresponding 𝛃̅ coefficient is negative. An example of the former is the teacher mentoring variable. This is higher in Vietnam than in other countries, and as expected mentoring of teachers is estimated to increase students’ test scores.24 In contrast, if Vietnam’s mean is higher but the corresponding 𝛃̅ coefficient is negative, or Vietnam’s mean is lower and the corresponding 𝛃̅ coefficient is positive, this widens the gap and thus makes the gap even harder to explain. For example, the mean years of schooling of mothers and of fathers is lower in Vietnam than in the other 62 countries, and as expected the corresponding β coefficients are positive, so the parent education variables cannot explain why Vietnamese students’ scores are higher than those of other countries’ students, and so these variables “increase the burden” on other variables to explain that gap. Table 9A provides the results of the Oaxaca-Blinder decomposition for the 2012 PISA mathematics test. Recall that the overall gap to explain is 53.7 points. The differences in the x variables, which are expressed as the βoʹ(𝐱̅vn –𝐱̅o) component of the decomposition, cannot explain the gap. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? Indeed, summing over all of the x variables shows that the values of the x 29 variables lead one to expect a slightly larger gap, with an overall contribution of -1.6 (see the bottom of the second to last column in Table 9A). Instead, the decomposition indicates that the entire gap is due to the difference in the β coefficients; on average, Vietnam is “more efficient” in “converting” x variables into higher test scores; this is seen in the last column in Table 9A. Given that it is the differences in the β coefficients that “explain” Vietnam’s remarkable performance on the 2012 PISA assessment, it is of interest to see which specific variables seem to contribute the most to this component of the decomposition. The variable in the last column of Table 9A that plays the most important role is the percentage of students in grade 10, which accounts for about seven eighths (88%) of this differential efficiency of the β coefficients. Quite simply, on average for other 2012 PISA countries, moving a student from grade 9 to grade 10 without changing any other characteristic increases his or her test score by 18.9 points, but in Vietnam this increases a students’ score by 85.9 points. This almost certainly reflects the fact that movement from grade 9 to grade 10 in Vietnam is a selection process based on province- level exams, which removes the grade 9 students with the weakest academic performance. But it is also likely that, apart from this selection effect, an additional year of school in Vietnam leads to more learning than an addition year in other countries; recall from Section II that correcting for the very low “coverage rate” in Vietnam explained only a small part of its exceptional performance. Moreover, as seen in the next paragraph, additional days of attendance have a larger impact in Vietnam than in the other 2012 PISA countries (on average). The other variables for which the higher β coefficient for Vietnam explains a substantial portion of the gap (a contribution greater than 20 points) are: 1. Using student performance as part of the formal process to evaluate teachers’ effectiveness; 2. Number of days attended in the past two weeks; and 3. Proportion of teachers who are “qualified”. 25 In fact, Vietnam is more likely to use student performance to evaluate teachers, but the effect of this variable’s difference in means (second to last column of Table 9A) is much smaller (3.0) than its differential effect (25.0). IV. What Can Be Learned from Oaxaca-Blinder Decompositions? It is important to keep in 30 mind that these effects do not reflect that these variables have higher mean values in Vietnam; the 25 point impact using student performance to assess teacher effectiveness in the last column of Table 9A does not reflect that Vietnam is more likely to use student performance when assessing teachers’ performance,25 but rather the results suggest that this policy is more effective in Vietnam than in other countries. Similarly, the role played by student attendance is not so much due to higher student attendance in Vietnam, but due to students learning more per day of school attended. Finally, note that the third largest contributor is in fact negative: While, as one would expect, having more “qualified” teachers increases student learning, the estimated impact of such teachers in Vietnam (18.2) is much lower than the estimated impact for the other countries that participated in the 2012 PISA assessment (46.1). Table 10A yields similar results for the reading decomposition using the 2012 PISA data, although they are somewhat more difficult to interpret. The overall gap to be explained is 40.3 points. As with the math score, the differences in the x variables explain little, and in fact they slightly widen the gap to be explained by 1 point. In contrast, the “greater efficiency” of the x variables explains virtually all of the gap by accounting for 41.3 points in that gap. Two x variables stand out as making the biggest contribution to explaining this gap. First, as with the mathematical results, the differential efficiency of being in grade 10 can account for virtually all of the gap (42.7 points). Again, this may reflect not only differential efficiency but also a screening process between grades 9 and 10 that removes the weakest students from the overall population of 15-year-old students. Second, the differential impact of school attendance makes an even larger contribution, unlike the results for the math exam. The contribution of this differential school attendance, 80.5 points, is so large that it requires some variable to have a 31 large negative effect, and that variable is the constant term, which is much larger in the other countries than in Vietnam, a result also not seen with the math test. 26 The application of the Oaxaca-Blinder decomposition in this paper could be misleading in that the estimated β terms for Vietnam are based on only within-country variation in the variables, while the estimates of β for all other countries are based on both within-country and between-country variation. It is possible that the decomposition results could change if the latter estimates were also based on only within-country variation, which can be done by estimating βo using a country-fixed-effects specification. This was done, and the main results still hold, as seen in Appendix Tables B.4 and B.5. In particular, the contribution of the difference in the x variables, as measured by 𝛃̅ʹ(𝐱̅vn-𝐱̅o), remains very small. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? This somewhat puzzling result for the reading decomposition, and unfortunately it is difficult to interpret this large difference in the constant terms between Vietnam and the other 62 countries in the 2012 PISA assessment.26 These Oaxaca-Blinder decompositions were also done for the 2015 PISA data, and the results are similar. The relevant tables for these results are Tables 9B and 10B. Note first that, as seen in Table 8, there are seven fewer variables in the 2015 PISA with which to do this decomposition, but that still leaves 15 variables. The decomposition of the difference in the mathematics scores for the 2015 PISA assessment is shown in Table 9B. As in the 2012 PISA, the differences in the x variables do not explain the 44.6 point gap in the mean test score between Vietnam and the 65 other countries that participated in the 2015 PISA. Indeed, those differences add 16.7 points to the gap. Thus, all of the differences are due to the differences in the β terms for Vietnam and for the other countries. The decomposition of the difference in the mathematics scores for the 2015 PISA assessment is shown in Table 9B. As in the 2012 PISA, the differences in the x variables do not explain the 44.6 point gap in the mean test score between Vietnam and the 65 other countries that participated in the 2015 PISA. Indeed, those differences add 16.7 points to the gap. Thus, all of the differences are due to the differences in the β terms for Vietnam and for the other countries. The decomposition of the difference in the reading scores is shown in Table 10B, and the results are very similar to those in Table 9B; the differences in the x variables do not explain the 27.0 point gap in the mean test score between Vietnam and the 65 other countries, and instead they in effect widen the gap. Again, the entire explanation for the gap must come from the differences in the β terms between Vietnam and the other countries. The decomposition of the difference in the reading scores is shown in Table 10B, and the results are very similar to those in Table 9B; the differences in the x variables do not explain the 27.0 point gap in the mean test score between Vietnam and the 65 other countries, and instead they in effect widen the gap. IV. What Can Be Learned from Oaxaca-Blinder Decompositions? Again, the entire explanation for the gap must come from the differences in the β terms between Vietnam and the other countries. 32 To summarize this section, the Oaxaca-Blinder decompositions for both 2012 and 2015 indicate that Vietnam’s exceptional performance on the PISA assessment in both years is not due to Vietnam having “better” observable child, household or school characteristics. Instead Vietnam seems to be more effective in transforming those factors into test scores. In other words, these decompositions indicate that virtually all of Vietnam’s strong performance in these two PISA assessments comes from the “unexplained” portion of this decomposition method, and thus this decomposition sheds very little light on the underlying reasons for that performance. V. Conclusion Vietnam’s very high performance on the 2012 and 2015 PISA assessments has raised the question of why Vietnam does so well, and whether other countries can improve their student learning outcomes by applying what works well in Vietnam. This paper has used the 2012 and 2015 PISA data to do three types of analysis to explore the reasons behind Vietnam’s apparent success. The analysis done thus far has led to three sets of general results. First, one important, albeit partial, explanation of Vietnam’s very strong performance on the 2012 and 2015 PISA assessments is that the weakest students are excluded from grade 10. In particular, only about 66% of Vietnamese 15-year-olds participated in the 2012 and 2015 PISA assessments, presumably because most or all of the other 34% were no longer in school. (Note that the OECD reports even lower participation rates, but this paper corrects those erroneous rates.) Yet applying three different methods to adjust for Vietnam’s low coverage (enrollment) rates has little effect on its outlier status. Moreover, back-of-the-envelope calculations to account for possible higher motivation of Vietnamese students on the PISA account for at most less than half of Vietnam’s outlier status. A related point is that the 15-year-old students who 33 participated in the PISA assessments appear to be better off when compared to the 15-year-olds enrolled in school in the 2012, 2014 and 2016 Vietnam Household Living Standards Surveys. Yet even after adjusting Vietnam’s test scores for these differences between the two datasets, those scores are still well above what one would predict based on Vietnam’s income alone. A similar result also holds for the 2015 PISA scores. Thus, even after accounting for these apparent differences between the PISA and VHLSS data, Vietnam still performs unusually well on the PISA assessments relative to what one would expect given its income level. Second, taking the PISA data at face value, this paper has used regression methods to investigate which family, teacher or school characteristics in the PISA data can “explain” the high performance of Vietnamese students. The general finding of this analysis is that accounting for household level and school level variables in the PISA data explains at most only one fourth or one third of Vietnam’s high performance on the 2012 PISA relative to its income level. V. Conclusion Moreover, adding these variables to an initial regression of PISA test scores on household wealth does very little to Vietnam’s “outlier” status. Similar results are found for the 2015 PISA. Third, this paper has applied the Oaxaca-Blinder decomposition method to better understand the difference in average test scores between Vietnamese students and students in the other countries that participated in the 2012 and 2015 PISA assessments. Unlike the analysis discussed in the previous paragraph, this approach is more flexible in that it allows the impacts (the β’s) of the household and school variables (the x variables) to differ between Vietnam and all other countries that participated in the PISA assessments. The decompositions indicate that all of the gap in average test scores between Vietnam and the other countries (62 in 2012 and 65 in 2015) is due to the greater “productivity” of various household and school variables in Vietnam, relative to the “productivity” of those variables in other countries, especially the 34 greater “productivity” that occurs when grade 9 students in Vietnam are enrolled in grade 10. This could reflect the fact that only Vietnamese students who pass an entrance exam are allowed to proceed from grade 9 to grade 10, which is the first grade of the upper secondary cycle in Vietnam. This is most clear for the mathematics results; the reading results show a similar pattern but also have other factors at work (in particular, more productivity from each day of school attendance). In contrast, differences in household and school characteristics explain none of the gap between Vietnam and the other countries that participated in the PISA assessment; indeed, they add to the gap to be explained. While the analysis in this paper has shed some light on the reasons for Vietnam’s exceptional performance on the 2012 and 2015 PISA assessments, in the end the main contribution of this paper is that it has shown what does not explain that exceptional performance. In particular, it does not appear to be due to the low participation of Vietnam’s 15- year-olds, nor to any possible selection of “better” students (as measured by urban location, parental education and household wealth) into the PISA assessments. V. Conclusion Observable child, household, and school characteristics explain little or none of the differential performance; in particular the Oaxaca-Blinder decompositions attribute none of the gap in test scores to such factors, and instead attribute all to the “unexplained” differences between Vietnam and the PISA participants in the coefficients that determine the impacts of these observed factors. Future research on Vietnam’s exceptional performance will need to use different data, and perhaps different methodologies, to understand that country’s impressive performance in education. 35 References Blinder, Alan. 1973. “Wage Discrimination: Reduced Form and Structural Estimates”. Journal of Human Resources 8(4):436-455. Dang, Hai-Anh, and Paul Glewwe. 2018. “Well Begun, But Aiming Higher: A Review of Vietnam’s Education Trends in the Past 20 Years and Emerging Challenges”. Journal of Development Studies 54(7):1171-1195. Fortin, Nicole, Thomas Lemieux and Sergio Firpo. 2011. “Decomposition Methods in Economics”, in O. Ashenfelter and D. Card, eds., Handbook of Labor Economics, Volume 4A. North-Holland: Amsterdam. Fryer, Roland, and Steven Levitt. 2004. “Understanding the Black-White Test Score Gap in the First Two Years of School”. Review of Economics and Statistics 86(2):447-464. General Statistics Office. 2010. The 2009 Vietnam Population and Housing Census: Completed Results. Hanoi: Vietnam. Available online at: https://www.gso.gov.vn/default_en.aspx?tabid=515&idmid=5&ItemID=10799 Gneezy, Uri, John A. List, Jeffrey A. Livingston, Xiangdong Qin, Sally Sadoff, and Yang Xu. 2019. “Measuring Success in Education: The Role of Effort on the Test Itself.” American Economic Review: Insights 1(3): 291-308. Jacob, Brian, and Steven Levitt. 2003. “Rotten Apples: An Investigation of the Prevalence and Predictors of Teacher Cheating”. Quarterly Journal of Economics 118(3):843-877. Jann, Ben. 2008. “The Blinder-Oaxaca Decomposition for Linear Regression Models”. The Stata Journal 8(4):453-479. Oaxaca, Ronald. 1973. “Male-Female Differentials in Urban Labor Markets”. International Economic Review 3(4):693-709. OECD. 2014a. PISA 2012 Results: What Students Know and Can Do – Student Performance in Mathematics, Reading and Science, Volume I (Revised Edition). Paris: Organization for Economic Cooperation and Development. OECD. 2014b. PISA 2012 Technical Report. Paris: Organization for Economic Cooperation and Development. OECD. 2016. PISA 2015 Results: Excellence and Equity in Education, Volume I. Paris: Organization for Economic Cooperation and Development. Singh, Abhijeet. 2019. “Learning More with Every Year: School Year Productivity and International Learning Divergence”. Journal of European Economic Association. https://doi.org/10.1093/jeea/jvz033. 36 Thanh Nien News. 2013. “Vietnam Deputy Education Minister not Convinced by Global Test”. December 7, 2013. http://www.thanhniennews.com/education-youth/vietnam-deputy- education-minister-not-convinced-by-global-test-18276.html. Waldow, Florian and Gita Steiner-Khamsi. 2019. Understanding PISA’s Attractiveness: Critical Analyses in Comparative Policy Studies. V. Conclusion Bloomsbury Academic, London. World Bank. 2013. “Skilling up Vietnam: Preparing the workforce for a modern market economy”. Vietnam Development Report 2014. World Bank, Washington, DC. . World Development Indicators Online database. World Bank, Washington, DC. 37 37 Figure 1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita gure 1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP 38 Figure 3. Mean Age 15 Math Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 3. Mean Age 15 Math Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 4. Mean Age 15 Language Scores in 2015 PISA, by 2015 Log Real GDP/capita Mean Age 15 Math Scores in 2015 PISA, by 2015 Log Real GDP/capita 015 PISA, by 2015 Log Real GDP/capita Figure 4. Mean Age 15 Language Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 4. Mean Age 15 Language Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 4. Mean Age 15 Language Scores in 2015 PISA, by 2015 Log Real GDP 39 Figure 5. Mean Age 15 Top 50% Math Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 6. Mean Age 15 Top 50% Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 5. Mean Age 15 Top 50% Math Scores in 2012 PISA, by 2010 Log Real GDP/capita an Age 15 Top 50% Math Scores in 2012 PISA, by 2010 Log Real GDP/capita 40 Figure 6. Mean Age 15 Top 50% Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita Figure 6. Mean Age 15 Top 50% Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita an Age 15 Top 50% Reading Scores in 2012 PISA, by 2010 Log Real GDP/capi Figure 6. Mean Age 15 Top 50% Reading Scores in 2012 PISA, by 2010 Log R 40 41 Figure 7. V. Conclusion Mean Age 15 Top 50% Math Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 8. Mean Age 15 Top 50% Reading Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 7. Mean Age 15 Top 50% Math Scores in 2015 PISA, by 2015 Log Real GDP/capita an Age 15 Top 50% Math Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 8. Mean Age 15 Top 50% Reading Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 8. Mean Age 15 Top 50% Reading Scores in 2015 PISA, by 2015 Log R igure 8. Mean Age 15 Top 50% Reading Scores in 2015 PISA, by 2015 Log Real GDP/capita Figure 8. Mean Age 15 Top 50% Reading Scores in 2015 PISA, by 2015 Log Real GDP/capita 41 42 Figure 9: Lower and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 2012 Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. Figure 10: Lower and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 2015 Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. Figure 9: Lower and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 2012 Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. Figure 9: Lower and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 2012 and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 2012 Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. es are sorted in an increasing order of the upper bound (observed mean from PISA participants) of dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. V. Conclusion Figure 10: Lower and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 2015 Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. Figure 10: Lower and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 2015 Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. wer and Upper (observed) Bounds of Math Scores for All 15-year-olds, PISA 20 Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. Note: All countries are sorted in an increasing order of the upper bound (observed mean from PISA participants) of test scores. The dots represent the observed mean test scores. Vietnam is indicated by the larger diamond. 42 Figure 11: Midpoint of Upper and Lower Bounds of 2012 PISA Math Scores, by Log of GDP/capita Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. Midpoint of Upper and Lower Bounds of 2012 PISA Math Scores, by Log of Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. Figure 12: Midpoint of Upper and Lower Bounds of 2012 PISA Reading Scores, by Log of GDP/capita Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. Figure 12: Midpoint of Upper and Lower Bounds of 2012 PISA Reading Scores, by Log of GDP/ it Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. 43 Figure 13: Midpoint of Upper and Lower Bounds of 2015 PISA Math Scores, by Log of GDP/capita Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. V. Conclusion Figure 13: Midpoint of Upper and Lower Bounds of 2015 PISA Math Scores, by Log of Midpoint of Upper and Lower Bounds of 2015 PISA Math Scores, by Log of Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. Figure 14: Midpoint of Upper and Lower Bounds of 2015 PISA Reading Scores, by Log of GDP/capita N t Adj t d t t f h t th id i t l f th b d t t d th th ti l Figure 14: Midpoint of Upper and Lower Bounds of 2015 PISA Reading Scores, by Log of GDP/capita Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. point of Upper and Lower Bounds of 2015 PISA Reading Scores, by Log of Figure 14: Midpoint of Upper and Lower Bounds of 2015 PISA Reading Figure 14: Midpoint of Upper and Lower Bounds of 2015 PISA Reading Scores, by Log of GDP/capita Note: Adjusted test scores for each country are the mid-point value of the observed test scores and the theoretical lower bounds based on Proposition 1. 44 Table 1: Student Characteristics in 2012 (born in 1996) and 2015 (born in 1999): PISA vs. V. Conclusion VHLSS 2012 PISA and 2012 VHLSS 2015 PISA and 2014 & 2016 VHLSS PISA VHLSS (PISA-eligible only) PISA VHLSS (PISA-eligible only) Variable (1) All (2) Mar.-July (3) Difference (3) – (1) (4) All (5) Mar.-July (6) Difference (6) – (4) Urban 50.3% 26.0% 25.3% -24.9*** 49.6% 30.5% 28.6% -21.0*** (4.2) (2.3) (3.2) (5.2) (4.0) (1.9) (2.7) (4.9) Female 53.8% 51.7% 51.7% -2.1 51.4% 51.4% 47.1% -4.3 (0.8) (2.6) (3.5) (3.6) (1.0) (1.9) (2.6) (2.8) Current grade: 10 or higher 86.1% 84.3% 75.7% -10.4*** 85.5% 90.5% 84.3% -1.2 (2.6) (1.8) (3.0) (3.9) (3.0) (1.0) (1.8) (3.5) Current grade: 9 or lower 10.3% 14.0% 22.2% 11.9*** 9.0% 8.5% 15.1% 6.2** (2.2) (1.7) (2.8) (3.6) (2.2) (1.0) (1.8) (2.8) Current grade: unknown/othera/ 3.6% 1.7% 2.1% -1.5 5.5% 1.0% 0.6% -4.9** (1.5) (0.7) (1.3) (2.0) (2.3) (0.4) (0.4) (2.3) Father’s years of schooling 8.95 7.18 7.19 -1.76*** 8.4 7.1 6.9 -1.47*** (0.17) (0.22) (0.32) (0.37) (0.17) (0.17) (0.23) (0.29) Mother’s years of schooling 8.34 6.80 6.93 -1.41*** 7.9 6.6 6.4 -1.41*** (0.19) (0.19) (0.26) (0.32) (0.20) (0.15) (0.22) (0.29) Owns an air-conditioner 16.0% 7.1% 7.1% -8.8*** 20.7% 19.2% 15.2% -5.5** (2.1) (1.4) (2.1) (3.0) (1.6) (1.7) (2.2) (2.8) Owns a motorbike 93.1% 91.0% 90.7% -2.4 93.9% 94.0% 93.8% -0.2 (0.5) (1.4) (2.0) (2.1) (0.5) (0.8) (1.3) (1.4) Owns a car 7.3% 0.7% 1.0% -6.3*** 7.9% 2.0% 2.6% -5.3*** (0.8) (0.3) (0.7) (1.1) (0.7) (0.5) (0.9) (1.1) Owns a computer 39.1% 24.5% 25.1% -14.1*** 44.1% 29.5% 28.5% -15.6*** (2.2) (2.3) (3.2) (3.9) (1.9) (1.8) (2.4) (3.1) Number of televisions owned 1.39 1.00 1.00 -0.38*** 1.42 1.09 1.05 -0.36*** (0.03) (0.02) (0.03) (0.04) (0.03) (0.02) (0.03) (0.04) Sample size 4,771 455 236 5687 849 415 PISA coverage/eligibility rate 56% 75% 78% 49% 76.4% 77.8% Robust standard errors, clustered at school level in the PISA sample and at commune level in the racteristics in 2012 (born in 1996) and 2015 (born in 1999): PISA vs. VHLSS Robust standard errors, clustered at school level in the PISA sample and at commune level in the VHLSS sample, are shown in parentheses. The difference column reports mean differences between the PISA sample and the VHLSS subsample interviewed from March to July, as well as their standard errors; t-tests are conducted to test whether the mean difference of each variable is significantly different from zero, for which: *** p<0.01, ** p<0.05, * p<0.1. V. Conclusion The difference column reports mean differences between the PISA sample and the VHLSS subsample interviewed from March to July, as well as their standard errors; t-tests are conducted to test whether the mean difference of each variable is significantly different from zero, for which: *** p<0.01, ** p<0.05, * p<0.1. a/ In the PISA sample, this category consists of observations originally categorized as “Ungraded”, with no further information; in the VHLSS sample, this category consists of observations originally categorized as “Attending vocational schools”. a/ In the PISA sample, this category consists of observations originally categorized as “Ungraded”, with no further information; in the VHLSS sample, this category consists of observations originally categorized as “Attending vocational schools”. 45 Table 2: Predicted PISA Math Scores Based on VHLSS Data, Decomposed by Variable (Using March – July Means of VHLSS data) A. 2012 PISA Data and 2012 VHLSS Data Variable Variable Means Difference in Means Math Coeff. Math Coefficient Multiplied by: PISA VHLSS PISA Mean VHLSS Mean Difference in Means Rural 0.497 0.747 -0.250 -18.04 -9.0 -13.5 4.5 Female 0.538 0.517 0.021 -16.58 -8.9 -8.6 -0.4 Grade 10 0.861 0.757 0.104 105.8 91.0 80.1 11.0 Dad Yrs. Sch. 8.81 7.19 1.62 2.231 19.7 16.0 3.6 Mom yrs. sch. 8.23 6.93 1.306 1.879 15.5 13.0 2.4 Air condit. 0.160 0.071 0.089 5.456 0.9 0.4 0.5 Car 0.094 0.010 0.084 -6.723 -0.6 -0.1 -0.6 Computer 0.391 0.251 0.140 17.35 6.8 4.4 2.4 TVs 1.39 1.00 0.39 0.526 0.7 0.5 0.2 Constant 1.000 1.000 0.000 396.7 396.7 396.7 0.0 Column sum -- -- -- -- 512.7 489.0 23.7 Table 2: Predicted PISA Math Scores Based on VHLSS Data, Decomposed by Variable (Using March – July Means of VHLSS data) edicted PISA Math Scores Based on VHLSS Data, Decomposed by Variable (Using March – July Means of VHLSS data) B. 2015 PISA Data and 2014 and 2016 VHLSS Data B. 2015 PISA Data and 2014 and 2016 VHLSS Data Variable Variable Means Math Coefficient Multiplied by: Difference Math PISA VHLSS Difference PISA VHLSS in Means Coeff. Mean Mean in Means Rural 0.504 0.714 -0.210 -9.822 -5.0 -7.0 2.1 Female 0.514 0.471 0.043 -8.461 -4.3 -4.0 -0.4 Grade 10 0.855 0.843 0.012 74.61 63.8 62.9 0.9 Dad yrs. sch. 8.40 6.446 1.410 2.041 17.1 9.4 2.1 Mom yrs. sch. 7.86 6.932 1.467 1.460 11.5 14.2 3.0 Air condit. V. Conclusion 0.207 0.152 0.055 -2.685 -0.6 -0.4 -0.15 Motorbike 0.939 0.938 0.002 6.451 6.1 6.0 0.01 Car 0.079 0.026 0.053 -1.249 -0.1 0.0 -0.1 Computer 0.441 0.285 0.156 23.40 10.3 6.7 3.7 TVs 1.416 1.054 0.363 6.734 9.5 7.1 2.4 Constant 1.000 1.000 0.000 386.4 386.4 386.4 0.0 Column sum 494.7 481.2 13.6 46 Table 3 Predicted Reading Scores Based on VHLSS Data, Decomposed by Variable (Using March – July Means for the VHLSS data) A. 2012 PISA and 2012 VHLSS Data Variable Variable Means Difference in Means Reading Coeff. Reading Coefficient Multiplied by: PISA VHLSS PISA Mean VHLSS Mean Difference in Means Rural 0.497 0.747 -0.250 -11.56 -5.7 -8.6 2.9 Female 0.538 0.517 0.021 24.61 13.2 12.7 0.5 Grade 10 0.861 0.757 0.104 95.14 81.9 72.0 9.9 Dad Yrs. Sch. 8.81 7.19 1.62 1.536 13.5 11.0 2.5 Mom yrs. sch. 8.23 6.93 1.30 1.661 13.7 11.5 2.2 Air condit. 0.160 0.071 0.089 -0.626 -0.1 -0.0 -0.1 Car 0.094 0.010 0.084 -3.442 -0.3 -0.0 -0.3 Computer 0.391 0.251 0.140 10.86 4.2 2.7 1.5 TVs 1.39 1.00 0.39 2.977 4.1 3.0 1.1 Constant 1.000 1.000 0.000 385.2 385.2 385.2 0.0 Column sum -- -- -- -- 509.8 489.5 20.3 B. 2015 PISA Data and 2014 and 2016 VHLSS Data Variable Variable Means Reading Coefficient Multiplied by: Difference Reading PISA VHLSS Difference PISA VHLSS in Means Coeff. Mean Mean in Means Rural 0.504 0.714 -0.210 -18.86 -9.5 -13.5 4.0 Female 0.514 0.471 0.043 15.97 8.2 7.5 0.7 Grade 10 0.855 0.843 0.012 69.85 59.7 58.9 0.9 Dad yrs. sch. 8.40 6.446 1.410 1.646 13.8 5.8 1.3 Mom yrs. sch. 7.86 6.932 1.467 0.893 7.0 11.4 2.4 Air condit. 0.207 0.152 0.055 -0.712 -0.1 -0.1 -0.04 Motorbike 0.939 0.938 0.002 15.83 14.9 14.8 0.03 Car 0.079 0.026 0.053 5.202 0.4 0.1 0.3 Computer 0.441 0.285 0.156 16.61 7.3 4.7 2.6 TVs 1.416 1.054 0.363 7.284 10.3 7.7 2.6 Constant 1.000 1.000 0.000 376.9 376.9 376.9 0.0 Column sum 489.0 474.3 14.7 Table 3 Predicted Reading Scores Based on VHLSS Data, Decomposed by Variable (Using March – July Means for the VHLSS data) A. 2012 PISA and 2012 VHLSS Data Variable Variable Means Difference in Means Reading Coeff. V. Conclusion Reading Coefficient Multiplied by: PISA VHLSS PISA Mean VHLSS Mean Difference in Means Rural 0.497 0.747 -0.250 -11.56 -5.7 -8.6 2.9 Female 0.538 0.517 0.021 24.61 13.2 12.7 0.5 Grade 10 0.861 0.757 0.104 95.14 81.9 72.0 9.9 Dad Yrs. Sch. 8.81 7.19 1.62 1.536 13.5 11.0 2.5 Mom yrs. sch. 8.23 6.93 1.30 1.661 13.7 11.5 2.2 Air condit. 0.160 0.071 0.089 -0.626 -0.1 -0.0 -0.1 Car 0.094 0.010 0.084 -3.442 -0.3 -0.0 -0.3 Computer 0.391 0.251 0.140 10.86 4.2 2.7 1.5 TVs 1.39 1.00 0.39 2.977 4.1 3.0 1.1 Constant 1.000 1.000 0.000 385.2 385.2 385.2 0.0 Column sum -- -- -- -- 509.8 489.5 20.3 Table 3 Predicted Reading Scores Based on VHLSS Data, Decomposed by Variable (Using March – July Means for the VHLSS data) Table 3 Predicted Reading Scores Based on VHLSS Data, Decomposed by Variable (Using March – July Means for the VHLSS data) B. 2015 PISA Data and 2014 and 2016 VHLSS Data Variable Variable Means Reading Coefficient Multiplied by: Difference Reading PISA VHLSS Difference PISA VHLSS in Means Coeff. Mean Mean in Means Rural 0.504 0.714 -0.210 -18.86 -9.5 -13.5 4.0 Female 0.514 0.471 0.043 15.97 8.2 7.5 0.7 Grade 10 0.855 0.843 0.012 69.85 59.7 58.9 0.9 Dad yrs. sch. 8.40 6.446 1.410 1.646 13.8 5.8 1.3 Mom yrs. sch. 7.86 6.932 1.467 0.893 7.0 11.4 2.4 Air condit. 0.207 0.152 0.055 -0.712 -0.1 -0.1 -0.04 Motorbike 0.939 0.938 0.002 15.83 14.9 14.8 0.03 Car 0.079 0.026 0.053 5.202 0.4 0.1 0.3 Computer 0.441 0.285 0.156 16.61 7.3 4.7 2.6 TVs 1.416 1.054 0.363 7.284 10.3 7.7 2.6 Constant 1.000 1.000 0.000 376.9 376.9 376.9 0.0 Column sum 489.0 474.3 14.7 B. 2015 PISA Data and 2014 and 2016 VHLSS Data 47 PISA Assessment Country 2012 Rankings, Overall and Top 50% of Overall 48 Table 4A: PISA Assessment Country 2012 Rankings, Overall and Top 50% of Overall Population Math (all students) Reading (all students) Math (top 50% of pop.) Reading (top 50% of pop.) Rank Country Avg. score Country Avg. score Country Avg. score Country Avg. V. Conclusion score 1 Singapore 573 Hong Kong 545 Singapore 648 Singapore 612 2 Hong Kong 561 Singapore 542 Taiwan 639 Japan 607 3 Taiwan 559 Japan 538 Hong Kong 623 Hong Kong 599 4 South Korea 554 South Korea 536 South Korea 622 South Korea 594 5 Macao 538 Finland 524 Japan 602 Belgium 587 6 Japan 536 Canada 523 Belgium 596 Finland 585 7 Liechtenstein 535 Taiwan 523 Macao 595 Taiwan 585 8 Switzerland 531 Ireland 523 Netherlands 592 New Zealand 585 9 Netherlands 523 Poland 518 Liechtenstein 589 France 584 10 Estonia 521 Liechtenstein 516 Switzerland 586 Ireland 583 11 Finland 519 Estonia 516 Germany 586 Netherlands 580 12 Poland 518 New Zealand 512 Poland 583 Poland 580 13 Canada 518 Australia 512 Czech Republic 583 Germany 575 14 Belgium 515 Netherlands 511 Estonia 578 Estonia 573 15 Germany 514 Macao 509 Finland 573 Norway 572 16 Vietnam 511 Belgium 509 Austria 569 Czech Republic 571 17 Austria 506 Switzerland 509 New Zealand 568 Canada 569 18 Australia 504 Vietnam 508 France 566 Israel 567 19 Ireland 501 Germany 508 Canada 563 Australia 567 20 Slovenia 501 France 505 Ireland 560 United Kingdom 565 21 Denmark 500 Norway 504 Iceland 559 Liechtenstein 565 22 New Zealand 500 United Kingdom 499 Slovakia 558 Sweden 561 23 Czech Republic 499 United States 498 Australia 557 Switzerland 560 24 France 495 Denmark 496 United Kingdom 556 United States 560 25 United Kingdom 494 Czech Republic 493 Luxembourg 556 Luxembourg 560 26 Iceland 493 Austria 490 Spain 555 Macao 558 27 Latvia 491 Italy 490 Norway 553 Italy 557 28 Luxembourg 490 Latvia 489 Italy 551 Spain 555 29 Norway 489 Spain 488 Slovenia 549 Austria 553 30 Portugal 487 Luxembourg 488 Portugal 548 Iceland 553 31 Italy 485 Portugal 488 Denmark 547 Hungary 551 32 Spain 484 Hungary 488 Latvia 547 Latvia 549 33 Russian Federation 482 Israel 486 Sweden 544 Portugal 548 34 Slovakia 482 Croatia 485 Vietnam 543 Denmark 546 35 United States 481 Iceland 483 Russian Federation 543 Croatia 546 36 Lithuania 479 Sweden 483 United States 542 Slovakia 542 37 Sweden 478 Slovenia 481 Israel 541 Greece 541 38 Hungary 477 Greece 477 Hungary 540 Russian Federation 538 39 Croatia 471 Lithuania 477 Lithuania 536 Vietnam 537 40 Israel 466 Turkey 475 Croatia 533 Lithuania 534 41 Greece 453 Russian Federation 475 Greece 510 Slovenia 530 42 Serbia 449 Slovakia 463 Romania 504 Turkey 512 43 Turkey 448 Serbia 446 Serbia 503 Chile 511 44 Romania 445 United Arab Emirates 442 Bulgaria 492 Bulgaria 509 45 Bulgaria 439 Chile 441 Chile 499 Romania 505 46 United Arab Emirates 434 Costa Rica 441 United Arab Emirates 486 Serbia 503 47 Kazakhstan 432 Thailand 441 Turkey 486 United Arab Emirates 499 48 Thailand 427 Romania 438 Thailand 482 Thailand 492 49 Chile 423 Bulgaria 436 Kazakhstan 471 Montenegro 482 50 Malaysia 421 Mexico 424 Malaysia 468 Qatar 470 51 Mexico 413 Montenegro 422 Montenegro 460 Tunisia 464 52 Montenegro 410 Uruguay 411 Uruguay 453 Argentina 462 53 Uruguay 409 Brazil 410 Qatar 447 Uruguay 461 54 Costa Rica 407 Tunisia 404 Mexico 443 Mexico 456 55 Albania 394 Colombia 403 Argentina 440 Jordan 455 56 Brazil 391 Jordan 399 Tunisia 438 Malaysia 449 57 Argentina 388 Malaysia 398 Jordan 430 Colombia 443 58 Tunisia 388 Indonesia 396 Brazil 414 Costa Rica 441 59 Jordan 386 Argentina 396 Albania 412 Brazil 437 60 Qatar 376 Albania 394 Colombia 410 Kazakhstan 437 61 Colombia 376 Kazakhstan 393 Peru 406 Peru 429 62 Indonesia 375 Qatar 388 Costa Rica 406 Indonesia 423 63 Peru 368 Peru 384 Indonesia 399 Albania 419 Table 4A: PISA Assessment Country 2012 Rankings, Overall and Top 50 P l ti 48 49 Table 4B: PISA Assessment 2015 Country Rankings, Overall and Top 50% of Overall Population Math (all students) Reading (all students) Math (Top 50% of pop.) Reading (Top 50% of pop.) Rank Country Avg score Country Avg score Country Avg score Country Avg score 1 Singapore 564 Singapore 533 Singapore 631 Singapore 603 2 Hong Kong 547 Canada 527 Hong Kong 609 Finland 595 3 Macao 543 Hong Kong 527 Japan 596 Germany 585 4 Japan 533 Finland 527 Macao 595 Hong Kong 584 5 South Korea 524 Ireland 520 South Korea 593 Ireland 583 6 Switzerland 520 Estonia 519 Switzerland 588 South Korea 583 7 Estonia 519 Japan 515 Netherlands 583 Norway 582 8 Canada 516 South Korea 515 Belgium 580 Japan 582 9 Netherlands 513 Norway 514 Estonia 576 New Zealand 582 10 Denmark 512 Germany 509 Germany 572 Estonia 582 11 Finland 511 New Zealand 509 Finland 572 France 580 12 Slovenia 510 Macao 508 Poland 565 Netherlands 580 13 Belgium 507 Poland 506 France 565 Canada 577 14 Germany 505 Slovenia 505 Czech Rep. V. Conclusion 564 Belgium 575 15 Poland 505 Netherlands 503 Malta 564 Sweden 573 16 Ireland 503 Australia 503 Canada 562 Poland 569 17 Norway 500 Denmark 500 Slovenia 562 Czech Rep. 567 18 Austria 496 Sweden 500 Ireland 562 Australia 567 19 New Zealand 494 Belgium 499 Norway 557 Israel 565 20 Sweden 494 France 498 Sweden 557 Switzerland 563 21 Australia 494 Portugal 498 New Zealand 557 Macao 562 22 Russia 494 United Kingdom 497 Denmark 557 United States 560 23 France 494 United States 496 Austria 557 Slovenia 560 24 Vietnam 493 Russia 495 Russia 553 Spain 558 25 Czech Rep. 492 Spain 495 Iceland 553 Russia 557 26 Portugal 492 Switzerland 492 Australia 551 United Kingdom 556 27 United Kingdom 492 Latvia 489 Portugal 550 Luxembourg 553 28 Italy 489 Vietnam 488 Luxembourg 550 Portugal 553 29 Iceland 487 Czech Rep. 487 United Kingdom 549 Iceland 552 30 Luxembourg 487 Croatia 487 Italy 548 Denmark 551 31 Spain 486 Italy 487 Spain 547 Croatia 551 32 Latvia 482 Austria 486 Hungary 546 Austria 550 33 Lithuania 479 Iceland 481 Israel 545 Italy 547 34 Malta 477 Luxembourg 480 Slovakia 542 Latvia 546 35 Hungary 477 Israel 479 Lithuania 535 Hungary 543 36 Slovakia 475 Lithuania 472 Latvia 534 Greece 542 37 United States 470 Hungary 469 Vietnam 528 Malta 541 38 Israel 468 Greece 466 United States 526 Lithuania 535 39 Croatia 463 Chile 458 Croatia 525 Slovakia 524 40 Greece 455 Slovakia 453 Greece 524 Chile 521 41 Romania 443 Malta 448 Romania 504 Vietnam 518 42 Bulgaria 442 Uruguay 438 Bulgaria 501 United Arab Em. 507 43 United Arab En. 427 Romina 433 United Arab Em. 494 Bulgaria 504 44 Chile 423 United Arab Em. 432 Chile 483 Romania 501 45 Turkey 421 Bulgaria 431 Moldova 482 Montenegro 489 46 Uruguay 420 Turkey 429 Qatar 473 Qatar 486 47 Moldova 419 Trinidad & Tob. 428 Montenegro 472 Moldova 485 48 Trinidad & Tob. 419 Costa Rica 427 Trinidad & Tob. 469 Trinidad & Tob. V. Conclusion 485 49 Montenegro 416 Montenegro 426 Albania 464 Uruguay 485 50 Thailand 415 Colombia 425 Uruguay 460 Colombia 478 51 Albania 412 Mexico 423 Georgia 458 Jordan 470 52 Mexico 408 Moldava 417 Turkey 456 Turkey 465 53 Georgia 405 Thailand 408 Thailand 455 Albania 464 54 Qatar 402 Brazil 408 Lebanon 439 Georgia 462 55 Costa Rica 400 Jordan 408 Macedonia 436 Costa Rica 454 56 Lebanon 398 Albania 405 Jordan 436 Mexico 451 57 Colombia 390 Qatar 403 Mexico 434 Thailand 447 58 Indonesia 387 Georgia 402 Colombia 434 Peru 444 59 Peru 386 Indonesia 398 Peru 427 Brazil 443 60 Jordan 381 Peru 397 Tunisia 423 Indonesia 433 61 Brazil 377 Tunisia 359 Costa Rica 422 Macedonia 420 62 Macedonia 372 Dominican Rep. 358 Indonesia 421 Tunisia 416 63 Tunisia 365 Macedonia 352 Brazil 407 Dominican Rep. 397 64 Kosovo 362 Algeria 348 Algeria 395 Lebanon 394 65 Algeria 360 Kosovo 347 Kosovo 393 Algeria 385 66 Dominican Rep. 329 Lebanon 347 Dominican Rep. 360 Kosovo 381 49 Table 5. Regressions of PISA Test Scores on Log(GDP)/capita or Wealth/capita: Student-Level Data (1) (2) (3) (4) (5) (6) (7) (8) Variables Math Reading Math Reading Math Reading Math Reading A. 2012 PISA Assessment Log of per capita GDP 34.14*** 31.53*** (1.57) (1.44) Wealth (national average) 28.84*** 26.63*** (1.10) (1.04) Wealth (student specific) 22.35*** 20.82*** 16.26*** 15.16*** (0.51) (0.50) (0.52) (0.46) Constant 126.08*** 159.47*** 454.86*** 463.16*** 458.27*** 466.98*** -- -- (15.47) (14.13) (1.18) (1.11) (1.06) (0.99) Vietnam residual (average) 135.8 119.0 111.6 96.7 98.2 83.6 82.8 73.4 Residual rank 1 1 2 1 4 2 5 3 More highly ranked none none HK none HK S. Korea Singap. HK HK S. Korea Singap. Taiwan HK S. Korea Observations 473,236 473,236 473,236 473,236 455,971 455,971 455,971 455,971 R-squared 0.117 0.103 0.126 0.111 0.155 0.140 0.350 0.280 B. 2015 PISA Assessment Log of per capita GDP 34.41*** 34.95*** (1.242) (1.170) Wealth (national average) 30.53*** 30.58*** (1.004) (0.951) Wealth (student specific) 22.89*** 23.03*** 16.24*** 16.22*** (0.445) (0.440) (0.476) (0.443) Constant 118.1*** 123.4*** 445.9*** 456.3*** 450.5*** 461.1*** (12.06) (11.29) (1.096) (1.042) (0.957) (0.901) Vietnam residual (average) 114.6 99 106.2 89.7 89.7 72.8 73.2 63.4 Residual rank 1 1 1 1 3 1 4 3 More highly ranked None none none none HK Singap. none Singap. HK Macao HK Singap. V. Conclusion Observations 464,518 464,518 460,701 460,701 428,716 428,716 428,716 428,716 R-squared 0.128 0.127 0.140 0.136 0.170 0.167 0.331 0.262 Country fixed effects No No No No No No Yes Yes Robust standard errors, clustered at the school level, in parentheses *** p<0.01, ** p<0.05, * p<0.1 For fixed effects regressions, residual = fixed effect – constant in regression without fixed effects. Table 6A: Regressions of 2012 Test Scores on Wealth/capita and Student and Household Variables Variables Math Reading Math Reading Math Reading Math Reading Wealth index 15.92*** 14.66*** 9.998*** 9.548*** 15.77*** 14.49*** 5.694*** 5.080*** (0.52) (0.48) (0.433) (0.406) (0.53) (0.48) (0.384) (0.355) Girl -8.705*** 33.31*** -15.39*** 26.55*** (0.767) (0.757) (0.697) (0.677) Sibling index -1.905*** -2.457*** -1.930*** -2.392*** (0.524) (0.542) (0.506) (0.514) Sibling index missing -19.59*** -15.66*** -17.54*** -13.51*** (0.798) (0.843) (0.763) (0.799) Mom years school 2.978*** 2.872*** 1.800*** 1.702*** (0.142) (0.143) (0.131) (0.130) Dad years school 3.310*** 3.065*** 2.046*** 1.841*** (0.131) (0.133) (0.120) (0.121) Grade10 22.87*** 23.87*** (1.36) (1.379 Years of preschool 10.74*** 10.05*** (0.680) (0.693) Educational input index 7.432*** 7.985*** (0.286) (0.306) Attendance (past 2 weeks) 7.710*** 7.638*** (0.365) (0.364) Books at home 0.069*** 0.0595*** (0.003) (0.003) Hours of study 3.170*** 3.017*** (0.094) (0.089) Extra math classes (tutored) -0.558* (0.212) Extra math variable missing -2.929*** (0.544) Extra read. classes (tutored) -4.440*** (0.2331) Extra read. variable missing -3.052*** (0.577) Vietnam fixed effect 78.2 68.3 80.6 70.7 79.1 68.9 65.0 55.1 Fixed effect rank 5 3 6 2 5 3 5 3 More highly ranked: HK S. Korea Singap. Taiwan HK S. Korea HK Macao Singap. S. Korea Taiwan HK HK S. Korea Singap. Taiwan HK S. Korea HK Macao Singap. Taiwan Finland HK Observations 401,489 401,489 401,489 401,489 393,730 393,730 393,730 393,730 R-squared 0.366 0.295 0.399 0.350 0.360 0.291 0.464 0.421 Robust standard errors, clustered at the school level, in parentheses. All regressions use country fixed effects. *** p<0.01, ** p<0.05, * p<0.1 Table 6A: Regressions of 2012 Test Scores on Wealth/capita and Student and of 2012 Test Scores on Wealth/capita and Student and Household Variables Variables Wealth index 1 Girl Sibling index Sibling index missing Mom years school Dad years school Grade10 Years of preschool Educational input index Attendance (past 2 weeks) Books at home Hours of study Extra math classes (tutored) Extra math variable missing Extra read. classes (tutored) Extra read. V. Conclusion variable missing Vietnam fixed effect Fixed effect rank More highly ranked: S S T Observations 4 R-squared Robust standard errors, cl 51 Table 6B: Regressions of 2015 Test Scores on Wealth/capita and Student and Household Variables Table 6B: Regressions of 2015 Test Scores on Wealth/capita and Student and Household Variables Variables Math Reading Math Reading Math Reading Math Reading Wealth Index 16.13*** 16.03*** 10.26*** 10.76*** 16.02*** 15.89*** 7.042*** 7.222*** (0.482) (0.452) (0.397) (0.391) (0.480) (0.453) (0.373) (0.378) Girl -6.680*** 23.62*** -9.667*** 20.45*** (0.671) (0.691) (0.636) (0.669) Mom years school 2.863*** 2.658*** 2.070*** 1.841*** (0.122) (0.131) (0.116) (0.126) Dad years school 3.151*** 2.990*** 2.327*** 2.129*** (0.121) (0.127) (0.114) (0.122) Grade 10 24.58*** 25.17*** (1.429) (1.447) Educational input index 7.580*** (0.283) 8.756*** (0.302) Books at home 0.063*** 0.059*** (0.002) (0.003) Vietnam fixed effect 73.0 62.4 82.6 71.8 72.2 61.4 68.7 57.0 Fixed effect rank 5 3 4 2 5 3 4 3 More highly ranked: Singap. HK Macao Japan HK Singap. Singap. HK Macao HK Singap. HK Macao Japan HK Singap. Macao HK Singap. HK Finland Observations 389,472 389,472 389,472 389,472 387,092 387,092 387,092 387,092 R-squared 0.341 0.276 0.372 0.314 0.338 0.273 0.404 0.348 Robust standard errors, clustered at the school level, in parentheses. All regressions use country fixed effects. *** p<0.01, ** p<0.05, * p<0.1 ons of 2015 Test Scores on Wealth/capita and Student and Household Variable Table 6B: Regressions of 2015 Test Scores on Wealth/capita and Student a Variables Math Reading Math Reading Math Reading Math Reading Wealth Index 16.13*** 16.03*** 10.26*** 10.76*** 16.02*** 15.89*** 7.042*** 7.222*** (0.482) (0.452) (0.397) (0.391) (0.480) (0.453) (0.373) (0.378) Girl -6.680*** 23.62*** -9.667*** 20.45*** (0.671) (0.691) (0.636) (0.669) Mom years school 2.863*** 2.658*** 2.070*** 1.841*** (0.122) (0.131) (0.116) (0.126) Dad years school 3.151*** 2.990*** 2.327*** 2.129*** (0.121) (0.127) (0.114) (0.122) Grade 10 24.58*** 25.17*** (1.429) (1.447) Educational input index 7.580*** (0.283) 8.756*** (0.302) Books at home 0.063*** 0.059*** (0.002) (0.003) Vietnam fixed effect 73.0 62.4 82.6 71.8 72.2 61.4 68.7 57.0 Fixed effect rank 5 3 4 2 5 3 4 3 More highly ranked: Singap. HK Macao Japan HK Singap. Singap. HK Macao HK Singap. HK Macao Japan HK Singap. Macao HK Singap. HK Finland Observations 389,472 389,472 389,472 389,472 387,092 387,092 387,092 387,092 R-squared 0.341 0.276 0.372 0.314 0.338 0.273 0.404 0.348 Robust standard errors, clustered at the school level, in parentheses. All regressions use country fixed effects. p p p and household variables not shown. All regressions use country fixed effects. V. Conclusion *** p<0.01, ** p<0.05, * p<0.1 52 52 Table 7A: Regressions 2012 Test Scores on Wealth/capita and Student, Household and School Variables Variables Math Reading Math Reading Wealth 15.32*** 13.75*** 5.436*** 3.869*** (0.57) (0.51) (0.414) (0.387) Class size (student/teacher ratio) 0.094 0.271*** (0.087) (0.082) Ratio qualified teachers 13.28*** 10.44*** (3.33) (3.30) Qual. tchr. ratio missing -1.370 -2.833 (3.320) (3.038) Square root of computers/pupil -2.087 -0.710 (3.211) (2.989) Stud. perf. used to assess tchrs 1.728 2.049 (1.841) (1.803) Teacher absenteeism -3.302*** -2.961*** (0.959) (0.969) Parents pressure teachers 11.59*** 11.33*** (1.25) (1.22) Principal observes teachers -2.741 0.117 (1.988) (1.889) Inspector observes teachers -4.735*** -6.698*** (1.746) (1.790) Tchr pay linked to stud perf -2.232** -2.501*** (0.947) (0.911) Teacher mentoring index 5.244*** 5.906*** (1.745) (1.784) Vietnam fixed effect 76.7 66.1 58.1 44.7 Fixed effect rank 5 4 8 4 More highly ranked: HK S. Korea Singap. Taiwan HK S. Korea Singap. HK Liecht. Macao S. Korea Singap. Switz. Taiwan Finland HK Liecht. Observations 341,409 341,409 341,409 341,409 R-squared 0.354 0.286 0.460 0.405 Robust standard errors, clustered at the school level, in parentheses. *** p<0.01, ** p<0.05, * p<0.1 Note: Student and household variables not shown. All regressions use country fixed effects. gressions 2012 Test Scores on Wealth/capita and Student, Household and bl Note: Student and household variables not shown. All regressions use country fixed effects. 53 Table 7B: Regressions of 2015 Test Scores on Wealth/capita and Student, Household and School Variables Variables Math Reading Math Reading Wealth Index 15.37*** 15.12*** 6.174*** 6.302*** (0.532) (0.488) (0.401) (0.402) Class size (student/teacher ratio) 0.574*** 0.704*** (0.104) (0.108) Ratio qualified teachers 9.323*** 7.501*** (3.031) (2.905) Qual. tchr. ratio missing -4.036 -4.125 (3.894) (4.371) Square root of computers/pupil -0.435 0.759 (2.884) (2.856) Stud. perf. used to assess tchrs 3.996 1.316 (2.538) (2.479) Teacher absenteeism -4.579*** -3.815*** (1.122) (1.093) Principal observes teachers -1.984 0.675 (2.471) (2.615) Inspector observes teachers -0.966 -2.204 (1.764) (1.885) Teacher mentoring index 0.449 0.276 (1.161) (1.162) Vietnam fixed effect 71.4 60.6 59.2 46.5 Fixed effect rank 4 3 4 8 More highly ranked: Singap. HK Macao HK Singap. Macao HK Singap. Switz. Finland HK Germany Ireland N. Zealand Estonia Singap. Observations 317,006 317,006 317,006 317,006 R-squared 0.320 0.262 0.391 0.341 Robust standard errors, clustered at the school level, in parentheses. *** p<0.01, ** p<0.05, * p<0.1 Note: Student and household variables not shown. All regressions use country fixed effects. V. Conclusion gressions of 2015 Test Scores on Wealth/capita and Student, Household and bl Regressions of 2015 Test Scores on Wealth/capita and Student, Household and riables Note: Student and household variables not shown. All regressions use country fixed effects. 54 gression Variables, for Vietnam and for Other Countries, 2012 and 2015 Table 8: Means of Regression Variables, for Vietnam and for Other Countries, 2012 and 2015 2012 PISA Assessment 2015 PISA Assessment Variable (x) Vietnam Other PISA Countries Vietnam Other PISA Countries Math test score 516.5 462.8 501.2 456.6 Reading test score 512.8 472.5 495.1 468.1 Wealth 4.143 6.101 3.21 5.63 Grade 10 0.874 0.584 0.89 0.59 Sibling index 1.048 1.086 -- -- Sibling index missing 0.149 0.238 -- -- Mom years schooling 8.313 10.98 8.03 11.45 Dad years schooling 8.883 11.09 8.53 11.54 Years preschool enrollment 1.600 1.487 -- -- Education inputs index (desk, books) 4.680 5.154 4.87 5.48 Books in home 57.59 114.1 69.17 113.15 Days attended in past 2 weeks 9.849 9.622 -- -- Hours of study per week 5.756 5.362 -- -- Extra reading classes (tutoring), hours/week 1.290 0.944 -- -- Extra reading classes variable missing 0.337 0.358 -- -- Extra math classes (tutoring), hours/week 2.741 1.325 -- -- Extra math classes variable missing 0.336 0.358 -- -- Class size 44.81 32.61 40.61 31.08 Proportion of teachers who are qualified 0.800 0.834 0.85 0.80 Proportion qualified teacher missing 0.069 0.188 0.07 0.08 Square root of computers/pupil 0.417 0.623 0.44 0.65 Student performance used to assess teachers 0.992 0.708 0.99 0.88 Teacher absenteeism 0.692 0.778 1.60 1.83 Parents pressure teachers 1.311 0.957 -- -- Principal observes teachers 0.965 0.802 0.99 0.87 Outside Inspector observes teachers 0.847 0.406 0.77 0.57 Teacher pay linked to student performance 1.487 0.703 -- -- Teachers are mentored 0.845 0.684 1.81 1.46 Sample size 4,421 336,988 4,895 312,111 Notes: 1. Averages over countries are weighted by country populations. 2. The following variables were not collected for all countries, or not for Vietnam, in 2015, and so are excluded from the analysis for that year: siblings, years in pre-school, days attended, hours of study per week, extra classes, parents pressure teachers, and teacher pay is linked to student performance. 2. The following variables were not collected for all countries, or not for Vietnam, in 2015, and so are excluded from the analysis for that year: siblings, years in pre-school, days attended, hours of study per week, extra classes, parents pressure teachers, and teacher pay is linked to student performance. Sample size 2. The following variables were not collected for all countries, or not for Vietnam, in 2015, and so are excluded from the analysis for that year: siblings, years in pre-school, days attended, hours of study per week, extra classes, parents pressure teachers, and teacher pay is linked to student performance. 55 Table 9A: Math Decomposition, 2012 (diff = 516.54– 462.80 = 53.74) Table 9A: Math Decomposition, 2012 (diff = 516.54– 462.80 = 53.74) Variable βvn 𝐱̅vn βvnʹ𝐱̅vn βo 𝐱̅o βoʹ𝐱̅o 𝛃̅ (= (βvn+βo)/2 𝛃̅ʹ(𝐱̅vn-𝐱̅o) (βvn - 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo) Wealth 6.764*** 4.143 28.02 9.633*** 6.101 58.77 8.198 -16.05 -14.69 Grade 10 85.85*** 0.874 75.01 18.93*** 0.584 11.05 52.39 15.19 48.76 Sibling index 3.152* 1.048 3.30 -1.697*** 1.086 -1.84 0.728 -0.03 5.17 Sibling index missing -0.576 0.149 -0.09 -17.87*** 0.238 -4.25 -9.225 0.82 3.35 Mom years schooling 0.962** 8.313 8.00 1.786*** 10.975 19.60 1.374 -3.66 -7.95 Dad years schooling 1.511*** 8.883 13.42 2.390*** 11.086 26.50 1.950 -4.30 -8.78 Years in preschool 6.533*** 1.600 10.45 13.07*** 1.487 19.43 9.799 1.10 -10.08 Education inputs index 4.397*** 4.680 20.58 7.337*** 5.154 37.81 5.867 -2.78 -14.46 Books in home 0.0089 57.59 0.51 0.0882*** 114.07 10.07 0.049 -2.74 -6.81 Days attend past 2 wks 10.43*** 9.849 102.72 8.094*** 9.622 77.88 9.261 2.10 23.74 Hours study per week 2.920*** 5.756 16.81 2.425*** 5.362 13.00 2.672 1.05 2.75 Extra math class, hrs/wk 3.904*** 2.741 10.70 -0.633** 1.325 -0.84 1.636 2.32 9.22 Extra math class missing 8.890*** 0.336 2.98 -3.188*** 0.358 -1.14 2.851 -0.06 4.19 Class size 0.0643 44.81 2.88 0.148* 32.61 4.82 0.106 1.29 -3.24 Proport. qualified tchrs 18.18** 0.800 14.55 46.08*** 0.834 38.42 32.13 -1.09 -22.79 Prop. qual. tchr. missing -17.15 0.069 -1.18 -23.14*** 0.188 -4.35 -20.14 2.40 0.77 Square root comp/pupil -0.0392 0.417 -0.02 4.925 0.623 3.07 2.443 -0.50 -2.58 Stud perf. to assess tchrs 25.08*** 0.992 24.89 -4.267** 0.708 -3.02 10.40 2.96 24.95 Teacher absenteeism -0.759 0.692 -0.53 -6.600*** 0.778 -5.13 -3.679 0.32 4.29 Parents pressure tchrs 15.71*** 1.311 20.60 6.686*** 0.957 6.40 11.20 3.97 10.24 Principal observes tchrs 14.12 0.965 13.63 -3.816** 0.802 -3.06 5.154 0.84 15.85 Inspector observes tchrs -16.73 0.847 -14.17 -10.15*** 0.406 -4.12 -13.44 -5.93 -4.13 Tchr pay link stud. perf. Sample size 2.209 1.487 3.28 -2.279** 0.703 -1.60 -0.035 -0.03 -4.92 Teachers are mentored 6.766 0.845 5.72 7.722*** 0.684 5.28 7.244 1.17 -0.73 Constant 154.46*** 1.000 154.46 160.07*** 1.000 160.07 157.26 0.00 -5.61 Column sum: -- -- 516.54 -- -- 462.80 -- -1.62 55.36 Table 9B: Math Decomposition, 2015 (diff = 501.23– 456.61 = 44.62) Variable βvn 𝐱̅vn βvnʹ𝐱̅vn βo 𝐱̅o βoʹ𝐱̅o 𝛃̅ (= (βvn+βo)/2 𝛃̅ʹ(𝐱̅vn-𝐱̅o) (βvn - 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo)ʹ𝐱̅o Wealth 6.573*** 2.275 14.95 10.590*** 4.198 44.47 8.581 -16.51 -13.00 Grade 10 79.641*** 0.894 71.20 22.482*** 0.592 13.30 51.061 6.80 51.10 Mom years schooling 1.214** 8.030 9.75 2.493*** 11.449 28.55 1.853 -8.53 -10.27 Dad years schooling 2.059*** 8.532 17.57 3.005*** 11.537 34.67 2.532 -9.03 -8.07 Education input index 8.585*** 4.031 34.61 7.360*** 4.415 32.49 7.972 -3.05 5.17 Books in home -0.021** 69.165 -1.42 0.089*** 113.154 10.10 0.034 -3.93 -7.59 Class size 0.045 40.612 1.81 -0.033* 31.084 -1.02 0.006 -0.31 3.14 Proport. qualified tchrs -5.666* 0.850 -4.82 26.023*** 0.805 20.94 10.178 1.19 -26.95 Prop. Qual. tchr. missing -5.084 0.070 -0.36 -11.814*** 0.085 -1.00 -8.449 0.17 0.47 Square root comp/pupil 11.477** 0.442 5.07 10.850*** 0.649 7.05 11.163 -2.25 0.28 Stud perf. Sample size to assess tchrs -19.137* 0.990 -18.94 -5.876*** 0.879 -5.16 -12.507 -0.65 -13.13 Teacher absenteeism -2.328 1.604 -3.74 -7.533*** 1.833 -13.81 -4.931 1.72 8.35 Principal observes tchrs 42.505** 0.994 42.26 1.178** 0.875 1.03 21.842 0.14 41.09 nspector observes tchrs 4.869* 0.766 3.73 -1.833*** 0.568 -1.04 1.518 -0.36 5.14 Teacher are mentored 12.247*** 1.808 22.15 -1.167*** 1.462 -1.71 5.540 -0.40 24.26 Constant 307.41*** 1.000 307.41 287.75*** 1.000 287.75 297.58 0.00 19.66 Column sum: -- -- 501.23 -- -- 456.61 -- -16.69 61.31 Table 9B: Math Decomposition, 2015 (diff = 501.23– 456.61 = 44.62) 57 Table 10A: Reading Decomposition, 2012 (diff = 512.82– 472.52 = 40.30) Variable βvn 𝐱̅vn βvnʹ𝐱̅vn βo 𝐱̅o βoʹ𝐱̅o 𝛃̅ (= (βvn+βo)/2 𝛃̅ʹ(𝐱̅vn-𝐱̅o) (βvn - 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo Wealth 4.748*** 4.143 19.67 9.305*** 6.101 56.77 7.026 -13.75 -23.34 Grade 10 79.18*** 0.874 69.18 20.58*** 0.584 12.01 49.88 14.46 42.70 Sibling index 4.045** 1.048 4.24 -1.736*** 1.086 -1.89 1.154 -0.04 6.17 Sibling index missing -0.428 0.149 -0.06 -12.01*** 0.238 -2.86 -6.217 0.55 2.24 Mom years schooling 0.721** 8.313 5.99 1.083*** 10.975 11.88 0.902 -2.40 -3.49 Dad years schooling 0.694** 8.883 6.17 1.877*** 11.086 20.81 1.286 -2.83 -11.81 Years in preschool 4.884** 1.600 7.81 10.98*** 1.487 16.34 7.933 0.89 -9.41 Education inputs index 5.657*** 4.680 26.47 8.061*** 5.154 41.55 6.859 -3.25 -11.82 Books in home 0.00231 57.59 0.13 0.0741*** 114.07 8.45 0.038 -2.16 -6.16 Days attend past 2 wks 16.08*** 9.849 158.34 7.806*** 9.622 75.11 11.94 2.71 80.52 Hours study per week 2.335*** 5.756 13.44 2.786*** 5.362 14.94 2.651 1.01 -2.51 Extra reading class hr/wk -1.547*** 2.741 -1.99 -4.887*** 1.325 -4.61 -3.217 -1.11 3.73 Extra reading class miss. 0.712 0.336 0.24 -3.434*** 0.358 -1.23 -1.361 0.03 1.44 Class size 0.258 44.81 11.58 0.358*** 32.61 11.67 0.308 3.76 3.85 Proport. qualified tchrs 16.22*** 0.800 12.98 35.92*** 0.834 29.95 26.07 -0.88 -16.09 Prop. qual. tchr. missing -17.21*** 0.069 -1.19 -16.85*** 0.188 -3.17 -17.03 2.03 -0.05 Square root comp/pupil -4.467 0.417 -1.86 7.049*** 0.623 4.40 1.291 -0.27 -5.99 Stud perf. to assess tchrs 1.901 0.992 1.89 -4.253** 0.708 -3.01 -1.176 -0.33 5.23 Teacher absenteeism -1.489 0.692 -1.03 -5.874*** 0.778 -4.57 -3.681 0.32 3.22 Parents pressure tchrs 9.980** 1.311 13.08 8.313*** 0.957 7.96 9.146 3.24 1.89 Principal observes tchrs 34.74*** 0.965 33.53 -1.893 0.802 -1.52 16.42 2.68 32.37 Inspector observes tchrs -18.02** 0.847 -15.26 -11.80*** 0.406 -4.79 -14.91 -6.57 3.90 Tchr pay link stud. perf. Sample size Yet these 1,717,966 individuals would be 18 years old in 2012, not 15 years old. The same census report shows 1,450,815 12-year-olds in 2009, and these individuals would then be 15 in 2012. Thus the correct PISA coverage rate for 2012 should be 65.9% (956,517/1,450,815). Appendix A: Further Derivations and Proof Appendix A: Further Derivations and Proof A1. Re-calculations of the PISA Coverage Rates The 55.7% coverage rate in the 2012 PISA report was obtained by taking Ministry of Education and Training (MoET) records, which showed a “weighted number of participating students” of 956,517 students enrolled in school who were 15 years old, divided by 1,717,996 15-year-olds in Vietnam (see Table 11.1 in OECD, 2014b). The 1,717,996 figure was obtained from the 2009 Census (General Statistics Office, 2010, Table 3); it is the number of 15-year-olds in Vietnam in 2009, and an implicit assumption was made that this number would be the same in 2012. Yet these 1,717,966 individuals would be 18 years old in 2012, not 15 years old. The same census report shows 1,450,815 12-year-olds in 2009, and these individuals would then be 15 in 2012. Thus the correct PISA coverage rate for 2012 should be 65.9% (956,517/1,450,815). The discrepancy for the 2015 PISA is even larger. The OECD report states that there were 1,803,552 15-year-olds in Vietnam in 2015 (OECD, 2016, Table A2.1), which seems to be based on an assumption of slow population growth based on the 1,717,966 figure used for 2012. Yet the 2009 census shows only 1,332,822 9-year-old children in 2009, and these are the individuals who would have been 15 years old in 2015. The 2015 PISA report (OECD, 2016, Table A2.1) shows a “weighted number of participating students” of 874,859 students enrolled in school who were 15 years old in 2015, and dividing this figure by 1,803,552 gives a coverage rate of only 48.5% (874,859/1,803,552). Yet the correct coverage rate should be 65.6% (874,859/1,332,822). A2. Proof of Proposition 1 p Assume that the true test scores follow a normal distribution, with mean 𝜇 and standard deviation 𝜎. The truncated mean from below is given by 𝑇̅𝑏= 𝐸(𝑇|𝑇> 𝜏). The given school enrollment rate is r. Sample size 3.676 1.487 5.47 -4.785*** 0.703 -3.36 -0.555 -0.43 9.27 Teachers are mentored 9.211 0.845 7.78 7.342*** 0.684 5.02 8.276 1.34 1.43 Constant 136.21*** 1.000 136.21 186.68*** 1.000 186.61 161.45 0.00 -50.47 Column sum: -- -- 512.82 -- -- 472.52 -- -1.02 41.32 Table 10A: Reading Decomposition, 2012 (diff = 512.82– 472.52 = 40.30) 58 Table 10B: Reading Decomposition, 2015 (diff = 495.09– 468.06 = 27.03) Table 10B: Reading Decomposition, 2015 (diff = 495.09– 468.06 = 27.03) Variable βvn 𝐱̅vn βvnʹ𝐱̅vn βo 𝐱̅o βoʹ𝐱̅o 𝛃̅(=(βvn+βo)/2 𝛃̅ʹ(𝐱̅vn-𝐱̅o) (βvn - 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo)ʹ𝐱̅o Wealth 7.217*** 2.275 16.42 11.487*** 4.199 48.23 9.352 -17.99 -13.82 Grade 10 90.124*** 0.894 80.57 21.122*** 0.592 12.50 55.623 6.38 61.69 Mom years schooling 0.522* 8.030 4.19 2.399*** 11.449 27.47 1.461 -8.20 -15.07 Dad years schooling 1.716*** 8.532 14.64 2.252*** 11.537 25.98 1.984 -6.77 -4.57 Education input index 6.883*** 4.032 27.75 8.435*** 4.415 37.24 7.659 -2.93 -6.55 Books in home -0.019** 69.165 -1.30 0.077*** 113.154 8.71 0.029 -3.39 -6.63 Class size 0.066 40.612 2.69 0.134*** 31.084 4.17 0.100 1.28 -2.75 Proport. qualified tchrs 0.242 0.850 0.21 15.364*** 0.805 12.36 7.803 0.70 -12.86 Prop. Qual. tchr. missing -12.390*** 0.070 -0.87 -5.350*** 0.085 -0.45 -8.870 0.08 -0.49 Square root comp/pupil 23.429*** 0.442 10.36 18.271*** 0.649 11.87 20.850 -3.79 2.28 Stud perf. to assess tchrs -28.768** 0.990 -28.48 -2.588*** 0.879 -2.27 -15.678 -0.29 -25.91 Teacher absenteeism 1.231 1.604 1.98 -3.973*** 1.833 -7.28 -1.371 0.91 8.35 Principal observes tchrs 37.289** 0.994 37.08 -0.191 0.875 -0.17 18.549 -0.02 37.27 Inspector observes tchrs 0.567 0.766 0.43 -4.498*** 0.568 -2.55 -1.966 -0.89 3.88 Teacher are mentored 11.366*** 1.808 20.56 -0.563** 1.462 -0.82 5.402 -0.20 21.57 Constant 308.871*** 1.000 308.87 293.091*** 1.000 293.09 300.981 0.000 15.78 Column sum: -- -- 495.09 -- -- 468.06 -- -16.96 44.00 59 Appendix A: Further Derivations and Proof A1. Re-calculations of the PISA Coverage Rates The 55.7% coverage rate in the 2012 PISA report was obtained by taking Ministry of Education and Training (MoET) records, which showed a “weighted number of participating students” of 956,517 students enrolled in school who were 15 years old, divided by 1,717,996 15-year-olds in Vietnam (see Table 11.1 in OECD, 2014b). The 1,717,996 figure was obtained from the 2009 Census (General Statistics Office, 2010, Table 3); it is the number of 15-year-olds in Vietnam in 2009, and an implicit assumption was made that this number would be the same in 2012. and 𝜇𝑢𝑡 =𝑇̅𝑎+ 𝜎 𝜆𝑎(𝛼) 𝜇𝑢𝑡 =𝑇̅𝑎+ 𝜎 𝜆𝑎(𝛼) Combining Equations (1.1) and (1.11), we can solve for 𝜎 in this case as ̅ Combining Equations (1.1) and (1.11), we can solve for 𝜎 in this case as 𝜏−𝑇̅𝑎 (1 12) 𝜎= 𝜏−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 (1.1 𝜎= 𝜏−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 𝜎= 𝜏−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 𝑎( ) Plugging this result into Equation (1.1), we have the stated result ̅ 𝑎( ) Plugging this result into Equation (1.1), we have the stated result 𝑇̅ 𝜇𝑢𝑡 = 𝑇̅𝑎+ 𝜆𝑎(𝛼) 𝜏−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 (1.13) 𝜇𝑢𝑡 = 𝑇̅𝑎+ 𝜆𝑎(𝛼) 𝜏−𝑇𝑎 𝜆𝑎(𝛼)+𝛼 (1.13) Note that the truncation point 𝜏 can be empirically estimated with the maximal observed test score in the data Tmax, which results in the following estimating equation for Equation (1.13) Note that the truncation point 𝜏 can be empirically estimated with the maximal observed test score in the data Tmax, which results in the following estimating equation for Equation (1.13) 𝜇𝑢𝑡 = 𝑇̅𝑎+ 𝜆𝑎(𝛼) 𝑇𝑚𝑎𝑥−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 (1.14) 𝜇𝑢𝑡 = 𝑇̅𝑎+ 𝜆𝑎(𝛼) 𝑇𝑚𝑎𝑥−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 (1.14) (1.14) 𝜎= 𝜏−𝑇̅𝑎 𝜆𝑎(𝛼)+𝛼 A2. Proof of Proposition 1 However, since we now assume that the PISA students are all worse-performing children, we need to rewrite Equation (1.2) to reflect this assumption as follows For equation (2) in Proposition 1, define 𝜆𝑎(𝛼) instead as 𝜙(𝛼) Φ(α), also using Theorem 19.2 in Greene (2018), Equation (1.1) still holds for the case of the truncation from above. However, since we now assume that the PISA students are all worse-performing children, we need to rewrite Equation (1.2) to reflect this assumption as follows 𝑃(𝑇≤) (1 9) 𝑃(𝑇≤𝜏) = 𝑟 (1.9) 𝑃(𝑇≤𝜏) = 𝑟 (1.9) Again, making use of the assumption that T follows a normal distribution, after some similar straightforward manipulations for Equation (1.9) as with the proof for Proposition 1.1, we have 𝛼= Φ−1(𝑟) (1.10) Again, making use of the assumption that T follows a normal distribution, after some similar straightforward manipulations for Equation (1.9) as with the proof for Proposition 1.1, we have 𝛼= Φ−1(𝑟) (1.10) A2. Proof of Proposition 1 Define 𝛼 as 𝜏−µ 𝜎 and 𝜆𝑏(𝛼) as 𝜙(𝛼) 1−Φ(𝛼) , using Theorem 19.2 in Greene (2018), we have 𝜎 𝑏( ) 1−Φ(𝛼) , g ( ), 𝜇𝑙𝑡 = 𝑇̅𝑏−𝜎 𝜆𝑏(𝛼) (1.1) Let r represent the given school enrollment rate, we also have 𝑃(𝑇> 𝜏) = 𝑟 (1.2) Since T follows a normal distribution, subtracting the two sides of Equation (1.2) from unity yields Φ ( 𝜏−µ 𝜎) ≡Φ(𝛼) = 1 −𝑟 (1.3) This leads to the following results 𝛼= Φ−1(1 −𝑟) (1.4) and 𝜇𝑙𝑡= 𝜏−𝜎𝛼 (1.5) Combining Equations (1.1) and (1.5), we can solve for 𝜎 as 𝜎= 𝑇̅𝑏−𝜏 𝜆𝑏(𝛼)−𝛼 (1.6) Plugging this result into Equation (1.1), we have the stated result 𝜇𝑙𝑡 = 𝑇̅𝑏− 𝜆𝑏(𝛼) 𝑇̅𝑏−𝜏 𝜆𝑏(𝛼)−𝛼 (1.7) 1 Φ(𝛼) 𝜇𝑙𝑡 = 𝑇̅𝑏−𝜎 𝜆𝑏(𝛼) (1.1) ( ) 𝜇𝑙𝑡 = 𝑇̅𝑏−𝜎 𝜆𝑏(𝛼) t the given school enrollment rate, we also have 𝑃(𝑇> ) (1 2) Let r represent the given school enrollment rate, we also have 𝑃(𝑇> 𝜏) = 𝑟 (1.2) Let r represent the given school enrollment rate, we also have 𝑃(𝑇> 𝜏) = 𝑟 (1.2) , 𝑃(𝑇> 𝜏) = 𝑟 b i h 𝑃(𝑇> 𝜏) = 𝑟 (1.2) Since T follows a normal distribution, subtracting the two sides of Equation (1.2) s a normal distribution, subtracting the two sides of Equation (1.2) from unity yields g Φ ( 𝜏−µ 𝜎) ≡Φ(𝛼) = 1 −𝑟 Φ ( 𝜏−µ 𝜎) ≡Φ(𝛼) = 1 −𝑟 Φ ( µ 𝜎) ≡Φ(𝛼) = 1 −𝑟 (1.3) This leads to the following results 𝛼= Φ−1(1 −𝑟) (1.4) and 𝜇𝑙𝑡= 𝜏−𝜎𝛼 Combining Equations (1.1) and (1.5), we can solve for 𝜎 as ̅ 𝜇𝑙𝑡= 𝜏−𝜎𝛼 (1.5) Combining Equations (1.1) and (1.5), we can solve for 𝜎 as 𝜎= 𝑇̅𝑏−𝜏 (1 6) 𝜎= 𝑇̅𝑏−𝜏 𝜆𝑏(𝛼)−𝛼 𝜎= 𝑇̅𝑏−𝜏 𝜆𝑏(𝛼)−𝛼 𝑏( ) Plugging this result into Equation (1.1), we have the stated result ̅ 𝑏( ) Plugging this result into Equation (1.1), we have the stated result ̅ ) 𝜇𝑙𝑡 = 𝑇̅𝑏− 𝜆𝑏(𝛼) 𝑇̅𝑏−𝜏 𝜆𝑏(𝛼)−𝛼 (1.7) (1.7) 𝑏( ) Note that the truncation point 𝜏 can be empirically estimated with the minimal observed test score in the data Tmin, which results in the following estimating equation for Equation (1.7) ̅ 𝑏( ) Note that the truncation point 𝜏 can be empirically estimated with the minimal observed test score in the data Tmin, which results in the following estimating equation for Equation (1.7) ̅ 𝜇𝑙𝑡 = 𝑇̅𝑏− 𝜆𝑏(𝛼) 𝑇̅𝑏−𝑇𝑚𝑖𝑛 𝜆𝑏(𝛼)−𝛼 (1.8) 𝜇𝑙𝑡 = 𝑇̅𝑏− 𝜆𝑏(𝛼) 𝑇̅𝑏−𝑇𝑚𝑖𝑛 𝜆𝑏(𝛼)−𝛼 (1.8) (1.8) For equation (2) in Proposition 1, define 𝜆𝑎(𝛼) instead as 𝜙(𝛼) Φ(α), also using Theorem 19.2 in Greene (2018), Equation (1.1) still holds for the case of the truncation from above. A3. Procedure to Adjust PISA Test Scores with Young Lives Data The adjustments to the PISA scores using the Young Lives data were done as follows. First, the Young Lives sample was sorted into 10 deciles based on the average test scores over the math and reading comprehension tests, where Decile 1 has the 10% of the Young Lives sample with the lowest scores, Decile 2 has the 10% with the next lowest test scores, and so forth, up to Decile 10, which has the 10% of the sample with the highest scores. For all 10 deciles, the proportion of Young Lives students who were still in school was calculated, which ranged from 0.582 for Decile 1 to 1.000 for Decile 10. These proportions are shown in column (1) of Table B3 in Appendix B. These proportions serve two purposes. First, they are used to assign students in the Vietnam PISA sample to deciles (ranked by student performance) of the distribution of all 15-year-olds, including those not in school. An initial step is to adjust the proportions in school (for the Young Lives sample) in column (1) so that they have a mean of 1.000; this shows how 15-year-olds in that sample who are in school are distributed across the deciles of the distribution of academic performance for all 15-year-olds. Thus column (2) in Table B3 shows that, of all 15-year-olds in school, 7.01% are in the bottom decile of the distribution of the academic performance of all 15- year-olds, 7.76% are in the second decile of that distribution, and so forth, and finally 12.03% are in the top decile; note that these percentages sum to 100%. Then the bottom 7.01% (in terms of academic performance on the PISA) of the 15-year-old PISA participants are assigned to the bottom decile of this “all 15-year-olds” distribution, the next 7.76% are assigned to the second decile, and so forth. The second purpose of the proportions in column (1) is to generate “inflation factors” for the PISA students assigned to these deciles. These inflation factors, which are shown in column (3) and are 61 the inverses of the proportions in column (2), are applied to the PISA participants assigned to these deciles to approximate the distribution of test scores that would have been generated if the entire population of 15-year-olds in Vietnam had participated in the PISA. A3. Procedure to Adjust PISA Test Scores with Young Lives Data For example, the 7.01% of the PISA participants assigned to the first decile are given a weight of 1.427 (= 1/7.01) so that they represent the bottom 10% of the entire population of 15-year-olds. This is last step in the adjustment, which is to assume that the mean scores of 15-year-olds not in school in each decile of the population are equal to the mean scores of the 15-year-olds in the respective deciles who are in school, and thus participated in the PISA. The means for the latter (which by this assumption are also assigned to the former) for the 2012 PISA are shown in column (4) of Table B3, separately for mathematics and reading. They can be compared to the actually means, by decile, in the 2012 PISA, which are shown in column (5). The overall results show that this adjustment decreases the 2012 PISA scores for math by only 12.8 points and the 2012 PISA scores for reading by only 11.3 points. The same adjustments for the 2015 PISA are shown in columns (6) and (7); they show that this this adjustment decreases the 2015 PISA scores for math by only 12.4 points and the 2015 PISA scores for reading by only 10.9 points. 62 62 Appendix B: Additional Tables and Figures Figure B1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita 63 Appendix B: Additional Tables and Figures Figure B1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita Figure B2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita Appendix B: Additional Tables and Figures Figure B1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita Appendix B: Additional Tables and Figures Figure B1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita pp g Figure B1. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real PPP G 63 re B2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita igure B2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita ean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real PPP GDP/capita e B2. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real PPP GDP/ 63 Figure B3. A3. Procedure to Adjust PISA Test Scores with Young Lives Data Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Asia Only i 4 A 1 i S i 2012 SA 2010 G / i MYS IDN THA MAC JPN HKG KOR SGP VNM 350 400 450 500 550 600 7 8 9 10 11 lgdppc2010real PISA 2012 Avg. Math Score Fitted values Figure B3. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita, East ean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Figure B3. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Asia Only Figure B4. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Asia Only MYS IDN THA MAC JPN HKG KOR SGP VNM 350 400 450 500 550 600 7 8 9 10 11 lgdppc2010real PISA 2012 Avg. Math Score Fitted values MYS IDN THA MAC JPN SGP HKG KOR VNM 400 450 500 550 Figure B3. Mean Age 15 Math Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Asia Only Figure B4. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Asia Only MYS IDN THA MAC JPN HKG KOR SGP VNM 350 400 450 500 550 600 7 8 9 10 11 lgdppc2010real PISA 2012 Avg. Math Score Fitted values 9 lgdppc2010real Fitted values Figure B4. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita, Ea Asia Only MYS IDN THA MAC JPN SGP HKG KOR VNM 400 450 500 550 7 8 9 10 11 lgdppc2010real PISA 2012 Avg. Reading Score Fitted values Figure B4. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Asia Only MYS IDN THA MAC JPN SGP HKG KOR VNM 400 450 500 550 7 8 9 10 11 lgdppc2010real PISA 2012 Avg. Reading Score Fitted values ean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita, East Figure B4. Mean Age 15 Reading Scores in 2012 PISA, by 2010 Log Real GDP/capita, East 9 lgdppc2010real 64 64 Figure B5. Distributions of 2015 PISA Scores for Four Countries with High Enrollment Rates igure B5. Distributions of 2015 PISA Scores for Four Countries with High Enr Figure B5. A3. Procedure to Adjust PISA Test Scores with Young Lives Data Distributions of 2015 PISA Scores for Four Countries with High Enrollment Rates ns of 2015 PISA Scores for Four Countries with High Enrollment Rates or Four Countries with High Enrollment Rates Figure B5. Distributions of 2015 PISA Scores f 65 Figure B6. Distribution of Test Scores, Truncated from Below Figure B6. Distribution of Test Scores, Truncated from Below Students in the sample (enrolled in school) Students not in the sample (not enrolled in school) Test Score Truncation Point 66 Table B1: Predictors of 2012 PISA Scores in Vietnam Variables Math Reading Rural -18.04*** -11.56** (6.775) (5.699) Female -16.58*** 24.61*** (2.317) (2.009) Grade 10 105.8*** 95.14*** (6.809) (6.077) Father years of schooling 2.231*** 1.536*** (0.495) (0.395) Mother years of schooling 1.879*** 1.661*** (0.489) (0.422) Owns an air conditioner 5.456 -0.626 (6.279) (4.450) Owns a car -6.723 -3.442 (4.645) (3.892) Owns a computer 17.35*** 10.86*** (3.511) (2.810) Number of televisions owned 0.526 2.977 (2.425) (2.187) Constant 396.7*** 385.2*** (8.881) (8.545) Observations 4771 4771 R-squared 0.310 0.341 Robust standard errors, clustered at the school level, in parentheses *** p<0.01, ** p<0.05, * p<0.1 Table B1: Predictors of 2012 PISA Scores in Vietnam 67 Table B2: Predictors of 2015 PISA Scores in Vietnam Table B2: Predictors of 2015 PISA Scores in Vietnam Table B2: Predictors of 2015 PISA Scores in Vietnam Variable Math Reading Rural -18.86*** -9.822 (4.98) (5.908) Female 15.97*** -8.461*** (2.05) (2.272) Grade 10 69.85*** 74.61*** (7.19) (6.07) Mother years of schooling 0.893** 1.460*** (0.408) (0.541) Father years of schooling 1.646*** 2.041*** (0.328) (0.373) Owns an air conditioner -0.712 -2.685 (4.126) (4.971) Owns a motorbike 15.83*** 6.451 (5.01) (5.974) Owns a car 5.202 -1.249 (4.758) (5.950) Own a computer 16.61*** 23.39*** (2.611) (3.34) Number of televisions owned 7.284*** 6.734** (2.141) (2.601) Constant 376.9*** 386.4*** (9.31) (10.41) Observations 5687 5687 R2 0.274 0.207 Robust standard errors, clustered at the school level, in parentheses * p < 0.10, ** p < 0.05, *** p < 0.01 68 Table B3. A3. Procedure to Adjust PISA Test Scores with Young Lives Data Adjusted PISA 2012 and 2015 Test Scores Using Young Lives Attrition Data Test Score Decile (1) Proportion in School in Young Lives Data (2) Proportions Divided by 0.831 ((1) ÷ 0.831) (3) Inflation Factor for PISA Sample (1/(2)) (4) Adjusted 2012 PISA Scores, by Decile (all 15- year-olds) (5) Original 2012 PISA Scores, by Decile (in school only) (6) Adjusted 2015 PISA Scores, by Decile (all 15- year-olds) (7) Original 2015 PISA Scores, by Decile (in school only) Math Reading Math Reading Math Reading Math Reading 1 0.582 0.701 1.427 358.0 363.4 364.3 370.3 340.8 349.0 352.2 359.6 2 0.646 0.776 1.289 409.4 419.2 421.5 432.1 391.2 396.1 405.2 409.9 3 0.746 0.897 1.115 442.1 449.6 454.3 461.7 419.9 424.8 434.6 438.7 4 0.761 0.915 1.093 463.2 472.1 477.2 484.8 444.1 447.2 458.9 460.1 5 0.849 1.022 0.978 483.2 492.6 498.7 502.7 466.3 467.0 479.8 478.8 6 0.885 1.065 0.939 507.2 509.4 521.2 520.1 488.0 485.7 502.5 497.4 7 0.920 1.106 0.904 530.1 528.2 543.8 539.5 512.7 506.1 525.7 516.3 8 0.951 1.144 0.874 555.4 548.1 568.5 558.8 539.6 527.9 550.9 537.4 9 0.973 1.171 0.854 586.6 570.8 600.6 583.2 571.5 556.1 580.7 564.5 10 1.000 1.203 0.831 648.7 615.9 662.6 630.1 635.6 608.7 643.6 614.8 Average 0.831 1.000 498.4 496.9 511.2 508.2 481.0 476.9 493.4 487.8 Adjusted PISA 2012 and 2015 Test Scores Using Young Lives Attrition Data Table B.4: Math Decomposition Using Fixed-Effects Estimates of βo (diff = 516.54– 462.80 = 53.74) Variable βvn 𝐱̅vn βvnʹ𝐱̅vn βo 𝐱̅o βoʹ𝐱̅o 𝛃̅ (= (βvn+βo)/2 𝛃̅ʹ(𝐱̅vn-𝐱̅o) (βvn - 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo)ʹ𝐱̅o Wealth 6.764*** 4.143 28.02 5.316*** 6.101 32.43 6.040 -11.83 7.42 Grade 10 85.85*** 0.874 75.01 19.34*** 0.584 11.29 52.595 15.25 48.49 Sibling index 3.152* 1.048 3.30 -2.343*** 1.086 -2.54 0.405 -0.02 5.86 Sibling index missing -0.576 0.149 -0.09 -18.19*** 0.238 -4.33 -9.383 0.84 3.41 Mom years schooling 0.962*** 8.313 8.00 1.657*** 10.975 18.19 1.310 -3.49 -6.70 Dad years schooling 1.511*** 8.883 13.42 1.991*** 11.086 22.07 1.751 -3.86 -4.79 Years in preschool 6.533*** 1.600 10.45 9.972*** 1.487 14.83 8.253 0.93 -5.31 Education inputs index 4.397*** 4.680 20.58 6.858*** 5.154 35.35 5.628 -2.67 -12.10 Books in home 0.00887 57.59 0.51 0.0677*** 114.07 7.72 0.038 -2.16 -5.05 Days attend past 2 wks 10.43*** 9.849 102.72 7.040*** 9.622 67.74 8.735 1.98 33.00 Hours study per week 2.920*** 5.756 16.81 2.882*** 5.362 15.45 2.901 1.14 0.21 Extra math class, hrs/wk 3.904*** 2.741 10.70 -0.858*** 1.325 -1.14 1.523 2.16 9.68 Extra math class missing 8.890*** 0.336 2.98 -2.590*** 0.358 -0.93 3.150 -0.07 3.98 Class size 0.0643 44.81 2.88 0.0657*** 32.61 2.14 0.065 0.79 -0.05 Proport. A3. Procedure to Adjust PISA Test Scores with Young Lives Data qualified tchrs 18.18*** 0.800 14.55 12.62*** 0.834 10.53 15.400 -0.52 4.54 Prop. qual. tchr. missing -17.15*** 0.069 -1.18 -0.486 0.188 -0.09 -8.818 1.05 -2.14 Square root comp/pupil -0.0392 0.417 -0.02 -0.782 0.623 -0.49 -0.411 0.08 0.39 Stud perf. to assess tchrs 25.08** 0.992 24.89 1.708*** 0.708 1.21 13.394 3.80 19.87 Teacher absenteeism -0.759 0.692 -0.53 -3.475*** 0.778 -2.70 -2.117 0.18 2.00 Parents pressure tchrs 15.71*** 1.311 20.60 11.23*** 0.957 10.75 13.470 4.77 5.08 Principal observes tchrs 14.12** 0.965 13.63 -2.586*** 0.802 -2.07 5.767 0.94 14.76 Inspector observes tchrs -16.73*** 0.847 -14.17 -4.317*** 0.406 -1.75 -10.524 -4.64 -7.78 Tchr pay link stud. perf. 2.209 1.487 3.28 -2.397*** 0.703 -1.69 -0.094 -0.07 5.04 Teachers are mentored 6.766** 0.845 5.72 5.030*** 0.684 3.44 5.898 0.95 1.33 Constant 154.46*** 1.000 154.46 227.4*** 1.000 227.39 190.93 0.00 -72.93 516.54 462.80 5.55 48.21 mposition Using Fixed-Effects Estimates of βo (diff = 516.54– 462.80 = 53.74) Table B.5: Reading Decomposition Using Fixed-Effects Estimates of βo (diff = 512.82– 472.52 = 40.30) Variable βvn 𝐱̅vn βvnʹ𝐱̅vn βo 𝐱̅o βoʹ𝐱̅o 𝛃̅ (= (βvn+βo)/2 𝛃̅ʹ(𝐱̅vn-𝐱̅o) (βvn - 𝛃̅)ʹ𝐱̅vn + (𝛃̅ – βo)ʹ𝐱̅ Wealth 4.748*** 4.143 19.67 3.859*** 6.101 23.54 4.3035 -8.43 4.55 Grade 10 79.18*** 0.874 69.18 21.15*** 0.584 12.35 50.165 14.55 42.30 Sibling index 4.045*** 1.048 4.24 -2.214*** 1.086 -2.40 0.9155 -0.03 6.68 Sibling index missing -0.428 0.149 -0.06 -12.56*** 0.238 -2.99 -6.494 0.58 2.35 Mom years schooling 0.721** 8.313 5.99 1.297*** 10.975 14.23 1.009 -2.69 -5.55 Dad years schooling 0.694** 8.883 6.17 1.702*** 11.086 18.87 1.198 -2.64 -10.06 Years in preschool 4.884*** 1.600 7.81 10.28*** 1.487 15.29 7.582 0.86 -8.33 Education inputs index 5.657*** 4.680 26.47 8.447*** 5.154 43.54 7.052 -3.34 -13.72 Books in home 0.00231 57.59 0.13 0.0572*** 114.07 6.52 0.029755 -1.68 -4.71 Days attend past 2 wks 16.08*** 9.849 158.34 7.325*** 9.622 70.48 11.7025 2.66 85.23 Hours study per week 2.335*** 5.756 13.44 3.225*** 5.362 17.29 2.78 1.10 -4.95 Extra reading class hr/wk -1.547*** 2.741 -1.99 -4.460*** 1.325 -5.91 -3.0035 -4.25 5.92 Extra reading class miss. 0.712 0.336 0.24 -3.047*** 0.358 -1.09 -1.1675 0.03 1.30 Class size 0.258*** 44.81 11.58 0.261*** 32.61 8.51 0.2595 3.17 -0.12 Proport. qualified tchrs 16.22*** 0.800 12.98 9.841*** 0.834 8.21 13.0305 -0.44 5.21 Prop. qual. tchr. missing -17.21*** 0.069 -1.19 -2.079*** 0.188 -0.39 -9.6445 1.15 -1.94 Square root comp/pupil -4.467 0.417 -1.86 0.639 0.623 0.40 -1.914 0.39 -2.66 Stud perf. A3. Procedure to Adjust PISA Test Scores with Young Lives Data to assess tchrs 1.901 0.992 1.89 2.067*** 0.708 1.46 1.984 0.56 -0.14 Teacher absenteeism -1.489 0.692 -1.03 -3.003*** 0.778 -2.34 -2.246 0.19 1.11 Parents pressure tchrs 9.980*** 1.311 13.08 11.22*** 0.957 10.74 10.6 3.75 -1.41 Principal observes tchrs 34.74*** 0.965 33.53 -0.136 0.802 -0.11 17.302 2.82 30.81 Inspector observes tchrs -18.02*** 0.847 -15.26 -6.500*** 0.406 -2.64 -12.26 -5.41 -7.22 Tchr pay link stud. perf. 3.676*** 1.487 5.47 -2.740*** 0.703 -1.93 0.468 0.37 7.03 Teachers are mentored 9.211*** 0.845 7.78 5.721*** 0.684 3.91 7.466 1.20 2.67 Constant 136.21*** 1.000 136.21 237.0*** 1.000 236.97*** 186.59 0.00 -100.76 512.82 472.52 4.45 38.04 71
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Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates
Scientific reports
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Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates Received: 15 December 2017 Accepted: 25 April 2018 Published: xx xx xxxx L. He1, E. J. Sayers   1, P. Watson 2 & A. T. Jones   1 The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors. Cell penetrating peptides (CPPs) are a group of short sequences typically containing 5–30 amino acids that have been extensively investigated as carriers for intracellular delivery of various cargos including genetic material, peptides, proteins and nanoparticles1–4 Numerous efforts have been made to unveil the mechanisms of CPP trans- location to the cytoplasm and cytosol of cells, and it is now well accepted that two modes of cell entry exist: direct membrane translocation, which may be energy and temperature independent, and uptake via one or more energy dependent endocytic pathways5,6. The propensity for uptake via these mechanisms is dependent on the peptide sequence, choice of cargo, in vitro model and can be influenced by experimental factors, including incubation temperature and the presence or absence of serum in media7. In a number of CPP studies an intact actin cytoskel- eton has been proposed to be required for cell internalisation and CPPs inside and outside of cells can modify the actin cytoskeleton to influence cellular processes including CPP entry8–11. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Materials and Methods The cells were then incubated for 1 hr (or 30 min for TF-647) at 37 °C/5% CO2 before washing 3x with PBS (TF-647 or Dex-647) or PBS 0.5 mg/mL heparin (R8-conjugates). They were then washed once with PBS, prior to the addition of 2 mL of imaging medium (phenol red free RPMI, 20 mM HEPES pH 7.4) and analysis by live cell confocal microscopy. Actin inhibition and confocal microscopy of endocytic probes and R8 conjugates. Cells were seeded on MatTek dishes as previously described and were washed with PBS before preincubation at 37 °C with diluent control (DMSO) or actin inhibitors: 10 µM Cyt D for 15 min; 0.1–1 µM Lat B for 30 min; 0.2–4 µM JAS for 45 min; 50–100 µM Y27632 for 4 hr in serum-free D-MEM at 37 °C/5% CO2. Endocytic probes (TF-647, Dex-647) or R8-conjugates were then incubated with the cells in the absence (control) or presence of the inhibitors and further incubated at 37 °C/5% CO2 for times specified in figure legends prior to analysis by confocal microscopy. For pulse chase experiments, A431 cells were incubated with 0.1 mg/ml Dex-647 for 2 hr (pulse) followed by washing and a further 4 hr (chase) in fresh complete growth medium. EGFP-R8 (2 µM) was then added for 1 hr in the absence or presence of 10 µM Cyt D. EGFP-R8 and Dex-647 subcellular distribution was then analysed in live cells using confocal microscopy. For low temperature experiments, cells were preincubated with Cyt D as above and then placed on ice for 2 min prior to addition of ice cold EGFP-R8. The cells were then maintained under these conditions for 1 hr prior to analysis by confocal microscopy. Confocal microscopy. Analysis was performed on a Leica SP5 confocal laser scanning microscope, equipped with laser lines: 405 Blue Diode (Excitation wavelength 405 nm for Hoechst 33342) Argon (Excitation wavelength 488 nm for Alexa 488 or EGFP), Helium Neon Laser 1 (Excitation wavelength 543 nm for Tetramethyl-Rhodamine Phalloidin (Rh-P)) and Laser 2 (Excitation wavelength 633 nm for Alexa 647 con- jugates). All images presented in this study were obtained using a HCX PL APO 63x 1.4 NA oil immersion objective with Leica Type F immersion oil. Parameters “Gain” and “Off-set” of the individual photomultiplier tubes were adjusted between experiments in order to obtain optimal image acquisition avoiding saturation. Materials and Methods Cell culture. HeLa (cervical carcinoma, epithelial) cells (ATCC Code CCL-2) or A431 (skin epidermal) cells (ATCC Code CRL-1555) were maintained in growth Dulbecco’s Modified Eagle’s medium (D-MEM) supple- mented with foetal bovine serum (10%, final volume) and penicillin/streptomycin (100 units/ml and 100 μg/ml respectively), herein referred to as complete medium in a humidified 5% CO2/37 °C incubator as a subconfluent monolayer. Cells were obtained from the ATCC and were routinely tested for mycoplasma. Peptide Labelling. Peptide octaarginine (R8), extended with GC at its C-terminal (RRRRRRRR-GC), was synthesized by American Peptide Company (California, USA) and labelled with Alexa Fluor® 488 C5 maleimide salt (Invitrogen, Paisley, UK) as previously described30. Labelling and conjugate mass (2126.5 Da) was confirmed by matrix-assisted laser-desorption ionization–time-of-flight spectrometry and quantified by UV spectrometry. All labelled peptides were resuspended to a stock concentration of 1 mM in autoclaved distilled water, aliquoted and frozen at −80 °C until needed. Expression and purification of recombinant proteins. Plasmids encoding histidine tagged - EGFP-octaarginine, or - EGFP referred to here as EGFP-R8 or EGFP, were a kind gift from Professor Shiroh Futaki, University of Kyoto, Japan. To produce the plasmid the DNA fragment encoding EGFP was amplified from pEGFP-N1 (Clontech) using primers possessing the sequences for NdeI followed by His6, and R8 and stop codon followed by EcoRI. The fragment was cleaved by NdeI and EcoRI and cloned into a pET3b (Novagen) derivative without N-terminal T7.Tag sequence to construct E. Coli expression vector pEV3b31 that has NdeI and EcoRI sites within the multi cloning site. Proteins were produced in E. coli BL21 (DE3) cells and assessed for purity and molecular weight as described in Supplementary Information and shown in Supplementary Fig. 1. Live cell imaging of endocytic probes and R8 conjugates. HeLa or A431 cells were seeded on 35 mm MatTek dishes (Ashland, US) at respective densities of 5 × 105 cells/dish and 1.0 × 106 cells/dish and maintained in 2.5 mL D-MEM containing 10%, 100 IU/mL penicillin and 100 µg/mL streptomycin. Cells were then allowed to adhere for 24 hr at 37 °C/5% CO2 to reach a confluence of ~80%. On the day of experimentation, they were washed with PBS and then incubated with 100 µl serum-free D-MEM (thus reducing well described serum binding effects32) containing either 2 µM CPP conjugate (R8-Alexa488 or EGFP-R8), 5 µg/ml transferrin-Alexa 647 (TF-647) or 0.1 mg/ml dextran (10 kDa)-Alexa 647 (Dex-647). Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates yl g y One endocytic pathway that is absolutely reliant on actin is macropinocytosis. When activated this process has the capacity to form large plasma membrane derived intracellular vesicles termed macropinosomes12–15. Classically macropincytosis is induced in response to growth factor activation such as epidermal growth factor (EGF) binding to the EGF receptor, initially leading to extensive actin-dependent ruffling on the plasma mem- brane. This induces a “gulping effect” manifest as an increased uptake of extracellular fluid13,14,16. Much of the information known regarding growth factor induced and actin dependent macropinocytosis comes from studies on high EGFR expressing A431 skin epithelia cells and their response to EGF13,17,18. Of interest are observations that some CPPs under defined experimental conditions may induce plasma membrane effects similar to that seen upon growth factor activation19–21 and in line with this that they promote the concomitant uptake of dextran, a well characterised marker of fluid phase endocytosis22–24. Dextran itself, in addition to being widely used as a fluid phase endocytic probe has been extensively investigated as a drug delivery vector25. Tools used routinely to examine the roles of the actin cytoskeleton in various cellular processes, including endocytosis and CPP entry are pharmacological/chemical inhibitors. The most notable such agent is the fungal 1Cardiff School of Pharmacy and Pharmaceutical Sciences, Redwood Building, Cardiff University, Cardiff, Wales, CF10 3NB, UK. 2Cardiff School of Biosciences, The Sir Martin Evans Building, Cardiff University, Cardiff, Wales, CF10 3AX, UK. L. He and E. J. Sayers contributed equally to this work. Correspondence and requests for materials should be addressed to P.W. (email: watsonpd@cardiff.ac.uk) or A.T.J. (email: jonesat@cardiff.ac.uk) Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 1 www.nature.com/scientificreports/ metabolite cytochalasin D (Cyt D) which disrupts actin polymerisation and is a well characterised inhibitor of var- ious endocytic mechanisms26–28. Other natural compounds and synthetic products such as Latrunculin B (Lat B) and Jasplakinolide (JAS) have been identified or developed to target the actin directly or indirectly and to disrupt its organisation and function29. Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates Very few studies have investigated the effects of these other actin disrupters on CPP uptake though it is generally recognised that actin disruption universally inhibits CPP entry.f p g g y g p y y Here we show that the effects of actin disruption on uptake of CPPs and dextran is cell type dependant and in A431 skin epithelia, in complete contrast to HeLa cells, leads to a dramatic increase in uptake of EGFP-R8 and dextran but inhibits the uptake of R8-Alexa488. Together the data indicate that actin organisation has very different influences on uptake of these octaarginine and fluid phase conjugates and that actin could be targeted to enhance cellular uptake of drug delivery vectors. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 Results ff Cyt D effects on actin and endocytosis in HeLa and A431 cells. Cyt D binds to the fast-growing ends of actin nuclei and filaments, preventing addition of monomeric actin to these sites. In our previous studies using flow cytometry we showed that Cyt D inhibited the uptake of Alexa488-CPPs R8 and Tat in two different cell mod- els, HeLa and A43133. But interestingly, the effect of actin disruption with this agent on the uptake of dextran was highly dependent on the choice of cell line as there was no effect in HeLa cells and an increased uptake in A431 cells. Here we further explored these contradictory effects by also performing uptake studies with recombinant EGFP-R8 following purification from E. Coli as described in Supplementary methods and Supplementary Fig. 1. g pi pp y pp y g At 2 µM extracellular concentration, both R8 conjugates, in the two cell lines, labelled vesicular structures scattered throughout the cytoplasm as expected (Supplementary Figs 2 and 3), no cell associated fluorescence was observed for EGFP alone. We then performed the same experiments in cells incubated with Cyt D, focusing on effects on endocytosis and also cell morphology. 10 μM of the inhibitor was used as this concentration has previously been shown by different groups to inhibit the uptake of cationic CPPs such as HIV-Tat and R8 linked to different cargoes33–35. DIC microscopy highlighted the dramatic morphological effects on both cell lines incu- bated with this agent (Fig. 1 and Supplementary Fig. 4). Cyt D inhibited the internalisation of R8-Alexa 488 in HeLa cells (Fig. 1A), confirming our published data using flow cytometry33. There was no clear evidence of Cyt D mediated inhibition of EGFP-R8 but the drug caused a scattering of the fluorescent structures compared to untreated cells that usually displayed strong juxtanuclear labelling (Fig. 1B). In contrast to HeLa cells there was less of a noticeable reduction in uptake of R8-Alexa488 in A431 cells but a clear change in subcellular distribution from evenly scattered throughout the cytoplasm in control cells to a largely clustered phenotype in treated cells (Fig. 1C). In contrast to the results obtained with R8-Alexa 488, Cyt D dramatically enhanced the internalisation of EGFP-R8 into small and large cytoplasmic structures (Fig. 1D). This data was confirmed in A431 cells using flow cytometry showing a highly significant 4.5 fold increase in intracellular fluorescence (Fig. Materials and Methods For multi-channel image acquisition, the channels were scanned in a sequential recording mode to avoid spectral cross-talk caused by overlapping excitation and/or emission spectra of fluorophores. Acquisition mode “XYZ” and image resolutions (pixels/image) of 512 × 512 or 1024 × 1024 were selected and pinhole size was set to 1 Airy Unit. Depending on acquisition requirements the scan speed was set as either 400 or 700 Hz and for single section images, the same optical section was scanned 3x to generate a line average image. Captured images were Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 2 www.nature.com/scientificreports/ then analysed using ImageJ. In some cases (live cell imaging) the same cells were also imaged using Differential Imaging Contrast microscopy (DIC). Actin cytoskeleton visualisation. A recently described method was utilised giving information on actin architecture at different regions of the cells termed basal and cell body and apex (CBA). For actin staining, HeLa (0.5 × 106 cells/well) or A431 (1.0 × 106 cells/well) cells in 2.5 mL D-MEM containing 10% FBS were seeded on round borosilicate No.1.5 cover slips in 6 –well plates and allowed to adhere for 24 hr at 37 °C/5% CO2, to reach a confluence of ~80%. The cells were then treated with actin disrupters as described above, washed 3x with PBS and fixed for 15 min at room temperature with 3% PFA in PBS. Cells were then washed 3x with PBS and permeabilised with 0.2% Triton X-100 in PBS at room temperature for 5 min, washed 3x with PBS and stained for cell nuclei with 1.0 µg/ml Hoechst 33342 in PBS and for polymerised actin with 1.0 µg/ml Rh-P in PBS for 15 min at room temperature. The cells were finally washed 3x with PBS and once with distilled water, coverslips were mounted in DAKO mounting medium on glass microscope slides and imaged by confocal microscopy19. Flow Cytometry. A431 cells were seeded onto 12-well plates and cultured for 24 hr to be ~80% confluent on the day of experiment. Cells were washed twice with PBS and then incubated with diluent control (DMSO/dH2O) or actin inhibitors as previously described. They were then loaded with serum-free DMEM containing either 2 µM EGFP-R8, 5 µg/ml TF-647 or 0.1 mg/ml Dex-647 in the presence or absence of actin inhibitors for times stipulated in the figure legends. Materials and Methods The plates were then placed on ice to prevent further uptake and washed 3x with either ice-cold PBS or 0.5 mg/ml heparin in PBS. To remove the plasma membrane bound TF, cells were washed 3x with ice-cold PBS followed by an incubation for 1 min in ice-cold acid wash (0.2 M acetic acid, 0.2 M NaCl, pH 2.0)33. All cells were then washed twice with PBS at room temperature before trypsinisation at 37 °C for 5 min before placing the detached cells in centrifuge tubes and centrifuging at 4 °C for 3 min at 800 × g. The obtained cell suspension was washed 3x with ice-cold PBS and finally resuspended in 300 µl ice-cold PBS for measurement of cell-associated fluorescence (cell gate 50,000 cells) by flow cytometry on a BD FACSVerse utilising a 488 laser and 527/32 filter for EGFP analysis and a 633 nm laser and 660/10 filter for Alexa 647. Cells were gated using FSC-A/ SSC-A to isolate single cell populations and analysed using FlowJo. Statistics. Flow cytometry measurements represent the mean fluorescence from three independent experi- ments performed in duplicate, where each individual data point was the geometric mean. A two-tailed unpaired Students t-test was used to test for differences within experiments. Significant differences were classed as having a P value of < 0.05. Data Availability. The datasets generated during the current study are available from the corresponding author on reasonable request. Results ff 2), P = 0.0036.f lil In these two cell lines, we also investigated the effects of Cyt D on the uptake of dextran, that enters cells via fluid phase uptake, and transferrin that enters via clathrin mediated endocytosis. In agreement with our previous flow cytometry and microscopy studies with 40 KDa FITC and Alexa488 dextran33, uptake of dextran-Alexa647 was dramatically increased in A431 cells (Supplementary Fig. 5A, C).lf Cell associated transferrin fluorescence was not markedly different between control and Cyt D treated HeLa cells (Supplementary Fig. 5B) confirming previous reports performed at the same concentration in this cell line36. Transferrin fluorescence in Cyt D treated HeLa cells was, however, more prominent on the plasma membrane com- pared to a strong perinuclear enrichment of fluorescence in control cells. Flow cytometry utilising acid washing of surface label confirmed that there was no difference in fluorescence uptake of transferrin in A431 cells treated with this drug (Supplementary Fig. 6), confirming previous studies in this cell line27. We focused our further investigations on the A431 cell line and EGFP-R8, as this was the conjugate that gave unexpected Cyt D mediated uptake effects. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 3 www.nature.com/scientificreports/ Figure 1. Cytochalasin D effects on the cellular uptake of R8 conjugates in HeLa or A431 cells. HeLa (A,B) or A431 (C,D) were preincubated with 10 µM Cyt D or diluent control for 15 min prior to incubation with 2 µM R8 conjugates (R8-Alexa 488, A,C, or EGFP-R8, B,D) for 1 hr in the presence or absence of 10 µM Cyt D before washing in heparin and analysis by confocal microscopy. Shown are single projection images of fluorescence only (R8-Alexa 488/EGFP-R8), DIC and merges of fluorescence and DIC of the same cells. Scale bars 10 µm. Figure 1. Cytochalasin D effects on the cellular uptake of R8 conjugates in HeLa or A431 cells. HeLa (A,B) or A431 (C,D) were preincubated with 10 µM Cyt D or diluent control for 15 min prior to incubation with 2 µM R8 conjugates (R8-Alexa 488, A,C, or EGFP-R8, B,D) for 1 hr in the presence or absence of 10 µM Cyt D before washing in heparin and analysis by confocal microscopy. Shown are single projection images of fluorescence only (R8-Alexa 488/EGFP-R8), DIC and merges of fluorescence and DIC of the same cells. Scale bars 10 µm. Figure 2. Results ff Cells on coverslips were treated either with diluent control (0.1% DMSO) or 0.1–1.0 μM Lat B for 30 min before fixing and staining with Rh-P and Hoechst. Single sections (A) show the overall distribution of the actin relative to the nucleus. (B) Actin arrangement from the basal, CBA regions and composite (Merge) of basal and CBA. Scale bars 10 μm. To try and identify the subcellular compartments occupied by EGFP-R8 we performed pulse/chase experi- ments with Dex-647: a 2 hr pulse labelled endolysosomal organelles was followed by a 4 hr chase to specifically label late endosomes and lysosomes37. EGFP-R8 was then incubated with the cells for 1 hr in the presence or absence of Cyt D. Control cells (Supplementary Fig. 8A, top row, indicated by arrows) show colocalisation of the two fluorophores demonstrating trafficking of some EGFP-R8 to dextran labelled late endosomes and lysosomes. However, EGFP only structures were also clearly visible suggesting that this fraction had not yet entered these structures or were trafficking to a different location. To verify that the EGFP fluorescence was being emitted from inside the cells we captured images of the Cyt D treated cells through the Z axis starting 1.44 µm from the glass surface to a height of 14.5 µm. This allowed us to visualise the dextran and EGFP-R8 structures at different focal planes and the data confirmed that the EGFP fluorescence was of intracellular origin (Supplementary Fig. 8B). Effects of other actin reagents on actin and cellular uptake of EGFP-R8. Lat B, binds to and com- plexes with actin monomers at a 1:1 molar ratio and prevents polymerisation38. We initially investigated the concentration dependent effects of this drug on the actin cytoskeleton. At the lowest concentration (0.1 μM) the drug caused the formation of small actin aggregates at both the basal and cell body and apex (CBA) sections of the cells (Fig. 3). At 0.5 μM, these aggregates were extremely prominent especially at the basal section. At 1 μM the drug caused major disruption of the actin organisation manifest as amorphous structures scattered throughout the cytoplasm. The effects of this drug on cell morphology is further highlighted in Supplementary Fig. 4. yhf g gy g g y g We subsequently performed CPP uptake experiments in A431 cells pre-incubated with 0.5 µM Lat B prior to addition of EGFP-R8. The effect of Lat B (Fig. Results ff Cytochalasin D treatment increases the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with 10 µM Cyt D or diluent control for 15 min prior to incubation with 2 µM EGFP-R8 in the absence or presence of 10 µM Cyt D for 1 hr. Cells were then washed, trypsinised and analysed by flow cytometry. Data represent the geometric means ± S.D. from three independent experiments. Figure 2. Cytochalasin D treatment increases the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with 10 µM Cyt D or diluent control for 15 min prior to incubation with 2 µM EGFP-R8 in the absence or presence of 10 µM Cyt D for 1 hr. Cells were then washed, trypsinised and analysed by flow cytometry. Data represent the geometric means ± S.D. from three independent experiments. To identify whether the enhanced cellular uptake of EGFP-R8 in A431 cells by Cyt D treatment was an energy dependant process, the same experiments were performed with cells pre-incubated at 37 °C with the drug, then placed on ice and maintained at 4 °C throughout the subsequent incubation with EGFP-R8. There was very little evidence above background fluorescence that EGFP-R8 entered cells at 4 °C in the presence or absence of Cyt D (Supplementary Fig. 7) confirming that the enhanced EGFP-R8 internalisation induced by the inhibitor was energy dependent. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 4 www.nature.com/scientificreports/ Figure 3. Effects of Lat B on the actin architecture in A431 cells. Cells on coverslips were treated either with diluent control (0.1% DMSO) or 0.1–1.0 μM Lat B for 30 min before fixing and staining with Rh-P and Hoechst. Single sections (A) show the overall distribution of the actin relative to the nucleus. (B) Actin arrangement from the basal, CBA regions and composite (Merge) of basal and CBA. Scale bars 10 μm. Figure 3. Effects of Lat B on the actin architecture in A431 cells. Cells on coverslips were treated either with diluent control (0.1% DMSO) or 0.1–1.0 μM Lat B for 30 min before fixing and staining with Rh-P and Hoec Single sections (A) show the overall distribution of the actin relative to the nucleus (B) Actin arrangement fr Figure 3. Effects of Lat B on the actin architecture in A431 cells. Results ff At 2 µM JAS the actin staining appeared in large clumps, previously identified as aggresomes43, or was absent, and was completely lost from all cells treated at 4 µM (Fig. 5A). This loss of fluorescence may be due to the fact that this drug has been shown to be a competitive inhibitor of phalloidin-actin binding39. Supplementary Fig. 4 shows the significant morphological effects of this drug on the cells. f l d k d ll ( ) d h d d cells, no visible changes in actin organisation were observed in both basal and CBA sections of cells until the con- centration of JAS reached 0.2 µM (data not shown and Fig. 5). At this concentration, actin staining was more prom- inent on the cell periphery at both basal and CBA regions. At 2 µM JAS the actin staining appeared in large clumps, previously identified as aggresomes43, or was absent, and was completely lost from all cells treated at 4 µM (Fig. 5A). This loss of fluorescence may be due to the fact that this drug has been shown to be a competitive inhibitor of phalloidin-actin binding39. Supplementary Fig. 4 shows the significant morphological effects of this drug on the cells. Confocal microscopy dramatic increase in EGFP-R8 uptake in JAS treated cells (Fig. 6A) and the drug caused a pronounced clustering of fluorescence in the cytoplasm. Equivalent experiments analysed by flow cytom- etry (Fig. 6B) indicated that JAS induced a four-fold increase in fluorescence that was statistically significant (P = 0.0025). The diluent control experiments with JAS required adding DMSO to the cells at a final concentration of 0.4% v/v; the highest used in this study. This concentration did not affect uptake of EGFP-R8 or R8-Alexa488 (Supplementary Fig. 9). p g pp y g gi p gf g Confocal microscopy dramatic increase in EGFP-R8 uptake in JAS treated cells (Fig. 6A) and the drug caused a pronounced clustering of fluorescence in the cytoplasm. Equivalent experiments analysed by flow cytom- etry (Fig. 6B) indicated that JAS induced a four-fold increase in fluorescence that was statistically significant (P = 0.0025). The diluent control experiments with JAS required adding DMSO to the cells at a final concentration of 0.4% v/v; the highest used in this study. This concentration did not affect uptake of EGFP-R8 or R8-Alexa488 (Supplementary Fig. 9). Results ff 4) was comparable to that of Cyt D (Fig. 2): the inhibitor treated cells showing much higher fluorescence compared to controls (Fig. 4). Flow cytometry analysis confirmed these findings and the 6.23 fold increase in uptake was highly statistically significant (P = 0.0045).i i g p g y y gi ( ) An alternative actin inhibitor, Jasplakinolide (JAS) binds directly to filamentous actin and unlike Cyt D and Lat B induces stabilisation of the existing actin filaments thus inhibiting actin disassembly39–42. Compared with control Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 5 www.nature.com/scientificreports/ Figure 4. Effects of Lat B on the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with either with diluent control or 0.5 μM Lat B for 30 min prior to incubation with 2 µM EGFP-R8 for 1 hr in the absence (control) or presence of 0.5 µM Lat B and washed in heparin. (A) Cell associated fluorescence was analysed using confocal microscopy and shown are single projection images of fluorescence only (top rows) and merges of fluorescence and DIC of the same cells. Scale bars 10 µm. (B) Cells treated as in A were washed, trypsinised and analysed by flow cytometry. Data represent the mean of geometric means ± S.D. from three independent experiments. Figure 4. Effects of Lat B on the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with either with diluent control or 0.5 μM Lat B for 30 min prior to incubation with 2 µM EGFP-R8 for 1 hr in the absence (control) or presence of 0.5 µM Lat B and washed in heparin. (A) Cell associated fluorescence was analysed using confocal microscopy and shown are single projection images of fluorescence only (top rows) and merges of fluorescence and DIC of the same cells. Scale bars 10 µm. (B) Cells treated as in A were washed, trypsinised and analysed by flow cytometry. Data represent the mean of geometric means ± S.D. from three independent experiments. cells, no visible changes in actin organisation were observed in both basal and CBA sections of cells until the con- centration of JAS reached 0.2 µM (data not shown and Fig. 5). At this concentration, actin staining was more prom- inent on the cell periphery at both basal and CBA regions. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 Discussion Since the discovery of CPPs, considerable efforts have been made to elucidate their internalisation mechanisms. Focusing mainly on arginine rich variants, macropinocytosis has been proposed to be a major contributing path- way for delivery of low molecular weight fluorophores or larger cargo by these peptides20,24,49. This is based on studies utilising chemical macropinocytosis inhibitors including, and almost exclusively Cyt D for actin inhi- bition. It should however be noted that the involvement of actin activity has been shown for other endocytic pathways such as clathrin-mediated endocytosis (CME)27,50. As shown here and in several other studies, Cyt D at concentrations >5 µM, completely destroys actin organisation and, unsurprisingly, cell morphology is also compromised. A number of other natural compounds and synthetic products have been identified or developed to target the actin cytoskeleton directly or indirectly and to disrupt its arrangement/function. These include Lat B, JAS and Y27632 and these inhibitors have not been used to support Cyt D data for drug delivery endocytosis studies, including CPPs. To our knowledge this is the first study that has analysed CPP uptake with actin inhibi- tors employing different mechanisms of action.l p y gf By employing high content analysis of the actin architecture using confocal microscopy and then flow cytom- etry we were able to correlate actin disorganisation with cellular uptake of EGFP-R8 as a model protein-CPP con- jugate. EGFP has been shown to be delivered into cells by R8 and R935,51 but correlative information regarding the uptake mechanism of the proteins relative to that of the peptides attached to fluorophores is lacking. EGFP-Tat uptake has been proposed to be mediated via macropinocytosis and caveolae24,52. Both Tat peptide, octaarginine,  and shorter number of arginine residues (R7, R9) have also been shown to deliver proteins into cells to mediate biological effects53–55. gf In this study, HeLa cells reacted in a somewhat predictable manner in response to actin breakdown with Cyt D showing a reduction in uptake of R8-Alexa488 and dextran. In this cell line EGFP-R8 uptake was also inhibited by Cyt D treatment suggesting a similar mechanism of uptake. We have previously shown that cortical actin in A431 cells is particularly prominent and suggested that this may have a role in the uptake of dextran that, under normal conditions, enters cells via constitutive pinocytosis; also termed fluid phase endocytosis. This can be differentiated from stimulus induced macropinocytosis56,57. Results ff Actin dynamics and regulation are achieved through the concerted action of actin regulatory proteins whose activities are finely modulated by protein kinases. These include Rho-associated coiled-coil forming serine/thre- onine kinases ROCK I and II that have been identified as important downstream effectors of RhoA through their interaction with its GTP-bound form44,45. A number of chemical inhibitors of the Rho-ROCK signalling cascade have been developed, including compound Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride]. This has been shown to inhibit the kinase activity of a number of ROCKs by binding to their catalytic sites46,47. Initially, 1 and 10 μM concentrations of Y27632 were investigated on actin localisation based on published observations showing actin deformities at these concentrations46,48. Supplementary Fig. 10 demonstrates that no visible changes in the organisation of the actin cytoskeleton were detected up to 10 μM drug. Higher concentrations caused extensive actin deformities including the formation of a large number of actin needles, the majority of which ran perpendicular to the plasma membrane (Fig. 7). Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 6 www.nature.com/scientificreports/ Figure 5. Effects of JAS on the actin architecture in A431 cells. Cells on coverslips were treated either with diluent control (0.1% DMSO) or 0.2, 2.0 or 4.0 μM JAS for 45 min before fixing and staining with Rh-P and Hoechst. (A) Single sections show the overall distribution of the actin relative to the nucleus. (B) Actin arrangement from the basal, CBA regions and composite (Merge) of basal and CBA. Scale bars 10 μm. Figure 5. Effects of JAS on the actin architecture in A431 cells. Cells on coverslips were treated either with diluent control (0.1% DMSO) or 0.2, 2.0 or 4.0 μM JAS for 45 min before fixing and staining with Rh-P and Hoechst. (A) Single sections show the overall distribution of the actin relative to the nucleus. (B) Actin arrangement from the basal, CBA regions and composite (Merge) of basal and CBA. Scale bars 10 μm. In CPP uptake experiments, Y27632 treated cells displayed scattered EGFP-R8 fluorescence in small vesicles in contrast to the vesicle aggregates seen in cells incubated with the other actin disrupting agents (Figs 1,4 and 8A) while DIC microscopy did not show the gross morphological effects caused by the other actin inhibitors. Flow cytometry analysis indicated that cell uptake of EGFP-R8 uptake was significantly inhibited by Y27632 but only by ~20% (Fig. 8B P = 0.02). Results ff To exclude the possibility that any of the actin inhibitors was affecting plasma mem- brane permeability, we incubated A431 cells with the inhibitors at the concentrations and incubations used above in the presence of live cell impermeable dye - DRAQ7. Using Tritin X-100 as positive control, none of the inhibi- tors showed any evidence of inducing plasma membrane permeabilisation (Supplementary Fig. 4). Discussion This potential for the involvement of cortical actin was identified by discovering a rather unexpected and highly significant increase in the uptake of 10 and 40 KDa dextran in Cyt D treated A431 cells that was not observed with Alexa conjugates of transferrin, Tat and R833. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 7 www.nature.com/scientificreports/ Figure 6. Effects of JAS on the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with diluent control or 2–4 µM JAS for 45 min prior to incubation with 2 µM EGFP-R8 for 1 hr in the absence (control) or presence of 2–4 µM JAS and washed thoroughly using heparin. (A) Cell associated fluorescence was analysed using confocal microscopy and shown are single projection images of fluorescence only (top row) and merges of fluorescence and DIC of the same cells. Scale bars 10 µm. (B) Cells treated as in A with 4 µM JAS were then washed, collected and analysed by flow cytometry. Data represent the mean of geometric means ± S.D. from three independent experiments. Figure 6. Effects of JAS on the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with diluent control or 2–4 µM JAS for 45 min prior to incubation with 2 µM EGFP-R8 for 1 hr in the absence (control) or presence of 2–4 µM JAS and washed thoroughly using heparin. (A) Cell associated fluorescence was analysed using confocal microscopy and shown are single projection images of fluorescence only (top row) and merges of fluorescence and DIC of the same cells. Scale bars 10 µm. (B) Cells treated as in A with 4 µM JAS were then washed, collected and analysed by flow cytometry. Data represent the mean of geometric means ± S.D. from three independent experiments. Figure 6. Effects of JAS on the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with dilue In this study in we show that dextran and also EGFP-R8 uptake is significantly increased in A431 treated with Cyt D and contrasts to that observed for R8-Alexa488 (inhibition) and transferrin (no change). This strongly suggests that the process by which these two R8 conjugates interact with and/or enter cells is different. It should be noted that the structure of both conjugates differ in their cargo-CPP orientation and also the linkers utilised between them. Discussion We consider that it is highly unlikely that these factors mediate the differences in endocytic pro- files of these conjugates. Due to the similarities we see in entry for dextran and EGFP-R8, we hypothesise that the differences are driven by the cargo and not the orientation of the CPP. But this warrants further study, noting that EGFP alone does not enter these cells at the concentrations used here, demonstrating a clear role for R8 that may be interacting differently with cells when attached to a fluorophore or protein. In none of the experiments performed did we observe clearly defined cytosolic EGFP fluorescence in Cyt D treated cells demonstrating that the effects of this drug maintained the localisation of the protein within a membrane bound compartment.fi f g p p We have previously shown that R8 and Tat peptides as Alexa conjugates traffic rapidly by endocytosis to reach lysosomes30 and by performing pulse chase experiments we now show that the same is true for EGFP-R8, demonstrating that it is trafficked to the same degradative compartments. From these combined studies we also highlight that traffic to lysosomes of R8 attached to different cargo is also entirely dependent on functional actin.i reach lysosomes30 and by performing pulse chase experiments we now show that the same is true for EGFP-R8, demonstrating that it is trafficked to the same degradative compartments. From these combined studies we also highlight that traffic to lysosomes of R8 attached to different cargo is also entirely dependent on functional actin. Our findings prompted us to further study EGFP-R8 uptake in cells treated with inhibitors of actin that oper- ate via different mechanisms and affect actin dynamics in different ways. Like Cyt D, at effective actin disrupting concentrations, JAS and Lat B also affected cell morphology and increased EGFP-R8 uptake that was again highly enriched in large structures in particular regions of the cells. Previous studies in non-polarised epithelial MDCK cells demonstrated that JAS treated cells had much higher levels of internalised and retained dextran compared fif Our findings prompted us to further study EGFP-R8 uptake in cells treated with inhibitors of actin that oper- ate via different mechanisms and affect actin dynamics in different ways. Like Cyt D, at effective actin disrupting concentrations, JAS and Lat B also affected cell morphology and increased EGFP-R8 uptake that was again highly enriched in large structures in particular regions of the cells. Discussion Previous studies in non-polarised epithelial MDCK cells demonstrated that JAS treated cells had much higher levels of internalised and retained dextran compared Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 8 www.nature.com/scientificreports/ Figure 7. Effects of Y27632 on the actin architecture of A431 cells. Cells on coverslips were treated either with diluent control (D-MEM) or 50, 100 μM of Y27632 for 4 hr before fixing and staining with Rh-P and Hoechst. (A) Single sections show the overall distribution of the actin relative to the nucleus. (B) Actin arrangement from the basal, CBA regions and composite (Merge) of basal and CBA. Scale bars 10 μm. Figure 7. Effects of Y27632 on the actin architecture of A431 cells. Cells on coverslips were treated either with diluent control (D-MEM) or 50, 100 μM of Y27632 for 4 hr before fixing and staining with Rh-P and Hoechst. (A) Single sections show the overall distribution of the actin relative to the nucleus. (B) Actin arrangement from the basal, CBA regions and composite (Merge) of basal and CBA. Scale bars 10 μm. Figure 7. Effects of Y27632 on the actin architecture of A431 cells. Cells on coverslips were treated either with diluent control (D-MEM) or 50, 100 μM of Y27632 for 4 hr before fixing and staining with Rh-P and Hoechst. (A) Single sections show the overall distribution of the actin relative to the nucleus. (B) Actin arrangement from the basal, CBA regions and composite (Merge) of basal and CBA. Scale bars 10 μm. with control cells57. In polarised cells, however, this effect was limited to dextran entering via the basolateral surface and the retained dextran was observed in large structures as we observe for EGFP-R8. It remains to be determined whether EGFP-R8 would also behave in a similar manner when introduced to different surfaces of polarised cells and whether this is influenced by any differences in cortical tension between these surfaces. The nature of these large structures is unknown but may be formed by tubulation of the plasma membrane that has been shown to be caused by Cyt D58. The plasma membrane tension of mammalian cells is coordinated and main- tained through the actin network, and this profoundly influences endocytosis59,60. The reduced cortical tension resulting from actin disruption in MDCK basolateral surfaces57 and here in A431, cells may favour the formation of invaginations and tubulation to allow entry of macromolecules. Discussion Cells were preincubated with diluent control or 100 µM Y27632 for 4 hr prior to incubation with 2 µM EGFP-R8 for 1 hr in the absence (control) or presence of 100 µM Y27632 and washed with heparin. (A) Cell associated fluorescence was analysed using confocal microscopy and shown are single projection images of fluorescence only and merge of fluorescence and DIC of the same cells. Scale bars 10 µm. (B) Cells were washed, trypsinised and analysed by flow cytometry. Data represent the mean of geometric means ± S.D. from three independent experiments. Figure 8. Effects of Y27632 on the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with diluent control or 100 µM Y27632 for 4 hr prior to incubation with 2 µM EGFP-R8 for 1 hr in the absence (control) or presence of 100 µM Y27632 and washed with heparin. (A) Cell associated fluorescence was analysed using confocal microscopy and shown are single projection images of fluorescence only and merge of fluorescence and DIC of the same cells. Scale bars 10 µm. (B) Cells were washed, trypsinised and analysed by flow cytometry. Data represent the mean of geometric means ± S.D. from three independent experiments. Conclusions Interpretation of endocytosis data in in vitro models obtained using actin modifying agents is difficult as the net- work that this protein nucleates is a regulator of so many processes; global cell morphology is often affected by its perturbation. Here we highlight that disruption of actin in some cell types promotes cell uptake of specific mol- ecules whilst having no effects, or even inhibitory effects on others. From careful scrutiny of the literature there is precedent for this in other cell types and distinct plasma membrane regions in polarised cells. The possibility exists that these cells have a specific but common cortical organisation of actin that could be exploited to drive internalisation of drug delivery vectors carrying macromolecular therapeutics. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 Discussion This strongly supports studies showing an increased uptake of 70 KDa dextran and the folic acid receptor in Cyt D treated cells61,62. Both folic acid and dex- tran have been extensively utilised as, respectively, targeting and passive vectors for drug delivery25,63.h Through interaction with the Rho GTPase, ROCKs play a central role in actin network assembly and mem- brane association64. In A431 cells the effects of Y27632 on the actin cytoskeleton was more subtle than that observed with the agents that directly targeted actin. No change in cell morphology was observed in treated cells but actin distribution was very different at high 100 µM concentrations. Effects of this agent in human trabecular meshwork eye cell actin has been shown to be extremely prominent at much lower concentrations, showing a loss of filamentous actin65. In contrast to results observed with direct actin inhibitors, we observed a slight but signifi- cant decrease in the uptake of EGFP-R8 in cells treated with Y27632. To our knowledge no CPP studies have been performed with this drug though it has previously been shown to affect the internalisation of plasma membrane receptors and also to inhibit (~50%) the uptake of 70 KDa dextran in macrophages66–68. This again supports our suggestion that dextran and EGFP-R8 uptake is responsive to the same inhibition in A431 cells but that they may not necessarily enter via the same mechanism. This supports recent observations showing that cell uptake profiles of HIV-TAT peptide and dextran can be separated69. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 9 www.nature.com/scientificreports/ Figure 8. Effects of Y27632 on the cellular uptake of EGFP-R8 in A431 cells. Cells were preincubated with diluent control or 100 µM Y27632 for 4 hr prior to incubation with 2 µM EGFP-R8 for 1 hr in the absence (control) or presence of 100 µM Y27632 and washed with heparin. (A) Cell associated fluorescence was analysed using confocal microscopy and shown are single projection images of fluorescence only and merge of fluorescence and DIC of the same cells. Scale bars 10 µm. (B) Cells were washed, trypsinised and analysed by flow cytometry. Data represent the mean of geometric means ± S.D. from three independent experiments. Figure 8. Effects of Y27632 on the cellular uptake of EGFP-R8 in A431 cells. References 1. Guidotti, G., Brambilla, L. & Rossi, D. 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Mol Pharmacol 57, 976–983 (2000). 46. Ishizaki, T. et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 57, 976–983 (2000). ( ) 7. Uehata, M. et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389 990–994, https://doi.org/10.1038/40187 (1997). Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 11 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-25600-8.h Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Scientific REPorTS | (2018) 8:7318 | DOI:10.1038/s41598-018-25600-8 12
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The effect of corticosteroid versus platelet-rich plasma injection therapies for the management of lateral epicondylitis: A systematic review
SICOT-J
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1 The University of Salford, Salford, UK 2 y , , ma and Orthopaedics department, Central Manchester University Hospitals NHS Foundation Trust, UK 2 Trauma and Orthopaedics department, Central Manchester University Hospitals NHS Fo Received 27 April 2017, Accepted 29 December 2017, Published online 21 March 2018 Abstract -- Introduction: Lateral epicondylitis is a common musculoskeletal disorder of the upper limb. Corticosteroid injection has been widely used as a major mode of treatment. However, better understanding of the pathophysiology of the disease led to a major change in treating the disease, with new options including platelet-rich plasma (PRP) are currently used. ( ) y bjectives/research aim: To systematically evaluate the effect of corticosteroid versus PRP injections for the eatment of LE. Hypothesis: PRP injections provide longer-term therapeutic effect and less rate of complications compared to corticosteroid injection. j Level of evidence: Level 2 evidence (4 included studies are of level 1 evidence, 1 study of level 2 evidence). Design: Systematic Review (according to PRISMA guidelines). Methods: Eleven databases used to search for relevant primary studies comparing the effects of corticosteroid and PRP injections for the treatment of LE. Quality appraisal of studies performed using Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0, CASP Randomised Controlled Trial Checklist, and SIGN Methodology Checklist 2. Results: 732 papers were identified. Five randomised controlled trials (250 Patients) met the inclusion criteria. Clinical findings: Corticosteroid injections provided rapid symptomatic improvement with maximum effect at 6/8/8 weeks before symptoms recurrence, whereas PRP showed slower ongoing improvements up to 24/52/104 weeks (3 studies). Corticosteroid showed more rapid symptomatic improvement of symptoms compared to PRP up to the study end-point of 3 months (1 study). Comparable therapeutic effects of corticosteroid and PRP were observed at 6 weeks (1 study). Ultrasonographic Findings: (1) Doppler activity decreased more significantly in patients who received corticosteroid compared to PRP. (2) Reduced tendon thickness and more patients with cortical erosion noted in corticosteroid group whereas increased tendon thickness and less number of patients with common extensor tendon tears noted in PRP group. (3) Fewer patients reported Probe-induced tenderness and oedema in the common extensor tendon in both corticosteroid and PRP groups (2 studies). onclusion: Corticosteroid injections provide rapid therapeutic effect in the short-term with recurrence of ymptoms afterwards, compared to the relatively slower but longer-term effect of platelet-rich plasma. Key words: Tennis elbow, Lateral epicondylitis, Epicondylopathy, Epicondylalgia Corticosteroid, Stero injections, Platelet-rich plasma, PRP injections This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. *Corresponding author: W.O.Ben-Nafa@edu.salford.ac.uk SICOT-J 2018, 4, 11 © The Authors, published by EDP Sciences, 2018 https://doi.org/10.1051/sicotj/2017062 SICOT-J 2018, 4, 11 © The Authors, published by EDP Sciences, 2018 https://doi.org/10.1051/sicotj/2017062 Available online at: www.sicot-j.org Available online at: www.sicot-j.org REVIEW ARTICLE REVIEW ARTICLE The effect of corticosteroid versus platelet-rich plasma injection therapies for the management of lateral epicondylitis: A systematic review Walid Ben-Nafa1,2,* and Wendy Munro1 1 The University of Salford, Salford, UK 2 Methodology Study selection criteria Although the main focus was on RCTs, all interventional studies were eligible for inclusion in this study. The search strategy of this review was structured using Population, Intervention, Compari- son, Outcome framework (PICO). Free-text searching and Medical Subject Headings were also considered. j g Search strategy The search strategy was conducted and reported in accordance with PRISMA guidelines [37] and Cochrane Collaboration [38] whenever possible. It also adhered to the published guidance used when undertaking reviews in healthcare [39]. Eleven databases were systematically searched, namely, Web of science, Scopus, PubMed (MedLine), ScienceDirect, CINAHL, EMBASE, Ovid, NICE, Physiotherapy Evidence Data- base, Cochrane Library and ClinicalTrials database (Figure 1). As we aimed to search for only the recent and relevant evidence, only papers published from 2005 onwards were accessed, and hand-searched for their bibliographic references to look for any relevant refer- ences. The search was also enhanced using forward citation searching strategy, with searching by author and study title was also considered using Web of Science Service. Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, and database of abstracts of reviews of effectiveness were also consulted. Potentially relevant studies, which are labelled as ongoing studies, were searched for using International Clinical Trials Registry Platform, UK Clinical Research Network Study Portfolio, US National Library of Medicine Database of Clinical Trials and metaRegister of Controlled Trials. Database bias was eliminated as possible, by reviewing journals which were not indexed in major bibliographic databases. Finally, potentially relevant studies in the grey literature was also considered to identify any unpublished research or research produced outside the traditional distribution channels. With recent histological findings showing that tendinosis is not an acute inflammatory condition but rather a failure of normal tendon repair [18,19] researchers started to focus on the function of biologics in treating LE [20], including Platelet-rich Plasma (PRP) injection [21]. Firstly introduced by Whitman, Berry, & Green [22], PRP has a 3–5 fold increase of platelet concentration compared to whole blood levels [23], and includes many growth factors essential for bone-to-tendon healing, as well as other vascular, epidermal and connective tissue growth factors [24]. Different laboratory studies showedenhanced tenocyteproliferationafterinjection with platelet-released growth factors [25,26]. Introduction including pain and tenderness over the origin of extensor muscles of the wrist and fingers [3,4]. With a prevalence rate of more than 1% among the general population, and with a slight predominance among females [5,6] the disease mostly affects people aged between 35–50 years, who have a history of repetitive activities involving the upper limb [7,8]. While the exact pathophysiology behind the condi- tion is not yet clear, and despite the presence of inflammatory cells locally, there is a strong argument Tennis elbow or lateral epicondylitis (LE), is one of the most common and painful musculo-skeletal conditions, which has a significant impact on the healthcare industry and society in general [1,2]. Lateral epicondylitis is a common term used to describe a group of symptoms W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 2 that LE can be regarded as a degenerative process caused by muscle overuse, with subsequent tendinosis, micro- trauma and tear of the extensor carpi radialis brevis tendon [9,10]. injections, leaving a gap in the literature, with inadequate knowledge available to inform the relevant clinical practices. In this systematic review, we aim to systemati- cally accumulate the evidence available from the relevant primary studies about the effect of these two treatments. We hypothesise that PRP injection provides longer-term therapeutic effect and less rate of complications compared to corticosteroid injection. [ , ] The relatively large number of options currently available for the treatment of lateral epicondylitis [11,12], could be attributed to the scarce evidence available about the disease aetiology and the lack of agreement about a definitive treatment for the condition. Corticosteroid injection has been considered a major treatment option for LE [9]. As corticosteroid mainly aims to reduce inflammation, it is apparently questionable whether it has any possible long-term healing potential with the disease’s degenerative changes [10]. Besides the known local and systemic side effects associated with its use [11,13–16], some researchers believe that corticoster- oid injections may even delay the natural recovery expected with watchful waiting or other management options [17], in addition to the high rate of symptoms recurrence noted with its use. Methodology However, and with the insufficient evidence available to support its clinical use [27], PRP injections continues to be debated in the literature, especially with the lack of its comparable rapid therapeutic effect compared to other pharmacological treatments [28,29]. Although a number of randomised controlled trials compared the effect of corticosteroid and PRP injection for treating LE, an up-to-date systematic review of these primary studies is lacking within the literature. While some reviews [17,30] assessed the effect of various injection therapies for LE, none of these studies investigated the effect of PRP injection as a comparative therapy for the treatment of LE. On the other hand, and while a number of systematic reviews [31–33] focused on the effect of PRP injections, none of these compared the outcome of this injection therapy with other injection modalities, includ- ing corticosteroid injections. Some reviews and meta- analyses recently included more primary studies about the effect of PRP and steroid injections [34–36], but again, none of these reviews focused or specifically compared the effect of steroid and PRP injection therapies. This attributed to the fact that these reviews were aiming at collectively comparing the effects of different injection therapies to obtain a general conclusion about their effect in LE. As a result, this has hindered for a conclusion to be drawn about the effects peculiar to steroid and PRP Only Papers published in English language were included in this review, although it was aimed initially to consider papers written in non-English languages too, including two papers in Turkish language. Nevertheless, and as the accuracy of quality assessment and data extraction could be affected when translating non-English papers, it was finally decided to restrict the work on papers written in English language. This was also supported by the evidence that language restriction does not appear to change the results of systematic reviews conducted on RCTs of conventional interventions [40]. 3 W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 7 study papers considered 732 potentially relevant study papers identified by search strategy: 199 (Web of science) 84 (PubMed: Including MEDLINE) 109 (Scopus) 340 (Other databases - refer to the methodology section) 689 articles were excluded because of the following reasons: - Duplicate papers. - Review articles. - Conference papers, periodicals, editorials, etc. Methodology 43 papers selected 36 study papers were excluded (after reading the papers abstracts) because of the following reasons: - Protocol-only study papers - No comparative assessment of interventions - Comparison of irrelevant treatment interventions 2 study papers written in non-English language (Turkish) 5 primary studies included in this systematic review Figure 1. PRISMA Flow Chart showing the studies’ selection process which led to the inclusion of the five study. 732 potentially relevant study papers identified by search strategy: 199 (Web of science) 84 (PubMed: Including MEDLINE) 109 (Scopus) 340 (Other databases - refer to the methodology section) 7 study papers considered 2 study papers written in non-English language (Turkish) 5 primary studies included in this systematic review Figure 1. PRISMA Flow Chart showing the studies’ selection process which led to the inclusion of the five study. reviewers before embarking on the data extraction process, to test the comprehensiveness and objectivity of the extraction form. Some of the experts in the field pertinent to this review, including some of the included studies’ authors, were contacted to inquire about any ongoing or unpublished relevant studies. No relevant studies were declared by the authors. To objectively illustrate all the potential biases of the studies in a comparative style, a table has been compiled to evaluate the included studies against different checklist elements used to assess potential biases in RCTs [41–44]. The reviewers independently appraise and eventually agreed on the quality of the included studies (Table 1). The process of inclusion of relevant studies was independently conducted by the two authors, and consen- sus has been reached between the two reviewers with no need for a third opinion. Exclusion criteria included old studies conducted before 2005, study population with elbow pain/pathologies other than LE, and injection materials other than corticosteroid or PRP, including autologus blood and autologous conditioned plasma injections, which were excluded due to their irrelevancy. Results Eleven electronic databases were searched up to February 2017 without language, publication, or study- designs restrictions (Figure 1). Out of 732 papers identified in the body of literature, 43 study papers were considered after excluding duplicate papers, review articles, confer- ence papers and other irrelevant articles. Five studies [29,45–48], including 250 patients, were finally considered The data extraction process was performed before assessing the quality of the studies, to enhance the objectivity in collecting data regardless of the quality. The process of data extraction was carried out indepen- dently by the two reviewers as well. A pilot data extraction form [41] was created and tested by the two W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 4 Y: Yes; N: No; C: Consecutive sampling; U: Unclear; RCT: Randomised controlled trials; NS: Not stated; DD: Demographic details; DC: Disease characteristics; P: Population; I: Intervention; O: Outcomes; OM: Outcome measures; DSL: Different study locations; HD: Hand dominance; SH: Smoking history; SD: Symptoms durations; PI: Previous interventions; IPB: Inter-personal bias. Table 1. The result of quality assessment of the included studies. Study authors Internal validity Items External validity items Summary of biases Study design Were groups similar at the start of the trial? SelecƟon bias Performance bias Blinding of outcome assessors? Co-intervenƟon bias Incomplete outcome data Likelihood of aƩriƟon bias? + (Drop-out raƟo) SelecƟve reporƟng (ReporƟng bias) ReporƟng details are clear Groups analysed according to their original allocaƟon (intenƟon-to-treat analysis)? Other sources of bias Confounding factors Sampling method Are ParƟcipants representaƟve of the wider populaƟon? DD DC Random sequence generaƟon AllocaƟon concealmen Blinding of parƟcipants Blinding of treatment providers Peerbooms et al. (2010) RCT Y N Y Y Y U U Y N N (6:100) N P Y Y/N - PI - IPB DSL C Y - Different demographic variables. - Possible performance, inter-personal and co- intervenƟon biases. I Y O Y Gosens et al. (2011) RCT Y N Y Y Y U U Y N N (6:100) N P Y Y/N - PI - IPB DSL C Y As above I Y I Y Krogh et al. (2013) RCT N N Y Y Y N Y Y Y Y (44:60) at 1 year Y P Y Y/N - PI - HD - SH - SD C /U Y - Different demographic variables. - Possible performance, aƩriƟon, co-intervenƟon and reporƟng biases. - confounding factors. I Y O Y Gautam et al. Results (2015) NS/ RCT U U U U U U U + Y Y N U N P N U - PI U U Possible selecƟon, performance, co- intervenƟon and sampling biases. I Y O Y Omar et al. (2012) RCT U Y/U U U U U U Y N U N P Y / U U - PI U Y/U As above I Y O Y Y: Yes; N: No; C: Consecutive sampling; U: Unclear; RCT: Randomised controlled trials; NS: Not stated; DD: Demographic details; DC: Disease characteristics; P: Population; I Intervention; O: Outcomes; OM: Outcome measures; DSL: Different study locations; HD: Hand dominance; SH: Smoking history; SD: Symptoms durations; PI: Previou interventions; IPB: Inter-personal bias. Internal validity Items W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 5 Study Peerbooms et al. (2010) Gosen et al. (2011) Krogh et al. (2013) Gautam et al. (2015) Omar et al. (2012) Study Ɵtle PosiƟve Effect of an Autologous Platelet Concentrate in Lateral EpicondyliƟs in a Double-Blind Randomized Controlled Trial: Platelet-Rich Plasma Versus CorƟcosteroid InjecƟon With a 1-Year Follow-up. Ongoing PosiƟve Effect of Platelet-Rich Plasma Versus CorƟcosteroid InjecƟon in Lateral EpicondyliƟs: A Double-Blind Randomized Controlled Trial With 2-year Follow-up. Treatment of Lateral EpicondyliƟs With Platelet-Rich Plasma, GlucocorƟcoid, or Saline: A Randomized, Double- Blind, Placebo-Controlled Trial. Platelet-rich plasma versus corƟcosteroid injecƟon for recalcitrant lateral epicondyliƟs: clinical and ultrasonographic evaluaƟon. Local injecƟon of autologous platelet rich plasma and corƟcosteroid in treatment of lateral epicondyliƟs and plantar fasciiƟs: Randomized clinical trial. When trial conducted? May 2006 To January 2008 May 2006 - January 2008 January 2009 To July 2010 May 2011 To October 2012 October 2009 – May 2010 Study design RCT RCT RCT Not menƟoned. (Most probably an RCT, as per study details) RCT Conflict of interest No PotenƟal conflict of interest : One or more of the authors declared that Biomet has sponsored the study and supplied the Recover system used at a discounted rate. However, Biomet did not have any influence on the collecƟon and analysis of the data from this study. PotenƟal conflict of interest : One or more of the authors declared that: 1) The Danish RheumaƟsm AssociaƟon has supported the primary invesƟgator (T.P.K.) with a 6-month grant. 2) The Musculoskeletal StaƟsƟcs Unit at the Parker InsƟtute is sponsored by the Oak FoundaƟon. 3) Biomet Biologics Inc. has provided the Recover GPS II Platelet Concentrate SeparaƟon Kit and donated an unrestricted grant to the Region Hospital Silkeborg in Denmark. No No Funding (eg, commercial research suppor The study was sponsored by Biomet, Dordrecht, The Netherlands. (There was no involvement in the trial phases by the sponsor). - The Danish RheumaƟsm AssociaƟon - Oak FoundaƟon - Biomet Biologics Inc. None stated Not menƟoned IntervenƟons used - PRP(1) injecƟons - CS(2) injecƟons - corƟcosteroid injecƟons - PRP injecƟons - Saline - corƟcosteroid injecƟons - PRP injecƟons - corƟcosteroid injecƟons - PRP injecƟons Post- intervenƟon treatments - Acetaminophen only if necessary. - Standardized stretching protocol to follow for 2 weeks under the supervision of a physiotherapist. - A formal eccentric muscle- and tendon-strengthening program was iniƟated aŌer this stretching protocol. Internal validity Items - Acetaminophen when necessary. - Ice packs or Paracetamol rather than non-steroidal anƟ- inflammatory drugs. - Acetaminophen only for pain, but not to use non-steroidal anƟ-inflammatory medicaƟon. any re- intervenƟons or other treatments given to paƟents aŌer the trial - Yes (18 parƟcipants received re-intervenƟons): - IntervenƟons in CS group: 6 operaƟons, 1 re-injecƟon CS, 6 injecƟons with PRP. - IntervenƟons in PRP group: 3 operaƟons, 2 injecƟons with CS. (P: 0.970) Yes (20 parƟcipants received re- intervenƟons). IntervenƟons in CS group: 6 operaƟons, 1 re- injecƟon CS, 7 injecƟons with PRP. IntervenƟons in PRP group: 3 operaƟons, 3 injecƟons with CS. Yes (42 paƟents received further treatments). 26 paƟents chose PRP, 12 paƟents received CS, 2 had surgical treatment and 2 received sclerosing injecƟons. (Unclear what their originaƟng group was). Not menƟoned Not menƟoned Was the study completed (All stages) Yes Yes No (Priori calculaƟons were originally made on an anƟcipated 12- month results, which weren’t establish because of high level of paƟents' drop-out at 12-month follow-up). Study was ended at 3- month follow-up. Yes Yes Drop-out rate among parƟcipants - CS group: 1 patient. - PRP group: 3 patients lost to follow-up temporarily. (P: 0.700) - 3 patient from each group lost to follow- up temporarily. 44/60 dropped out & chose alternative treatments. Not menƟoned Not menƟoned W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 6 Table 3. Population characteristics of the review studies. Study Number of parƟcipants Mean age of parƟcipants (± SD) (1) ParƟcipants’ Gender raƟo (M:F) Ethnic groups of parƟcipants Clinical problem (disease) Disease duraƟon Co- morbidiƟes in Previous treatments received CS (2) PRP (3) Saline CS PRP Saline Peerbooms et al. (2010) 100 47.3 (±7.6) 46.9 (±8.4) IR (4) 25:26 23:26 IR NM (5) LE (6) ≥ 6 M (7) NM - Cast immobilizaƟon - CS InjecƟons - Physiotherapy (All were received ≥ 6 M before study intervenƟons). (P: 0.797) (P: 0.840) Gosen et al. (2011) 100 47.3 (±7.8) 46.8 (±8.5) IR 23: 26 23: 28 IR NM LE ≥ 6 M NM The same as above. (P: 0.780) (P: 0.712) Krogh et al. (2013) 60 (Only 40 trialled for PRP Vs CS) 43.9 (+ 8.7) 47.6 (+ 7.1) 44.7 (+ 7.9) 11:9 9:11 9:11 NM LE 23.1 M (±38.) NM - CS for 35 out of 60 paƟents ( Given ≥ 3M before the intervenƟon) Gautam et al. Internal validity Items (2015) 30 NM NM IR NM NM IR NM LE ≥ 6 M NM - Oral medicaƟons. - Non-invasive treatments: (not menƟoned what they were specifically). paƟents’ ages were between (18- 60) Omar et al. (2012) 60 (only 30 trialled for LE) 37.5 (±17.5) 40.5 (± 15.5) IR 5:10 6:9 IR NM LE NM NM No treatments were given at least 4 weeks before the intervenƟon. (1) SD: Standard deviation. (2) CS: corticosteroid. (3) PRP: platelet-rich plasma. (4) IR: Irrelevant. (5) NM: Not mentioned. (6) LE: Lateral Epicondylitis. (7) M: Month. (P: P W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 7 7 Table 4. Details of steroid and PRP preparations used by the included studies. Steroid preparaƟons Study Type of corƟcosteroid used Dose and concentraƟon Type, dose & concentraƟon of local anaestheƟc used Peerbooms et al. (2010) Triamcinolone acetonide 1 ml (40 mg/ml) Bupivacaine hydrochloride 0.5% + Epinephrine (1:200000) Gosens et al. (2011) Triamcinolone acetonide 1 ml (40 mg/ml) Bupivacaine hydrochloride 0.5% + Epinephrine (1:200000) Krogh et al. (2013) Triamcinolone 1 ml (40 mg/ml) 2 ml lidocaine 10 mg/mL Gautam et al. (2015) Methylprednisolone 2 ml (40mg/ml) NIG (1) Omar et al. (2012) NIG NIG NIG PRP preparaƟons Study volume Platelets concentraƟon (compared to platelets concentraƟon in blood) DuraƟon between blood collecƟon and PRP injecƟon Exogenous platelets acƟvaƟon Buffering agent Peerbooms et al. (2010) 3 ml NIG Around 30 minutes No 8.4% sodium bicarbonate + bupivacaine hydrochloride 0.5% + epinephrine (1:200000) Gosens et al. (2011) 3 ml NIG Around 30 minutes No As above Krogh et al. (2013) 3- 3.5 ml 8 folds Injected directly aŌer preparaƟon NIG 8.4% sodium bicarbonate Gautam et al. (2015) 2 ml NIG NIG NIG NIG Omar et al. (2012) NIG At least 2 folds NIG NIG NIG Table 4. Details of steroid and PRP preparations used by the included studies. (1) NIG: No information given has slower but longer-term clinical effect with no recurrence of symptoms over the follow-up periods included. This was almost the opposite with the use of corticosteroid injections. in this systematic review After reading full-text papers. The quality appraisal of the included studies is summar- ised in Table 1. In addition, Studies’ characteristics, demographic details and details of treatment preparations have also been listed (Tables 2–4). (1) NIG: No information given Internal validity Items Safety of interventional materials used Besides some of the known local adverse effect [49,50], no other complications or systematic effects were reported (Table 8). Except for one study [29], The lack of details regarding the rate/number of complications incidence ( ) Analytic data of the included studies With careful observation of the studies’ outcome results (Tables 5–7), it can be concluded that there was a general trend by most of the studies showing that PRP W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 8 Table 5. Values for Visual analogue scale (VAS) & Disabilities of the Arm, Shoulder and Hand (DASH) as reported by the included studies. (1) SD: Standard deviation; (2) CS: Corticosteroid; (3) PRP: Platelet-rich plasma. (P: P value). Visual analogue scale (VAS) Follow-up intervals study name & intervenƟons used Baseline Value ±SD(1) 2 Weeks 1 month (4weeks) 6 weeks 8 weeks 3 months (12 weeks) 6 months (26 Weeks) 52 weeks 104 weeks Scale used Peerbooms et al. (2010) CS(2) 65.8 ±13.8 44.2 ±26.4 42.9 ±29.2 44.2 ±27.1 56.6 ±23.2 50.1 ±28.1 (0 - 100) PRP(3) 70.1 ±15.1 55.4 ±24.2 46.9 ±24.9 38.7 ±27.2 32.6 ±31.5 25.3 ±31.2 Gosen et al. (2011) (Data according to intenƟon-to- treat analyses) CS 66.2 ± 14.0 (P: 0.340) 44.3 ± 26.3 ( P: 0.023) 43.4 ± 28.9 ( P: 0.411) 45.5 ± 27.1 ( P: 0.319) 55.8 ± 24.1 ( P: < 0.001) 48.8 ±27.0 ( P: < 0.001) 42.4 ±26.8 ( P: < 0.001) (0 - 100) PRP 69.0 ± 15.9 55.7 ± 24.1 47.7 ± 25.0 40.2 ± 27.5 32.9 ± 30.8 25.9 ± 30.6 21.3 ±28.1 Krogh et al. (2013) CS N o t a p p l i c a b l e PRP Gautam et al. (2015) CS 7.0 ±0.8 (P: 0.650) 2.1 ±0.7 (P: 0.000) 1.4 ±0.5 (P: 0.000) 1.7 ±0.5 (P: 0.493) 2.9 ±1.2 (P: 0.001) (0 - 10) PRP 7.1 ±0.8 4.5 ±1.1 2.7 ±0.8 1.8 ±0.6 1.6 ±0.5 Omar et al. (2012) CS 8.6 ±1.6 4.3 ±2.1 (P:<0.001) (0 - 10) PRP 8.0 ±1.4 3.8 ±1.9 (P:<0.001) DisabiliƟes of the Arm, Shoulder, and Hand QuesƟonnaire (DASH) Follow-up Intervals Study name and interventions used Baseline ±SD 2 Weeks 1 month (4 weeks) 6 weeks 8 weeks 3 months (12 weeks) 6 months (26 Weeks) 52 weeks 104 weeks Peerbooms et al. (1) SE: Standard error. (2) CS: Corticosteroid. (3) PRP: Platelet-rich plasma. (2) CS: Corticosteroid. (3) PRP: Platelet-rich plasma. Internal validity Items (2010) CS 131.2 ± 58.2 97.4 ± 69.0 84.7 ± 73.4 92.2 ± 68.7 117.3 ± 75.6 108.4 ± 82.2 PRP 161.3 ± 62.4 135.9 ± 78.0 113.4 ± 79.6 92.0 ± 78.8 79.5 ± 80.3 54.7 ± 73.2 Gosen et al. (2011) (Data according to intenƟon-to- treat analysis) CS 43.3 ± 16.1 ( P: 0.002) 31.2 ± 20.8 ( P: 0.005) 28.3 ± 22.2 (P: 0.060) 32.3 ± 21.7 (P: 0.813) 37.6 ± 23.1 (P: 0.037) 36.8 ± 24.0 (P: < 0.001) 36.5 ± 23.8 (P: < 0.001) PRP 54.3 ± 19.5 43.1 ± 21.6 37.2 ± 24.7 21.3 ± 22.0 27.8 ± 24.7 20.0 ± 23.5 17.6 ± 24.0 Krogh et al. (2013) CS PRP Gautam et al. (2015) CS 67.5 ±6.9 ( P: 0.378) 39.7 ±6.7 (P: 0.000) 32.7 ±4.1 (P: 0.003) 34.3 ±3.3 (P: 0.675) 39.6 ±1.0 (P: 0.012) PRP 69.7 ±6.1 51.6 ±6.8 38.6 ±5.7 33.6 ±5.1 32.0 ±4.5 Omar et al. (2012) CS 57.3 ±10.3 20.2 ±14.0 (P<0.001) PRP 58.9 ±10.5 19.9 ±12.9 (P<0.001) 1) SE: Standard error. (2) CS: Corticosteroid. (3) PRP: Platelet-rich plasma. 8 W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 Table 5. Values for Visual analogue scale (VAS) & Disabilities of the Arm, Shoulder and Hand (DASH) as reported by the included studies. (1) SD: Standard deviation; (2) CS: Corticosteroid; (3) PRP: Platelet-rich plasma. (P: P value). Table 5. Values for Visual analogue scale (VAS) & Disabilities of the Arm, Shoulder and Hand (DASH) as r studies. (1) SD: Standard deviation; (2) CS: Corticosteroid; (3) PRP: Platelet-rich plasma. (P: P value). W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 9 Table 6. Outcome values for Patient-Rated Tennis Elbow Evaluation (PRTEE), Oxford elbow score, Modified Mayo score and Hand grip strength at different follow-up intervals. es for Patient-Rated Tennis Elbow Evaluation (PRTEE), Oxford elbow score, Modified Mayo score and Hand t follow-up intervals. Table 6. Outcome values for Patient-Rated Tennis Elbow Evaluation (PRTEE), Oxford elbow score, Modifi grip strength at different follow-up intervals. Outcome measure used Patient-Rated Tennis Elbow Evaluation (PRTEE) Krogh et al. tandard error. (2) CS: Corticosteroid. (3) PRP: Platelet-rich plasma. (4) SD: Standard deviation. (P:P value Internal validity Items (2015) Corticosteroid injections Platelet- rich plasma Corticosteroid injections Total:15 Patients Platelet-rich plasma Total:15 Patients DA (1) (Mean Value) ±SD (α) 0 (A) (3.2) ± 0.9 (3.8) ± 0.4 Δ DA (2) (Mean Value) SE (β) 3 M (B) (- 3.0) 0.2 (- 0.4) 0.2 6 M (C) TT (3) (Mean Value) ±SD 0 (5.1) ± 0.8 (5.4) ± 0.6 Δ TT (4) (Mean Value) SE 3 M (- 0.2) 0.1 (0.3) 0.1 6 M No. & (%) TCET (5) 0 5 (33) 10 (67) 3 M 4 (27) 8 (53) 6 M 5 (33) 4 (27) No. & (%) OCET (6) 0 7 (47) 7 (47) 3 M 3 (20) 6 (40) 6 M 2 (13) 1 (7) No. & (%) RTCET (7) 0 2 (13) 3 (20) 3 M 4 (27) 2 (13) 6 M 12 (80) 1 (7) No. & (%) PIT(8) 0 15 (100) 15 (100) 3 M 9 (60) 10 (67) 6 M 10 (67) 6 (40) No. & (%) CELE (9) 0 9 (60) 14 (93) 3 M 11 (73) 14 (93) 6 M 11 (73) 14 (93) A D l i i (G d 4) ( ) D DA Ch i D l i i ( ) TT Thi k f d (i (1) DA: Doppler activity (Grades 0–4); (2) D DA: Change in Doppler activity; (3) TT: Thickness of common extensor tendon (in millimetres); (4) D TT: Change in Thickness of common extensor tendon; (5) No. and (%) TCET: Number and percentage of patients who had tears in the common extensor tendon; (6) No. and (%) OCET: Number and percentage of patients who had oedema in the common extensor tendon; (7) No. and (%) RTCET: Number and percentage of patients who had reduced thickness of the common extensor tendon; (8) No. and (%) PIT: Number and percentage of patients who had probe-induced tenderness; (9) No. and (%) CELE: Cortical erosion at the lateral epicondyle. (a) SD: Standard deviation; (b) SE: Standard error. (A) 0: baseline assessment; (B) 3M: 3 month; (C) 6M: 6 months. the two interventions at 2 years, by Gosens and co- researchers. However, and as the two studies were not conducted by exactly the same researchers, and with two among the participants prevents any quantitative analysis to assess the prevalence of these complications among studies’ participants. Internal validity Items (2013) Follow-up intervals Baseline 1 month 3 months Outcome measure elements (Mean Pain score: 0-50) & SE (1) (Mean FuncƟonal score: 0- 100) & SE (Mean Change in Pain) & SE (Mean Change in Disability) & SE (Mean Change in Pain) & SE (Mean Change in in Disability) & SE Interventions CS (2) (28.0) 8.0 (51.1) 22.3 (- 9.8) 2.2 (- 21.9) 4.3 (- 7.1) 2.2 (- 13.8) 4.3 PRP (3) (27.5) 7.5 (51.5) 19.1 (- 0.5) 2.2 (- 5.2) 4.3 (- 6.0) 2.2 (- 16.6) 4.3 Saline (25.0) 7.3 (47.1) 22.3 (- 1.7) 2.2 (- 3.4) 4.3 (- 3.3) 2.2 (- 7.6) 4.3 Outcome measure used Oxford Elbow Score (Value) ±SD (4) Gautam et al. (2015) Follow-up intervals Baseline 2 weeks 6 weeks 3 months 6 months Interventions CS (31.2) ±4.1 (P: 0.015) (39.7) ±3.4 (P: 0.001) (41.5) ±2.5 (P: 0.045) (41.7) ±2.4 (P: 0.029) (36.3) ±5.9 (P: 0.007) PRP (27.4) ±3.9 (34.7) ±4.3 (39.3) ±3.1 (39.3) ±3.3 (41.2) ±2.7 Outcome measure used Modified Mayo Score (Value) ±SD Gautam et al. (2015) Follow-up intervals Baseline 2 weeks 6 weeks 3 months 6 months Interventions CS (56.8) ±5.4 (P: 0.770) (68.5) ±3.9 (P: 0.000) (70.4) ±3.2 (P: 0.017) (69.6) ±3.5 (P: 0.578) (61.5) ±5.8 (P: 0.000) PRP (56.1) ±6.9 (61.3) ±3.1 (67.7) ±2.6 (70.2) ±2.2 (70.7) ±3.0 Outcome measure used Hand grip strength (Value) ±SD Gautam et al. (2015) Follow-up intervals Baseline 2 weeks 6 weeks 3 months 6 months Interventions CS (19.2) ±4.6 (P: 0.683) (25.5) ±4.9 (P: 0.159) (25.5) ±6.0 (P: 0.976) (25.8) ±6.7 (P: 0.884) (23.3) ±6.5 (P: 0.258) PRP (18.5) ±5.1 (22.5) ±6.6 (25.5) ±6.3 (25.5) ±5.6 (25.9) ±6.2 SE: Standard error. (2) CS: Corticosteroid. (3) PRP: Platelet-rich plasma. (4) SD: Standard deviation. (P:P value) W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 10 Table 7. Ultrasonographic outcome data reported by 2 studies. Table 7. Ultrasonographic outcome data reported by 2 studies. 7. Ultrasonographic outcome data reported by 2 studies. Study name & Interventions used The Outcome measures used (at 3 different follow-up periods) Krogh et al. (2013) Gautam et al. Internal validity Items distinctive articles with study titles, it appears that the authors in the first study did not originally plan to measure the patient’s outcomes at 2 years. This have entailed some changes in the studies settings, including the use of two different variants of one of the main outcome measures used (DASH). This was declared by one of the authors who stated that all DASH items were summed in the first study, yet the core elements only were used to calculate the DASH score in the second study. Secondly, the two studies were conducted at two teaching hospitals, with no details about which patients Discussion Five primary studies were included in this systematic review (Table 2). All these studies were high in the hierarchy of evidence and followed the randomised controlled trials (RCTs) design [51]. Two of these studies [45,46] were merely a two-stage trial rather than two distinctive studies. Initially, Peerbooms and colleagues investigated the effect of corticosteroid versus PRP injections, with an end-point assessment at 1 year, which was followed by an assessment of the longer-term effect of W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 11 Table 8. Adverse effects of injection therapies as reported by the review studies. Study Adverse effects Peerbooms et al. (2011) Local inflammation at the injection site, which caused increased pain 3 to 4 weeks after the injection Gosens et al. (2011) Initial worsening of localised pain (1 - 2 weeks). Krogh et al. (2013) Generic adverse effects PRP Corticosteroid post injection pain - persistent pain (4 subjects) - Reduced elbow movement (3 subjects) - localised skin rash (1 subject) - local skin atrophy (3 subjects) - loss of skin pigmentation (1 subject) - Persistent pain (1 subject) Gautam et al. (2015) NIG Omar et al. (2012) NIG (NIG: No information given) (NIG: No information given) were assessed in each hospital, and how much the hospital settings were similar, and thus more details could have eliminated any potential bias. [52,53], the variation in this demographic element (smoking) can be considered as a confounding factor especially if we observe the comparable therapeutic effect noted in saline (placebo) group compared to corticosteroid and PRP groups. While consecutive sampling was adopted by two studies [45,46], no details provided by the other studies [29,47,48]. Population’s details, which are important to assess the external validity, varied widely among the included studies (Table 3). Age and gender ratios of participants were generally representative of the popu- lation, although no demographic data were provided by one study [47]. Also, and while Krogh and co-researchers (2013) [29] provided the most extensive details of patient’s demographic data including smoking history, the percentage of patients who were current smokers varied widely among the three study arms, which ranged between 10 % in saline group, to around one-third in PRP and glucocorticoid groups. W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 Additionally, and although Krogh and co- researchers avoided this potentially confounding element by using saline as a placebo injection, the saline material itself could be considered more than an inactive compara- tor substance, with its possible beneficial/harmful effects as well. Regarding the cost-effectiveness of the injection treatments used, PRP injections may not be of interest to healthcare providers with an overall cost of around ($ 840–1000), compared to approximately one-third of this for corticosteroid injections [45,54]. However, the higher rate of symptoms recurrence among the relatively cheap corticosteroid injections means that the expenses of new treatments, including the higher costs of surgical treat- ments for example [54], which might be required after these cheap modalities fail, may entail higher cost than a single PRP injection. The use of different types, doses and concentrations of the interventional steroid preparations (Table 4) should be noted, especially with the known difference of the therapeutic effect of these preparations [55,56]. Addi- tionally, the combined use of local anaesthetics with steroid preparations in some studies [29,45] can be a source of bias as well, as these anaesthetic agents add volume to the injectate and help distributing the corticosteroid substance within the affected tissues [57], with possible changes in the effect of corticosteroid injections. This kind of heterogeneity can be seen more profoundly with the use of PRP injections, where different preparation methods, various commercial centrifugation/separation systems as well as different concentrations of platelets, growth factors and other blood products in the PRP preparations were used [58– 63] (Table 4). With no ideal concentrations of platelets and growth factors in the PRP preparations has been established yet, these sources of variability were clearly observed in the included studies. This in addition to technical details including centrifugation speed, buffer- ing agents used to achieve physiological pH, and the time needed for the centrifugation of blood. In all of the included studies, the participants were provided with some forms of post-injection therapies, including different physiotherapeutic modalities [29,45,46]. It should be noted here that these modalities are known for their therapeutic effect in lateral epicondyl- opathy [66,67], and again this might have created a source of uncertainty when assessing the real effect of the injected treatments. Outcome measures used by the review studies It is clearly seen the degree of heterogeneity when observing the various outcome measures used by the included studies (Table 5). W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 12 have resulted in more pain in the early follow-ups compared to those who received single-injection cortico- steroid. This difference in injection technique may have distributed the injectates differently in the study arms, with possible subsequent different therapeutic effects in vivo. Additionally, the same study [29] adopted the practice of injecting local anaesthetic in the peritendon (the connective tissue sheath around the tendon) before administering the interventional treatments, with possible subsequent detrimental effects on tendon cells viability and thus change in treatment effect [64]. Also, the possible mix of lidocaine with PRP at nearly the same site, could have reduced the therapeutic effect of PRP, as it is shown that local anaesthetics reduce the positive effects of PRP [65]. It is unclear how much these details could have affected the treatment delivery and subsequently the therapeutic effect of the used treatments. Finally, and with the inter-personal bias in mind, Peerbooms and co- researchers [45] reported that treatments were given by more than one person of different level of experience (consultant and resident doctors), which might entail that treatments could have been given differently. This confounding factor has not been ruled out by two of included studies [47,48]. Treatments received by the participants before start- ing the trials is another important element. Although most studies reported that patients received no treatments in the last 3–6 months before the interventions, there is still a potential long-term effect of any treatments given before that, this in addition to the possible treatment-treatment interactions after receiving the new interventional treat- ments. This might be of real significance if we noted, for instance, that steroid injection was received by as high as 58 % of patients received corticosteroid injections in the trial by Krogh and co-researchers [29]. Additionally, and with the considerable variation of the disease duration before the treatment interventions, it is unclear how far this kind of heterogeneity has a role when assessing the comparative effect of the treatments given. Injection materials used While the use of cortico- steroid as a control injection can be acceptable to compare the effect of PRP, the use of such medicinal therapeutic agent itself may undermine the accurate assessment of the effect of PRP as a treatment option. For example, the probable deleterious effects of corticosteroid [17] may falsely exaggerate the beneficial effect of PRP in comparison. Discussion With the known correlation between smoking and delayed tissue healing The randomisation process and allocation of study population was not reported by two studies [47,48], which entails a potentiality of selection bias, and questions the quality of these two high-level evidence RCTs. Addition- ally, the lack of blinding of outcome assessors was one of the main limitations faced by most of the included studies. Krogh and co-researchers [29] were the best who reported these elements. Peerbooms and co-researchers [45] only reported the blinding of patients to treatment interven- tions, Gautam and colleagues [47] only reported the blinding of ultrasound operator(s), while the study by Omar and co-researchers [48] lacked the information about all the elements of the blinding process. W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 When assessing the outcome measures used by the first two studies [45,46], and while there were minimal variations in the baseline VAS values between corticosteroid and PRP study arms, significant differences in the DASH baseline scores were noted (Table 5). This disparity necessitates careful interpreta- tion of the DASH outcome results by the two studies. While PRTEE has sufficient psychometric properties [68,69], it may lack the objectivity and thus the accurate assessment, as it depends solely on the subjective patient-rated evaluation, beside the fact that this was the only clinical outcome measure used by Krogh and Although all of the included studies used one-phase of treatment delivery of one injection, different injection sites, techniques and methods were used. While peppering technique (multiple tendon perforations) was adopted by two studies [45,47], Krogh and colleagues used 1 direct injection for corticosteroid while adopted peppering technique for PRP and saline injections [29], which could W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 13 colleagues [29] (Table 6). This also demonstrates the difficulty in comparing the clinical outcomes of this trial [29] with the other 4 studies which used VAS and DASH in their outcome measurement. corticosteroid injections could be attributed to the fact that these patients usually experience relatively rapid improvement of symptoms which encourage them to rely on the affected limb prematurely, with the possible re- injury of the newly treated tendon and subsequently a recurrence of symptoms [29,46]. The role of this factor can be investigated in the future, by observing the participant’s return to daily activities and assessing/ restricting their degree of reliance on the affected limb after receiving the treatment. Finally, and despite the strong argument which states that LE is a degenerative non-inflammatory process, the rapid therapeutic effect provided by corticosteroid injections needs to be justified here, which could hypothesise that the disease itself could be caused by a combined pathophysiological mechanisms, which could involve both inflammatory and degenerative processes. Drop-out of participants Variable drop-out rates were reported by the included studies. While (6%) reported by Gosens and colleague [46] by the end of their trials at 102 weeks, Krogh and colleagues [29] reported a 0% of drop-out at 3 months. W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 However, considering the high drop-out which faced Krogh and colleagues at 12 months (around 73%), it can be argued here that this could be the main reason for these researchers to limit the study follow- up to 3-month only. Interestingly, and with careful observation of this study, it can be seen that the drop- out rate among patients who received PRP was signifi- cantly less than that in corticosteroid group beyond the 6- month period. This could have been caused by the late ongoing therapeutic effect of PRP, which might have encouraged the patients to adhere to their original interventional treatment option (PRP). Limitations of this review The relatively small number of included studies, the ambiguity of some studies’ methodological details, and the potential biases noted in many of the trial’s aspects are the main limitations of this review. Additionally, and while the quantitative analysis (meta-analysis) was considered in this review, the varied outcome measures used and the different timings of follow- ups have hindered the chance to conduct a meta-analysis to draw a large-scale summative conclusion. Finally, and with the above reported data in mind, a few final observations should be elaborated here. Firstly, although most of the included studies followed the trend of rapid reduction then a regaining increase in the VAS and DASH values for the use of corticosteroid, there was a unique trend observed at 52 weeks and 104 weeks by the two consecutive studies [45,46]. This trend shows that the VAS and DASH values in the corticosteroid group started to fall again, which means that there were improvements in pain and function at these two follow-ups. Although this might be interpreted as an ongoing long-term improvement using corticosteroid, it should be remem- bered that this could have resulted from the fact that 13 out of 20 patients on the corticosteroid group have received further treatments (re-intervention) after the original treatments. Secondly, Krogh and co-researchers [29] reported that no superior outcomes can be observed with the use of PRP compared to saline and corticosteroid injections. This can be attributed to the fact that the study did not assess the longer term effects of PRP beyond 3 months, which could have shown improvement in the PRP group compared to corticosteroids group, especially that more clinical improvement started to appear in the PRP group in the period between the 1- and 3-month follow-ups. Funding for this research work No funding, whether from commercial or non-com- mercial bodies, was received to support this research work. ( ) 16. Wong MW, Tang YY, Lee SK, Fu BS, Chan BP, Chan CK (2003) Effect of dexamethasone on cultured human tenocytes and its reversibility by platelet-derived growth factor. J Bone Jt Surg (American Volume) 85-A(10) 1914– 1920. Conclusion 8. Küçüksen S, Yilmaz H, Salli A, Ugurlu H (2013) Muscle energy technique versus corticosteroid injection for man- agement of chronic lateral epicondylitis: randomized controlled trial with 1-year follow-up. Arch Phys Med Rehabil 94(11), 2068–2074. Platelet-rich plasma demonstrated better, although delayed, therapeutic effects compared to corticosteroid injections, with no subsequent regression of their positive clinical findings for up to 2 years, and with no reported adverse effects except for local discomfort felt at the injection site, as report by a single study. This conclusion can be also supported by the fact that no long gaps can be observed between the follow-ups conducted by all of the included studies, and thus minimal chance of missed data, which might demonstrate different change in effect, can be assumed. 9. Osborne H (2010) Stop injecting corticosteroid into patients with tennis elbow, they are much more likely to get better by themselves!. J Sci Med Sport 13(4), 380–381. 10. Walz DM, Newman JS, Konin GP, Ross G (2010) Epicondylitis: Pathogenesis, Imaging, and Treatment 1. Radiographics 30(1), 167–184. ( ) 11. Ahmad Z, Siddiqui N, Malik SS, Abdus-Samee M, Tytherleigh-Strong G, Rushton N (2013) Lateral epicondy- litis: a review of pathology and management. Bone Jt J 95-B (9), 1158–1164. ( ), 12. Chesterton LS, Mallen CD, Hay EM (2011) Management of tennis elbow. Dovepress J 2011(2), 53–59. W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 14 Conflict of interest ( ) 13. Carofino B, Chowaniec DM, McCarthy MB, Bradley JP, Delaronde S, Beitzel K, Mazzocca AD (2012) Corticoste- roids and local anesthetics decrease positive effects of platelet-rich plasma: an in vitro study on human tendon cells. Arthroscopy: J Arthroscopic Related Surg 28(5), 711– 719. The two authors of this systematic review declare no conflict of interest. Data sharing statement Additional details about this review, including the full original manuscript, are available on request by contact- ing the corresponding author. 17. Coombes BK, Bisset L, Vicenzino B (2010) Efficacy and safety of corticosteroid injections and other injections for management of tendinopathy: a systematic review of randomised controlled trials. Lancet 376(9754) 1751–1767. ( ) 18. Ciccotti MC, Schwartz MA, Ciccotti MG (2004) Diagnosis and treatment of medial epicondylitis of the elbow. Clin Sports Med 23(4) 693–705. Ethical approval 14. Han SH, An HJ, Song JY, Shin DE, Do Kwon Y, Shim JS, Lee SC (2012) Effects of corticosteroid on the expressions of neuropeptide and cytokine mRNA and on tenocyte viability in lateral epicondylitis. J Inflamm 9(40), 1–9. As a systematic review, no ethical approval was needed to conduct this research work, as there was no involvement or contact with human subjects. ( ) 15. Liu D, Ahmet A, Ward L, Krishnamoorthy P, Mandelcorn ED, Leigh R, Kim H (2013) A practical guide to the monitoring and management of the complications of systemic corticosteroid therapy. Allergy, Asthma & Clin Immunol 9(1), 1–25. W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 Additionally, the selective reporting by the same researchers [29] is difficult to be excluded here, especially that the researchers who clearly reported the higher clinical improvement in corticosteroid groups in the period up to 1-month follow-up, did not similarly acknowledge that this trend has reversed in favour of PRP in the subsequent follow-up. Thirdly, despite the comparatively longer-term therapeutic effects of PRP, the self-limited nature of LE should always be remem- bered, as this might have a role in the condition improvement on the mid- to long-term [70], although this effect was not observed in the corticosteroid groups who experienced deterioration in their symptoms on the mid- to long-term. Fourthly, the short-term therapeutic effect and high rate of symptoms recurrence following Recommendations for future research With the wide variation in using the injection treatments, there is a growing need to carefully assess and compare many of these treatments settings, aiming to adopt a unified preparation/technique, especially with PRP injections, where different separation systems, concentrations of blood components and injection techniques are used. For example, with the different injection methods used by various studies, the adoption a computer-guided injection technique [54] can be utilised to enhance needle placement and to avoid potential variability during the injection process. It can be also suggested that a unified injection protocol can possibly be used to standardise the mode of injection, including for instance, the specific use of peppering or single-dose injection, agreeing on a pre- determined injection site, defining specific numbers of injections, and to determine whether local injections will be used before/with corticosteroid and PRP injection. Furthermore, the use of post-injection therapeutic modal- ities should be regulated, by possibly avoiding or at least adopting a unified post-injection protocol. That also applies to the outcome measures used, as this can considerably help comparing the clinical outcomes more efficiently. These variables, if considered, can eliminate any potential variations and thus help towards unbiased assessment of the effect of the injection treatments. Finally, it should be noted here that none of the included studies reported any details about the ethnicity of the studies’ population, where different ethnic groups might have different response to different treatments. Investi- gating this variable in the future could reveal further information about the disease pathophysiology and its management. W. Ben-Nafa and W. Munro: SICOT-J 2018, 4, 11 Gautam VK, Verma S, Batra S, Bhatnagar N, Arora S (2015) Platelet-rich plasma versus corticosteroid injection for recalcitrant lateral epicondylitis: clinical and ultrasono- graphic evaluation. J Orthop Surg 23(1), 1–5. 32. de Vos RJ, Windt J, Weir A (2014) Strong evidence against platelet-rich plasma injections for chronic lateral epicondy- lar tendinopathy: a systematic review. Br J sports med 48 (12), 952–956. 48. 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Krogh TP, Fredberg U, Stengaard-Pedersen K, Christensen R, Jensen P, Ellingsen T (2013) Treatment of lateral epicondylitis with platelet-rich plasma, glucocorticoid, or saline: a randomized, double-blind, placebo-controlled trial. Am J Sports Med 41(3), 625–635. 46. Gosens T, Peerbooms JC, van Laar W, den Oudsten BL (2011) Ongoing Positive Effect of Platelet-Rich Plasma Versus Corticosteroid Injection in Lateral Epicondylitis: A Double-Blind Randomized Controlled Trial With 2-year Follow-up. Am J Sports Me, 39(6), 1200–1208. p ( ), 30. Gaujoux-Viala C, Dougados M, Gossec L (2009) Efficacy and safety of steroid injections for shoulder and elbow tendonitis: a meta-analysis of randomised controlled trials. Annals rheumatic diseases 68(12), 1843–1849. 31. Sheth U, Simunovic N, Klein G, Fu F, Einhorn TA, Schemitsch E, Bhandari M (2012) Efficacy of autologous platelet-rich plasma use for orthopaedic indications: a meta- analysis. J Bone & Jt Surg 94(4), 298–307. 47. References p ( ) 19. 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MLTJ Muscles, Ligaments Tendons J 4(1), 3–9. ( ) 69. MacDermid J (2005) Update: the patient-rated forearm evaluation questionnaire is now the patient-rated tennis elbow evaluation. J Hand Ther 18(4), 407–410. ( ), 70. Scher DL, Wolf JM, Owens BD (2009) Lateral epicondylitis. Orthopedics 32(4), 276–282.
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Nutritional status and food intake of patients with systemic psoriasis and psoriatic arthritis associated
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Nutritional status and food intake of patients with systemic psoriasis and psoriatic arthritis associated Marina Yazigi Solis1, Nathalia Stefani de Melo2, Maria Elisa Moschetti Macedo2, Fabiana Prata Carneiro2, Cid Yazigi Sabbag3, Antonio Hebert Lancha Junior4, Vera Silvia Frangella5 ORIGINAL ARTICLE ORIGINAL ARTICLE ABSTRACT an increased risk for chronic diseases related to obesity, worsening of the psoriatic lesions, and poor quality of life. Objective: To identify the nutritional status and food intake of individuals with systemic psoriasis and psoriatic arthritis associated. Methods: This is an exploratory and cross-sectional study with 34 men aged between 19 and 60 years seen at a Psoriasis Center. Participants were divided into systemic psoriasis group and arthritic- systemic psoriasis associated group. For nutritional assessment we used anthropometry, bioelectrical impedance analysis and whole- body plethysmography. Clinical and nutritional information were assessed using the clinical and nutritional history-taking, and the 24-hour dietary recall. For statistics the general linear model test (p < 0.05) was used. Results: According to the body mass index 29.4% patients (n = 10) were eutrophic, 41.2% (n = 14) overweight and 29% (n = 10) obese. Almost all individuals (60%; n = 21) had body fat percentage above normal levels (> 25%) and a high risk for metabolic complications according to the waist circumference and the obesity index, however, there were no statistically significant differences between groups. The mean food intake, total fat, calories and protein were above recommended levels, being 58.8% for lipids (319.17 ± 241.02 mg of cholesterol and 17.42 ± 11.4 g saturated fatty acids); 29.4% for calories and 67.6% for proteins. Thus, regardless of the psoriasis type, an excessive consumption of calories, lipids, fatty acids, cholesterol and a higher incidence of overweight were found. Conclusion: The sample showed an abnormal nutritional condition, Keywords: Psoriasis/complications; Obesity/complications; Feeding; Food consumption; Nutritional status; Risk factors Study carried out at the Brazilian Psoriasis Study Center – CBEP, São Paulo (SP), Brazil. 1 Nutrition and Metabolism Applied to Motor Activities Laboratory – São Paulo (SP), Brazil; Physical Education and Sports School, Universidade de São Paulo – USP, São Paulo (SP), Brazil; Brazilian Psoriasis Study Center – CBEP, São Paulo (SP), Brazil. 2 Centro Universitário São Camilo – São Paulo (SP), Brazil. 3 Brazilian Psoriasis Study Center – CBEP, São Paulo (SP), Brazil; Hospital Ipiranga – São Paulo (SP), Brazil. 4 Physical Education and Sports School, Universidade de São Paulo – USP, São Paulo (SP), Brazil; Nutrition and Metabolism Applied to Motor Activities Laboratory – São Paulo (SP), Brazil. 5 Centro Universitário São Camilo – São Paulo (SP), Brasil; Metabolism and Nutrition Institute – IMeN, São Paulo (SP), Brazil. Corresponding author: Marina Yazigi Solis – Praça Amadeu Amaral, 47 - 4º andar – Conjunto 47 – Paraíso – Zip code: 01327-010 – São Paulo (SP), Brazil – Phone: (11) 3285-1273 – E-mail: ma_yazigi@hotmail.com Received on: Jun 22, 2011 – Accepted on: Jan 20, 2011 Conflict of interests: None Study carried out at the Brazilian Psoriasis Study Center – CBEP, São Paulo (SP), Brazil. 2 Centro Universitário São Camilo – São Paulo (SP), Brazil. y Center – CBEP, São Paulo (SP), Brazil. Activities Laboratory – São Paulo (SP), Brazil; Physical Education and Sports School, Universidade de São Paulo – USP, São Paulo (SP), Brazil; Brazilian Psoriasis RESUMO Objetivo: Identificar o estado nutricional e o consumo alimentar de indivíduos com psoríase sistêmica e artropática associada. Métodos: Pesquisa exploratória e transversal, na qual avaliaram-se 34 homens, de 19 a 60 anos, atendidos em um Centro de Psoríase, separando-os em Grupo PS (com psoríase sistêmica) e Grupo PAS (com sistêmica mais artropática). A avaliação nutricional deu-se pelo emprego da antropometria; bioimpedância e plestimografia de corpo inteiro. Aspectos clínicos e nutricionais foram investigados pela anamnese clínica, nutricional e recordatório de 24 horas. Empregou-se o teste General Linear Model (p < 0,05) para avaliação estatística. Resultados: Segundo Índice de Massa Corporal, 29,4% (n = 10) apresentaram-se eutróficos; 41,2% (n = 14) com sobrepeso e 29% (n = 10) com obesidade. A maioria dos avaliados (60%; n = 21) apresentou valor da porcentagem de gordura (avaliada pela antropometria, bioimpedância e plestimografia de corpo inteiro) acima da normalidade (> 25%) e com risco alto para complicações einstein. 2012;10(1):44-52 Nutritional status and food intake of patients with systemic psoriasis 45 metabólicas segundo CC e índice de obesidade, sem diferença estatística significativa entre os grupos. O consumo alimentar médio de lipídio, calorias e proteína apresentou-se acima do recomendado, sendo 58,8% para os lípides (319,17 ± 241,02 mg de colesterol e 17,42 ± 11,4 g de ácidos graxos saturados); 29,4% para as calorias e 67,6% para as proteínas. Assim, independentemente do tipo de psoríase, encontrou-se consumo excessivo de calorias, lípides, colesterol e ácidos graxos, além de maior ocorrência de excesso de peso. Conclusão: A amostra apresentou estado nutricional comprometido, aumento do risco para doenças crônicas relacionadas à obesidade, agravamento das lesões e má qualidade de vida. psoriasis, observed that 30% of them also presented joint problems. The PsA is characterized by T and B cell infiltration leading to increased concentrations of IL-1, IL-6, IL-12, IL-15, IL-17, IL-18, interferon-γ and TNF-α in tissues that line the inner part of the joints and are known as synovial tissue. Because of the chronic inflammation provoked by the skin and joint disease, it is believed that individuals with psoriasis are subjected to systemic changes in the organism, such as insulin resistance, changes in the lipid profile, obesity and increased cardiovascular risk(7). Many studies point out a wide relationship between psoriasis and the development of associated chronic disease as dyslipidemia, hypertension, non-alcoholic fatty liver disease and type 2 diabetes mellitus, besides that, a high risk for heart diseases and metabolic syndromes. INTRODUCTION Psoriasis is a demartosis that affects about 2 to 3% of the world population and occurs in both sexes. Although it can happen at any age, studies indicate a high prevalence between 20 and 30 years, and also between 50 and 60 years of age. The incidence of psoriasis is higher in countries such as Finland, Iceland, Norway and Germany, and less common in dark-skinned persons of East Africa, Indians and Eskimo people. In the United States it is estimated that 8 million of persons have psoriasis, in Brazil approximately 2% of population has this disease(1). The literature reports that nutritional treatment applied to psoriasis patients (associated to the control of anthropometric and biochemical variables) gives more clinical stability, preventing related non- transmissible chronic diseases (NTCD), and provides long-term quality of life. In other words, the weight control improves the prognosis of psoriasis(2). On the other side, other studies indicate that dietary pattern established associated to life style may contribute to the development of psoriasis. Therefore, nutrition can influence psoriasis in two different ways, as the cause of the metabolic disorder or as the treatment and prevention. This disorder can affect the whole skin surface, but it is most common on the extensor surface of the limbs, scalp, nails, sacral and palmo-plantar regions. Psoriasis is classified according to the site of the lesion. When it appears as a chronic disease in plaque forms it is known as psoriasis vulgaris, if presented as drops on the skin it is called psoriasis guttata, which is usually found in young people. In addition, there is the inverse psoriasis found in the folds of the skin; the palmo-plantar psoriasis found in palms and sole of feet; and there is also the erythrodermic psoriasis that affects most of the body(2,3). However, despite the fact that nutrition is considered a tool for the treatment of psoriasis there are no national or international guidelines that recommend an adequate diet for such patients. Some authors suggest that several active compounds in the human diet perform an important role in the physiopathology of psoriasis, having the same impact as the monitorization of the diet energy supply and the total fat and saturated fat intake on the control of NTCDs. RESUMO The prevalence of metabolic syndrome in people with PsA or other psoriasis type was high when compared with those without psoriasis as suggested by Raychaudhuri et al.(8) and Cohen et al.(9). Descritores: Psoríase/complicações; Obesidade/complicações; Alimentação; Consumo alimentar; Estado nutricional; Fatores de risco INTRODUCTION Psoriasis can also be considered an autoimmune disease mediated by defense cells known as T lymphocytes (CD4 and CD8) that provoke a high proliferation of proinflammatory cytokines, such as interferon-γ, interleukins (IL) 1 and 6 and tumor necrosis factor-alpha (TNF-α), which increase skin lesions and causing also a chronic inflammatory status(4). Among these nutrients some vitamins and minerals (vitamins A, E, C and D and folic acid), omega 3 polyunsaturated fatty acids besides low-energy diets(10) are mentioned. It is believed that some vitamins (A, E and C), carotenoids and minerals (iron, copper, manganese, zinc and selenium) have antioxidants ability, which decrease oxidative stress and the production of reactive oxygen species, mainly in systemic inflammation like psoriasis(10). In 1818 a study by Alibert verified that psoriasis patients with skin lesions could also develop joint problems, which he named as psoriatic arthritis (PsA) (5). An epidemiologic study done in 2002 by Zachariae et al.(6) with 5,000 patients from Denmark, Finland, Norway and Sweden who had different types of einstein. 2012;10(1):44-52 Solis MY, Melo NS, Macedo ME, Carneiro FP, Sabbag CY, Lancha Junior AH, Frangella VS 46 In addition, food fibers also play an important role in systemic inflammation, decreasing oxidative stress produced by large amounts of sugar, also improving glycemic, insulin and lipidemic control. of health risk, assurance of anonymity and privacy of personal information; the possibility of leaving the study whenever they decided to do so without any kind of uneasiness or punishment; they were informed that their participation did not imply in expenses or payment; if leaving the study at any time as desired would have no influence in the treatment or loss of any of the acquired benefits. All individuals who decided to join the study signed the Informed Consent Form (ICF), which included a statement authorizing the authors to report results in congresses or scientific journals if applicable. Population, sample, inclusion and exclusion criteria The center where the research was carried out receives about 30 to 60 new cases of psoriasis each month of both sexes aged between 20 and 65 years. The sample included men with systemic psoriasis associated with psoriatic arthritis and who fit the following inclusion criteria: aged between 19 and 60 years, male gender (to evaluate the natural cycle of psoriasis without the changes of the aging process, of gestation hormones or of menstrual cycle), be diagnosed with psoriasis for at least three years because at this time the clinical features will be well defined, using laser therapies, topical medication, oral medicines like methotrexate, cyclosporine and biological medicines, being diagnosed with systemic psoriasis associated with psoriatic arthritis. - cultural and socioeconomic background, such as name, age, city of birth, place of birth, address, occupation, marital status, use of medicines and practice of physical activity; - family background: parents, grandparents and uncles health status; - clinical background: (prior and current disease): prior health problems, allergies, medicine currently used, test results, clinical or surgical treatment performed; - nutriticional background (food or dietary): weight gain or loss, anthropometric measures as weight, height, circumference and skinfolds; We excluded women, those patients aged less than 19 years and more than 60 years, who had mild psoriasis forms, and did not fit psoriasis classification as defined in the study. - 24-hour dietary recall: it enabled to define and measure food and drink intake in the 24 hours before the interview. To do so, we provided booklets with examples of utensils and portions to help volunteers. OBJECTIVE To define, identify and associate the nutritional status and food intake of patients with systemic psoriasis and associated psoriatic arthritis seen at a Psoriasis Center. Design and Setting The psoriasis area severity index (PASI) was used by the responsible physician to classify and describe the disease. The total score of the PASI was measured based on the evaluation of four body surface areas (head, chest, upper and lower limbs) using the method described by Harari et al(11). The selected sample had only individuals who presented PASI ≥ 18, which is classified as a high severity. After selecting the patients, all participants were interviewed to gather clinical and nutritional history containing the following issues: This is cross-sectional, quantitative, analytical study of exploratory approach performed during 1 year and 5 months that started on December 2009. Data collection was done at the Brazilian Psoriasis Study Center in São Paulo city. einstein. 2012;10(1):44-52 Ethical issues We also evaluated the lack or the use of medicines regarding dosage and administration hours before the appointment at the center. The study was approved by the Research Ethics Committee of the Centro Universitário São Camilo, registered under the number 193/09, which addresses the resolution 196 of October 10, 1996 stated by the Brazilian National Health Council that supervises researches using human beings. To evaluate body composition three different methods of nutritional assessment were applied, and made available in distinct moments to the subjects, mainly those that did not show an uncontrolled risk or danger to the participants. All participants were informed of the study purpose, as well as the procedures to be done, the lack Nutritional status and food intake of patients with systemic psoriasis 47 - Anthropometry: the volunteers’ weight was checked using a digital balance (Plenna®) with 150 kg weighting capacity and division of 100 g placed on the floor level. For height (m) we used a metric stadiometer (Sanny®) of 150 cm placed on the wall. These measures used to calculate the body mass index (BMI) given by weigh/height², and the result was classified according to the World Health Organization (WHO)(12). Using the techniques described in the literature we measured the patients’ right hemi body, the arm circumference, waist and hip circumference with an inelastic tape from Sanny®(13). After gathering these measures the relations hip waist/hip circumference was calculated and the results classified as stated by WHO(14). To evaluate the regional body fat or topographic evaluation the method proposed by Lohmann et al. was used(15). Finally, the skinfold measures were taken by a fat caliper from Lange® of 1.0 mm of precision and maximum amplitude of 65 mm. The body areas measured followed the standards proposed in the literature, and were taken by a skilled evaluator. Each fold was measured in three different times and the final results determined by the mean values. We assessed skinfolds at biceps, triceps, subscapular and suprailiac. From the obtained results we calculated the fat percentage based on the equation of Durnin et al.(16). model® was used. In the analysis the participants were positioned on the stretcher in dorsal decubitus without watches or any metallic objects. Before placing skin-electrodes, we cleaned points of contact with alchool 70%. Patients rested for 3 minutes before the measurement was performed. waist circumference (m) C index = C index = Ethical issues The equipment report provided: (1) fat mass (fat percentage and body fat (Kg)); (2) thin mass (muscles, bones and viscera); (3) whole body water (liters and water percentage in thin mass); ( ) Whole body plethysmography (Air Displacement Plethysmography, BOD POD®, body composition system; Life Measurement Instruments, Concord, CA). The evaluation was done using the criteria described in the manual of Fields et al.(19). After the equipment calibration, participants were evaluated in a seated position, wearing minimal clothes, a swimming cap, and without any metallic objects like earrings, rings, necklace, etc. Variations between pressure and volume were measured to define body density of each subject using the equation of Siri(20). When the data obtained were insufficient, the software itself performed a new evaluation until it got the adequate screening. Statistical analysis i i l l i Statistical analysis was performed using the General Linear Model test (GLM) for multivariate and unbalanced analysis comparing PS group (systemic psoriasis) with SPA group (systemic psoriatic arthritis associated). A significance level of p<0.05 was used. In addition, Shapiro-Wilk test was adopted to verify the normal sample distribution. The conicity index (C index) was calculated to determine obesity, body fat distribution and risk to diseases associated to excessive weight by the equation of Valdez et al.(17): C index = waist circumference (m) 0109 × √ body weight (kg) ÷ height (m) RESULTS Relationship between length of time of psoriasis and related diseases Group Length of time of psoriasis (years) Evidence of related diseases (%) PS group 14.96 ± 7.24 36 yes 64 no SAP group 16.75 ± 9.59 44.4 yes 55.6 no Total 15.41 ± 7.93 38.2 yes 61.8 no PS: systemic psoriasis; PAS: systemic arthropathy associated. Figure 1. Patients’ distribution by length of time of psoriasis and related Table 1. Relationship between length of time of psoriasis and related diseases Group Length of time of psoriasis (years) Evidence of related diseases (%) PS group 14.96 ± 7.24 36 yes 64 no SAP group 16.75 ± 9.59 44.4 yes 55.6 no Total 15.41 ± 7.93 38.2 yes 61.8 no PS: systemic psoriasis; PAS: systemic arthropathy associated. Figure 1. Patients’ distribution by length of time of psoriasis and related Table 1. Relationship between length of time of psoriasis and related diseases Group Length of time of psoriasis (years) Evidence of related diseases (%) PS group 14.96 ± 7.24 36 yes 64 no SAP group 16.75 ± 9.59 44.4 yes 55.6 no Total 15.41 ± 7.93 38.2 yes 61.8 no PS: systemic psoriasis; PAS: systemic arthropathy associated. Table 1. Relationship between length of time of psoriasis and related diseases Group Length of time of psoriasis Evidence of related Table 1. Relationship between length of time of psoriasis and related diseases The mean BMI was 28.01± 4.42 kg/m². In the PS group it was 27.86 ± 4.55 kg/m² and in the PAS group it was 28.42 ± 3.99 kg/m² without significant differences regarding BMI in both groups (p> 0.05). Based on the WHO20 classification, 10 patients (29.4%) showed eutrophy using the BMI as a parameter, 14 (41.2%) were overweight, 7 (20.6%) were obese class I, 2 (5.9%) obese class II, and 1 (2.9%) obese class III. These results showed that 24 patients (70.6% of the sample) had excessive weight. In the PS group 7 individuals (28%) had eutrophy, 12 (48) were overweight, 4 (16%) were obese class I, 1 (4%) obese class II, and 1 (4%) obese class III. The SAP group had 3 individuals (33.3%) with eutrophy, 2 (22.2%) were overweight, 3 were (33.3%) were obese class and 1 (11.1%) obese class II (Table 2). Figure 1. Patients’ distribution by length of time of psoriasis and related Figure 1. RESULTS The sample had 34 patients divided into two groups. The PS group had 25 individuals (73.5%) and the SAP group had 9 (26.5%) patients. The participants mean age was 40.94± 11.19 years. The PS group mean age was 38.84 ± 10.57 years and in the SAP group it was 46.78 ± 10,77 years. No statistical significance was observed between groups as related to age (p>0.05). In both groups, a total of 25 individuals (73.5%) were sedentary and 9 (26.5%) did some physical activity. Both groups had sedentary individuals, which comprised 76% of PS group and 66.7% of SAP group. Therefore, in our study sedentarism was a characteristic of the psoriasis patients, which constituted 73.5% of all patients of the sample. The index of central obesity (ICO), a new parameter for obesity, was also calculated. Some studies have considered the waist circumference is not sufficient to measure the total body fat, mainly because height differs in each race and sex. Therefore, the ICO is considered the best parameter to determine central obesity, which is calculated by dividing the waist circumference (m) by the height (cm). This study cutoff point took the weight mean as used in many countries and the waist circumference as suggested by the International Diabetes Federation (IDF) for metabolic syndrome(18), being 1.18 for women and 1.25 for men. - Bioelectrical impedance (BIA): this methods enabled to obtain body water composition and fat mass of the participants. To do so a Biodynamics 310 - Bioelectrical impedance (BIA): this methods enabled to obtain body water composition and fat mass of the participants. To do so a Biodynamics 310 A total of 12 patients (32.4%) used biological medicines (Stelara®, Humir®a, Enbrel®), being 8 (26.5%) einstein. 2012;10(1):44-52 einstein. 2012;10(1):44-52 Solis MY, Melo NS, Macedo ME, Carneiro FP, Sabbag CY, Lancha Junior AH, Frangella VS 48 32.4% high risk and 41.2% no risk, no significant differences between groups was found (p > 0.05). immunosuppressive agents (methotrexate). Fourteen patients (41.2%) did not use oral medicines. Among the individuals from the PS group 7 participants (28%) used biological medicines, 8 (32%) immunosuppressive agents and 10 (40%) did not take medicines. In the SAP group 4 individuals (44.4%) used biological medicines, 1 (11.1%) immunosuppressive agents and 4 (44.4%) did not take medicines. The waist-hip ratio for all participants, PS group, SAP group was 0.91 ± 0.07; 0.90 ± 0.08 and 0.92 ± 0.06, respectively. einstein. 2012;10(1):44-52 RESULTS Among all individuals evaluated, 24 (70.6%) did not present risk for cardiovascular disease and 10 (29.4%) had risk based on WHO classification(14). The same risk proportion was found in the PS and SAP groups, however, in the SAP group, using the same parameters, no patient had risk for heart disease. A total of 13 patients (38.2%) presented associated diseases like hypertension, diabetes mellitus, dyslipidemia, heart disease and cancer. From this total, 9 individuals (36%) were in PS group and 4 (44.4%) in SAP group (Table 1). According to ICO the total mean of the sample was 0.55 ± 0.08 (0.54 ± 0.08 in PS group and 0.57 ± 0.07 in SAP group). Considering this mean based on the ICO value proposed by IDF, both groups had no risk to metabolic syndrome. We did not find statistical differences between the groups (p > 0.05). For C index, the total mean for the sample was 25 ± 0.08, PS group and SAP group had 1.24 ± 0.09 and 1.28 ± 0.07, respectively, without statistical differences (p > 0.05). The mean length of time of psoriasis was 15.41 ± 7.93 years. Those in PS and SAP group had mean length of disease time of 14.96 ± 7.24 years and 16.75 ± 9.59 years, respectively. There was no statistical significance on length of time of the disease between the groups (p>0.05). We stratified the sample into two other categories considering the length of the disease for individuals with up to 20 years of psoriasis and those with more than 20 years with the disease. We observed among the participants that 24 had psoriasis for up to 20 and 10 had the disease for more than 20 years, and 5 of these (50%) had one or more associated disease (Figure 1). Hence, there were more comorbidities associated to psoriasis in the group affected longer by the disease. We found by the anthropometric technique after summing the measurement values of the biceps, triceps, subscapular and suprailiac skinfolds that body fat was 32.07 ± 6.52%, although more than 2/3 (n=25) of the Table 1. RESULTS Patients’ distribution by length of time of psoriasis and related Besides BMI we considered other central measures to verify the possible risks. For example, the waist circumference mean was 95 ± 13.45 cm (94.6 ± 14.45 cm in PS group and 95.9 ± 10.27 cm in SAP group). As a result based on the metabolic syndrome council classification(18), 23.5% of all participants presented extremely high risk to develop metabolic complications, re 1. Patients’ distribution by length of time of psoriasis and relate Figure 1. Patients’ distribution by length of time of psoriasis and related Nutritional status and food intake of patients with systemic psoriasis 49 sample had fat percentage above the normal range with risk to acquire associated disease. The fat percentage in the PS group and SAP group was 30.85 ± 5.96% and 35.44 ± 6.81%, respectively. Finally, when fat percentage was measured using plethysmography (BOD POD®) the mean values were 27.43 ± 8.99% in the sample (27.02 ± 9.49% in the PS group and 28.49 ± 7.48% in the SAP group). In addition, 24 participants had fat percentage above normal ranges, being 17 in the PS group. In statistical analysis no significant differences were found (p>0.05) for this parameters between groups. A total of 17 patients (68%) from the PS group and 9 from the SAP group had fat percentage risk of associated diseases to obesity. We did not find statistical differences between groups regarding fat percentage (p > 0.05). Generally, we verified that fat percentage was above normal ranges using the anthropometric methods in the PS (72%) and SAP (88%) groups. According to BIA and BOD POD®, the PS group had 64% and the SAP group 78%, without significant differences. In addition, we could observe that both groups had fat percentage above normal ranges, and it was present in more than 64% of the participants (n=21) (Table 3). The mean of body fat using the BIA technique was 24.14 ± 5.91% (23.68 ± 6.2% in PS group and 25.32 ± 4.86% in SAP group). Among the 34 patients, 67% were above normal range with associated risk for disease, in which 16 were from PS group and 7 from SAP group. In the comparison using this technique no statistical significant differences was found between groups (p > 0.05). Patients’ food intake was distributed in calories and macronutrients. PS: systemic psoriasis; PAS: systemic arthropathy associated. einstein. 2012;10(1):44-52 RESULTS The mean caloric consumption was 2,031.87 ± 728.96 Kcal (2,196.62 ± 565.28 Kcal in PS group and 1,578.81 ± 913.27 Kcal in SAP group). In the SAP group 4 (44.4%) individuals had caloric consumption lower than recommended. We verified significant statistical differences between groups related to caloric consumption (p < 0.05). Table 2. Classification of body composition, according to BMI related to psoriasis Table 2. Classification of body composition, according to BMI related to psoriasis Diagnosis for BMI PS group SAP group Total n % n % n % Eutrophy 7 28 3 33.3 10 29.4 Overweight 12 48 2 22.2 14 41.2 Class I obesity 4 16 3 33.3 7 20.6 Class II obesity 1 4 1 11.1 2 5.9 Class III obesity 1 4 0 0 1 2.9 BMI: body mass index; PS: systemic psoriasis; PAS: systemic arthropathy associated. The sample presented consumption of 43.3 ± 12.24% of carbohydrates; 19.4 ± 5.43% of proteins and 35.8 ± 10.02% of lipids. The mean consumption in the PS group was 42.8 ± 12.53% of carbohydrates, 20.1 ± 5.78% of proteins, 35.4 ± 9.6% of lipids. The PAS group presented Table 3. Comparison of body composition of psoriasis patients using different methods Methods Diagnosis (%) PS group SAP group Total n % n % n % Anthropometry Morbid obesity (> 40) 2 8 3 33.3 5 14.7 High obesity (35-40) 4 16 1 11.1 5 14.7 Moderate obesity (30-35) 7 28 1 11.1 8 23.5 Mild obesity (25-30) 5 20 3 33.3 8 23.5 Above mean (16-24) 4 16 0 0 4 11.8 Mean (15) 0 0 0 0 0 0 Bioeletrical impedance Risk (> 30) 4 16 2 22.2 6 17.6 Excessive adiposity (20-30) 12 48 5 55.6 17 50 Moderate thin mass (12-20) 7 28 2 22.2 9 26.5 Adequate (8-12) 0 0 0 0 0 0 Too thin (5-8) 0 0 0 0 0 0 Whole body plethysmography Risk (> 30) 10 40 4 44.4 14 41.2 Excessive adiposity (20-30) 7 28 3 33.3 10 29.4 Moderate thin mass (12-20) 4 16 2 22.2 6 17.6 Adequate (8-12) 2 8 0 0 2 5.9 Too thin (5-8) 0 0 0 0 0 0 PS: systemic psoriasis; PAS: systemic arthropathy associated. Table 3. Comparison of body composition of psoriasis patients using different methods DISCUSSION The number of patients with joint problems resembled the total observed in an epidemiological study done by Zachariae et al.(6) with individuals from Demark, Finland, Norway and Sweden. The number of patients with joint problems resembled the total observed in an epidemiological study done by Zachariae et al.(6) with individuals from Demark, Finland, Norway and Sweden. Obesity cases observed followed by hypertension and type 2 diabetes mellitus were almost the same as those described in the study by Cohen et al.(9), which evaluated 340 Israeli patients with psoriasis vulgaris. Another study by Altobelli(21) also determined the occurence of NTCD in more than 1,300 individuals with psoriasis from 21 dermatology departments in Italy. These studies stated that the risk for NTCD increases according to age, more specifically between 35 and 50 years, which seems to be due to exposition to chemical pollution and bad life habits as to food intake and by the lack of physical activity. A chronic inflammatory status, common in psoriasis, inappropriate food intake and the lack of physical activity may trigger the development of psoriais and related NTCD, as well as increase the inflammatory process(23). However, it should be remembered interestingly, that nutrition can influence psoriais in two different ways: as cause of metabolic disorders or as treatment and prevention. The literature reports that nutritional treatment of psoriasis patients, associated to the control of biochemical variables and anthropometrical control, assures more clinical stability for individuals with psoriasis, preventing commonly related NTCD and providing long-term quality of life(24). A cohort study done in 1996 by Naldi et al.(22), evolving around 78,000 nurses from the Nurse’s Health Study II between 1991 and 2005 assessed the relationship among BMI, waist and hip circumference and waist-hip ratio, and revealed that high BMI represent high relative risk (RR) to develop psoriasis. Therefore, patients with BMI between 23.0 e 24.9 kg/m² have a RR of 1.19, those with 25.0 to 29.9 kg/m² have a RR of 1.40, patients with 30.0 to 34.9 kg/m² have RR of 1.48, and those with ≥ 35.0 kg/m² a RR of 2.69. They also found a tendency in the relationship between the increase of waist and hip circumference and the appearance of psoriasis. Methods einstein. 2012;10(1):44-52 Solis MY, Melo NS, Macedo ME, Carneiro FP, Sabbag CY, Lancha Junior AH, Frangella VS 50 50 occurrence of obesity was two times higher than the expected for the general population. consumption of 44.8 ± 11.28% of carbohydrates; 17.8 ± 3.83% of proteins; and 36.8 ± 11.02% of lipids. There were no statistic differences regarding carbohydrates and lipids consumption between groups (p > 0.05), but the PS group had a significantly higher consumption of proteins (p = 0.006). The three methods of body composition used pointed to excessive adiposity, without significant statistical difference between groups, indicating the efficacy of these methods to evaluate fat percentage of psoriasis patients. We found after the analysis the lipid intake quality that 40% of the sample (n=14) had an inappropriate consumption of cholesterol (39.17 ± 241.02 mg), saturated fat (17.42 ± 11.4 g) mono (22.12 ± 15.11 g) and polysaturated (14.71 ± 10.71 g). Besides that, the analyzed subjects had low intake of fibers (13.47 ± 8.91 g), low consumption of fruits and vegetables, which helped to reduce in both groups the amounts of vitamins (A, B, C, D and B-complex), minerals (manganese and selenium) in almost 50% for all micronutrients. The excessive weight was the most common nutritional diagnosis in both studied groups, being obesity more frequent in SAP group. It could be explained by the fact that the individuals in the SAP group had a systemic inflammation and joint inflammation. However, such hypothesis could not be supported by the literature and more studies should be conduct to confirm this finding. Evidences in the literature and the findings of this study confirm the positive relationship between psoriasis and obesity. It is important to note that first one is also considered an inflammatory disease and obesity presents high production of pre-inflammatory cytokines like TNF-α, IL-1, IL-6 e IL-8 by the adipose tissue. So, psoriasis and obesity can induce hyperglycemia, decreased sensibility to insulin and hypertension, which enables the occurrence of the metabolic syndrome when the individual had central adiposity(1,7), that we also found in this study. einstein. 2012;10(1):44-52 REFERENCES 1. Kremers HM, McEvoy MT, Dann FJ, Gabriel SE. Heart disease in psoriasis. J Am Acad Dermatol. 2007;57(2):347-54. 2. Wolters M. Diet and psoriasis: experimental data and clinical evidence. Br J Dermatol. 2005;153(4):706-14. 3. Christophers E. Psoriasis--epidemiology and clinical spectrum. Clin Exp Dermatol. 2001; 26(4):314-20. This study results enabled to create a nutritional profile of the participants, to identify the occurrence of excessive weight no matter the psoriasis type. Therefore, these findings seem to be consistent with other results reported in the literature regarding the relationship between psoriasis and obesity, and the risk to develop other comorbidities, like hypertension, diabetes mellitus, dyslipidemia and heart diseases. 4. Zhang X, Wang H, Te-Shao H, Yang S, Wang F. Frequent use of tobacco and alcohol in Chinese psoriasis patients. Int J Dermatol. 2002;41(10):659-62. 5. Henseler T, Christophers E. Disease concomitance in psoriasis. J Am Acad Dermatol. 1995;32(6):982-6. 6. Zachariae H, Zachariae R, Blomqvist K, Davidsson S, Molin L, Mork C, et al. Quality of life and prevalence of arthritis reported by 5,795 members of the Nordic Psoriasis Associations. Data from the Nordic Quality of Life Study. Acta Derm Venereol. 2002;82(2):108-13. 7. Sterry W, Strober BE, Menter A. Obesity in psoriasis: the metabolic, clinical and therapeutic implications. Report of an interdisciplinary conference and review. Br J Dermatol. 2007;157(4):649-55. In addition, when the psoriasis types were analyzed trying to understand possible metabolic differences between them, we verified that both had the same behavior as to metabolic changes and increased risk to develop related diseases. 8. Raychaudhuri SK, Chatterjee S, Nguyen C, Kaur M, Jialal I, Raychaudhuri SP. Increased prevalence of the metabolic syndrome in patients with psoriatic arthritis. Metab Syndr Relat Disord. 2010;8(4):331-4. 9. Cohen AD, Sherf M, Vidavsky L, Vardy DA, Shapiro J, Meyerovitch J. Association between psoriasis and the metabolic syndrome. A cross-sectional study. Dermatology. 2008;216(2):152-5. This study also indicated different methods and tools that could be used in clinical practice to evaluate and follow-up psoriasis patients. 10. Voutilainen S, Nurmi T, Mursu J, Rissanen TH. Carotenoids and cardiovascular health. Am J Clin Nutr. 2006;83(6):1265-71. 11. Harari M, Shani J, Hristakieva E, Stanimirovic A, Seidl W, Burdo A. Clinical evaluation of a more rapid and sensitive Psoriasis Assessment Severity Score (PASS), and its comparison with the classic method of Psoriasis Area and Severity Index (PASI), before and after climatotherapy at the Dead-Sea. Int J Dermatol. 2000;39(12):913-8. CONCLUSION The psoriasis patients showed an abnormal nutritional condition, an increased risk for chronic diseases related to obesity, worsening of the psoriatic lesions, and poor quality of life. There were no significant differences between PS and SAP groups as related to dietary intake. The psoriasis patients showed an abnormal nutritional condition, an increased risk for chronic diseases related to obesity, worsening of the psoriatic lesions, and poor quality of life. There were no significant differences between PS and SAP groups as related to dietary intake. DISCUSSION Hence, these results corroborate the findings of the present study as regarding to enlarged waist circumference and elevated BMI, that almost all participants evaluated showed as well as having excessive weight, indicating any degree of overweight and obesity. Besides that, the Results of food intake showed a heterogeneous consumption between groups, however without statistical differences. Also, there is the possibility of under report in this population. According to Scagliusi et al.(25), an overweight and obese population have higher under reporting when describing its nutrition. The WHO(26) suggests, as values of percentile distribution of macronutrients, proteins (10 to 15%) and lipids (15 to 30%), although, in this study the mean of protein and fat intake were higher among the participants. On the other hand, carbohydrates were lower than the recommended intake, between 50% and 60%. This study results corroborate the findings einstein. 2012;10(1):44-52 Nutritional status and food intake of patients with systemic psoriasis 51 must be stimulated, following the recommendations proposed by Brazilian Dietary Guidelines of daily intake of such foods(29). of Willet(27), which verified a direct relation between hyperlipidemic diet and the worsening of chronic disease, like obesity and dyslipidemia. We think the high intake of lipids, cholesterol and saturated fat was due to the high consumption of animal proteins as reported by the participants. Besides, these individuals had low intake of fibers, low consumption of fruits and vegetables, which also contributed to reduced amount of vitamins (A, C, D and B-complex), minerals (manganese, zinc and selenium) in both groups. In a study with 316 psoriasis patients done by Naldi et al.(22) that assessed food intake using a semi-quantitative questionnaire, it was found a low consumption of vitamins and minerals in all participants. The same authors suggested that consuming food with carotenoids, flavonoids, selenium, vitamins A, C and E is extremely relevant to psoriasis patients because it can reduce the production of reactive oxygen species and tissue inflammation, providing stability of cell membrane and recovery of skin lesions. Food inadequacies according to Mobbs et al.(28) might be explained by the increase of industrial and urbanization process in Brazil and around the world, increasing the consumption of fat/sugar rich diets, sugar drinks and refined food, besides reducing the consumption of complex carbohydrates and fibers. ACKNOWLEDGMENT We thank all volunteers of the research to be part of this analysis, the Brazilian Psoriasis Study Center and Instituto Vita to provide installations to perform the investigation. REFERENCES These results indicate that nutritionists should closely follow the amount and quality of food intake of these patients. Particularly regarding the intake of calories, lipids, vitamins (A, C, E a B-complex) and minerals (manganese, zinc and selenium) making nutritional interventions to maintain an ideal weight, decrease the production of free radicals and tissue inflammation, providing cell membrane stability and skin lesions repair. In addition, the consumption of food rich in antioxidants such as vegetables and fruits 12. Karim R. WHO in the 1970s and 1980s--a users view. World Health Forum. 1998;19(4):365-8. 13. Monteiro I, Vaz Almeid MD. [Dietary fat and ischemic stroke risk in Northern Portugal]. Acta Med Port. 2007;20(4):307-18. Portuguese. 14. European food and nutrition policies in action. Introduction. WHO Reg Publ Eur Ser. 1998;73:1-7. 14. European food and nutrition policies in action. Introduction. WHO Reg Publ Eur Ser. 1998;73:1-7. 15. Lohmann G, Dolch N. [Intensive medical care from the viewpoint of patients and their families. Study of an internal medical intensive care unit]. Krankenpflege (Frankf). 1988;42(11):508-11. einstein. 2012;10(1):44-52 Solis MY, Melo NS, Macedo ME, Carneiro FP, Sabbag CY, Lancha Junior AH, Frangella VS 52 23. Withers RT, LaForgia J, Pillans RK, Shipp NJ, Chatterton BE, Schultz CG, et al. Comparisons of two-, three-, and four-compartment models of body composition analysis in men and women. J Appl Physiol. 1998;85(1): 238-45. 16. Durnin JV, Womersley J. Body fat assessed from total body density and its estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years. Br J Nutr. 1974;32(1):77-97. 17. Valdez R, Seidell JC, Ahn YI, Weiss KM. A new index of abdominal adiposity as an indicator of risk for cardiovascular disease. A cross-population study. Int J Obes Relat Metab Disord. 1993;17(2):77-82. 24. Bjorneboe A, Smith AK, Bjorneboe GE, Thune PO, Drevon CA. Effect of dietary supplementation with n-3 fatty acids on clinical manifestations of psoriasis. Br J Dermatol. 1988;118(1):77-83. 18. I Diretriz Brasileira de Diagnóstico e Tratamento da Síndrome Metabólica. Arq Bras Cardiol. 2005;84(1):3-28. 18. I Diretriz Brasileira de Diagnóstico e Tratamento da Síndrome Metabólica. Arq Bras Cardiol. 2005;84(1):3-28. 25. Scagliusi FB, Polacow VO, Artioli GG, Benatti FB, Lancha AH Jr. Selective underreporting of energy intake in women: magnitude, determinants, and effect of training. J Am Diet Assoc. 2003;103(10):1306-13. 19. Fields DA, Hunter GR. Monitoring body fat in the elderly: application of air-displacement plethysmography. Curr Opin Clin Nutr Metab Care. 2004;7(1):11-4. REFERENCES 26.Organização Mundial da Saúde (OMS). Necessidades de energia e proteína. São Paulo: Roca, 1998. Série de relatos técnicos, 724. 20. Siri G. [Mental pathology]. Policlinico Prat. 1961;68:1722-4. Italian. 27. Willett WC. Invited commentary: comparison of food frequency questionnaires. Am J Epidemiol. 1998;148(12):1157-9; discussion 62-5. 21. Altobelli E, Petrocelli R, Maccarone M, Altomare G, Argenziano G, Giannetti A, et al. Risk factors of hypertension, diabetes and obesity in Italian psoriasis patients: a survey on socio-demographic characteristics, smoking habits and alcohol consumption. Eur J Dermatol. 2009;19(3):252-6. 28. Mobbs CV, Mastaitis J, Yen K, Schwartz J, Mohan V, Poplawski M, et al. Low- carbohydrate diets cause obesity, low-carbohydrate diets reverse obesity: a metabolic mechanism resolving the paradox. Appetite. 2007;48(2):135-8. 22. Naldi L, Parazzini F, Peli L, Chatenoud L, Cainelli T. Dietary factors and the risk of psoriasis. Results of an Italian case-control study. Br J Dermatol. 1996; 134(1):101-6. 29. Philippi ST, latterza AR, Cruz AT, Ribeiro lC. Pirâmide alimentar adaptada: guia para escolha dos alimentos. Rev Nutr. 1999; 12(1): 65-80. einstein. 2012;10(1):44-52 einstein. 2012;10(1):44-52
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Ituglanis agreste, a new catfish from the rio de Contas basin, northeastern Brazil (Siluriformes: Trichomycteridae)
Neotropical ichthyology/Neotropical Ichthyology
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Neotropical Ichthyology, 11(3):513-524, 2013 Copyright © 2013 Sociedade Brasileira de Ictiologia Neotropical Ichthyology, 11(3):513-524, 2013 Copyright © 2013 Sociedade Brasileira de Ictiologia 1Universidade Federal do Rio Grande do Norte, Laboratório de Ictiologia Sistemática e Evolutiva, Departamento de Botânica, Ecologia e Zoologia. 59978-970 Natal, RN, Brazil. smaialima@gmail.com (SMQL), cinarapinheironeves@hotmail.com (CPN) 2Rua Professor Joaquim Louzada, 300, Casa 120, Bairro Camaquã, 91920-050 Porto Alegre, RS, Brazil. rafamcpaiva@gmail.com Ituglanis agreste, a new catfish from the rio de Contas basin, northeastern Brazil (Siluriformes: Trichomycteridae) Sergio M. Q. Lima1, Cinara P. Neves1 and Rafael M. Campos-Paiva2 Ituglanis agreste, a new species of catfish, is described from a tributary stream of the rio Gongogi drainage, rio de Contas basin, Bahia State, northeastern Brazil, from a transition area between the Atlantic Rain Forest and the semi-arid Caatinga savanna. This species is distinguished from its congeners by the following characters: elongate interopercle plate with high number of odontodes (26-30), high number of ribs (5-6), fewer vertebrae (36), number of branchiostegal rays (7), number of pectoral-fin rays (i,6) and absence of s1 pore. Comparisons with other Ituglanis species and putative plesiomorphic characters are presented. Some comments about conservation of Ituglanis species from northeastern Brazil are also made. Ituglanis agreste, espécie nova, é descrita de um tributário da drenagem do rio Gongogi, bacia do rio de Contas, Bahia, nordeste do Brasil, de uma área de transição entre dois biomas, a Caatinga e a Mata Atlântica. Esta espécie distingue-se de todas as demais espécies do gênero pelas seguintes características: placa interopercular alongada com elevado número de odontódeos (26-30), número elevado de costelas (5-6), número reduzido de vértebras (36), número de raios branquiostégios (7), número de raios da nadadeira peitoral (i,6), e ausência do poro s1. Comparações com outras espécies de Ituglanis e a presença de supostos caracteres plesiomórficos são apresentados. Alguns comentários sobre conservação das espécies de Ituglanis do nordeste do Brasil também são feitos. Key words: Atlantic Forest, Biome transition, Semi-arid Caatinga, Serra da Ouricana, Taxonomy. Introduction Copionodontinae, and Trichomycterinae) as well as the specialized forms that comprise the TSVSG clade (Tridentinae, Stegophilinae, Vandelliinae, Sarcoglanidinae, and Glanapteryginae), which all share a smaller number of ribs (2-7) (e.g., I. passensis Fernandez & Bichuette). The genus Ituglanis Costa & Bockmann is a monophyletic assemblage of small trichomicterid catfishes, previously included in Trichomycterus Valenciennes (Trichomycterinae) due to its superficial similarity with the members of this genus. Thus, a new taxon was proposed to accommodate species that share three synapomorphies present in the neurocranium: sphenotic directed anteriorly, parieto-supraoccipital fontanel reduced to a small orifice or completely closed (e.g., in I. macunaima Datovo & Landim, and some specimens of I. epikarsticus Bichuette & Trajano and I. mambai Bichuette & Trajano; Bichuette & Trajano, 2008), and autopalatine with a deep concavity in the inner medial portion (Costa & Bockmann, 1993). The Ituglanis assemblage has been considered essential for understanding the emergence of the morphological and ecological adaptations in the trichomycterids (de Pinna, 1998). This is due to the intermediary phylogenetic position between both the more generalized forms (Trichogeninae, Currently comprised of twenty nominal valid species (Sarmento-Soares et al., 2006; Campos-Paiva & Costa, 2007; Bichuette & Trajano, 2008; Wosiacki et al., 2012), and there- fore the second most speciose trichomycterid genus, Ituglanis has a wide distribution in the cisandine Neotropi- cal region, from the Guianas to Uruguay (de Pinna & Keith, 2003), including five troglobitic species found in Central Brazil (Bichuette & Trajano, 2008) and miniaturized forms as I. macunaima from Araguaia Basin (Datovo & Landim, 2005). However, only recently two species of coastal basins of northeastern Brazil were described: I. cahyensis Sarmento- Soares, Martins-Pinheiro, Aranda & Chamon and I. paraguassuensis Campos-Paiva & Costa, respectively, from the rio Cahy and rio Paraguaçu coastal basins, in Bahia state (Sarmento-Soares et al., 2006, Campos-Paiva & Costa, 2007). 513 514 New Ituglanis from Brazil Laboratório de Sistemática e Evolução de Peixes Teleósteos, Universidade Federal do Rio de Janeiro, Rio de Janeiro; UFRN, Universidade Federal do Rio Grande do Norte, Natal, and USNM, National Museum of Natural History, Smithsonian Institution, Washington, D.C. The coastal watersheds of eastern Brazil north of the rio Pardo have their upper reaches within the temporary rivers of the Caatinga biome, while the lower reaches are in the Atlantic Forest biome. In contrast, watersheds south of the rio Jequitinhonha are entirely in the Atlantic Forest biome (Langeani et al., 2009). Material and Methods Paratypes. MNRJ 40197, 5, 32.4-40.8 mm SL; UFBA 7134, 5, 33.4-41.7 mm SL; UFRN 29, 6, 32.8-41.9 mm SL (3 c&s), all collected with holotype. MZUSP 102535, 6., 38.4-44.1 mm SL, same locality as holotype, 10 Feb 2009, S. M. Q. Lima, R. M. Campos-Paiva, P. Hollanda Carvalho & D. F. Almeida. Measurements and counts follow Costa (1992), and are presented as percentages of standard length (SL), except for subunits of head, which are presented as percentage of head length (HL). Terminology for osteology follows Adriaens et al. (2010), cephalic and lateral line systems follow Arratia & Huaquin (1995), and caudal-skeleton structures according to Bockmann et al. (2004). Counts of procurrent caudal-fin rays, vertebrae, branchiostegal rays, teeth and odontodes were made only in cleared and stained specimens (c&s) prepared according to Taylor & Van Dyke (1985). Vertebral counts do not include the Weberian complex or the compound caudal centrum. Numbers in parentheses indicate the number of specimens. Detailed osteological description focus on traits informative for future phylogenetic analyses of trichomycterid groups. Illustrations were prepared using a stereomicroscope with a camera lucida attachment. Morphological data of Ituglanis bambui Bichuette & Trajano; I. epikarsticus; I. guayaberensis Dahl; I. herberti (Miranda Ribeiro); I. ina Wosiacki, Dutra & Mendonça; I. macunaima; I. mambai; I. nebulosus de Pinna & Keith; I. parkoi (Miranda Ribeiro); I. passensis Fernández & Bichuette; I. ramiroi Bichuette & Trajano, were obtained in the original descriptions or in detailed redescriptions (Bichuette & Trajano, 2004, 2008; Canto, 2009; Dahl, 1960; Datovo & Landim, 2005; Eigenmann, 1918; Fernández & Bichuette, 2002; Miranda Ribeiro, 1940; de Pinna & Keith, 2003; Wosiacki et al., 2012). Data from caudal skeleton of I. proops, I. nebulosus, count of vertebrae and ribs of I. proops, I. nebulosus and I. laticeps were based on digital images of x-ray photographs available from the image base homepage of the All Catfish Species Inventory project (http:// acsi.acnatsci.org/base/image_list.html?mode=genus& genus=Pygidium, 26 December 2012) and the research tool of the online collection homepage of USNM (http:// collections.mnh.si.edu/search/fishes/, 16 January 2013). Diagnosis. Ituglanis agreste is distinguished from all species of the genus, except I. paraguassuensis by the reduced number of vertebrae 36 (vs. 38 or more in all other Ituglanis, except in I. paraguassuensis, I. nebulosus, I. bambui, I. epikarsticus, I. ramiroi, and I. passensis; unknown for I. guayaberensis); number of ribs (5-6) (vs. 2 or 3 in I. amazonicus, I. eichorniarum, I. gracilior, I. Introduction While Ituglanis paraguassuensis was only recorded in the semi-arid Caatinga, I. cahyensis is restricted to the pluvial Atlantic forest. This paper describes Ituglanis agreste, a new species of catfish from the rio de Contas basin, found in a transition area between the Atlantic Forest and the Caatinga savanna. Ituglanis agreste, new species Fig. 1 Holotype. MNRJ 40196, 40.5 mm SL, Brazil, Bahia, municipality of Boa Nova; rio de Contas basin, rio Gongogi drainage, rio Tarugo, tributary of rio Uruba, 14º22’08.95”S 40º11’45.33”W, 09 Jul 2008, S. M. Q. Lima, R. M. Campos- Paiva, P. Hollanda Carvalho & H. Lazzarotto. Material and Methods ina, I. macunaima, I. nebulosus, and I. parkoi ; 4 in I. laticeps and I. cahyensis; 7 in I. passensis); number of pectoral-fin rays i,6 (vs. i,4 in I. cahyensis, I. macunaima, and I. parahybae ; i,5 in I. amazonicus, I. eichorniarum, I. metae, and I. nebulosus; i,7 in I. bambui, I. epikarsticus, I. mambai, I. passensis; iii,5 in I. guayaberensis; i,8 in I. ramiroi); and skin covered by irregular brown blotches (vs. distinct color pattern in all other Ituglanis; absence in I. ina and the subterranean species, I. bambui, I. passensis, I. epikarsticus, and I. ramiroi). Ituglanis agreste is also easily distinguished from the subterranean species by large eyes (vs. minute eyes) and intense pigmentation (vs. pigmentation absent or almost lack). Ituglanis agreste is distinguished from I. paraguassuensis, its geographically closest species, by a higher number of odontodes in an elongate interopercle patch (26-30) (vs. reduced with 14-15 odontodes); seven branchiostegal rays (vs. 8) and pore s1 lacking (vs. s1 present). Description. Morphometric data for holotype and paratypes are given in Table 1. Body elongated, subcylindrical about to dorsal-fin origin, and gradually compressed in caudal peduncle. Dorsal and ventral side view straights, except in dorsal part of the head, which is slightly convex. Head depressed, longer than wide, rounded in dorsal view. Eyes rounded and small, without free orbital margin, covered by a thin translucid membrane, lightly located on anterior half Abbreviations for institutions are: LIRP, Laboratório de Ictiologia de Ribeirão Preto, Ribeirão Preto; MNRJ, Museu Nacional, Rio de Janeiro; MZUSP, Museu de Zoologia da Universidade de São Paulo, São Paulo; UFBA, Universidade Federal da Bahia, Salvador; UFRJ, 515 S. M. Q. Lima, C. P. Neves & R. M. Campos-Paiva S. M. Q. Lima, C. P. Neves & R. M. Campos Paiva 515 f h d B b l d h d d b i ill fi 13 (i 11 i) d l 13 (2) 14 (1) d Fig. 1. Ituglanis agreste, new species, holotype, MNRJ 40196, 40.5 mm SL, Brazil, Bahia State, municipality of Boa Nova, rio de Contas basin. a - lateral; b - dorsal; c - ventral views. of head. Barbels and head covered by minute papillae. fin rays 13 (i,11,i), procurrent dorsal rays 13 (2) or14 (1) and Fig. 1. Ituglanis agreste, new species, holotype, MNRJ 40196, 40.5 mm SL, Brazil, Bahia State, municipality of Boa Nova, rio de Contas basin. Material and Methods Maxilla curved with developed ventral process, smaller than premaxilla (Fig. 2a). Dentary almost straight with two regular rows of conical teeth and with pronounced coronoid process (Fig. 3). Holotype Paratypes Range Mean SD Standard length (mm) 40.5 32.9-41.9 38.0 3.35 Percentage of standard length Body depth 12.4 11.6-15.1 13.5 1.1 Caudal peduncle depth 10.8 8.9-11.1 10.1 0.9 Body width 7.5 6.4-9.3 7.4 1.9 Caudal peduncle width 4.2 3.7-4.7 4.7 0.6 Dorsal-fin base length 10.9 8.6-11.8 10.3 0.8 Anal-fin base length 7.3 6.2-11.6 8.3 1.4 Pectoral-fin length 12.8 11.0-15.1 13.5 1.1 Predorsal length 69.1 64.1-73-3 69.1 1.9 Head length 16.9 15.9-19.6 17.8 1.1 Percentage of head length Head depth 77.5 41.8-77.5 53.9 8.4 Head width 98.7 83.5-107.9 93.9 6.8 Interorbital width 18.9 14.4-22.8 19.7 2.4 Preorbital length 32.5 30.4-39.6 33.2 4.5 Eye diameter 9.8 8.1-14.5 9.9 1.9 Table 1. Morphometric data of holotype and 17 paratypes of Ituglanis agreste. SD = standard deviation. p p ( g ) Hyomandibula with an anterior laminar projection, a deep depression in dorsolateral portion and pores on an- terior and dorsomedian regions (Fig. 4). Anterior portion of quadrate laminar and pronounced. Metapterygoid large, laminar and somewhat trapezoidal, articulating to quadrate by a cartilaginous block and to hyomandibula by bone contact area. Interopercular plate broad and elongate bear- ing conspicuous posterior projection, with 26-30 odontodes placed in two rows, including in anteroventral projection (Fig. 4). Opercle with 16-17 odontodes. Odontodes conical, curved on the opercular patch of odontodes and thinner on interopercular patch of odontodes. Fig. 2. Neurocranium and Weberian capsule of Ituglanis agreste, UFRN 38, 41.8 mm SL; a, dorsal view; b, ventral view. Abbreviations: AOR, antorbital; APA, autopalatine; BAO+EXO, basioccipital-exoccipital bone; EPO, epioccipital; FRO, frontal; FSO, parieto-supraoccipital fontanel; i1-i11, infraorbital pores; ll1-ll3, lateral line pores; LET, lateral ethmoid; MAX, maxilla; MET, mesethmoid; ORB, orbitosphenoid; PAS, parasphenoid; PMX, premaxilla; po1, preopercular pore; PSO, parieto- supraoccipital; PSC, posttemporo-supracleithrum; PTE, pterotic; s1-s6, supraorbital pores; SPH+POT+PSF, sphenotic-prootic- pterosphenoid; SSO, sesamoid supraorbital; VOM, vomer; WEB, Capsule of Weberian apparatus. Scale bar = 1 mm. Fig. 2. Neurocranium and Weberian capsule of Ituglanis agreste, UFRN 38, 41.8 mm SL; a, dorsal view; b, ventral view. Material and Methods a - lateral; b - dorsal; c - ventral views. Fig. 1. Ituglanis agreste, new species, holotype, MNRJ 40196, 40.5 mm SL, Brazil, Bahia State, municipality of Boa Nova, rio de Contas basin. a - lateral; b - dorsal; c - ventral views. fin rays 13 (i,11,i), procurrent dorsal rays 13 (2) or14 (1) and ventral 10 (2) or14 (1). Caudal fin subtruncate. of head. Barbels and head covered by minute papillae. Mouth subterminal. Tip of nasal barbel reaching posterior edge of opercular patch of odontodes; tip of maxillary barbel reaching pectoral-fin base and tip of rictal barbel reaching posterior edge of interopercular patch of odontodes or the anterior edge of pectoral-fin. Mesethmoid with the anterior portion and shaft nearly straight, gradually tapering at proximal tip (Fig. 2a). Lateral ethmoid without lateral projections. Anterior fontanel restricted to small pit with small enlargement in posterior third of frontals. Posterior fontanel as small round opening on posterior portion of parieto-supraoccipital. Sesamoid supraorbital elongate, slender and curved, without lateral process, slightly longer than autopalatine; sphenotic- prootic-pterosphenoid narrow, with anterior portion anteriorly directed (Fig. 2a). Pterotic with posterolateral projection. Total vertebrae 36, 12-13 precaudal and 23-24 caudal. Ribs 5 (2) or 6 (1). Origin of dorsal-fin in a vertical through the 22nd vertebra. Origin of anal-fin in a vertical through the 23rd vertebra and through the base of the 7th ray of dorsal fin. Origin of pelvic fin in a vertical through the 17th- 18th vertebra. Pectoral-fin rays i,6, pectoral fin triangular, first ray simple and pectoral filament absent (holotype plus one specimen) or ranging from 5-25% (16) greater than the other rays of the pectoral fin. Pelvic-fin rays 5 (i,4). End of pelvic fin overreach the urogenital papilla. Dorsal-fin rays 11-12 (v-vi,6), anal-fin rays 10-11 (v-vi,5). Principal caudal- Autopalatine with a deep concavity on the internal medial border; posterior process moderate, about 50% of the length of the autopalatine (Fig. 2a). Posttemporo- supracleithrum with anterior and posterior processes. New Ituglanis from Brazil 516 Table 1. Morphometric data of holotype and 17 paratypes of Ituglanis agreste. SD = standard deviation. Vomer arrow-shaped, with thin lateral projections and long posterior process (Fig. 2b). Parasphenoid with two anterior and one posterior processes. Weberian capsule fused to basioccipital and exoccipital and with small lateral opening on each side. Premaxilla trapezoidal and curved with two rows of conical teeth. Material and Methods Abbreviations: AOR, antorbital; APA, autopalatine; BAO+EXO, basioccipital-exoccipital bone; EPO, epioccipital; FRO, frontal; FSO, parieto-supraoccipital fontanel; i1-i11, infraorbital pores; ll1-ll3, lateral line pores; LET, lateral ethmoid; MAX, maxilla; MET, mesethmoid; ORB, orbitosphenoid; PAS, parasphenoid; PMX, premaxilla; po1, preopercular pore; PSO, parieto- supraoccipital; PSC, posttemporo-supracleithrum; PTE, pterotic; s1-s6, supraorbital pores; SPH+POT+PSF, sphenotic-prootic- pterosphenoid; SSO, sesamoid supraorbital; VOM, vomer; WEB, Capsule of Weberian apparatus. Scale bar = 1 mm. S. M. Q. Lima, C. P. Neves & R. M. Campos-Paiva 517 P h l ith i t l f d l t Fig. 3. Left mandible of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Medial view. Abbreviations: AAR, angulo- articulo-retroarticular; CPR, coronoid process; DEN, dentary. Scale bar = 1 mm. Fig. 5. Left hyoid arch of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Ventral view. Abbreviations: ACH, anterior ceratohyal; BRR, branchiostegal rays; PCH, posterior ceratohyal; PUH, h l VHH t lh h l S l b 1 Fig. 5. Left hyoid arch of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Ventral view. Abbreviations: ACH, anterior ceratohyal; BRR, branchiostegal rays; PCH, posterior ceratohyal; PUH, parurohyal; VHH, ventral hypohyal. Scale bar = 1 mm. Fig. 3. Left mandible of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Medial view. Abbreviations: AAR, angulo- articulo-retroarticular; CPR, coronoid process; DEN, dentary. Scale bar = 1 mm. Fig. 3. Left mandible of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Medial view. Abbreviations: AAR, angulo- articulo-retroarticular; CPR, coronoid process; DEN, dentary. Scale bar = 1 mm. Fig. 5. Left hyoid arch of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Ventral view. Abbreviations: ACH, anterior ceratohyal; BRR, branchiostegal rays; PCH, posterior ceratohyal; PUH, parurohyal; VHH, ventral hypohyal. Scale bar = 1 mm. Parurohyal with conspicuous central foramen and lat- eral process laminar and elongated, gradually tapering from base to tip, with about same length of ceratohyal (Fig. 5). Ventral hypohyal with deep depressions for articulation with parurohyal condyles. Seven branchiostegal rays. anterior process; second with two small alternated pro- cess; third with one rounded posterior process; fourth flat- tened, somewhat rectangular; fifth absent or not evident. First and second pharyngobranchials absent; third and fourth stem-like, this last attached to tooth plate. Upper pharyngeal tooth plate with conical teeth arranged in two rows in ventromedial region (Fig. 6). Material and Methods Abbreviations: CLE, cleithrum; PTR, pectoral rays; PR1, proximal radial 1 (cartilaginous); PR2, proximal radial 2; SCO, scapulocoracoid. Scale bar = 1 mm. Fig. 6. Branchial skeleton of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Dorsal view (left dorsal elements and gill rakers not shown). Abbreviations: BB2-4, basibranchials 2 to 4; CB1-5, ceratobranchials 1 to 5; EB1-4, epibranchials 1 to 4; HB1-3, hypobranchials 1 to 3; PB3-4, pharyngobranchials 3 to 4; TPL, tooth plate. Scale bar = 1 mm. Fig. 6. Branchial skeleton of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Dorsal view (left dorsal elements and gill rakers not shown). Abbreviations: BB2-4, basibranchials 2 to 4; CB1-5, ceratobranchials 1 to 5; EB1-4, epibranchials 1 to 4; HB1-3, hypobranchials 1 to 3; PB3-4, pharyngobranchials 3 to 4; TPL, tooth plate. Scale bar = 1 mm. defined. Chromatophores more concentrated in dorsolat- eral region of head and opercular patch of odontodes. Nasal, maxillary, and rictal barbels with chromatophores irregularly distributed throughout its entire length. Ven- tral region of head with chromatophores forming conspicu- ous spot on lower lip and with irregular and scattered blotches in anterior region to isthmus and interopercle (Fig. 1c). Fins with few chromatophores, more often at base of these. Some individuals exhibit lighter color than others, but following same color pattern. Fig. 7. Left Pectoral girdle of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Ventral view. Abbreviations: CLE, cleithrum; PTR, pectoral rays; PR1, proximal radial 1 (cartilaginous); PR2, proximal radial 2; SCO, scapulocoracoid. Scale bar = 1 mm. Etymology. The specific name “agreste” (from latim agrestis, which means relative to land, field, wild, or rustic) refers to a semi-humid narrow strip parallel to the coast in north- eastern Brazil, encompassing the area between the Rio Grande do Norte State to the middle section of rio de Contas basin in Bahia State (Forattini et al., 1981), that marks the transition between two distinct biomes, the Atlantic For- est and the semi-arid Caatinga (Prado, 2003), where the new species was discovered. A noun in apposition. Distribution. Ituglanis agreste is only known from the type locality, in rio Tarugo, municipality of Boa Nova, Bahia, north- eastern Brazil. This river is a tributary of the rio Uruba, that flows into rio Gongogi, principal drainage of right margin on the middle section of rio de Contas basin (Fig. 10). Material and Methods First basibranchial absent, second and third basibranchials ossified with anterior and posterior carti- laginous tips; and fourth basibranchial consisting of elon- gated, flattened and rounded cartilage (Fig. 6). First hypo- branchial 1 ossified and stem-like; second elongate some- what trapezoidal, mostly cartilaginous except for antero- lateral process; third flattened cartilaginous with ossified anterolateral process. First, second, third, and fourth ceratobranchials ossified with cartilaginous tips, and with posterolateral laminar expansions; fifth one curved with small teeth on anterior half. First epibranchial with long Cleithrum flatenned, curved and slightly triangular. Scapulocoracoid trapezoidal, ossified with cartilaginous tip (Fig. 7). Pelvic girdle slender and mostly ossified, with cartilage restricted to posteromedial margins (Fig. 8). Upper caudal plates composed of pleurostyle and hypural plates (hypurals 3-5) fused in a single triangular element (1), or with hypural 3 and hypurals 4-5 not fused (2) (Fig. 9). Parhypural partially fused to hypurals 1-2, with distal portion forming a spine-like structure (Fig. 9). Fig. 4. Right suspensorium and opercular series of Ituglanis agreste, UFRN 38, 41.8 mm SL. Lateral view. The arrow indicates the interopercle plate broad and elongate. Abbreviations: HYO, hyomandibula; IOP, interopercle; MPT, metapterygoid; OPE, opercle; POP, preopercle; QUA, quadrate. Scale bar = 1 mm. Cephalic sensory and lateral line channels composed by simple tubes ending in single pore, continuous and interconnected on pterotic, sphenotic-prootic- pterosphenoid, and lateral border of frontals. All cephalic pores paired (18): supraorbital 2-3 (s2, s3, and s6; one speci- men with s2 absent), infraorbital 4-5 (i1, i3, i10, and i11; one specimen with i9), preopercular 1 and lateral line 3 (ll1, ll2, and ll3). Coloration. Body with diffuse and irregular blotches on yellow background. Chromatophores more concentrated in dorsal and lateral region of body, forming large and ir- regular blotches. In lateral region, chromatophores less concentrated and blotches more dispersed. Ventral region spotless from isthmus to pelvic-fin base, region posterior to pelvic fin with diffuse, irregular and scattered blotches. Caudal penducle with smaller irregular blotches, but more Fig. 4. Right suspensorium and opercular series of Ituglanis agreste, UFRN 38, 41.8 mm SL. Lateral view. The arrow indicates the interopercle plate broad and elongate. Abbreviations: HYO, hyomandibula; IOP, interopercle; MPT, metapterygoid; OPE, opercle; POP, preopercle; QUA, quadrate. Scale bar = 1 mm. 518 New Ituglanis from Brazil Fig. 7. Left Pectoral girdle of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Ventral view. Material and Methods Most Ituglanis species have a low rib count (2-3; Sarmento-Soares et al., 2006), but all cave species (I. bambui, I. epikarsticus, I. mambai, I. passensis, and I. ramiroi; Fernández & Bichuette, 2002; Bichuette & Trajano, 2004, 2008) and at least some other species (I. parahybae, I. paraguassuensis, and I. proops) present a large number of ribs (5-7) (de Pinna & Keith, 2003; Datovo & Landim, 2005). Trichomycterinae (Sarmento-Soares et al., 2006) and the large interopercular plate bearing numerous odontodes (de Pinna, 1998; Adriaens et al., 2010). Most Ituglanis species have a low rib count (2-3; Sarmento-Soares et al., 2006), but all cave species (I. bambui, I. epikarsticus, I. mambai, I. passensis, and I. ramiroi; Fernández & Bichuette, 2002; Bichuette & Trajano, 2004, 2008) and at least some other species (I. parahybae, I. paraguassuensis, and I. proops) present a large number of ribs (5-7) (de Pinna & Keith, 2003; Datovo & Landim, 2005). drifts. Substrate consists of rocks or sand. The most com- mon depth observed was about 0.5 m, but some parts had about 1.7 m. Some sites had underwater aquatic macro- phytes and riparian vegetation. The backwaters area had vegetation composed of Typha sp. The margins were de- forested and the environment was mainly composed of pastures. A reservoir without a lake formed by the dam of the river was observed above the collection site. Other species collected with Ituglanis agreste were: Astyanax bimaculatus (Linnaeus), Callichthys callichthys (Linnaeus), Geophagus brasiliensis (Quoy & Gaimard), Rhamdia quelen (Quoy & Gaimard) and Poecilia reticulata Peters, this last an exotic species. Ituglanis valid species usually show interopercle plate reduction and less odontodes (10-17), a derived character shared with the most specialized forms of the family, as- sembled in the large clade TSVSG (de Pinna, 1998). At least I. proops presents an elongate interopercle plate (de Pinna & Keith, 2003; Datovo & Bockmann, 2010), a character also observed in I. agreste. Ituglanis agreste also pre- sents some others putative plesiomorphic characters, ob- served in most species of Trichomycterus and Trichogenes Britski & Ortega: the few number of vertebrae (36) and anal-fin origin inserted on a vertical at 23rd vertebra. Both features are also present in I. paraguassuensis (Campos- Paiva & Costa, 2007), which suggests a basal position to these species within the genus. Material and Methods Nevertheless, no formal phylogenetic analysis among Ituglanis species has been made yet, and the relationships among them remain un- known (Datovo & Landim, 2005; Wosiacki et al., 2012). Material and Methods This is the transition zone of the Serra da Ouricana, where small remnants of a formerly extensive humid forest that delin- eates the limits of the Atlantic Forest domain in the region (Gonzaga et al., 1995). Just a few kilometers to the west, the landscape gives way to the semi-arid Caatinga region, typi- cally known for regular droughts. Thus, it is possible that the rio Tarugo represents the western geographical limit of distribution for this species. During a two year sampling (2008 - 2009) along a 325 km transect of the rio de Contas (between the municipalities of Ibuaçussê and Uruçuca), Ituglanis agreste was found only at one site. Ecological notes. Ituglanis agreste was found along a stretch of about 200 m in a mid-small size river with width up to 8 m, in a moderate slope with clear and cold water, with transparency of about 2 m (Fig. 7). The water flow was mainly turbulent, with alternating areas of currents and pools, though a predominance of moderate to strong 519 S. M. Q. Lima, C. P. Neves & R. M. Campos-Paiva Fig. 9. Caudal skeleton of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Left lateral view. Abbreviations: HS, hemal spines; H3, hypural 3; H4+H5, hypurals 4 and 5 fused; NS, neural spine; PC1+UR, pleural 1 and ural fused; PC2, pleural centrum 2; PH+HU1+HU2, parhypural partially fused to hypurals 1-2; UN, uroneural. Scale bar = 1 mm. Fig. 8. Pelvic girdle of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Dorsal view. Abbreviations: APS, anterior processes; AMP, anterior medium processes; BPT, basipterygium; PSP, pelvic splint; PVR, pelvic rays. Scale bar = 1 mm. Fig. 9. Caudal skeleton of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Left lateral view. Abbreviations: HS, hemal spines; H3, hypural 3; H4+H5, hypurals 4 and 5 fused; NS, neural spine; PC1+UR, pleural 1 and ural fused; PC2, pleural centrum 2; PH+HU1+HU2, parhypural partially fused to hypurals 1-2; UN, uroneural. Scale bar = 1 mm. Fig. 8. Pelvic girdle of Ituglanis agreste, paratype, UFRN 38, 41.8 mm SL. Dorsal view. Abbreviations: APS, anterior processes; AMP, anterior medium processes; BPT, basipterygium; PSP, pelvic splint; PVR, pelvic rays. Scale bar = 1 mm. Trichomycterinae (Sarmento-Soares et al., 2006) and the large interopercular plate bearing numerous odontodes (de Pinna, 1998; Adriaens et al., 2010). Discussion The new species is recognized as a member of Ituglanis because it possesses all three synapomorphies proposed for the genus by Costa & Bockmann (1993). However, among the genus, Ituglanis agreste has some characters that are apparently a plesiomorphic condition, character- istic of the most basal trichomycterid taxa: the high num- ber of ribs (5-6), since it is present in a highest number (more than eight) in Copionodontinae, Trichogeninae, and 520 New Ituglanis from Brazil Fig. 10. Geographic distribution of the three Ituglanis species in northeastern Brazil. The locality of Ituglanis agreste (rio Tarugo) represents the type locality. Fig. 10. Geographic distribution of the three Ituglanis species in northeastern Brazil. The locality of Ituglanis agreste (rio Tarugo) represents the type locality. If corroborated, these results indicate that the genus Ituglanis could be another example of the “biogeographic pattern B” proposed by Ribeiro (2006) for several freshwa- ter fish groups in the coastal drainages of eastern Brazil, including another trichomycterid genus, the Sarcoglanidinae Microcambeva Costa & Bockmann. Such pattern makes sister-group relationships found in the coastal basins of Brazil and subsequent irradiation with the most inclusive groups located inside the Brazilian Shield and the rio Amazonas basin (Ribeiro, 2006). paraguassuensis, from the rio Paraguaçu basin adjacent in the north; and I. cahyensis from rio Cahy basin in the south, the diagnostic features of the new species of rio de Contas basin gives enough evidence to differentiate from these two species formally described, confirming the re- stricted distribution of most species of the genus. The most conspicuous differences between I. agreste and I. paraguassuensis are found in interopercle patch of odontodes (elongate with 26-30 odontodes vs. reduced with 14-15 odontodes), and should not be related to ontoge- netic development once individuals of similar range size were observed (3 c&s, 32.8-41.9 mm SL vs. 3 c&s, 30.3-39.6 mm SL). According to Arratia et al. (1990) these integu- mentary teeth arise very early during ontogeny (Datovo & Bockmann, 2010), indicating that Ituglanis from rio de Contas and rio Paraguaçu are not conspecific. They can be also distinguished by the number of branchiostegal rays, (7 vs. 8) and lack of s1 pore (vs. presence). Nevertheless, they share some diagnostic features used to distinguish other Ituglanis, as 36 vertebrae, seven pectoral-fin rays (i,6) and six pair of ribs. Discussion (2010), in a study of freshwater fish promoting the expan- sion and implementation of conservation units in south- ern Bahia, conduct ichthyological surveys in the rio Cachoeira, Colônia, Contas, and Jequié basins, including these new protected areas in Boa Nova, near the type lo- cality (about 9 km east). These authors only recorded the presence of Ituglanis sp. about 120 km south of the type locality of I. agreste, in the rio Cachoeira basin. Because the taxonomic status of Ituglanis sp. collected in the rio Cachoeira basin remains uncertain, it could belong to a previously described species or a distinct new species. Regardless, I. agreste most likely occurs in the new con- servation areas in Boa Nova (PARNA and/or REVIS), since these protected areas are situated near the type locality where the new species was collected. The color pattern of Ituglanis agreste also differs clearly from I. cahyensis that presents longitudinal rows of coalescing blotches (Sarmento-Soares et al., 2006). They also differs by several features, as the higher number of pectoral fin-rays (i,6 vs. i,4), higher number of opercular (17 vs. 9-12) and interopercle (26-30 vs. 16) odontodes, fewer vertebrae (36 vs. 38 or more), higher number of branchiostegal rays (7 vs. 4-6), higher number of ribs (5-6 vs. 4), 13 caudal-fin rays (vs. 10-11), 10-11 anal-fin rays (vs. 7-8), the position of the pelvic fin, with its origin inserted on a vertical at 17th and 18th vertebra (vs. 20th) and metapterygoid large in contact with the quadrate and the hyomandibular (vs. metapterygoid short, laminar, almost rectangular joined only with the quadrate; Sarmento-Soares et al., 2006). Despite of that, they shared at least one char- acter, the absence of s1 pore. The type locality of Ituglanis agreste is located in the coastal strand of the Serra da Ouricana, which holds im- portant small remnants of a formerly humid forest (Gonzaga et al., 1995) and delineates the westernmost limits of the Atlantic Forest domain bordering with the Caatinga biome. In northeastern Brazil these transition zone are referred as “agreste”, which corresponds to a semi-humid climate (Fig. 10). The three species of Ituglanis described to the north- eastern Brazil were found in streams placed in distinct cli- mate and vegetation domains: I. paraguassuensis in the Caatinga, I. cahyensis in the Atlantic Forest and I. agreste was collected in a transition area between these two biomes. Discussion The number of vertebrae shared by them, was referred as an unusual condition among the genus (Campos-Paiva & Costa, 2007), but it is shared at least by other five species (I. nebulosus, I. passensis, I. ramiroi, I. epikarstikus and I. bambui; Fernández & Bichuette, 2002; de Pinna & Keith, 2003; Bichuette & Trajano, 2004). The troglomorphic species from Central The caudal skeleton of Ituglanis agreste presents two conditions: upper hypural 3, 4, and 5 fused (one specimen) or hypural 3 detached from 4 and 5 (two specimens). Both attributes occurring within the same species was also re- ported for I. ina (Wosiacki et al., 2012), Trichomycterus tupinamba Wosiacki & Oyakawa (Wosiacki & Oyakawa, 2005), and vary among the genus in the subfamily Trichomycterinae (Arratia et al., 1978; comparative mate- rial). Within Ituglanis, some species has the hypural 3 de- tached from 4 and 5 (I. amazonicus and I. eichorniarum), but at least eight species (I. paraguassuensis, I. parahybae, I. proops, I. macunaima, I. nebulosus, I. bambui, I. epikarticus, and I. ramiroi have the hypurals fused, also seen in the specialized species of the TSVSG clade (Bichuette & Trajano, 2004; Datovo & Landim, 2005). Although situated between two coastal basins of Ba- Although situated between two coastal basins of Ba- hia with Ituglanis species previously described: I. S. M. Q. Lima, C. P. Neves & R. M. Campos-Paiva S. M. Q. Lima, C. P. Neves & R. M. Campos-Paiva 521 Fig. 11. Type locality of Ituglanis agreste, Brazil, Bahia State, Boa Nova municipality, rio Tarugo, rio de Contas basin. Brazil and a few specimens of I. ina also presents a high number of interopercle odontodes (more than 24, except in I. epikarsticus) but differ in some aspects from I. agreste (pectoral-fin rays i,7 or i,8 and some degree of reduction of eyes and pigmentation in subterranean species or usually i,5 and homogeneous color pattern with dark vertical bar in caudal peduncle in I. ina vs. i,6 and spotted color pat- tern; Bichuette & Trajano, 2008; Wosiacki et al., 2012). Although the color pattern of I. agreste is similar to I. paraguassuensis, both with a yellow background body covered by blotches, in I. agreste the spots are more dif- fuse and irregular than I. paraguassuensis, that presents blotches more conspicuous (Campos-Paiva & Costa, 2007). The concentration of chromatophores is more intense in I. Discussion agreste, covering the dorsal and lateral region of the body, which gives a darker appearance to I. agreste, if compared to I. paraguassuensis, in which the pigmentation is slightly more concentrated near the eyes and opercular region. Fig. 11. Type locality of Ituglanis agreste, Brazil, Bahia State, Boa Nova municipality, rio Tarugo, rio de Contas basin. agriculture and cattle (Fig. 11) in Boa Nova adjacencies. According to Gonzaga et al. (1995) the forest environments in Serra da Ouricana are among the most neglected habi- tats for conservation. Only recently this region was recog- nized of a high importance for conservation (MMA, 2007), which led to the creation of new conservation units in 2010: Boa Nova National Park (PARNA) and Boa Nova Wildlife Refuge (REVIS), with the aim to preserve the natural eco- systems of the transition area between Atlantic forest and Caatinga, protect endangered species and maintain water sources/springs and streams (Brasil, 2010). Cetra et al. (2010), in a study of freshwater fish promoting the expan- sion and implementation of conservation units in south- ern Bahia, conduct ichthyological surveys in the rio Cachoeira, Colônia, Contas, and Jequié basins, including these new protected areas in Boa Nova, near the type lo- cality (about 9 km east). These authors only recorded the presence of Ituglanis sp. about 120 km south of the type locality of I. agreste, in the rio Cachoeira basin. Because the taxonomic status of Ituglanis sp. collected in the rio Cachoeira basin remains uncertain, it could belong to a previously described species or a distinct new species. Regardless, I. agreste most likely occurs in the new con- servation areas in Boa Nova (PARNA and/or REVIS), since these protected areas are situated near the type locality where the new species was collected. agriculture and cattle (Fig. 11) in Boa Nova adjacencies. According to Gonzaga et al. (1995) the forest environments in Serra da Ouricana are among the most neglected habi- tats for conservation. Only recently this region was recog- nized of a high importance for conservation (MMA, 2007), which led to the creation of new conservation units in 2010: Boa Nova National Park (PARNA) and Boa Nova Wildlife Refuge (REVIS), with the aim to preserve the natural eco- systems of the transition area between Atlantic forest and Caatinga, protect endangered species and maintain water sources/springs and streams (Brasil, 2010). Cetra et al. Acknowledgments We are grateful to Henrique Lazzarotto by helping to photograph the holotype and Erica Caramaschi for provid- ing logistical support. To Daniel Almeida, Henrique Lazzarotto, and Pedro Hollanda-Carvalho for helping on fieldwork. To Wilson Costa (UFRJ), Marcelo R. Britto (MNRJ) and Michel D. Gianeti (MZUSP) that provided the comparative material. To Ricardo Rosa, Telton Ramos and Robson Ramos (UFPB) by loan the steromicroscope with camera lucida. To Ali Ger that reviewed the English of a previous version of the manuscript. This study was sup- ported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico/ Ministério de Ciências e Tecnologia), Biodinâmica LTDA and Bahia Mineração LTDA. Collections were made under permits 010/2008 NUBIO/IBAMA - BA from Instituto Brasileiro do Meio Ambiente e Recursos Naturais Renováveis/ Ministério Brasileiro do Meio Ambiente. Comparative material examined. Trichogenes longipinnis, UFRJ 894, 9, 29.7-46.5 mm SL, Brazil, São Paulo State, Ubatuba municipality, affluent of rio Camburi; UFRJ 682, 1 c&s, 48.4 mm SL, São Paulo, Ubatuba, affluent of rio Camburi. Trichomycterus potschi, UFRJ 719, 2 c&s 33.4-47.6 mm SL, Brazil, Rio de Janeiro State, Itaguaí municipality; Trichomycterus giganteus, UFRJ 5999, holotype, 120.6 mm SL, Brazil, Rio de Janeiro State, Campo Grande municipality, rio Guandu-Mirim; UFRJ 5730, paratypes, 10, 116.2-138.7 mm SL, same locality. Ituglanis amazonicus, MNRJ 35401, 3, 38.8- 56.6 mm SL, Brazil, Pará State, Uruará municipality, tributary of rio Uruará; USNM 305468, 2, 31.6-31.7 mm SL, Bolivia, Beni, Ballivián, 40 km east San Borja; USNM 317738, 1 c&s, 42.3 mm SL, same locality; USNM 300990, 1, 33.2 mm SL, Peru, Madre de Dios, Manu National Park; USNM 263929, 1, 43.4 mm SL, Madre de Dios, Reserva Natural de Tambopata, on trail to laguna Cocococha; USNM 263930, 1, 34.0 mm SL, Madre de Dios, rio Tambopata. Ituglanis cahyensis, MNRJ 32080, 1, 39.30 mm SL, Brazil, Bahia State, Prado municipality, rio Cahy. Ituglanis eichorniarum, UFRJ 5474, 1, 24.3 mm SL, Brazil, Mato Grosso State, Porto Cercado, Corixo Santa Rosa in rio Cuiabá; UFRJ 5608, 1 c&s, 29.4 mm SL, same locality. Ituglanis gracilior, USNM 272287, 1, 50.8 mm SL, Venezuela, Amazonas, caño Temblador, in the road between San Carlos do Rio Negro and Solano; USNM 272288, 1 c&s, 56.2 mm SL, Amazonas, caño Loro, in the road between San Carlos do Rio Negro and Solano; USNM 272289, 1, 50.0 mm SL, Amazonas, caño Urami, upriver of Santa Lucia. Discussion Unlike rio Cahy basin, which is a small coastal basin fully inserted in the Atlantic forest domains, the rio Paraguaçu and Contas basins are perennial middle size riv- ers, with some intermittent tributaries in the upper and middle stretches in the Caatinga region, and lower stretches in the Atlantic Forest (Rosa et al., 2003). The protection of water courses and their riparian mar- gins through the creation of conservation units is among the most important measures concerning the future and diversity of fish species of the coastal basins of Brazil (Menezes et al., 2007). Fortunately, the three species of Ituglanis known from Bahia (including the one described herein), are found in hydrographic basins that pass through integral protection conservation units: I. paraguassuensis in the rio Paraguaçu basin that passes through the Chapada Diamantina National Park, I. cahyensis in the rio Cahy ba- sin that has part of it course protected by the The collection site was highly degraded due to anthro- pogenic impacts, mainly removal of riparian vegetation for 522 New Ituglanis from Brazil Paraguaçu; LIRP 5834, paratypes, 5, 34.8-41.3 mm SL; UFRJ 7209, paratype, 1, 36.5 mm SL; UFRJ 7282, paratype, 1 c&s, 39.6 mm SL; USNM 301016, paratypes, 5 (2 c&s), 33.7-41.6 mm SL, same locality. Ituglanis proops, MZUSP 39027, 2 c&s, 34.8-40.7 mm SL, Brazil, São Paulo State, Iporanga municipal- ity; Ituglanis sp., UFRJ 7237, 8, 31.6-40.7 mm SL, Brazil, Pará State, Primavera municipality, small stream on the road to Bragança; UFRJ 7238, 2 c&s, 29.8-32.6 mm SL; UFRJ 7239, 2 c&s, 28.3-33.4 mm SL, same locality. Ituglanis sp., MNRJ 37539, 4, 46.6-54.0 mm SL, Brazil, Mato Grosso State, Aripuanã mu- nicipality, rio Aripuanã, upstream Dardanelos waterfall. Microcambeva ribeirae, MZUSP 78617, paratypes, 5, 41.3- 48.1 mm SL, Brazil, São Paulo State, Pedro de Toledo munici- pality, rio São Lourencinho; MZUSP 68169, paratypes, 3 c&s, 37.7-41.8 mm SL, Brazil, São Paulo State, Juquiá, ribeirão Poço Grande. Listrura picinguabae, UFRJ 6111, 1, holotype, 48.6 mm SL, Brazil, São Paulo State, Ubatuba municipality, small tributary of rio da Fazenda, near Km 11 of the road BR-101 in Serra do Mar state Park; UFRJ 5951, 4 c&s, 31.3-35.5 mm SL, São Paulo State, Ubatuba municipality, small tributary of rio da Fazenda in Serra do Mar state Park. Descobrimento National Park and I. Discussion agreste in the rio de Contas basin with the type locality close to the recently created Boa Nova National Park and Boa Nova Wildlife Refuge. The discovery of this new species in the rio de Contas basin reinforces the necessity for ichthyofaunal invento- ries in northeastern Brazil, especially in the numerous iso- lated coastal watersheds south of rio São Francisco, where more undescribed species are expected to exist (Langeani et al., 2009). In the last three years, six new fish species were described for the rio de Contas basin: Cyphocharax pinnilepis Vari, Zanata & Camelier, Gymnotus interruptus Rangel-Pereira, Hasemania piatan Zanata & Serra, Hyphessobrycon brumado Zanata & Camelier, a distinct species from H. negodagua that inhabits the rio Paraguaçu basin, Leporinus brinco Birindelli, Britski & Garavello, Trichomycterus tete Barbosa & Costa (Vari et al., 2010; Zanata & Camelier, 2010; Zanata & Serra, 2010; Barbosa & Costa, 2011; Rangel-Pereira, 2012; Birindelli et al., 2013). Hence, the case of Ituglanis agreste, together with the recently discovered endemic fish above, suggests that the rio de Contas basin contains several endemic species (Zanata & Camelier, 2010). Acknowledgments Ituglanis laticeps, USNM 163907, 2, 56.7-73.2 mm SL, Ecuador, Pastaza, rio Bobonaza. Ituglanis metae, USNM 100765, 1, 54.3 mm SL, Colombia, Villavivencio. 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C. da Silva (Eds.). Ecologia e conservação da Caatinga. EDUFPE, Recife, Brasil. Datovo, A. & M. I. Landim. 2005. Ituglanis macunaima, a new catfish from the rio Araguaia basin, Brazil (Siluriformes: Trichomycteridae). Neotropical Ichthyology, 3: 455-464. Eigenmann, C. H. 1918. The Pygidiidae, a family of South American catfishes. Memoirs of the Carnegie Museum, 7: 259-398. Sarmento-Soares, L. M., R. F. Martins-Pinheiro, A. T. Aranda & C. C. Chamon. 2006. Ituglanis cahyensis, a new catfish from Bahia, Brazil (Siluriformes: Trichomycteridae). Neotropical Ichthyology, 4: 309-318. Fernández, L. & M. E. Bichuette. 2002. A new cave dwelling species of Ituglanis from the São Domingos karst, central Brazil (Siluriformes: Trichomycteridae). Ichthyological Exploration of Freshwaters, 13: 273-278. Taylor, W. R. & G. C. Van Dyke. 1985. Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. Cybium, 9: 107-119. 524 New Ituglanis from Brazil Vari, R. P., A. M. Zanata & P. Camelier. 2010. New species of Cyphocharax (Ostariophysi: Characiformes: Curimatidae) from the Rio de Contas drainage, Bahia, Brazil. Copeia, 2010: 382-387. Wosiacki, W. B., G. M. Dutra & M. B. Mendonça. 2012. Description of a new species of Ituglanis (Siluriformes: Trichomycteridae) from Serra dos Carajás, rio Tocantins basin. Neotropical Ichthyology, 10: 547-554. Wosiacki, W. B. & O. T. Oyakawa. 2005. Two new species of catfish genus Trichomycterus (Siluriformes: Trichomycteridae) from the rio Ribeira de Iguape Basin, Southeastern Brazil. Literature Cited Neotropical Ichthyology, 3: 465-472. Zanata, A. M. & P. Camelier. 2010. Hyphessobrycon brumado: a new characid fish (Ostariophysi: Characiformes) from the upper rio de Contas drainage, Chapada Diamantina, Bahia, Brazil. Neotropical Ichthyology, 8: 771-777. Zanata, A. M. & J. P. Serra. 2010. Hasemania piatan, a new characid species (Characiformes: Characidae) from headwaters of rio de Contas, Bahia, Brazil. Neotropical Ichthyology, 8: 21-26. Submitted October 17, 2012 Accepted June 6, 2013 by Marcelo Britto Published September 30, 2013
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Cross-cultural similarity in relationship-specific social touching
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Cross‑cultural similarity in relationship‑specific social touching Suvilehto, Juulia T.; Nummenmaa, Lauri; Harada, Tokiko; Dunbar, Robin I. M.; Hari, Riitta; Turner, Robert; Sadato, Norihiro; Kitada, Ryo Suvilehto, Juulia T.; Nummenmaa, Lauri; Harada, Tokiko; Dunbar, Robin I. M.; Hari, Riitta; Turner, Robert; Sadato, Norihiro; Kitada, Ryo 2019 Suvilehto, J. T., Nummenmaa, L., Harada, T., Dunbar, R. I. M., Hari, R., Turner, R., . . . Kitada, R. (2019). Cross‑cultural similarity in relationship‑specific social touching. Proceedings of the Royal Society B : Biological Sciences, 286(1901), 20190467‑. doi:10.1098/rspb.2019.0467 https://hdl.handle.net/10356/83249 © 2019 The Author(s). Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Downloaded on 24 Oct 2024 13:21:33 SGT Research 1Department of Neuroscience and Biomedical Engineering, 2Department of Computer Science, and 3Department of Art, Aalto University, Espoo, Finland 4Turku PET Centre, and 5Department of Psychology, University of Turku, Turku, Finland 6Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan 7Department of Experimental Psychology, University of Oxford, Oxford, UK 8Department of Neurophysics, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany 9National Institute for Physiological Sciences, Okazaki, Japan 10Division of Psychology, School of Social Sciences, Nanyang Technological University, 14 Nanyang Avenue, 637332 Singapore 8Department of Neurophysics, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany 9National Institute for Physiological Sciences, Okazaki, Japan 10Division of Psychology, School of Social Sciences, Nanyang Technological University, 14 Nanyang Avenue, 637332 Singapore 10Division of Psychology, School of Social Sciences, Nanyang Technological University, 14 Nanyang Avenue, 637332 Singapore royalsocietypublishing.org/journal/rspb Juulia T. Suvilehto1,4, Lauri Nummenmaa4,5, Tokiko Harada6, Robin I. M. Dunbar2,7, Riitta Hari3, Robert Turner8, Norihiro Sadato9 and Ryo Kitada10 Juulia T. Suvilehto1,4, Lauri Nummenmaa4,5, Tokiko Harada6, Research Cite this article: Suvilehto JT, Nummenmaa L, Harada T, Dunbar RIM, Hari R, Turner R, Sadato N, Kitada R. 2019 Cross-cultural similarity in relationship-specific social touching. Proc. R. Soc. B 286: 20190467. http://dx.doi.org/10.1098/rspb.2019.0467 Research Cite this article: Suvilehto JT, Nummenmaa L, Harada T, Dunbar RIM, Hari R, Turner R, Sadato N, Kitada R. 2019 Cross-cultural similarity in relationship-specific social touching. Proc. R. Soc. B 286: 20190467. http://dx.doi.org/10.1098/rspb.2019.0467 LN, 0000-0002-2497-9757; RIMD, 0000-0002-9982-9702; RK, 0000-0001-7446-4033 Received: 25 February 2019 Accepted: 4 April 2019 Subject Category: Behaviour Subject Areas: behaviour, evolution, neuroscience Keywords: social touch, cultural differences, emotion, bonding Author for correspondence: Ryo Kitada e-mail: ryokitada@ntu.edu.sg Electronic supplementary material is available online at http://dx.doi.org/10.6084/m9. figshare.c.4464836. Received: 25 February 2019 Accepted: 4 April 2019 Many species use touching for reinforcing social structures, and particularly, non-human primates use social grooming for managing their social networks. However, it is still unclear how social touch contributes to the maintenance and reinforcement of human social networks. Human studies in Western cultures suggest that the body locations where touch is allowed are associated with the strength of the emotional bond between the person touched and the toucher. However, it is unknown to what extent this relationship is culturally universal and generalizes to non-Western cultures. Here, we compared relationship-specific, bodily touch allowance maps across one Western (N ¼ 386, UK) and one East Asian (N ¼ 255, Japan) country. In both cultures, the strength of the emotional bond was linearly associated with permissible touch area. However, Western participants experienced social touching as more pleasurable than Asian participants. These results indicate a similarity of emotional bonding via social touch between East Asian and Western cultures. Subject Category: Behaviour Subject Areas: behaviour, evolution, neuroscience Keywords: social touch, cultural differences, emotion, bonding 1. Introduction Author for correspondence: Ryo Kitada e-mail: ryokitada@ntu.edu.sg Interpersonal touch is a critical part of human social communication. It contri- butes to cognitive and socioemotional development in childhood [1,2] and promotes relational, psychological and physical well-being in adulthood [3,4]. Given its importance, there has been growing interest in the effects of interper- sonal touch on human social behaviour and in the resulting social relationships. Non-human primates spend remarkable amounts of time in grooming others, well beyond the necessity of removing parasites or vegetation debris from the fur [5]. Social grooming thus plays a particularly important role in social bond- ing, and the psychological experience of increased social closeness is reflected in prosocial behaviours [5,6]. In individual female primates, the social grooming patterns are explained by factors such as attraction to high-ranking individuals, attraction to kin and competition for grooming partners [7,8], implying that variations in the relationship specificity of social touch might be correlated with differences in social structure. In our previous study, we asked 1368 people from Western countries (Finland, France, Italy, Russia and the UK) to indicate where on their body they would allow relatives, friends and strangers to touch them [9]. We also measured the emotional bond between them since such bonds are the best predictor for Electronic supplementary material is available online at http://dx.doi.org/10.6084/m9. figshare.c.4464836. & 2019 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. 1Department of Neuroscience and Biomedical Engineering, 2Department of Computer Science, and 3Department of Art, Aalto University, Espoo, Finland 4Turku PET Centre, and 5Department of Psychology, University of Turku, Turku, Finland 6Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan 7Department of Experimental Psychology, University of Oxford, Oxford, UK 8Department of Neurophysics, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany 9National Institute for Physiological Sciences, Okazaki, Japan 10Division of Psychology, School of Social Sciences, Nanyang Technological University, 14 Nanyang Avenue, 637332 Singapore Physiological Sciences, Japan. The English version of the online rating tool (emBODY, [9,22]; https://version.aalto.fi/gitlab/egler- ean/embody) was first translated into Japanese by a professional translator (ZENIS Co., Ltd, Tokyo Japan). Then two researchers (R.K. and T.H.) revised the translated materials by conducting back-translation and translation until both were satisfied with the result. engaging in social contact with someone and as they track the position of different individuals in one’s social network [10–12]. In each country, the topographic map of body areas that one is allowed to touch was associated with the strength of the emotional bond between the participant and the toucher. Thus, the relationship-specific patterns of social touch seem to be related to the establishment and maintenance of social structures and affective relationships among human adults. 2 Participants first provided background information about themselves and members of their social network. They were given a list of categories of relationship which may be found in one’s social network (e.g. ‘aunt’, ‘female friend’) and participants then indicated if they had one or more individuals from these categories in their own social network. If participants had multiple individuals in their social network fitting one category, they were instructed to pick one individual and to answer all the subsequent questions regarding this individual. We also added ‘female stranger’ and ‘male stranger’ to the list of social categories, to probe the acceptable social touch with strangers. However, these results cannot resolve whether bonding by social touch is a culturally universal phenomenon or specific to Western Europe which our previous study was limited to. Cross-cultural studies have shown that deeply embedded differences between Western and Eastern cultures strongly influence emotional processing, together with ideas regarding experiences such as emotions [13,14], the facially expressed emotions of others [15–17] and their integration with emotion- al voices [18]. Indeed, social touch varies according to culture [19–21]. For instance, North American students have more fre- quent physical contact than Japanese students with their friends and their parents [19]. Moreover, the US students’ social touch patterns in regard to their fathers and mothers were similar, whereas Japanese students were physically closer to their mothers than their fathers. However, this study did not examine the relationship between the touching pattern and the strength of the emotional bonding. Thus, it remains unclear to which extent this relationship is culturally universal and generalizes to non-Western cultures. Participants were then asked to give information about the sex (only for spouses) and ages of the selected members of their social network, as well as estimates of how long ago they last met them. We assumed that participants would encounter strangers on a daily basis and set time since the last meeting to 0 days for the strangers. We used participants’ own age for the age of the stran- gers, and in subsequent tasks asked partners to respond with respect to ‘a woman/man of your age whom you do not know’. Next, the participants reported their emotional bond with each net- work member (scale from 1 representing no emotional bond to 10 representing the strongest possible emotional bond) and gave an estimate of how pleasant they would find being touched by each member of their social network (scale from 1, not pleasant at all, to 10, extremely pleasant). Here we compared relationship-specific social touching patterns between one East Asian and one European culture. We used a high-resolution self-reporting tool (emBODY) to quantify relationship-specific maps of bodily regions where social touch is allowed. Participants in Japan and UK evalu- ated their emotional bonds with and drew touchable body regions for different social network members. We hypoth- esized that patterns of allowed interpersonal touch differ in Japan and in the UK but vary in a similar way as a function of social bond in both countries. y p ) After completing background questions, the participants were presented with the colouring task with the emBODY tool. They were asked to consider where on their bodies they would find it acceptable for different social network members to touch them in everyday situations. For each member of their social network, the participants were shown a body outline in both front and back view (electronic supplementary material, figure S1a) and asked to colour the bodily regions where each social network member would be allowed to touch them. The participants coloured the bodies using a computer mouse or, if they were com- pleting the task using a mobile device, using their finger. Painting was additive so that multiple strokes on the same region increased opacity; this information was, however, not considered in the analyses (see below). (c) Data analysis (i) Data preprocessing Th d fi h k The data were first checked for completeness. Data from the colour- ing tasks were then converted to Matlab (R2015b) two-dimensional matrices, where each cell represented a pixel on the screen. The size of the figures was 522  342 pixels (for front and back combined), out of which 89 129 pixels fell within the body outlines. The diam- eter of the painting tool was set to 17 pixels. The coloured images were binarized so that the amount of time a participant spent on colouring an area would not impact the results. Each participant completed between two and 15 individual touch area maps (TAMs), depending on the size of their social network. As the aim of the study was to compare data between different cultures, cultural background criteria were used to exclude some participants. Japanese participants were asked about their parents’ ethnicity, and whether they had spent an extended period of time abroad. We excluded any participants who did not report both of their parents’ ethnicity as ‘Japanese’ or who reported having spent more than a yearabroad. Altogether, 17 Japanese participants were excluded based on the background questions. In the English (a) Participants l h (a) a t c pa ts Altogether, 309 Japanese individuals and 622 British individuals participated in the study. The British sample was collected through Maximiles (data reported in [9]) and the Japanese sample was collected via MyVoice Communications, Inc., in 2016. As the popu- lation-level effect size was unknown, we aimed to have the sample in line with the first study reporting topographies of acceptable touch (altogether 1368 participants, average 274 per culture, [9]), which in turn were targeted to double the sample sizes from the earliest studies using this technology (total of 773 participants over seven separate experiments, [22]). After cultural background had been validated and quality control established (described in detail in the ‘Data analysis’ section), the data of 255 Japanese indi- viduals (124 male, 40.1+14.6 years) and 386 British individuals (214 male, 46.0+12.6. years) were analysed. The British sample was chosen for comparison [9], (1) because it was collected via a paid service similar to the Japanese sample and (2) because the age distributions were similar in both groups. royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 286: 2019046 3 (ii) Comparing the samples using two-proportion z-test We compared the acceptable touch areas of the two cultural samples by comparing each pixel in each image using a two- proportion z-test, with a two-tailed alternative hypothesis, with a ¼ 0.05, corrected for false detection rate with no correlation assumptions. To test the association between emotional bond and touchable body area, we first calculated a ‘touchability index’ (TI) as the proportion of coloured pixels within the body outline for each TAM [9]. To better quantify the differences in the topographies of acceptable touch, we then defined 13 anatom- ical regions of interest (ROIs) and calculated ROI-specific TIs as the proportion of coloured pixels within the ROI. We conducted mul- tiple linear regression analysis on these TIs for each social network member in both countries, with the emotional bond and cultural background as explanatory variables. This analysis was conducted using group mean data and individual data. pleasantness and touchability index pleasantness and touchability index Figure 2 depicts the correlations between TI, pleasantness and emotional bond, with TI as the proportion of pixels in the body that a particular member of the participant’s social network was allowed to touch (electronic supplementary material, figure S4). Linear multiple regression analysis with emotional bond, pleasantness of touch and culture as explanatory vari- ables revealed that together these variables explained 20% of the variance in the total touchable body area (adjusted R2 ¼ 0.20, F3,7375 ¼ 626, p , 10210). Bond (b ¼ 0.18, p , 10210), plea- santness (b ¼ 0.31, p , 10210) and culture (b ¼ 0.16, p , 10210) all predicted TI (see also electronic supplementary material, table S1 for a linear mixed effects model with subject as random effect). Partitioning R2 assigned 11.3% of the explana- tory power to pleasantness of touch, 8.6% to emotional bond and only 0.4% of explanatory power to the cultural background (electronic supplementary material, figure S5). For the data averaged such that each social network member was rep- resented by the average of responses about that person in each culture (figure 2), the adjusted R2 was 0.85, F3,26 ¼ 58, p , 10210. (a) Touch area maps for Japanese and British Figure 1 shows the mean TAMs for different social members in Japanese and British samples. The relationship-specific TAMs in the Japanese and UK populations were generally consistent. Specifically, the partner was allowed to touch basically anywhere on the body, and clo- sest acquaintances and relatives were allowed to touch over the head and upper torso. By contrast, strangers were restricted to touch only the hands. Direct comparison of TAMs from British and Japanese participants by two-proportion z-test revealed that Japanese allowed more touching from their female rela- tives than did British, especially in the lower extremities and the bottom (figure 1c). On the other hand, more British partici- pants allowed their partners to touch their bodies on the torso, face and legs. Moreover, British participants allowed their mother, aunt, female cousin and female friend to touch their heads more and male strangers to touch their hands more. Due to the high correlation between pleasantness and emotional bond, we also ran partial correlation tests. When controlling for pleasantness of the relationship, the association between emotional bond and TI was still significant, rs7377 ¼ 0.15, p , 10210, 95% CI [0.13, 0.17]. Similarly, when controlling for emotional bond, the association between pleasantness and TI remained significant, rs7377 ¼ 0.22, p , 10210, 95% CI [0.20, 0.24]. Thus, both pleasantness and emotional bond contributed independently to the relationship-specific TI. Altogether, the analysis showed that emotional bond and perceived pleasantness of touch explained around 20% of the variance in TI, with only a negligible (0.4%) contribution from the culture. A supplementary analysis with emotional bond treated as categorical variable confirmed that emotional bond explains the variance in the total touchable body area (electronic supplementary material, figure S8). Finally, we confirmed this result by conducting the same regression ana- lyses for both sexes in each culture (mean adjusted R2 ¼ 0.21, range [0.17, 0.28]). Patterns of TI as a function of emotional bond were consistent regardless of the culture or toucher’s sex (electronic supplementary material, figure S6). (i) Emotional bond and pleasantness ratings In both countries, an individual’s emotional bond was the strongest with their partner, followed by their closest family members and relatives. By contrast, participants reported the least emotional bond with strangers (electronic supplementary material, figure S2). The strengths of the reported emotional bonds with friends were between those for primary and extended family members in both samples. A Mann–Whitney U test (after Holm–Bonferroni correction) on emotional bond yielded a significant difference only for the partner (U ¼ 13 917, p  8  1029, Holm–Bonferroni corrected), such that the emotional bond with the partner was lower in Japanese than in British participants. (b) Data acquisition The data were collected online. Before beginning the study, partici- pants gave informed consent online. The Japanese study protocol was approved by the ethics committee at the National Institute for The participants reported that being touched by their partner elicited most pleasantness, followed by their close relatives (electronic supplementary material, figure S3). Touch pleasantness and emotional bond were significantly correlated in both cultures, with Spearman correlation coeffi- cient rs4402 ¼ 0.69 in the British sample and rs2977 ¼ 0.74 in the Japanese sample ( ps , 10210), The two cultures differed sig- nificantly in the degree of pleasantness of being touched by others; with the exception of sister and male stranger, the British reported finding social touch as more pleasant than did the Japanese (Mann–Whitney U test, ps in range [3  10216, 0.015], Holm–Bonferroni corrected). version, the sample consisted of only British nationals. The partici- pants were asked about their native (first) language and their cultural identity. We excluded all participants who did not report their first language as being English, or who did not identify them- selves as ‘British’, ‘English’, ‘Irish’, ‘Welsh’ or ‘Scottish’. Altogether, 159 British participants were excluded based on the background questions. Finally, we visually inspected the TAMs and excluded 38 participants from Japanese dataset and 77 participants from British dataset due to inappropriate drawing (e.g. doodling or excessive colouring). The remaining samples, consisting of 386 British and 255 Japanese participants, were used for analysis. version, the sample consisted of only British nationals. The partici- pants were asked about their native (first) language and their cultural identity. We excluded all participants who did not report their first language as being English, or who did not identify them- selves as ‘British’, ‘English’, ‘Irish’, ‘Welsh’ or ‘Scottish’. Altogether, 159 British participants were excluded based on the background questions. Finally, we visually inspected the TAMs and excluded 38 participants from Japanese dataset and 77 participants from British dataset due to inappropriate drawing (e.g. doodling or excessive colouring). The remaining samples, consisting of 386 British and 255 Japanese participants, were used for analysis. 3 (c) Sex differences We next examined if sex influences touch acceptance similarly in the UK and Japan. Figure 3 shows the relationship between touchable body area and sex of the toucher with respect to male d female participants (blue and red dots) in the UK d ll l h ff f partners, the sex of the partner was determined by whethe h l f l d h d ff partner back front back front back front father brother uncle mother sister aunt acq. acq. friend friend cousin cousin stranger stranger partner father brother uncle mother sister aunt acq. acq. friend friend cousin cousin stranger stranger partner father brother uncle mother sister aunt acq. acq. friend friend cousin cousin stranger stranger (a) Japan (b) UK (c) difference proportion of subjects 1.0 0.8 0.6 0.4 0.2 0 proportion of subjects 1.0 0.8 0.6 0.4 0.2 0 10 5 0 –5 –10 UK Z score Japan gure 1. Relationship-specific TAMs in (a) Japan and (b) the UK. The colouring displays the proportion of the sample reporting that being touched by this person s area is acceptable to them. (c) Comparison of the proportion of participants per culture who allow touching in different areas. Red colour in the maps indicate t Japanese participants reported that area more acceptable, blue colour indicates that British participants reported that area more acceptable. The data in (c) a esholded at p , 0.05, FDR corrected. After FDR correction, Z threshold with no correlation assumptions varied from 3.22 to 5.98, depending on the number o ticipants who had that particular individual in their social network. Red and blue labels indicate female and male members of the social network, respectivel partner back front front father brother uncle mother sister aunt acq. acq. friend friend cousin cousin stranger stranger partner father brother uncle mother sister aunt acq. acq. friend friend cousin cousin stranger stranger (a) Japan (b) UK proportion of subjects 1.0 0.8 0.6 0.4 0.2 0 ion of subjects 1.0 0.8 0.6 0 4 (c) difference back front partner father brother uncle mother sister aunt acq. acq. friend friend cousin cousin stranger stranger (c) difference 10 5 0 –5 –10 UK Z score Japan front back Figure 1. Relationship-specific TAMs in (a) Japan and (b) the UK. The colouring displays the proportion of the sample reporting that being touched by this person in this area is acceptable to them. (c) Sex differences 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 emotional bond pleasantness touchability index (TI) touchability index (TI) UK Japan country 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 7 8 9 10 emotional bond touchability index (TI) UK Japan country 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 7 8 9 10 pleasantness touchability index (TI) emotional bond variance in TI explained by 1 2 3 4 5 6 7 8 9 10 emotional bond 10 9 8 7 6 5 4 3 2 1 pleasantness pleasantness Figure 2. Correlations between touchability index (TI), emotional bond and pleasantness. Each dot represents the average response for one member of the social network in each culture (e.g. mother), with linear regression line and confidence interval for the regression fitted separately for each culture. Bottom right panel presents the relative importance of regressors in determining the TI in a linear model. 0.3 0.2 0.1 0 female male female male female male sex of toucher sex of subject mean TI UK Japan Figure 3. Interaction plot of the average TI for male and female participants (blue and red dots, respectively) with respect to male and female touchers for each culture. Error bars depict the 95% confidence interval. Red and blue lines indicate the interaction between toucher sex and the sex of the participant. Note: partners are excluded from the analyses, as the sex of the partner differs between the male and female participants. male sex of subject mean TI Figure 3. Interaction plot of the average TI for male and female participants (blue and red dots, respectively) with respect to male and female touchers for each culture. Error bars depict the 95% confidence interval. Red and blue lines indicate the interaction between toucher sex and the sex of the participant. Note: partners are excluded from the analyses, as the sex of the partner differs between the male and female participants. (c) Sex differences (c) Comparison of the proportion of participants per culture who allow touching in different areas. Red colour in the maps indicates that Japanese participants reported that area more acceptable, blue colour indicates that British participants reported that area more acceptable. The data in (c) are thresholded at p , 0.05, FDR corrected. After FDR correction, Z threshold with no correlation assumptions varied from 3.22 to 5.98, depending on the number of participants who had that particular individual in their social network. Red and blue labels indicate female and male members of the social network, respectively. and female participants (blue and red dots) in the UK and Japan. To statistically evaluate the effect of sex on TI, we conducted an ANOVA on the TIs of male and female par- ticipants and male and female touchers in both cultures. For partners, the sex of the partner was determined by whether the participant was male or female, and hence it was difficult to compare the effect of sex on TI between them. For this reason, we excluded the partner data from this analysis. 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 emotional bond emotional bond 10 9 8 7 6 5 4 3 2 1 pleasantness pleasantness variance in TI explained by pleasantness 43.5% bond 36.1% country 7.4% total R2 87.0% touchability index (TI) touchability index (TI) UK Japan country Figure 2. Correlations between touchability index (TI), emotional bond and pleasantness. Each dot represents the average response for one member of the social network in each culture (e.g. mother), with linear regression line and confidence interval for the regression fitted separately for each culture. Bottom right panel presents the relative importance of regressors in determining the TI in a linear model. 0.019, such that the parameter estimates were larger for female touchers (M ¼ 0.18, s.d. ¼ 0.24) than for male touchers (M ¼ 0.12, s.d. ¼ 0.19, t6739.3 ¼ 11.7, p , 10210, 95% CI [0.050 0.071], d ¼ 0.085). Thus, touch by female members of the social network was generally considered more acceptable across the whole social network (electronic supplementary material, figure S7). The effect of participant sex was not significant (F1,6945 ¼ 0.72, p ¼ 0.39). (c) Sex differences The grey intercept and slope terms mean no significant difference in the regression lines between the cultures. The coloured terms signal which culture ad higher intercept (a) or slope (b) values, with blue and red colours indicating the British and the Japanese, respectively. The visualization is presented with veraged data in the interest of clarity, but the significance of intercept and slope are calculated from the full (un-averaged) data. Proc. R. Soc. B 286: 20190467 differences in a (intercept) and b (slope): greater in Japan differences in a (intercept) and b (slope): greater in Japan emotional bond Figure 4. Visualization of ROI-specific cultural differences in TI versus emotional bond. Linear regression lines were fitted to each culture separately. Blue and red colours depict UK and Japan, respectively. Each dot represents the average response for one member of the social network (e.g. ‘British partner’ or ‘Japanese mother’). The grey intercept and slope terms mean no significant difference in the regression lines between the cultures. The coloured terms signal which culture had higher intercept (a) or slope (b) values, with blue and red colours indicating the British and the Japanese, respectively. The visualization is presented with averaged data in the interest of clarity, but the significance of intercept and slope are calculated from the full (un-averaged) data. All interactions were significant at the p , 0.05 significance level. Of the two-way interactions, the interaction between the toucher sex and participant sex was significant at F1,6945 ¼ 18.5, p  2  1025, h2 ¼ 0.003, such that the difference between TI for female touchers and male touchers was larger for female participants (M ¼ 0.08, s.d. ¼ 0.11) than for male participants (M ¼ 0.04, s.d. ¼ 0.09, t592.61 ¼ 5.7, p  2  1028, d ¼ 0.46). The interaction between the sex of the participant and the country was significant (F1,6945 ¼ 22.6, p  2  1026, h2 ¼ 0.003). The TIs reported by Japanese male participants (M ¼ 0.18, s.d. ¼ 0.26) were higher on average than those reported by Japanese women (M ¼ 0.15, s.d. ¼ 0.23), British men (M ¼ 0.14, s.d. ¼ 0.20), or British women (M ¼ 0.16, s.d. ¼ 0.20), two sample t-test t-scores t2712.5 ¼ 3.8, p ¼ 0.0002, d ¼ 0.14; t2329.3 ¼ 5.3, p  1027; and t2496.4 ¼ 3.4, p ¼ 0.0006, d ¼ 0.13 respectively. (c) Sex differences Three-way ANOVA (2 levels of cultures  2 levels of tou- cher sex  2 levels of participant’s sex) on the TI revealed a significant main effect of culture F1,6945 ¼ 12.6, p  4  1025, h2 ¼ 0.002, such that the TIs in the Japanese sample (M ¼ 0.17, s.d. ¼ 0.24) were larger than the TIs in the British sample (M ¼ 0.15, s.d. ¼ 0.20, t5316.8 ¼ 3.38, p ¼ 0.0007, 95% CI [0.008 0.029], d ¼ 0.085). The main effect of the sex of the toucher was also significant F1,6945 ¼ 137.9, p , 10210, h2 ¼ 0.019, such that the parameter estimates were larger for female touchers (M ¼ 0.18, s.d. ¼ 0.24) than for male touchers (M ¼ 0.12, s.d. ¼ 0.19, t6739.3 ¼ 11.7, p , 10210, 95% CI [0.050 0.071], d ¼ 0.085). Thus, touch by female members of the social network was generally considered more acceptable across the whole social network (electronic supplementary material, figure S7). The effect of participant sex was not significant (F1,6945 ¼ 0.72, p ¼ 0.39). 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 emotional bond Japan UK hair face shoulder (front) shoulder (back) shoulder (back) shoulder (front) torso (back) torso (front) arm hand crotch bottom foot leg (front) leg (back) a b a b a b a b a b a b a b a b a b a b a b a b a b ROI definitions hair arms face torso (front) torso (back) crotch bottom hands legs (front) legs (back) feet differences in a (intercept) and b (slope): greater in Japan greater in the UK no difference country touchability index per ROI igure 4. Visualization of ROI-specific cultural differences in TI versus emotional bond. Linear regression lines were fitted to each culture separately. Blue and red olours depict UK and Japan, respectively. Each dot represents the average response for one member of the social network (e.g. ‘British partner’ or ‘Japanese mother’). (c) Sex differences The interaction between the tou- cher sex and country was significant (F1,6945 ¼ 5.0, p ¼ 0.025, h2 ¼ 0.001). The TIs for Japanese female touchers (M ¼ 0.20, s.d. ¼ 0.26) were on average higher than those for British female touchers (M ¼ 0.17, s.d. ¼ 0.22) (t2758.4 ¼ 3.7, p ¼ 0.0003, d ¼ 0.13). The three-way interaction was also significant (F1,6945 ¼ 25.0, p  6  1027, h2 ¼ 0.003). This result seems to be mostly driven by the different responses of Japanese men and women with respect to male touchers (figure 3). Simi- lar results were obtained using a mixed-effects model with subject as a random effect (electronic supplementary material, table S2). (d) Region-of-interest analysis (d) Region-of-interest analysis Whole-body TAM analyses revealed cultural differences in touchability of specific body areas, such as face, hand, and arm. To further examine for area-specific cultural differences, we next conducted linear regression analyses on the regional TIs for each culture, with emotional bond as the explanatory variable (figure 4). Adjusted R2 values of fitted linear func- tions averaged across body regions ranged between 0.04 and 0.15. Comparing linear models fitted to ROI-wise TI data showed significantly higher baseline acceptability (inter- cept) of touch on hair (b ¼ 20.02, t7376 ¼ 22.96, p ¼ 0.003), feet (b ¼ 20.05, t7376 ¼ 27.3, p , 10210), legs (both front, b ¼ 20.03, t7376 ¼ 25.3, p  9  1028, and back, b ¼ 20.03, t7376 ¼ 25.7, p  2  1028), crotch (b ¼ 20.01, t7376 ¼ 23.0, p ¼ 0.003) and bottom (b ¼ 20.03, t7376 ¼ 25.1, p  3  1027) in the Japanese sample. The same test showed higher baseline acceptability of touch on arms (b ¼ 0.06, t7375 ¼ 3.4, p ¼ 0.0006), hands (b ¼ 0.14, t7375 ¼ 7.2, p , 10210) and face (b ¼ 0.07, t7376 ¼ 9.3, p , 10210) in the British sample. The same test showed a different rate of increase in TI for each unit of emotional bond (slope) on the arms (b ¼ 20.01, t7375 ¼ 23.5, p ¼ 0.0004) and hands (b ¼ 20.02, t7375 ¼ 27.3, p , 10210). In both of these ROIs, the TI was more responsive to changes in emotional bond (steeper slope) in the Japanese sample. However, a cross-sectional design cannot reveal whether pleasure derived from being touched by someone enhances the emotional bond to the toucher, or whether liking someone makes their touch feel more pleasant. 7 Social touch is important for group cohesion of non-human primates, and grooming relationships predict the level of support during conflict situations and when in need of help [8,31–34]. It is hypothesized that grooming-induced relaxed and pleasurable feelings could constitute the psychological mechanism for an individual’s willingness to offer subsequent help, thus forming the basis of mutual exchanges of social sup- port [5,34]. Because social touching increases prosocial behaviour [35–37], it is possible that human social touch increases emotional bonding with the individual being touched, enhancing their prosocial behaviour. (a) Gender difference in both cultures One possible explanation for the cross-cultural similarities in social touching is the effect of globalization. For instance, Japanese culture may be influenced by social customs in e.g. Western movies that are shown widely in Japan. How- ever, the relationship specificity of touchable areas (found here in European and Japanese cultures) is consistent with both our earlier study with Western cultures [9] as well as an older study looking at Japanese and North American cultures [19]. Thus, it is unlikely that the role of recently shared cultural elements plays a major role in the cross-cultu- rally similar association between social touch and emotional bond. Participants in both cultures allowed women to touch their bodies more than men, which accords with prior findings in Western cultures [9,47]. The preference to touch by women was comparable in British men and women. By contrast, responses of the Japanese participants showed a clear gender difference: Japanese men did not show a statistically significant preference for touch by women, whereas Japanese women showed a remarkably strong preference. Earlier studies have found opposite-sex touch to be prefer- able to same-sex touch [19,23,48–50]. Preference for female touch in the female participants might therefore seem to con- tradict earlier findings. On closer inspection, this seeming contradiction is caused by a difference in terminology. Most of the earlier studies discussing gender effects have inspected the difference of body accessibility for ‘same-sex friend’ and ‘opposite-sex friend’. Unfortunately, most of the older exper- iments [23,48–50] use the euphemism ‘opposite-sex friend’ to indicate a romantic partner (a usage made explicit in [51]), whereas ‘same-sex friend’ seems to refer to a platonic friend. This practice biases the comparison, as touching romantic part- ners is both qualitatively and quantitatively different from touching other people [9]. Social touch induces positive feelings and improves interpersonal evaluation [24–27]. For example, participants evaluate even a stranger more positively, if that person has inconspicuously touched them during an interaction [25,27–29]. This close connection between social touching and preference toward the source of touch suggests a causal role of touch on social bonding. There is some evidence of touching causally impacting bonding in romantic relationships [30]. However, it is not clear if this extends to other relationships and this cannot be directly addressed from the current cross- sectional data. We also found that emotional bonds are correlated with experienced pleasantness of social touch. (d) Region-of-interest analysis Neurophysiological studies have revealed that the un- myelinated afferent c-tactile nerve fibres are selectively responsive to light and slow touch [38–40]. The signals car- ried out by these fibres are indirectly transmitted to the insula [38], a part of the neural network responsible for affec- tive tactile interactions [40,41]. This pathway could thus support culturally universal hedonic nature of interpersonal touch. However, as shown in the present study, relationship information imposes top-down influences on this circuitry (e.g. how the individuals are touched and by whom they are touched, electronic supplementary material, figure S3). It is also known that physical contact with members of one’s social network attenuates brain activity associated with both the threat of physical pain stimulus [42] and aver- sive images [43]. Processing interpersonal aspects of touch may recruit additional brain networks [44,45], including the action observation network (AON) [46]. Thus, the positive and calming effect of interpersonal tactile interchanges may be generated by the interaction of these brain networks, med- iating the relationship between touchable area and emotional bonding, regardless of the different cultural norms. 4. Discussion The results show that although the touchable areas differed between the UK and Japan, they were similarly dependent on the strength of the emotional bond. Emotionally close individuals in the inner layers of the social network were allowed to touch larger bodily areas, whereas touching by strangers was primarily limited to the hands. The ROI analyses confirmed that most of the ROIs were similarly sensitive to emotional bond in both countries. This result suggests that the use of social touch for bonding purposes serves a similar function in East Asian and European cultures, rather than being merely culture-based normative behaviour. This interpretation accords with previous studies [9,19,23] suggesting cultural invariance in social touching. We have previously reported consistent social touching pat- terns in a large sample of European cultures [9]. Additionally, American and Japanese college students were asked where they had touched and been touched by their parents, a same- sex friend and an opposite-sex friend [19]. The relative frequencies of touch by these different members of the social network followed the same pattern in both cultures [19]. (c) Limitations O d (c) Limitations Our study was conducted online. Because the participants did not meet the experimenter, they could answer the questions without the sense of invasion of privacy (e.g. feeling embar- rassed that experimenters in front of them know their touching behaviour). While there is some experimental evi- dence suggesting that attitudes towards touch impact touching behaviour [55], it is possible that the data may not directly translate to real-life touching behaviour. Therefore, it will be important to validate the current findings by conduct- ing observational research of real-life touching in the future. Second, although the currently available evidence suggests that social touching is concordant across a wide range of Wes- tern, Orthodox and Japanese cultures (see also [9]), these data do not generalize to the other major cultures of the world [56]. However, already the present data demonstrate marked cross- cultural consistency in touch-dependent bonding across a wide range of cultures and geographical locations. Second, we also observed three notable differences in the topographies of acceptable touch. The British participants allowed their partners to touch their bodies more than the Japanese (figure 1). This finding accords with previous research showing that touch between partners is more frequent in most body areas in the Western (North American) culture than in Japan [19]. The British participants in the present study also reported stronger emotional bonds with their partners than did the Japanese participants, suggesting that the difference between touch allowances for the partner might not be merely a cultural norm in behaviour but indicative of a wider difference in the intimate relationships in the two cultures. Moreover, British participants allowed female family mem- bers and female friends to touch their faces more than did the Japanese participants. A previous study demonstrated that the British tend to touch their own faces more frequently than the Japanese do [53]. It is possible that such ‘accessibility’ to face may originate from gestures that are more specific to British than Japanese and their self-touching behaviour. By contrast, more Japanese participants reported that their female relatives are allowed to touch them on their legs and bottoms. Female members in the Japanese culture often take care of children in their network and have physical contact with them. It is con- sidered that British culture is more individualistic than Japan and that individuals in such culture assume responsibility only for themselves and their immediate family [54]. (c) Limitations O d Thus, the observed difference may be explained by the roles of the female relatives in child rearing in the two cultures. (b) Cultural differences between the Japanese and the British While the overall association of touching and social bonding was concordant across cultures, some differences were also found. First, the Japanese participants reported the overall pleasantness of being touched to be lower than the British did (electronic supplementary material, figure S3), although both cultures showed similar changes in emotional bond across different touchers (figure 2). This difference may be in part due to differences in wording (‘kokochiyoi’ in Japanese versus pleasant in British), but it can also reflect differences in daily non-verbal communication: Japanese conduct social touching (e.g. handshakes and hugs) much less frequently than Americans [19] and they do likely less than British. Funding. This work was supported by Emil Aaltonen Foundation grant and Alfred Kordelin Foundation grant to J.T.S.; by Academy of Finland grant (no. 294897) to L.N. and European Research Council grants to L.N. (no. 313000) & R.I.M.D. (no. 295663), by MEXT/JSPS (a) Gender difference in both cultures Taking this difference of nomenclature into account, our findings would in fact seem to be in line with earlier studies. For example, a similar preference for female touch has pre- viously been found with respect to parents: participants touch and are touched more by their mothers than fathers, both in the United States [23,52] and in Japan [19]. Similar to the current findings, both male and female Japanese participants touch and are touched by their mothers more than by their fathers, but the difference is greater for female participants [19]. Conversely, in the American sample, the preference for mother over father was of comparable size between male and female participants [19]. such as handshakes and hugs, are more common in Western countries than in East Asian countries, such as Japan. This is supported by the higher intercepts in arm and hand for the UK sample in the ROI-wise analysis. Thus, more emotional closeness may be necessary for the Japanese to engage in this type of social touching. 8 Finally, the difference between the touch allowances for female touchers and male touchers was larger for Japanese women than for Japanese men or British participants of either sex (culture  sex of toucher  sex of participant inter- action, figure 3). This culture-specific gender difference can be also associated with the above-mentioned points. More specifically, Japanese women had much smaller TIs for all males in their social network than for women with similar formal relationship (e.g. mother and father, see electronic sup- plementary material, figure S7). Collectively, although we found some differences, the associations between emotional bond, pleasantness and TI were concordant in both cultures. Competing interests. The authors declare no conflict of interest. Acknowledgements. We thank Enrico Glerean for his help in collecting the data. We acknowledge the computational resources provided by the Aalto Science-IT project. 5. Conclusion Relationship-specific emotional bonds account for the magni- tude of social touching from social network members similarly between Western and East Asian (Japanese) cul- tures. Pleasure derived from touch, however, depended on the culture. Because the relation-specific social touching pat- terns of Japanese and Western respondents are consistent, the relationship between emotional bonding and touchable body area may be largely biologically determined. Human social touch, like social grooming of non-human primates, may provide a scaffold for social bonding with the members of one’s social network. This supports the role of somatosen- sation and emotional feelings in the maintenance of social bonds in humans [57]. We also observed cultural differences in the relationship between the emotional bond and the touchable area in specific body parts such as the arms and hands. In these regions, the strength of the emotional bond with a social net- work member increased touchability more in the Japanese versus British sample, i.e. the Japanese had a steeper slope in the fitted linear function (figure 4). This difference might be explained in terms of cultural difference in daily gestures. For instance, gestures involving physical contacts, Data accessibility. The data are available from the Dryad Digital Reposi- tory: https://doi.org/10.5061/dryad.f0f2642 [58] and the code to produce all the figures and numbers are available at https://ver- sion.aalto.fi/gitlab/jtsuvile/cultural-similarity-touch. Funding. This work was supported by Emil Aaltonen Foundation grant and Alfred Kordelin Foundation grant to J.T.S.; by Academy of Finland grant (no. 294897) to L.N. and European Research Council grants to L.N. (no. 313000) & R.I.M.D. (no. 295663), by MEXT/JSPS Acknowledgements. We thank Enrico Glerean for his help in collecting the data. 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Nonverbal Behav. 13, 3–14. (doi:10.1007/BF01006469) 45. (doi:10.1073/pnas.1519231112) Kitada R, Yoshihara K, Sasaki AT, Hashiguchi M, Kochiyama T, Sadato N. 2014 The brain network underlying the recognition of hand gestures in the blind: the supramodal role of the extrastriate body area. J. Neurosci. 34, 10 096–10 108. (doi:10.1523/ JNEUROSCI.0500-14.2014) 50. Heslin R, Nguyen TD, Nguyen ML. 1983 Meaning of touch: the case of touch from a stranger or same sex person. J. Nonverbal Behav. 7, 147–157. (doi:10.1007/BF00986945) 56. Huntington SP. 1997 The clash of civilizations and the remaking of world order. Harmondsworth, UK: Penguin Books. 51. Nguyen T, Heslin R, Nguyen ML. 1975 The meanings of touch: sex differences. J. Commun. 25, 92–103. (doi:10.1111/j.1460-2466.1975.tb00610.x) 57. Nummenmaa L, Hari R, Hietanen JK, Glerean E. 2018 Maps of subjective feelings. Proc. Natl Acad. Sci. U.S.A. 115, 9198–9203. 46. Kilner JM. 2011 More than one pathway to action understanding. Trends Cogn. Sci. 15, 352–357. (doi:10.1016/j.tics.2011.06.005) 52. Rosenfeld HM. 1967 Nonverbal reciprocation of approval: an experimental analysis. J. Exp. Soc. Psychol. 3, 102–111. 58. Suvilehto JT, Nummenmaa L, Harada T, Dunbar RIM, Hari R, Turner R, Sadato N, Kitada R. 2019 Data from: Cross-cultural similarity in relationship-specific social touching. Dryad Digital Repository. (doi:10. 5061/dryad.f0f2642) 47. Tomita M. 2008 An exploratory study of touch zones in college students on two campuses. Calif. J. Health Promot. 6, 1–22. 53. Hatta T, Dimond SJ. 1984 Differences in face touching by Japanese and British people. Neuropsychologia 22, 531–534. (doi:10.1016/0028-3932(84)90050-2) 48. Rosenfeld LB, Kartus S, Ray C. 1976 Body accessibility revisited. J. Commun. 26, 27–30.
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РОЗРОБКА ЗАВДАНЬ МАЙБУТНІМИ ВЧИТЕЛЯМИ МАТЕМАТИКИ ЯК ЗАПОРУКА ЇХ ПРОФЕСІЙНОГО ЗРОСТАННЯ
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Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Випуск 21 УДК 373.51:377.1(075) Олександр Манченко здобувач другого (магістерського) рівня вищої освіти Харківський національний педагогічний університет імені Г.С. Сковороди https://orcid.org/0000-0002-5378-3841 Євгеній Войтов здобувач другого (магістерського) рівня вищої освіти Харківський національний педагогічний університет імені Г.С. Сковороди https://orcid.org/0000-0001-5259-7863 Оксана Жерновникова доктор педагогічних наук, професор, завідувач кафедри математики Харківський національний педагогічний університет імені Г.С. Сковороди https://orcid.org/0000-0002-5383-4493 РОЗРОБКА ЗАВДАНЬ МАЙБУТНІМИ ВЧИТЕЛЯМИ МАТЕМАТИКИ ЯК ЗАПОРУКА ЇХ ПРОФЕСІЙНОГО ЗРОСТАННЯ Анотація. Статтю присвячено проблемі розробки та створення математичних завдань майбутніми вчителями математики. Проаналізовано провідний досвід підгоовки майбутніх вчителів математик з Сполучених Штатів Америки щодо створення завдань на уроках математики. Виділені характерні особливості створення та підготовки математичних завдавань для підвищення ефективності занять. Ключові слова: математичні завдання, учні, вчителі математики Випуск 21 удосконалення професійної підготовки майбутнього вчителя РОЗРОБКА ЗАВДАНЬ МАЙБУТНІМИ ВЧИТЕЛЯМИ МАТЕМАТИКИ ЯК ЗАПОРУКА ЇХ ПРОФЕСІЙНОГО ЗРОСТАННЯ Анотація. Статтю присвячено проблемі розробки та створення математичних завдань майбутніми вчителями математики. Проаналізовано провідний досвід підгоовки майбутніх вчителів математик з Сполучених Штатів Америки щодо створення завдань на уроках математики. Виділені характерні особливості створення та підготовки математичних завдавань для підвищення ефективності занять. Ключові слова: математичні завдання, учні, вчителі математики Олександр Манченко Олександр Манченко здобувач другого (магістерського) рівня вищої освіти Харківський національний педагогічний університет імені Г.С. Сковороди https://orcid.org/0000-0002-5378-3841 Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Науково-дослідна робота студентів як чинник Ключові слова: математичні завдання, учні, вчителі математики Математичні завдання є важливою частиною навчання, оскільки вони є інструментами, які вчителі використовують, щоб допомогти учням засвоїти певні математичні концепції [7]. Завдання «визначають не тільки те, що (учні) вивчають, але й те, як вони починають думати про математику, розвивати, використовувати та розуміти її» [9]. Крім того, завдання, які здобувачі виконують на своїх курсах математики, «передають знання про те, що таке математика і що передбачає заняття 95 Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього Випуск 21 у р у удосконалення професійної підготовки майбутнього вчителя з математики» (Національна рада викладачів математики) [5, с. 24]. Оскільки «те, що учні вивчають, фундаментально пов’язане з тим, як вони це вивчають» [6, с. 18], типи завдань, які вчителі пропонують своїм учням для виконання, визначають навчальні можливості учнів. Таким чином, завдання, які вчителі використовують під час навчання, повинні бути вартими того, щоб витрачати навчальний час, не тільки тому, що вони впливають на те, як учень повинен мислити [10], що впливає на те, як учні навчатимуться, але також тому, що вони впливають на те, як учні сприймають природу математики. Розробка математичних завдань є одним із найважливіших обов’язків учителів математики [5]. Математичні завдання можуть бути більш-менш корисними для розвитку математичного розуміння залежно від характеристик, якими вони володіють. Проте дослідники виявили, що багато вчителів включають у свої програми завдання, які, на думку дослідників, мають меншу цінність, ніж хотілося б. Наприклад, Jacobs, J. та ін. [3] проаналізували відеодослідження TIMSS 1995 та 1999 років, щоб побачити, якою мірою вчителі математики у восьмих класах США викладали або не викладали згідно з стандартів, встановленими національною агенцією у 1995, 2000 роках. Ці дослідники вважали, що математичні завдання та те, як вони використовувалися під час уроків, є важливим показником того, чи вчителі викладають на основі стандартів. Більшість вчителів у своєму дослідженні використовували завдання, які не були складними і які можна було розв’язати за допомогою звичайної процедури рішення, що означає, що завдання були вправами, а не завданнями, які варті уваги. Багато вчителів, які брали участь у дослідженні, вірили, що вони викладають згідно з духом реформи освіти, але це не так. Отже, ймовірно, що багато вчителів зараз не розробляють варті уваги завдання, щоб включити їх у навчання, навіть якщо вони думають, що вони це роблять. Оскільки ми знаємо, що вчителі-практики рідко включають варті уваги математичні завдання у свої уроки, ми очікуємо, що вони не навчилися розробляти варті уваги завдання під час свого навчання. Ключові слова: математичні завдання, учні, вчителі математики Розглядаючи приклад Сполучених Штатів Америки можна зазначити, що на допомогу майбутнім вчителям математики прийшли курси підвищення кваліфікації. На цих курсах майбутні вчителі математики 96 Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбут Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчител Випуск 21 удосконалення професійної підготовки майбутнього вчителя покращували свої навички розробки математичних завдань та навчалися, як розробляти різнорівневі завдання для учнів з різною підготовкою. Цей досвід став першим кроком до вдосконалення їхніх навичок проектування завдань на якісно новому рівні. покращували свої навички розробки математичних завдань та навчалися, як розробляти різнорівневі завдання для учнів з різною підготовкою. Цей досвід став першим кроком до вдосконалення їхніх навичок проектування завдань на якісно новому рівні. Мета цього дослідження полягає в тому, щоб описати, розробку математичних завдань, як один з факторів підвищення професійного рівня майбутніх учителів математики. Дізнатися, на що звертають увагу майбутні учителі математики, коли вони намагаються розробити важливі математичні завдання, є цінним, оскільки це дає педагогам конкретну інформацію, яку вони можуть використати для роботи над покращенням свого професійного рівня. Здатність розробляти важливі математичні завдання стане неоціненним надбанням для майбутніх учителів математики, коли вони почнуть викладати, оскільки ця важлива навичка дасть їхнім учням доступ до знань, які дозволять їм розвинути глибоке розуміння математики. Математичне завдання – це «заняття в класі, метою якого є зосередити увагу учнів на певній математичній ідеї» [9]. Прикладами математичних завдань є проекти, задачі, програми та вправи [5]. Є три способи, якими майбутні учителі математики можуть розробляти математичні завдання. По-перше, вчителі можуть вибрати завдання з доступних ресурсів. Ці ресурси можуть включати підручники, які вони використовують, інші навчальні матеріали або Інтернет. По-друге, вчителі можуть адаптувати вже наявні завдання. Часто вчителі можуть знайти завдання, дослідити його та змінити завдання, щоб воно відповідало їхнім потребам. По-третє, вчителі можуть створити завдання з нуля як унікальний продукт який поєднує найкращі практики математичного проектування. У цьому дослідженні розробка завдання стосуватиметься всіх цих трьох підходів: вибору, адаптації та створення. Релевантні математичні завдання знайомлять студентів з важливими математичними ідеями та залучають їх до них. У професійних стандартах зазначено, що варте уваги математичне завдання має ґрунтуватися на «надійній та значущій математиці» [5, с. 25], а Hiebert J. та ін. [2] стверджував, що відповідні завдання повинні 97 Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Випуск 21 вимагати від студентів роздумів над важливими математичними ідеями і тільки тоді вони зможуть творчо імплементувати їх у свою майбутню професійну діяльність вчителя математики. Ключові слова: математичні завдання, учні, вчителі математики Окрім змісту завдання, головна увага в літературі також приділяється міркуванню, яке вимагає від учнів чіткого розуміння поставленої математичної цілі завдання. Незважаючи на те, що автори можуть описувати це міркування, використовуючи іншу термінологію, таку як вирішення проблем або когнітивна діяльність, очевидно, що життєво необхідно, щоб здобувачі думали, щоб вивчати математику з розумінням. Професійні стандарти стверджують, що завдання мають бути інтелектуально захоплюючими та вимагати від здобувачів міркування та вирішення проблем у різний спосіб [5]. Hiebert та ін. [2] стверджував, що відповідні завдання повинні бути справжньою проблемою для здобувачів, оскільки вони ще не знають, як їх вирішити. На мислення, яке потрібне учням для виконання завдання, впливають їхні попередні знання та досвід. Завдання мають бути складними, але доступними. Smith M. та Stein M. [8] припустили, що вчитель повинен враховувати вік, рівень класу, попередні знання та досвід своїх учнів, вирішуючи які завдання надавати для роботи у класі. Сміт і Штейн пояснили, що якби вчитель дав шестикласнику завдання, де він повинен скласти п’ять двозначних чисел і пояснив, як він це зробив, учень, швидше за все, використав би алгоритм і перерахував кроки, через які він пройшов, щоб його вирішити. Однак, якби вчитель дав задачу другокласнику, який не знав алгоритму та мав доступні знання, учень, швидше за все, досліджував би математичні поняття. Варте завдання вимагає від здобувачів використання наявних знань для вирішення нового завдання [2] і «створює потребу» [1, с. 318] у новій концепції, вимагаючи від них побудови нових знань. Є й інші характеристики, які допомагають зробити завдання вартим уваги. Професійні стандарти підготовки у США включали такі визначення: гідне завдання має передати, що математика є постійною діяльністю людини, вимагати від учнів спілкування про математику, допомагає учням встановлювати зв’язки між математичними ідеями та розвивати навички учнів разом із розумінням цих явишь та процесів. Професійні стандарти [5] (також запропонували, щоб вчителі зважали 98 Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Науково-дослідна робота студентів як чинник ф ій ї і йб Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Випуск 21 на рівень здобувачів при розробці завдань, рекомендуючи, щоб варті завдання базувалися на «знанні розуміння, інтересів і досвіду учнів» і «знанні ряду способів, за допомогою яких різні учні вивчають математику. ‖ (с. 25), і що корисні завдання повинні розвивати в учнів схильність до математики через розуміння, а не заучування. Smith M. та Stein M. Ключові слова: математичні завдання, учні, вчителі математики [8] також заявили, що завдання повинні вимагати від учнів глибокого мислення, але не повинні бути надто складними для учнів, щоб учні відчули, що вони можуть залучитися до завдання. Важливим фактором підвищення якості освіти є впровадження креативності у завдання, що значно підвищує зацікавленість здобувачів у вивченні різних предметів [4]. Таким чином, можна констатувати, що розробка математичних завдань є важливою складовою підвищення професійного рівня майбутніх учителів математики в умовах постійного розвиваючихся педагогічних технологій навчання. Математичні завдання це комплекс дій у класі які направлені на розуміння математичних догм та знань через сприйняття інформації у посильній мисленевій діяльності учнів. У майбутніх наукових розвідках планується дослідити питання впровадження диференційованих математичних завдань у процесі опанування основ математики. Список використаних джерел 1. Confrey J. Learning to see children's mathematics: Crucial challenges in constructivist reform. In K. Tobin (Ed.), The practice of constructivism in science education (pp. 299- 321). 1993. Hillsdale, NJ: Lawrence Erlbaum Associates. 2. Hiebert J., Carpenter T. P., Fennema E., Fuson K. C., Wearne D., Murray H., . . . Human P. Making sense: Teaching and learning mathematics with understanding. 1997. Portsmouth, NH: Heinemann. 3. Jacobs J. K., Hiebert J., Givvin K. B., Hollingsworth H., Garnier H., & Wearne D. Does eighth-grade mathematics teaching in the United States align with the NCTM Standards? Results from the TIMSS 1995 and 1999 video studies. Journal for Research in Mathematics Education. 2006. 37. P. 5-32. 99 Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Випуск 2 Науково-дослідна робота студентів як чинник удосконалення професійної підготовки майбутнього вчителя Випуск 21 4. Nalyvaiko O., & Ronzhes O. Creativity in digital environment. possibilities of application in the educational space. Electronic Scientific Professional Journal ―Open Educational E-Environment of Modern University‖. 2021. (11). P. 106-120. https://doi.org/10.28925/2414-0325.2021.119 4. Nalyvaiko O., & Ronzhes O. Creativity in digital environment. possibilities of application in the educational space. Electronic Scientific Professional Journal ―Open Educational E-Environment of Modern University‖. 2021. (11). P. 106-120. https://doi.org/10.28925/2414-0325.2021.119 5. National Council of Teachers of Mathematics. Professional standards for teaching mathematics. 1991. Reston, VA: Author. 5. National Council of Teachers of Mathematics. Professional standards for teaching mathematics. 1991. Reston, VA: Author. 6. National Council of Teachers of Mathematics. Mathematics teaching today (2nd ed.). 2007. Reston, VA: Author. 6. National Council of Teachers of Mathematics. Mathematics teaching today (2nd ed.). 2007. Reston, VA: Author. 7. Simon M. A. & Tzur R. Explicating the role of mathematical tasks in conceptual learning: An elaboration of the hypothetical learning trajectory. Mathematical Thinking and Learning. 2004. 6. P. 91-104 8. Smith M. & Stein M. Selecting and creating mathematical tasks: From research to practice. Mathematics Teaching in the Middle School. 1998. 3. P. 344-350. 9. Stein M., Grover B. & Henningsen M. Building student capacity for mathematical thinking and reasoning: An analysis of mathematical tasks used in reform classrooms. American Educational Research Journal. 1996. 33. P. 455-488. 10. Zhernovnykova O. A., Nalyvaiko O. O. & Chornous N. A. Intelectual competence: essence, components, levels of formation. Pedagogy and Psychology. 2017. 58. P. 32-41. doi: 10.5281/zenodo.1116977. O. Manchenko, Y. Voitov, O. Zhernovnykova Annotation. The article is devoted to the problem of developing and creating mathematical tasks by future teachers of mathematics. The leading experience of training future teachers of mathematics from the United States of America regarding the creation of tasks in mathematics lessons was analyzed. Characteristic features of 100
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O estágio supervisionado nos cursos de graduação em Educação Física: um desafio presente nesta formação
Dialogia
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Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. O estágio supervisionado nos cursos de graduação em Educação Física: um desafio presente nesta formação Enny Vieira Moraes Professora assistente – UESB; Doutoramento em História Social – PUC. São Paulo – SP[Brasil] ennymoraes@hotmail.com Alantiara Peixoto Cabral Graduada em Educação Física – Universidade Estadual do Sudoeste da Bahia. Luanda Nogueira Souza Graduada em Educação Física – Universidade Estadual do Sudoeste da Bahia. Manuela Soares de Alcântara Graduada em Educação Física – Universidade Estadual do Sudoeste da Bahia. Alantiara Peixoto Cabral Graduada em Educação Física – Universidade Estadual do Sudoeste da Bahia. Luanda Nogueira Souza Graduada em Educação Física – Universidade Estadual do Sudoeste da Bahia. Manuela Soares de Alcântara Graduada em Educação Física – Universidade Estadual do Sudoeste da Bahia. Neste artigo, discute-se o estágio supervisionado, como discipli­ na curricular obrigatória, nas licenciaturas em Educação Física, além dos desafios presentes na relação entre formação profissio­ nal e ética na escola pública. Essa proposta amplia-se quando a ótica adotada para discutir tais desafios envolve não apenas a visão de docentes, mas também a visão dos discentes. Concluiu- se que o estágio, um momento desafiador na formação, produz a consciência da importância do profissional da educação e de sua participação como agente questionador da realidade. Palavras-chave: Estágio supervisionado. Formação profissional. História da Educação Física Palavras-chave: Estágio supervisionado. Formação profissional. História da Educação Física Artigos 199 Outros autores, no século XIX, já escreviam sobre os exercícios físicos ou ginástica, como afirma Cunha Júnior (1998, p. 26): A prática de ensino nos cursos de licen­ ciatura em Educação Física traz à tona a dificuldade dos graduandos – que, por vezes, reflete a de alguns professores – inte­ grarem prática e teoria que, apesar de não serem idênticas, possuem interdependência (GONÇALVES JÚNIOR e RAMOS, 1998)1. Os professores civis também atuaram na escrita de manuais e livros sobre Educação Física/Ginástica, durante o século XIX. A formação profissional do grupo ocorreu em suas vivências nas escolas e colé­ gios em que haviam freqüentado aulas de Educação Física/Ginástica. Eram ex- alunos que, ao se destacarem nas aulas, recebiam convites de seus mestres e passa­ vam posteriormente a ministrá-las. Os professores civis também atuaram na escrita de manuais e livros sobre Educação Física/Ginástica, durante o século XIX. A formação profissional do grupo ocorreu em suas vivências nas escolas e colé­ gios em que haviam freqüentado aulas de Educação Física/Ginástica. Eram ex- alunos que, ao se destacarem nas aulas, recebiam convites de seus mestres e passa­ vam posteriormente a ministrá-las. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 1 Introdução A dissociação entre teoria e prática na Educação Física brasileira foi, durante anos, um pro­ blema que se refletia e, talvez se reflita até hoje, mais profundamente, na prática pedagógica dos profissio­ nais de ensino. Como conseqüência dessa ruptura, identificamos problemas em relação ao aspecto pe­ dagógico da área, que consistem num desafio que se apresenta e que se evidencia, primordialmente, nas disciplinas de estágio curricular obrigatório, presen­ te nas licenciaturas desses cursos. Também nesse período, publicações começa­ ram a surgir no País, principalmente em formato de manuais que “ensinavam” atividades físicas, pu­ blicados por autores nacionais e internacionais, que auxiliaram a despertar o interesse da população pela prática das atividades físicas, como afirma Cunha Júnior (1998) sobre Arthur Higgins, ex-jornalista que passou a se dedicar aos exercícios ginásticos, na condição de autor e professor. É nesse contexto que surge a Educação Física no Brasil. Assim, até hoje responder a questões, tais como o que é o estágio em Educação Física? Qual seu conteúdo específico? Quais as orientações e discus­ sões pedagógicas que baseiam o estágio curricular em Educação Física? São desafios não apenas para o profissional que está presente no espaço escolar, mas também para aquele em processo de formação. É nesse sentido que, com base em algumas expe­ riências de docentes e discentes no âmbito formal, buscamos sugerir direcionamentos introdutórios para essa discussão. Assim, a Educação Física surge, como uma prática corporal, impulsionada pela atuação de diferentes atores sociais – civis e mesmo imigran­ tes –, que não tinham, necessariamente, qualquer tipo de formação e, cuja prática tinha como base a sua própria experiência e, normalmente, ocorriam em diferentes locais, com objetivos e com base em elementos distintos para sustentar essas mesmas práticas. Pode-se dizer, então, que o surgimento da Educação Física brasileira tem como reflexo a po­ larização entre o conhecimento teórico e prático e que essa dicotomia caracterizou historicamente essa área por muitos anos. Desde que foi introduzida obrigatoriamen­ te nas escolas brasileiras, no fim dos anos 1920, a Educação Física caracterizou-se pela fragmentação entre o conhecimento da produção da medicina hi­ giênica e a prática dos profissionais que atuavam nas escolas e em outros espaços (MELO, 1996). Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 200 É sabido, entretanto, que a produção teórica da área dará um salto qualitativo no Brasil, durante os anos 1970 e 1980. 1 Introdução Entretanto, como mostra Daolio (1995), os professores de Educação Física que atuavam nas escolas durante esse período, não percebiam claramente a importância dessa disci­ plina no currículo escolar. Apesar de a atuação desses docentes parecer diferenciada em relação às demais disciplinas, em sua visão, eles mesmos ainda não apresentam uma argumentação precisa para definir sua área ou mesmo sua ação peda­ gógica. Essa indefinição se reflete também nos currículos dos cursos de graduação em Educação Física, ou melhor, na ausência de clareza sobre essa formação, demonstrando também a distância entre as discussões teóricas que se apresentavam e sua implementação no espaço escolar. fissionais que tinham como característica basilar a fragmentação de seu conhecimento. Realmente, existe um grande desafio a ser enfrentado na formação desses profissionais no que diz respeito a uma efetiva união entre o fazer pe­ dagógico, a clareza de sua opção metodológica e a reflexão filosófica desse fazer – complexidade que se apresenta, de forma mais transparente, nas discipli­ nas de estágio curricular ou estágio supervisionado. A Resolução CNE/CP 1, de 19 de fevereiro de 2002, publicada em 4 de março de 2002, contribuiu para que as disciplinas de estágio e as licenciatu­ ras, de modo geral, sofressem importantes altera­ ções tanto em relação à ampliação da carga horária quanto à definição dos espaços de atuação dos es­ tágios curriculares que, anteriormente, não eram definidos claramente. De acordo com a nova resolução e em con­ sonância com o Parecer 109/2002 do Conselho Nacional de Educação, homologado em 9 de maio de 2002 e publicado em 13 de maio de 2002 (sessão 1, p.21), no que se refere ao estágio curricular: Embora esse não seja o foco de nossa dis­ cussão, é importante refletir sobre as diversas concepções (higienista, militarista, tecnicista, crítico-superadora, entre outras) que marcaram a formação dos professores de Educação Física. Essas concepções nortearam, durante anos, a formação desses profissionais, e contribuíram para criar as lacunas pedagógicas dessa prática, o que se reflete no cotidiano do espaço escolar. Neste estudo, o objetivo não é afirmar que essas correntes histori­ camente não foram importantes; entretanto, é pos­ sível observar que, pedagogicamente, não deram respostas suficientes a questões que se encontram na base e no surgimento da Educação Física brasi­ leira – no caso específico deste trabalho, à prática pedagógica dessa formação. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 1 Introdução Cada Instituição de Ensino Superior, por­ tanto, deverá incluir no seu projeto pedagó­ gico como componente curricular obrigató­ rio, o estágio curricular supervisionado de ensino como um momento de capacitação em serviço de 400 horas, que deverá ocorrer em unidades escolares onde o estagiário, ao final do curso, assuma efetivamente, sob su­ pervisão, o papel de professor. Portanto, ficam definidos a carga horária mínima para os estágios e os espaços em que deverão ocorrer. No caso da Educação Física, como tal for­ mação, mesmo em licenciatura, incluía, em alguns cursos de nível superior, experiência nos espaços A ausência de definição do compromisso po­ lítico-social dos profissionais da área desnudou um problema central: a necessidade de unir o conheci­ mento teórico com a prática pedagógica desses pro­ Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. Artigos Artigos 201 cenário exige dos discentes soluções ou alternativas de soluções, permeadas de dúvidas (científicas e políti­ cas), constituindo-se num momento importante para qualquer formação profissional. não-formais, esse aspecto também sofreu alteração. Antes dessa resolução legal a docência poderia ser exercitada nas escolas e também em outros espaços como academias de ginástica, clubes etc. o que muda com a Lei é que os estágios, em Educação Física, vão se resumir apenas aos espaços formais. O futuro profissional deve ser levado a perce­ ber no estágio a possibilidade de se ver não como um manipulador de instrumentos ou simples executor de atividades, mas como construtor de propostas que contribuirão no processo de formação daqueles que se encontram no espaço escolar. Com base na refle­ xão concebida teoricamente, são abertas perspecti­ vas para o futuro educador exercer conscientemente posturas mais críticas no exercício de sua prática. Além de tudo, é importante que busque extrapolar a repetição, o que permitirá a identificação dos pro­ blemas que permeiam suas atividades e a fragilida­ de da ação no exercício da prática pedagógica. Para os estágios, o grande desafio na for­ mação é ser eixo articulador entre a capacitação teórico-científica e a atuação político-pedagógica dos futuros profissionais. No entanto, é importante lembrar que essa responsabilidade, especialmen­ te em relação à qualidade da formação, não deve nem pode se limitar apenas a uma ou duas disci­ plinas de estágio soltas no currículo, mas precisa estar presente em todos os momentos da graduação; caso contrário, apenas os estágios responderiam às demandas para essa formação, especificamente em se tratando das licenciaturas. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 1 Introdução O estágio supervisionado realizou-se de 23 de outubro a dezembro de 2007 nas turmas de 1° ano do ensino médio, do período vespertino, formadas, em média, por 35 alunos cada uma, com os alunos e alunas de uma faixa etária entre 15 a 16 anos. Convém salientar que, no fluxograma do curso de licenciatura plena em Educação Física da Universidade Estadual do Sudoeste da Bahia (UESB), campus Jequié, as disciplinas que oferecem um contato com o futuro local de trabalho, analisam e questionam os conhecimentos adquiridos durante o processo de ensino-aprendizagem, na busca de uma possível transformação do ambiente, são as discipli­ nas de Estágio Supervisionado I, II e III, com as te­ máticas: I (Atividades Físicas e Saúde), II (Educação Física Escolar) e III (Educação Física e Lazer). No momento do estágio, o acompanhamento, a supervisão e a orientação – atividades exercidas pelos professores orientadores – são fundamen­ tais. Esses profissionais, que exercem eticamente seu papel, servem de referência, não no sentido da cópia, mas pelo norte inicial de uma prática pro­ fissional pautada na seriedade e no compromisso No momento do estágio, o acompanhamento, a supervisão e a orientação – atividades exercidas pelos professores orientadores – são fundamen­ tais. Esses profissionais, que exercem eticamente seu papel, servem de referência, não no sentido da cópia, mas pelo norte inicial de uma prática pro­ fissional pautada na seriedade e no compromisso – elementos que, infelizmente, nem sempre estão presentes no setor educacional, de um modo geral. Entretanto, o reconhecimento dessa disciplina na formação é inquestionável. – elementos que, infelizmente, nem sempre estão presentes no setor educacional, de um modo geral. Entretanto, o reconhecimento dessa disciplina na formação é inquestionável. É importante frisar que os Estágios Supervisionados I e III eram realizados em espaços não-formais, porém, com a nova resolução, os con­ teúdos dessas disciplinas passaram a ser trabalha­ dos também na escola. Mesmo com inúmeros problemas e dificul­ dades que precisam ser superadas, o estágio é im­ portante no currículo tanto dos discentes quanto dos docentes (SOUZA; BONELA; PAULA, 2007). Por isso, abriu-se espaço, neste estudo, para a expla­ nação de relato de experiência de três discentes do curso de licenciatura plena em Educação Física da Universidade Estadual do Sudoeste da Bahia (Uesb), campus Jequié. Lembrando sempre que, para a efe­ tivação do estágio, de fato, é necessário que ele seja construído no cotidiano da escola, ouvindo todos os atores. 1 Introdução Nesse sentido, concor­ damos com Piconez (1991, p. 25), ao afirmar: Sabe-se que as dificuldades são muitas, prin­ cipalmente porque não há um fazer pedagógico pronto, completo. Essa disciplina tem como carac­ terística “o não ser/estar pronta”, ou seja, “o fazer-se constante na constância e na dinâmica do cotidiano do espaço escolar”. É nessa dinâmica, que é aquilo que trava, interrompe, amadurece, cria e recria pos­ sibilidades de um refazer e refazer-se constantemen­ te, que o estágio deve nortear-se. Acreditamos que a problematização da prática desenvolvida coletivamente pelas di­ ferentes disciplinas do currículo, portanto, articuladas, podem assegurar a unidade, favorecer a sistematização coletiva de novos conhecimentos e preparar o futuro profes­ sor para compreender os determinantes mais profundos de sua prática, com vistas a sua possível transformação. Além dos problemas que envolvem o fazer pe­ dagógico nas disciplinas de estágio, outros, de ordem prática, também se apresentam, entre os quais o número insuficiente de docentes na supervisão e no acompanhamento dos discentes no campo de estágio. Essa ausência transformada em impessoali­ dade gerou uma série de dificuldades, como afirma Silva (2005, p. 37): Nesse processo, é importante que as disciplinas do currículo levem o futuro profissional a questionar sua formação, a ter dúvidas e assumi-las de forma mais concreta. Já nas disciplinas que enfocam prática de ensino e o estágio supervisionado, esse questio­ namento surge, principalmente, no contato com a realidade contextual dinâmica, plural e contraditó­ ria que se instala, propondo desafios concretos. Esse Já se tornou tradicional em nosso país que a orientação de estágio seja encarada pelas IES como um procedimento mera­ Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 202 mente burocrático, cuja ação do profes­ sor-orientador é a cobrança e o controle de entrega de cartas de credenciamento de instituições concedentes de estágio, fichas de seguro, cômputo de horas de estágio e relatórios de estágio […] Paradoxalmente, a impessoalidade acaba tornando-se a tônica de um processo ide­ alizado para que o graduando construa sua futura identidade profissional, onde a personalização do acompanhamento da ação e reflexão sobre a realidade vivida deveria vir em primeiro lugar. – reflexão – ação, num movimento constante, pois a dinâmica da realidade exige esse esforço. A experiência relatada ocorreu no Colégio da Rede Pública Estadual, Luiz Eduardo Magalhães2, situado na cidade de Jequié (BA), e que oferece ensino médio nos três turnos: noturno, vesperti­ no e matutino. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 1 Introdução Nesse processo, os discentes e docentes precisam estar em constante diálogo sobre a ação Após essa discussão, propôs-se a inclusão, neste trabalho, de um relato da experiência dos discentes no encerramento da disciplina Estágio Supervisionado I, cujo conteúdo é atividade física e saúde. Esse relato foi uma sugestão de trabalho com o conteúdo programático dessa disciplina. Como foi produzido depois de finalizada a discipli­ na, não houve aferição de nota ou do conceito dado aos discentes e nem foi atribuído sentido avaliativo. Essa produção está associada ao compromisso do Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. Artigos Artigos 203 Desse modo, a disciplina de Estágio Supervisionado I, do curso de Licenciatura Plena em Educação Física da UESB, que será tratada no relato de experiência, tem como finalidade propor­ cionar aos discentes experiências pedagógicas, que lhes possibilitarão planejar, executar e avaliar situa­ ções de ensino-aprendizagem, durante as atividades físicas e de saúde, buscando focalizá-las como um importante conteúdo a ser trabalhado nas aulas de educação física. grupo em produzir conhecimento sobre o próprio fazer pedagógico. Assim, o relato que consiste na experiência de três discentes do curso de Educação Física da UESB foi resultado de um esforço que envolveu a participação de todos e, certamente, servirá de re­ ferência na formação dessas futuras licenciadas em Educação Física. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 2 Relato de experiência próprio professor regente não dá a devida impor­ tância a essa etapa, pulando-a e deixando que os estagiários se responsabilizem por seus discentes. Acreditamos que esse período seja essencial para a formação de futuros educadores, sendo necessário o comprometimento de ambas as partes. As disciplinas de estágio supervisionado no currículo são divididas em duas etapas: aulas na uni­ versidade e no campo de estágio (na escola) que, por sua vez, subdivide-se em três momentos: observação, co-participação e regência. No primeiro momento – observação – os estagiários se familiarizam com o colégio, conhecem sua estrutura e a(as) turma(as) a ser(em) trabalhada(s) e, por meio das observações das aulas do professor, analisam a prática docente e suas implicações na formação do sujeito. No segundo – co-participação – são vivenciadas experi­ ências docentes, de forma integrada e participativa, com o professor regente. Por fim, a regência, em que os estagiários assumem verdadeiramente a respon­ sabilidade pela turma, desenvolvendo atividades de docência adquiridas durante o curso, tais como pla­ nejamento, procedimentos metodológicos, seleção e aplicação de materiais didáticos, além da operacio­ nalização dos projetos desenvolvidos durante a dis­ ciplina, particularmente nas orientações práticas, por meio do contato com o docente da disciplina na universidade e o supervisor externo. Em relação à co-participação, é possível per­ ceber seu valor durante todo o processo de estágio, em que a atuação era conjunta (professor e estagiá­ rias) e, em cada momento de interação em que uma nova aprendizagem ocorria, contribuindo qualitati­ vamente para a futura atuação docente. Durante a regência, aprende-se que, ao entrar em uma sala de aula, é preciso ter segurança e pensar que, independentemente de qualquer coisa, não se deve esquecer de que sempre há uma troca constante de aprendizado entre estagiários e alunos, ou seja, cada um traz suas experiências pessoais para serem re-passadas, re-discutidas, re-pensadas. Nesse período também se compreende quanto é importante relacionar os conhecimentos a serem transmitidos para os alunos com os adqui­ ridos na vida cotidiana, e assim associar a necessi­ dade de adquirir esses conhecimentos para usá-los na vida diária. Nessa perspectiva, entende-se que é indispensável investigar, no processo educativo, a situação individual e social do grupo de alunos, os conhecimentos e experiências que trazem, de modo que, nas situações didáticas, ocorra a ligação entre os objetivos e conteúdos propostos pelo pro­ fessor e as condições de aprendizagem dos alunos (LIBÂNEO, 1994). 2 Relato de experiência Logo no início, verificamos algumas difi­ culdades nessa disciplina, como os problemas para encontrar as turmas pela falta de horário, uma vez que, nas escolas estaduais do município, as de 1° e 2° anos têm apenas duas aulas semanais de educa­ ção física, e as de 3° ano, apenas uma. Percebemos, assim, por essa e outras questões específicas de nossa realidade, que a educação física é uma dis­ ciplina pouco valorizada no currículo escolar, pois, em algumas escolas, há apenas uma aula de educa­ ção física semanal. O curso de Licenciatura Plena em Educação Física da Universidade Estadual do Sudoeste da Bahia (UESB), campus de Jequié, tem 11 anos de existência e propõe uma formação generalista aos seus estudantes. Em seu fluxograma, apresenta disciplinas do conhecimento biológico, profissiona­ lizante, socioantropológico, filosófico, psico-peda­ gógico, lingüístico e científico. Dessas as de estágio supervisionado tem por objetivo aproximar o gradu­ ando da realidade escolar, possibilitando que viven­ cie experiências de sua futura profissão, o que lhe possibilitará conhecer melhor sua área de atuação. Essa prática é de fundamental importância, pois in­ troduz o futuro professor no âmbito escolar, com o intuito de integrar teoria e prática pedagógica, favo­ recendo o desenvolvimento profissional. Após encontrar as turmas, outra dificuldade foi trabalhar com Atividade Física e Saúde, conte­ údo a ser abordado no Estágio Supervisionado I, pois o professor regente – e supervisor externo – já havia abordado esse conteúdo com suas turmas. Desse modo, foram ministradas poucas aulas com a temática proposta, uma vez que o professor au­ torizou realizar o estágio nas turmas, desde que fosse dada continuidade às atividades planejadas e desenvolvidas por ele. Nesse sentido, concordamos com Pimenta (2004) quando afirma que, […] no estágio dos cursos de formação de professores, compete possibilitar que os futuros professores compreendam a com­ plexidade das práticas institucionais e das ações aí praticadas por seus profissionais como alternativa no preparo para sua in­ serção profissional. Em razão disso, foi preciso reorganizar o estágio e adequá-lo ao planejamento do docente que estava preparando uma gincana com jogos popula­ res, com todas as turmas dos primeiros anos do res­ pectivo colégio. Diante dessa situação, observou-se que a universidade deveria dialogar mais com a co­ Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 204 munidade externa, neste caso a escola, para tentar minimizar esses problemas. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 2 Relato de experiência A observação do estágio Supervisionado I foi de fundamental importância para o processo de ensino- aprendizagem, pois proporcionou um entrosamento com a turma e com o professor regente. Além disso, possibilitou que se criticassem as atitudes do professor, tanto dentro quanto fora da sala de aula. Por meio dessa análise, foi possível absorver certos compor­ tamentos didáticos, considerados necessários para um docente, que resultaram em uma reflexão mais apurada sobre a prática educativa, como também maneiras de atuar no espaço escolar. De acordo com essa experiência, também se percebe que o planejamento e a metodologia de ensino são de suma importância para o proces­ so educativo, e que as aulas nem sempre ocorre­ rão exatamente da forma planejada. Ainda com relação ao mesmo autor, concordamos com seu É sabido que, muitas vezes, os estagiários se restringem a apenas “observar” as aulas, esque­ cendo de analisá-las criticamente. Outras vezes, o Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. Artigos Artigos 205 ponto de vista, quando afirma que o planejamen­ to tem a função de orientar a prática pedagógica, porém não pode ser um documento rígido e abso­ luto, pois uma das características do processo de ensino é que está sempre em movimento, sofrendo modificações ante as condições reais. só as dimensões biológicas dos indivíduos, mas também, as dimensões sociais e pedagógicas, sensibilizando as pessoas sobre a importância de adotar um estilo de vida ativo. Ao longo de sua história, a Educação Física teve uma relação direta com a saúde, seja voltada para a educação higiênica e eugênica da popula­ ção, ou para o desenvolvimento da aptidão física. Sua inclusão nos currículos escolares ocorreu com fortes influências médicas, que pretendiam restau­ rar a saúde da população, adotando hábitos higiê­ nicos com uma visão eminentemente biologicista do homem. Em seguida, vieram as influências militar e desportiva, também com uma visão bio­ lógica, como afirma Moraes (1996). Diante disso, podemos verificar que todas as etapas foram de importância incalculável para o processo de ensino-aprendizagem, e que, a cada momento, no ambiente escolar, tentávamos extrair o máximo de cada aluno, contribuindo, assim, para discussão sobre a formação daqueles futuros profissionais. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 2 Relato de experiência 206 quantidade mínima de professores supervisores ou mesmo sua insuficiência em relação ao número de estagiários, ou até mesmo falta de compromisso de alguns professores, são algumas das queixas fre­ qüentemente apresentadas pelos alunos. na formação dos estudantes, para que possam ser cidadãos que reivindiquem sobre seus direitos e re­ flitam sobre eles buscando melhorias para a popu­ lação que integram e são sujeitos. Atualmente há nas escolas, quando muito, duas aulas de Educação Física de 50 minutos cada uma. Diante disso, constata-se que, para um estu­ dante, é muito difícil obter os benefícios de uma prática de atividade física apenas em aulas regula­ res. Por isso, acredita-se que o principal objetivo do professor esteja focalizado na reflexão do conteúdo trabalhado e em seus métodos. Nesse contexto, seria possível criar situações em que os alunos possam, com a população, reivindicar seus direitos para a melhoria de condições de vida, incluindo aí políticas públicas para uma melhor qualidade de saúde. Nesse sentido, Silva (2005) relata que, quando a experiência dos estágios curriculares é bem orien­ tada, seguramente, gera não apenas benefícios ao graduando, mas também serve de instrumento de avaliação, retroalimentação e aperfeiçoamento do próprio curso de graduação. Dessa forma, percebemos que a presença e o acompanhamento dos supervisores são fundamen­ tais, uma vez que suas orientações e apoio con­ tribuíram para a construção de outras formas de práxis pedagógicas, além de auxiliar nos estudos e na superação das dificuldades que sempre se apre­ sentaram durante todo o processo de estágio. Assim, não se trata de desvirtuar a prática de atividade física como componente que exerce influ­ ência sobre a melhoria do estado de saúde, quando praticada corretamente. No entanto, é preciso não se deixar levar por afirmações simplistas como a de que, atividade física, por si só, é capaz de gerar saúde. A atividade física deve ser compreendida como um componente de inúmeros fatores impres­ cindíveis (capacidade econômica, habitação, tempo livre, acesso a escolarização, cultura, nutrição ade­ quada etc), que concorrem para promoção de saúde. (SCHNEIDER, 2001). Acredita-se ainda que seja necessário estar na escola no começo do ano letivo participando da semana pedagógica e de outras atividades que fazem parte da prática docente, auxiliando desde seu planejamento, até o desenvolvimento das ati­ vidades sem interromper a proposta do docente. É sabido que, para isso, várias barreiras devem ser superadas. 2 Relato de experiência Assim, tanto alguns docentes, professores das universidades e de instituições dos diversos graus de ensino, quanto os próprios estagiários precisam mudar sua maneira de ver o processo de ensino-aprendizagem, buscando aproveitar todas as oportunidades que surgem, para construir uma prática educativa realmente comprometida com o meio social do qual todos nós integramos. No entanto, acreditamos que o professor deva superar essas visões, abordando a questão da ativi­ dade física relacionada à saúde, de forma diferencia­ da, não levando em consideração apenas o aspecto biológico. O professor deve levar em conta que o ser humano é resultado de inúmeras determinações que vão desde o aspecto biológico, o sócio-histórico, e cultural. De outro modo, e como historicamen­ te fizemos, estaríamos apenas reproduzindo uma visão única da realidade. Quanto ao conteúdo relativo à atividade física e saúde, ou mesmo do lazer e da educação física escolar, acreditamos ser fundamental que os profissionais o discutam anteriormente, para que sua aplicação na escola ocorra efetivamente – de forma crítica. Especificamente os conteú­ dos da área da atividade física e saúde, poucas pessoas, principalmente as oriundas das classes desfavorecidas socialmente, não têm muito acesso a esse tipo de informação, e a escola pode ser uma instituição social importante para levar esse conhecimento aos atores presentes no espaço escolar. Nesse sentido, a Educação Física se torna a maior responsável pela transmissão desse con­ teúdo, pois o tem enquanto conteúdo curricu­ lar, e o acesso a esse conhecimento no ambiente escolar se faz necessário para contemplar não Quanto ao conteúdo relativo à atividade física e saúde, ou mesmo do lazer e da educação física escolar, acreditamos ser fundamental que os profissionais o discutam anteriormente, para que sua aplicação na escola ocorra efetivamente Diante disso, concordamos com Matsudo et al. (2003), quando afirmam que não basta apenas falar para as crianças e adolescentes praticarem atividade física, o que trará vários benefícios para sua saúde, mas também deixar claro que vários fatores influen­ ciam na execução dessa prática, contribuindo para que os estudantes possam refletir sobre a sociedade, questionando as informações adquiridas. Portanto, é importante que, em aulas de Educação Física, os estudantes possam analisar o sistema em que a so­ ciedade vive e suas possíveis formas de intervenção. Acredita-se que o professor, agindo dessa forma, poderá contribuir, de maneira significativa, Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. The supervised training in the postgraduate studies in physical education: a challenge of this process The supervised training in the postgraduate studies in physical education: a challenge of this process sibilitaram maior reflexão sobre a prática docente, além de reforçar nossa percepção de que sua execu­ ção vai muito além das salas de aula. É importante ressaltar que essa instituição de ensino em que se realizou este estágio se diferen­ cia das outras e, provavelmente, é essa participação dos docentes que faz a diferença, não apenas da Educação Física, mas também de outras áreas, nas citadas reuniões e em outras atividades da escola. A atuação dos profissionais desta instituição é um exemplo do interesse e do compromisso de todos, agindo realmente coletivamente. In this article, it is discussed the supervising period of probation as a compulsory discipline in the Physical Education bachelor’s degree, besides the challenges of the relation between professional training and ethics in the public school. This proposal gets bigger when the point of view to discuss these challenges involves not only the academician vision, but also the stu­ dents’ point of view. Then, it is concluded that the period of probation, a challenger moment in the graduation, produces the conscience of the importance of the professional’s education and his participation as an agent that analyses the reality. 3 Tentando concluir… Fica registrada, portanto, com essa experi­ ência, que a participação dos futuros professores em espaços escolares contribui significativamente para sua identidade profissional, e que, apesar das indefinidas dificuldades encontradas, o estágio auxilia expressivamente em sua formação, pois é só por meio da vivência no meio escolar que conse­ guiremos constituir uma base de formação profis­ sional e pessoal. O estágio é, como afirma Pimenta (2004, p. 34), “[…] uma atitude investigativa que envolve a reflexão e a intervenção na vida da escola, dos professores, dos alunos e da sociedade.” Key words: History of Physical Education. Professional training. Supervised probation. 2 Relato de experiência Uma delas é levar os estudantes a entender que o meio escolar ultrapassa as ativi­ dades realizadas em salas de aulas, chegando aos corredores em conversas com professores, alunos e funcionários. Desse modo, a Educação Física, como dis­ ciplina escolar, não pode abrir mão do conteúdo atividade física e saúde em sua proposta pedagógi­ ca nas escolas. No entanto, essa incorporação não pode ocorrer acriticamente e, muito menos, des­ vinculada da realidade. Apesar dos impasses verificados durante o estágio, muitos aspectos importantes foram deba­ tidos nas reuniões de planejamento pedagógico (aulas de coordenação – AC), que ocorreram sema­ nalmente, possibilitando um aprendizado significa­ tivo pela troca de conhecimentos entre os estagiários e os docentes mais experientes, que integravam o quadro de efetivos da escola. Esses momentos pos­ Sobre o supervisionamento dos estágios3, muitas vezes, eles não acontecem de maneira correta, ficando o estudante, por diversos motivos, sem orientação e supervisão adequadas. Uma Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. Artigos Artigos 207 Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. Referências CUNHA JÚNIOR, C. F. F. A produção teórica brasileira sobre educação física/ginástica no século XIX: autores, mercado e questões de gênero. In: NETO, A. F. (Org.). v. 3. Pesquisa histórica em educação física brasileira. Aracruz, ES: Faculdade de Ciências Humanas de Aracruz, 1998, p. 26. MORAES, E. V. A presença do ideário higienista no curso de formação em Educação Física da Universidade Federal de Alagoas. Dissertação de mestrado, Universidade Gama Filho, 1996. DAOLIO, J. Da cultura do corpo. Campinas, São Paulo: Papirus, 1995. Notas 1 Embora de forma sintética, nesse trabalho surgem importantes questionamentos sobre o estágio como espaço reconstrutivo da ação pedagógica em educação física e, por esse motivo, sugerimos sua consulta. 2 Gostaríamos de agradecer a todos os profissionais desse estabelecimento de ensino, seus funcionários e alunos pela forma como nos receberam e nos trataram durante todo o percurso do estágio - espaço e local onde todos podemos refletir, discutir, recriar e aprender. Outro aspecto importante é percebermos que essa disciplina se faz em seu percurso, embora, an­ teriormente, tentemos buscar definir sua estrutura, seus objetivos e ações. No entanto, outra análise é possível e necessária: se todos se comprometerem, será possível construir uma experiência rica, não apenas para aqueles em formação, mas também para os docentes, alunos e demais envolvidos, pois é necessário compreender que somos todos incomple­ tos, eternos aprendizes. 3 Como foi citado, existe a figura do supervisor externo de estágio, aquele que se encontra no espaço escolar, e o outro, que é o docente professor da disciplina estágio supervisionado, ocupante dessa cadeira na universidade. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. 208 MELO, V. A. de. Escola Nacional de Educação Física e Desportos: um estudo histórico, a ‘história’ de um estudo e o estudo da história. In NETO, A. F. (Org.). Pesquisa histórica em educação física brasileira. Vitória: UFES. Centro de Educação Física e Desportos, 1996. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. DAOLIO, J. Da cultura do corpo. Campinas, São Paulo: Papirus, 1995. PIMENTA, S.G. L.; LUCENA, M.S. Estágio e docência. São Paulo: Cortez, 2004. FAZENDA; RIBEIRO; BIZZO et al. A prática de ensino e o estágio supervisionado. 2.ed. Campinas: Papirus, 1994 SILVA, S. A. P. S. Estágios curriculares na formação de professores de educação física: o ideal, o real e o possível. Revista Digital, Buenos Aires, ano 10, n. 82, mar. 2005. GONÇALVES J. L.; RAMOS, G. N. S. A prática de ensino e o estágio supervisionado nos cursos de licenciatura em Educação Física. Revista da Unicastelo, São Paulo, v. 1, n. 1, p.13-15, 1998. GONÇALVES J. L.; RAMOS, G. N. S. A prática de ensino e o estágio supervisionado nos cursos de licenciatura em Educação Física. Revista da Unicastelo, São Paulo, v. 1, n. 1, p.13-15, 1998. SOUZA, J. C. A.; BONELA, L.A.; PAULA, A. H. de. A importância do estágio supervisionado na formação do profissional de educação física: uma visão docente e discente. MOVIMENTUM. Revista digital de Educação Física – Ipatinga; Unileste- MG, v. 2, nº 2, ago, dez. 2007. SCHNEIDER, O. Educação Física como promoção da saúde: Contradições de um discurso. Revista Brasileira de Ciências do Esporte, v. 21, n. 1, 2001. SOUZA, J. C. A.; BONELA, L.A.; PAULA, A. H. de. A importância do estágio supervisionado na formação do profissional de educação física: uma visão docente e discente. MOVIMENTUM. Revista digital de Educação Física – Ipatinga; Unileste- MG, v. 2, nº 2, ago, dez. 2007. LIBÂNEO, J. C. Didática. São Paulo: Cortez, 1994. LIBÂNEO, J. C. Didática. São Paulo: Cortez, 1994. MATSUDO, V. K. R.; ANDRADE, D. R.; MATSUDO, S. M. M.; ARAUJO, T. L.; ANDRADE, E.; OLIVEIRA, L.C.; BRAGGION, G; RIBEIRO, M. A. “Construindo” saúde por meio da atividade física em escolares. R. Bras. Ci e Mov. Brasília, DF, v. 11, n.4, p.111-118, out./dez. 2003. SCHNEIDER, O. Educação Física como promoção da saúde: Contradições de um discurso. Revista Brasileira de Ciências do Esporte, v. 21, n. 1, 2001. Artigos recebido em jun. 2008 / aprovado em ago. 2008 Para referenciar este texto: MORAES, E. V. et al. O estágio supervisionado nos cursos de graduação em educação física: um desafio presente nesta formação. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. recebido em jun. 2008 / aprovado em ago. 2008 Para referenciar este texto: MORAES, E. V. et al. O estágio supervisionado nos cursos de graduação em educação física: um desafio presente nesta formação. Dialogia, São Paulo, v. 7, n. 2, p. 199-209, 2008. Artigos 209
https://openalex.org/W4382897447
http://masujournal.org/store_file/archive/85-10-12-556-557.pdf
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EFFECT OF INOCULATION OF Acetobacter diazotrophicus (ACN) WITH DIFFERENT LEVELS OF NITROGEN ON THE YIELD OF SUGARCANE VARIETY CoC-92061.
Madras Agricultural Journal
1,998
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14
htt //d i /10 29321/MAJ 10 A00798 htt //d i /10 29321/MAJ 10 A00798
https://openalex.org/W2884298444
https://europepmc.org/articles/pmc6116249?pdf=render
English
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Distribution of Paramagnetic Fe2O3/SiO2–Core/Shell Nanoparticles in the Rat Lung Studied by Time-of-Flight Secondary Ion Mass Spectrometry: No Indication for Rapid Lipid Adsorption
Nanomaterials
2,018
cc-by
12,400
Received: 10 July 2018; Accepted: 24 July 2018; Published: 26 July 2018 Abstract: Amorphous silica nanoparticles comprise a class of widely used industrial nanomaterials, which may elicit acute inflammation in the lung. These materials have a large specific surface to which components of the pulmonary micro-milieu can bind. To conduct appropriate binding studies, paramagnetic Fe2O3/SiO2 core/shell nanoparticles (Fe-Si-NP) may be used as an easy-to-isolate silica surrogate, if several prerequisites are fulfilled. To this end, we investigated the distribution of Fe, Si, protein and phosphatidylcholine (PC) by Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) in cryo-sections from the rat lungs to which Fe-Si-NP had been administered for 30 min. Regions-of-interest were identified and analyzed with incident light and enhanced dark-field microscopy (DFM). Fe-Si-NP particles (primary particle size by electron microscopy: 10–20 nm; aggregate size by tracking analysis: 190 ± 20 nm) and agglomerates thereof were mainly attached to alveolar walls and only marginally internalized by cells such as alveolar macrophages. The localization of Fe-Si-NP by DFM was confirmed by ToF-SIMS signals from both, Fe and Si ions. With respect to an optimized signal-to-noise ratio, Fe+, Si+, CH4N+ and the PC head group (C5H15NO4P+) were the most versatile ions to detect iron, silica, protein, and PC, respectively. Largely congruent Fe+ and Si+ signals demonstrated that the silica coating of Fe-Si-NP remained stable under the conditions of the lung. PC, as a major lipid of the pulmonary surfactant, was colocalized with the protein signal alongside alveolar septa, but was not detected on Fe-Si-NP, suggesting that silica nanoparticles do not adsorb lipids of the lung surfactant under native conditions. The study shows that ToF-SIMS is a valuable technique with adequate spatial resolution to analyze nanoparticles together with organic molecules in the lung. The paramagnetic Fe-Si-NP appear well suited to study the binding of proteins to silica nanomaterials in the lung. Keywords: ToF-SIMS; dark-field microscopy; lipid adsorption; amorphous silica; core/shell nanoparticles; lung tissue; protein corona formation; nanotoxicology; rat nanomaterials nanomaterials Lothar Veith 1 ID , Antje Vennemann 2, Daniel Breitenstein 1, Carsten Engelhard 3, Birgit Hagenhoff 1 and Martin Wiemann 2,* Lothar Veith 1 ID , Antje Vennemann 2, Daniel Breitenstein 1, Carsten Engelhard 3, Birgit Hagenhoff 1 and Martin Wiemann 2,* 1 Tascon GmbH, Mendelstraße 17, 48149 Münster, Germany; lothar.veith@tascon-gmbh.de (L.V.); daniel.breitenstein@tascon-gmbh.de (D.B.); birgit.hagenhoff@tascon-gmbh.de (B.H.) daniel.breitenstein@tascon-gmbh.de (D.B.); birgit.hagenhoff@tascon-gmbh.de (B.H.) 2 IBE R&D Institute for Lung Health gGmbH, Mendelstraße 11, 48149 Münster, Germany; vennemann@ibe-ms.de 3 Department of Chemistry & Biology, University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany engelhard@chemie.uni-siegen.de 3 Department of Chemistry & Biology, University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany; engelhard@chemie.uni-siegen.de * Correspondence: martin.wiemann@ibe-ms.de; Tel.: +49-251-9802340 www.mdpi.com/journal/nanomaterials nanomaterials nanomaterials 1. Introduction Amorphous silica (SiO2) is among the most widely used industrial nanomaterials, which can be found in products such as tires, plastics, and lacquers. It is also used as a food additive to prevent Nanomaterials 2018, 8, 571; doi:10.3390/nano8080571 www.mdpi.com/journal/nanomaterials 2 of 14 Nanomaterials 2018, 8, 571 clogging or to ensure fluidity [1]. The high abundance of SiO2 nanomaterials in everyday life products may lead to an increased exposure of humans to this type of nanomaterial. With respect to the workplace situation, the lung is a major route for a non-intentional uptake of SiO2. In contrast to crystalline silica (quartz and cristobalite), which elicit inflammation and progressive fibrosis in the lung, effects of amorphous silica appear to be confined to a transient inflammation of the lung, even at comparably high doses [2,3]. With respect to the mode of action, it is important that amorphous silica nanomaterials have a large specific surface of up to several hundred square meters per gram. It has been shown that the inflammatory effect of amorphous silica on the lung and also the cytotoxic effect on macrophages increases with surface size [4]. Of note, the inflammatory effect of colloidal SiO2 can be reduced by surface coating with amino or phosponate residues [5,6]. Since these surface coatings have no major influence on particle size or agglomeration behavior of the SiO2 nanoparticles, it is highly likely that the inflammatory effects of SiO2 on the lung involve an early interaction with biomolecules accessible in the lung micro-milieu. The analysis of biomolecules interacting with the surface of silica nanomaterials under in vivo conditions is, therefore, of pivotal interest. Nanosized SiO2 particles, which are deposited alongside the alveolar wall, most likely bind to molecules of the lipid-rich pulmonary surfactant and/or the protein containing hypophase underneath [7]. The pulmonary surfactant covers the inner surface of lung alveoli and reduces the surface tension. It is a thin layer mainly composed of (phospho) lipids (90% by mass), with phosphatidylcholine being a major component [8]. The lung surfactant also contains specific surfactant proteins, some of which (e.g., SP-A) may bind to bacteria and, therefore, contribute to the first line of defense against invading microorganisms [9]. The lung surfactant is produced by type-2 epithelial cells and are stored in lamellar bodies. Upon secretion of lamellar bodies, the surfactant spreads and self-organizes as a monolayer whose non-polar lipids chains are directed towards the alveolar space [8]. 2.2. Sample Preparation and Animal Experiments Fe-Si-NP were diluted with sterile distilled H2O to a concentration of 6 mg/mL and ultrasonicated with a probe (VibraCellTM, Sonics & Materials, Danbury, CT, USA) adjusted to 50 W (20 kHz) for 10 s. This suspension was used as the final instillation fluid. Animal experiments were conducted at the animal facility of the University Clinics of Münster, Germany, and ethically approved by LANUV (Dortmund, Germany, Accession No. 84–02.04.2022.A157). Female Wistar rats (Charles River Laboratories, Sulzfeld, Germany), weighing 200–220 g, were maintained at a 12 h lights-on lights-off cycle; food and water were provided ad libitum. To administer Fe-Si-NP into the lung, animals were briefly anaesthetized with isoflurane and intratracheally instilled with 500 µL of instillation fluid containing 3 mg (w/v) Fe-Si-NP using a Penn Century Microsprayer. After 30 min, rats were deeply anaesthetized with ketamine/xylazine and bled via the descending aorta. A cannula was inserted into the trachea and the lung was lavaged five times with 5 mL 0.9% NaCl, to obtain lavage fluid for an accompanying investigation (data not included). Thereafter, the lung was inflated with 5 mL Cryomatrix (Thermo Shandon Ltd., Runcorn, UK), resected, snap frozen in liquid nitrogen, and stored at −80 ◦C for histological studies. Transverse sections were cut from the hilar region of the left lung with a cryo-microtome (HM 500, MICROM International GmbH, Walldorf, Germany). For ToF-SIMS analysis, 7 µm thick sections were dried onto ITO-coated glass slides (Sigma Aldrich, Germany) and stored at −20 ◦C to investigate Fe-Si-NP. Of note, the lavage procedure as carried out here leaves a considerable number of e.g., cells (>70%) within the lung parenchyma [14]. The same is true for particles attached to the alveolar walls as shown below. 2.3. Enhanced Dark-Field Microscopy Enhanced dark-field microscopy (DFM) makes use of highly intense white light and allows to visualize particles and NPs in cells and tissues with strong light scattering properties down to a size of 20–30 nm. To apply the technique, air-dried cryo-sections were fixed in formaldehyde for 10 min, washed in phosphate buffer and cover-slipped using an aqueous mounting medium (Shandon Immu-Mount, Thermo Fisher Scientific, Bremen, Germany). Sections were viewed with an upright microscope (Olympus BX51) and digital bright-field images were captured using a RETIGA 2000R camera (Q Imaging, Surrey, BC, Canada). Areas of interest were then imaged with a DFM microscope, equipped with a 40-fold UPlanApo Oil Iris objective (Olympus, Hamburg, Germany), and a CytoViva enhanced dark-field condenser. All DFM components were purchased from CytoViva, Inc. (Auburn, AL, USA). 1. Introduction Considering the spatial organization of surfactant components and the fact that nanoparticles bathed in biological fluids such as plasma rapidly acquire a protein corona [10], it is reasonable to assume that SiO2 nanoparticles, once deposited inside alveoli, form a corona, which, at least in vitro, can be composed of surfactant lipids and/or proteins [11]. However, because non-polar (phospho) lipid residues are facing the alveolar space, the corona of biomolecules formed around polar SiO2 nanomaterials under in vivo conditions has not been investigated and may be different from the corona attracted by non-polar nanoobjects such as carbon nanotubes [12]. The analysis of the protein corona formed around NP inside the lung is a challenging task. However, isolation of protein-laden nanoparticles (NP) would be easier, if NP could be rapidly isolated from the broncho-alveolar lavage fluid, freed from cells and non-bound components, and eventually be analyzed for their protein corona. A tempting tool for this task is paramagnetic Fe2O3/SiO2 core/shell nanoparticles (Fe-Si-NP). While their maghemite iron (Fe) core allows for a rapid magnetic isolation, their shell is composed of amorphous SiO2, to which biomolecules can bind. In this investigation, we explored commercially available paramagnetic Fe-Si-NP for the purpose of analyzing the protein corona in vivo. The main questions were: (i) How are Fe-Si-NP distributed in the lung if they are applied via intratracheal instillation? (ii) Is the SiO2 shell stable under the conditions of the lung? (iii) Are there any hints for a binding or accumulation of surfactant (phospho) lipids? To answer all these questions with a single investigation, we applied Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) to sections of Fe-Si-NP-laden rat lungs. ToF-SIMS is an ideal technique to detect inorganic as well as organic materials with detection limits in the femtomole range. A lateral resolution down to 30 nm may be reached and the technique has recently been shown to unambiguously detect SiO2 nanomaterial in lung tissue [13]. Here, we detect Si, Fe and PC by mass spectrometry in a three-dimensional approach. Regions of interest were identified by light microscopic techniques such as enhanced dark-field microscopy, and incident light microscopy. The results of this study confirm our assumption that Fe-Si-NP are a useful tool to describe the early process of protein corona formation in the lung. 3 of 14 Nanomaterials 2018, 8, 571 2.1. Particle Characterization and Sample Preparation 2.1. Particle Characterization and Sample Preparation The Fe-Si-NP were purchased from Kisker Biotech GmbH, Steinfurt, Germany (order No. PMSI-H.25–5). According to manufacturer, particles are composed of a maghemite Fe2O3 core and an amorphous SiO2 shell and had a surface area of 50 m2/g, according to the Brunauer–Emmet–Teller (BET) method. To further characterize particle size and shape by transmission electron microscopy (TEM), we dried 0.5 µL of the aqueous suspension, as used for intratracheal instillation (6 mg/mL H2O), onto carbon-coated copper grids. TEM analysis was carried out with a Tecnai G2 (ThermoFisher Scientific, Waltham, MA, USA). The size distribution of these aggregates (in H2O) was measured with a NanoSight LM10 instrument equipped with a green laser (532 nm), an Andor CCD camera, and NTA software 2.1 (Malvern Instruments GmbH, Herrenberg, Germany). 2.5. ToF-SIMS Analysis y software using the ex S S ft To confirm the chemical identity of the nanoparticles and their Fe/Si core/shell structure a droplet of the aqueous Fe-Si-NP nanoparticle suspension was dried onto aluminum foil. ToF-SIMS spectra were acquired in the spectrometry mode at a TOF.SIMS5 (IONTOF, Münster, Germany) for the pre-characterization of the nanoparticles. The raster size was 120 × 120 µm2 at 128 × 128 pixels. 25 keV Bi3 ions were applied at an ion dose of 1.5 × 108 ions and a cycle time of 200 µs. The corresponding mass range reached from 1 Da up to 1650 Da. SensoScan software (Version 3.5.2). 2.5. ToF-SIMS Analysis To confirm the chemical identity of the nanoparticles and their Fe/Si core/shell structure a droplet of the aqueous Fe-Si-NP nanoparticle suspension was dried onto aluminum foil. ToF-SIMS spectra were acquired in the spectrometry mode at a TOF.SIMS5 (IONTOF, Münster, Germany) for the pre-characterization of the nanoparticles. The raster size was 120 × 120 µm² at 128 × 128 pixels. Measurements using O2 sputtering were performed using Bi3 at 25 keV with an ion dose of about 2.2 × 1010 ions applied to an analysis field-of-view of 300 × 300 µm2 with a pixel raster of 512 × 512 pixels. O2+ was used as a sputter ion at an energy of 1 keV. A total sputter dose of 8.0 × 1014 ions was applied to a sputter raster size of 700 × 700 µm2. The delayed extraction mode in combination with non-interlaced sputtering was used at a cycle time of 80 µs resulting in a mass range from 1 Da to 550 Da. Three replicate analyses were conducted to assure the validity of the obtained data. p p µ p 25 keV Bi3 ions were applied at an ion dose of 1.5 × 108 ions and a cycle time of 200 µs. The corresponding mass range reached from 1 Da up to 1650 Da. Measurements using O2 sputtering were performed using Bi3 at 25 keV with an ion dose of about 2.2 × 1010 ions applied to an analysis field-of-view of 300 × 300 µm² with a pixel raster of 512 × 512 pixels. O2+ was used as a sputter ion at an energy of 1 keV. A total sputter dose of 8.0 × 1014 ions was applied to a sputter raster size of 700 × 700 µm². 2.6. Correlation Analysis Da. Three replicate an Image processing was carried out with the FIJI distribution for ImageJ (National Institute of Health, Bethesda, MD, USA) [16,17]. The colocalization threshold plugin was used for the colocalization analysis [18]. The image registration was executed with the Turboreg [19] plugin (Biomedical Imaging Group, EPFL Lausanne, Switzerland). 2.6. Correlation Analysis Image processing was carried out with the FIJI distribution for ImageJ (National Institute of Health, Bethesda, MD, USA) [16,17]. The colocalization threshold plugin was used for the colocalization analysis [18]. The image registration was executed with the Turboreg [19] plugin d l I E l d 2.5. ToF-SIMS Analysis y software using the ex S S ft The delayed extraction mode in combination with non-interlaced sputtering was used at a cycle time of 80 µs resulting in a mass range from 1 Da to 550 2.4. Topographic Analysis DFM imaged slides were cautiously immersed and rinsed in H2O to remove the coverslip and embedding medium, respectively. The sections were air-dried again and analyzed with an optical profiler 4 of 14 Nanomaterials 2018, 8, 571 instrument (PLu neox, Sensofar-Tech, Barcelona, Spain). A 50-fold EPI-objective (numerical aperture: 0.80) was used to acquire images with a resolution of 768 × 576 pixels for the selected area. The corresponding dimensions of a single pixel are 330 × 330 µm2, which corresponds to a pixel density of 3.03 pixels/mm. Up to 33 consecutive images were acquired at a focal distance of 0.2 µm by an automated routine. The image stack was converted into a single sharp image with ImageJ software using the extended depth of field plugin [15] or used to generate a topographic image using SensoScan software (Version 3.5.2). Nanomaterials 2018, 7, x FOR PEER REVIEW 4 of 14 aperture: 0.80) was used to acquire images with a resolution of 768  576 pixels for the selected area. The corresponding dimensions of a single pixel are 330  330 µm², which corresponds to a pixel density of 3.03 pixels/mm. Up to 33 consecutive images were acquired at a focal distance of 0.2 µm by an automated routine The image stack was converted into a single sharp image with ImageJ 3. Results and Discussion ( g g 3 Results and Discussio The e e e of Fe u o i a y io bo ba d e t u e t that the ToF SIMS i fo atio While the findings illustrate the need for an extensive pre-characterization of nanoparticles before their use in toxicology studies, they also indicate several characteristic secondary ions (28Si+, 30Si+, 54Fe+, 56Fe+, and 56FeOH+) that can be used for the detection of the nanoparticles in the tissues. While the findings illustrate the need for an extensive pre-characterization of nanoparticles before their use in toxicology studies, they also indicate several characteristic secondary ions (28Si+, 30Si+, 54Fe+, 56Fe+, and 56FeOH+) that can be used for the detection of the nanoparticles in the tissues. The presence of Fe upon primary ion bombardment suggests that the ToF SIMS information While the findings illustrate the need for an extensive pre-characterization of nanoparticles before their use in toxicology studies, they also indicate several characteristic secondary ions (28Si+, 30Si+, 54Fe+, 56Fe+, and 56FeOH+) that can be used for the detection of the nanoparticles in the tissues. The presence of Fe upon primary ion bombardment suggests that the ToF-SIMS information depth might be greater than the thickness of the Si shell. This finding can be explained by the highly energetic primary ion beam, which induces significant amounts of damage to nanoparticles and sometimes may even disrupt the particle. Sandoval et al. found an increased secondary ion yield for nanoparticles compared to bulk materials [21]. Increases in the secondary ion yield are always to be expected from nanoparticles depending on their sizes, because they have a larger surface area than bulk materials. However, the performance of the collision cascade is affected, if the size of the particles is in the same order of magnitude as the collision cascade. Obviously, this can also lead to increased intermixing of the particle components. Yang et al. observed a deformation of nanoparticles upon ion bombardment [22], which could also explain the simultaneous detection of core and shell components observed here for Fe-Si-NP. In any case, the simultaneous detection of the Si and Fe signals without the need for sputtering was beneficial for the detection of the core/shell particles in the tissue The presence of Fe upon primary ion bombardment suggests that the ToF-SIMS information depth might be greater than the thickness of the Si shell. 3. Results and Discussion ( g g 3 Results and Discussio 3.1. Electron Microscopic and ToF-SIMS Characterization of the Nanoparticles 3 1 El t Mi i d T F SIMS Ch t i ti f th N ti l TEM analysis showed that Fe-Si-NP consisted of nanoparticles with a size of ca. 10–20 nm, which mainly formed larger aggregates (Figure 1, inset). Fe-Si-NP dispersed in H2O showed two maxima at 168 nm and 228 nm, as analyzed with optical tracking analysis; mean and mode values amounted to 217.5 ± 7.8 nm and 190.3 ± 19.6 nm, respectively (Figure 1). 3.1. Electron Microscopic and ToF SIMS Characterization of the Nanoparticles TEM analysis showed that Fe-Si-NP consisted of nanoparticles with a size of ca. 10–20 nm, which mainly formed larger aggregates (Figure 1, inset). Fe-Si-NP dispersed in H2O showed two maxima at 168 nm and 228 nm, as analyzed with optical tracking analysis; mean and mode values amounted to 217.5 ± 7.8 nm and 190.3 ± 19.6 nm, respectively (Figure 1). Figure 1. Size distribution of Fe-Si nanoparticles (Fe-Si-NP) in aqueous suspension as used for intratracheal instillation. Measurements were carried out by NanoSight optical tracking analysis. The inset shows electron microscopic images of Fe-Si-NP as typically found in the instilled suspension. Figure 1. Size distribution of Fe-Si nanoparticles (Fe-Si-NP) in aqueous suspension as used for intratracheal instillation. Measurements were carried out by NanoSight optical tracking analysis. The inset shows electron microscopic images of Fe-Si-NP as typically found in the instilled suspension. Figure 1. Size distribution of Fe-Si nanoparticles (Fe-Si-NP) in aqueous suspension as used for intratracheal instillation. Measurements were carried out by NanoSight optical tracking analysis. The inset shows electron microscopic images of Fe-Si-NP as typically found in the instilled suspension. Figure 1. Size distribution of Fe-Si nanoparticles (Fe-Si-NP) in aqueous suspension as used for intratracheal instillation. Measurements were carried out by NanoSight optical tracking analysis. The inset shows electron microscopic images of Fe-Si-NP as typically found in the instilled suspension. 5 of 14 5 of 14 Nanomaterials 2018, 8, 571 Na o ate ial 2018 7 FO The mass spectrum, as revealed by ToF-SIMS, shows several prominent Si- and Fe-containing species (Figure 2), from which 28Si+, 30Si+, 54Fe+, 56Fe+ and 56FeOH+ were selected as relevant secondary ions for the detection of Fe-Si-NP in lung tissue sections. 3. Results and Discussion ( g g 3 Results and Discussio Besides the often detected hydrocarbons [20], B+, Na+ and Ca+ were detected with notable intensities and these substances are most likely associated with the particle production process. The mass spectrum, as revealed by ToF-SIMS, shows several prominent Si- and Fe-containing species (Figure 2), from which 28Si+, 30Si+, 54Fe+, 56Fe+ and 56FeOH+ were selected as relevant secondary ions for the detection of Fe-Si-NP in lung tissue sections. Besides the often detected hydrocarbons [20], B+, Na+ and Ca+ were detected with notable intensities and these substances are most likely associated with the particle production process. Figure 2. Mass spectrum of the Fe-Si-NP with a Fe/Si core/shell structure. Particles were deposited on an aluminum substrate and analyzed by ToF-SIMS. The particle components SixOyHz+ and FexOyHz+ and their respective isotopes are detected along with hydrocarbons (CH3+ and C2H3+), substrate components (Al+), and other residues possibly linked to the production process (B+, Na+, and Ca+). Figure 2. Mass spectrum of the Fe-Si-NP with a Fe/Si core/shell structure. Particles were deposited on an aluminum substrate and analyzed by ToF-SIMS. The particle components SixOyHz+ and FexOyHz+ and their respective isotopes are detected along with hydrocarbons (CH3+ and C2H3+), substrate components (Al+), and other residues possibly linked to the production process (B+, Na+, and Ca+). Figure 2. Mass spectrum of the Fe-Si-NP with a Fe/Si core/shell structure. Particles were deposited on an aluminum substrate and analyzed by ToF-SIMS. The particle components SixOyHz+ and FexOyHz+ and their respective isotopes are detected along with hydrocarbons (CH3+ and C2H3+), substrate components (Al+), and other residues possibly linked to the production process (B+, Na+, and Ca+). Figure 2. Mass spectrum of the Fe-Si-NP with a Fe/Si core/shell structure. Particles were deposited on an aluminum substrate and analyzed by ToF-SIMS. The particle components SixOyHz+ and FexOyHz+ and their respective isotopes are detected along with hydrocarbons (CH3+ and C2H3+), substrate components (Al+), and other residues possibly linked to the production process (B+, Na+, and Ca+). While the findings illustrate the need for an extensive pre-characterization of nanoparticles before their use in toxicology studies, they also indicate several characteristic secondary ions (28Si+, 30Si+, 54Fe+, 56Fe+, and 56FeOH+) that can be used for the detection of the nanoparticles in the tissues. 3. Results and Discussion ( g g 3 Results and Discussio This finding can be explained by the highly energetic primary ion beam, which induces significant amounts of damage to nanoparticles and sometimes may even disrupt the particle. Sandoval et al. found an increased secondary ion yield for nanoparticles compared to bulk materials [21]. Increases in the secondary ion yield are always to be expected from nanoparticles depending on their sizes, because they have a larger surface area than bulk materials. However, the performance of the collision cascade is affected, if the size of the particles is in the same order of magnitude as the collision cascade. Obviously, this can also lead to increased intermixing of the particle components. Yang et al. observed a deformation of nanoparticles upon ion bombardment [22], which could also explain the simultaneous detection of core and shell components observed here for Fe-Si-NP. In any case, the simultaneous detection of the Si and Fe signals without the need for sputtering was beneficial for the detection of the core/shell particles in the tissue. 3 2 Inspection of Fe Si NP Laden Lung Tissue by Light Microscopy 3.2. Inspection of Fe-Si-NP-Laden Lung Tissue by Light Microscopy 3.2. Inspection of Fe Si NP Laden Lung Tissue by Light Microscopy Light microscopic techniques are increasingly used to detect nanoparticles in tissue sections [23]. Especially enhanced dark-field microscopy can detect light scattering particles down to a size of 20– 30 nm [24]. Furthermore, metallic or metal oxidic nanoparticles often have plasmonic, reflective and diffractive properties, which allow for their differentiation against translucent tissue [25], or, in ideal cases, for identification via hyperspectral imaging [26]. Here, bright-field microscopy combined with DFM was used to localize Fe Si NP in the lung tissue prior to ToF SIMS analysis Light microscopic techniques are increasingly used to detect nanoparticles in tissue sections [23]. Especially enhanced dark-field microscopy can detect light scattering particles down to a size of 20–30 nm [24]. Furthermore, metallic or metal oxidic nanoparticles often have plasmonic, reflective and diffractive properties, which allow for their differentiation against translucent tissue [25], or, in ideal cases, for identification via hyperspectral imaging [26]. Here, bright-field microscopy combined with DFM was used to localize Fe-Si-NP in the lung tissue prior to ToF-SIMS analysis. 6 of 14 Nanomaterials 2018, 8, 571 Although the microscopic detection of SiO2 nanomaterials in tissue sections is hampered by low optical contrast, the iron core of Fe-Si-NP caused a “rust”-like color facilitating the identification of larger agglomerates in bright-field images (Figure 3A–F). DFM confirmed this distribution but additionally revealed smaller yellow or blue dots in, or attached to, the hardly light scattering tissue structure (Figure 3C,D). Although the detection of nanoparticles in tissues by DFM is generally a straightforward approach, it is obvious that any light scattering inhomogeneity might lead to misinterpretation. Nanomaterials 2018, 7, x FOR PEER REVIEW 6 of 14 larger agglomerates in bright-field images (Figure 3A–F). DFM confirmed this distribution but additionally revealed smaller yellow or blue dots in, or attached to, the hardly light scattering tissue structure (Figure 3C,D). Although the detection of nanoparticles in tissues by DFM is generally a straightforward approach, it is obvious that any light scattering inhomogeneity might lead to misinterpretation Figure 3. Light microscopic analyses of a Fe-Si-NP-laden cryo-section from rat lung. Overview (A,C,E,G) and detail (B,D,F,H) of the boxed area shown on the left. (A–D) Routine images of the coverslipped section taken with bright-field optics (A,B) and DFM (C,D); Fe-Si-NP agglomerates appear as brownish patches at alveolar septa or in macrophages. 3 2 Inspection of Fe Si NP Laden Lung Tissue by Light Microscopy 3.2. Inspection of Fe-Si-NP-Laden Lung Tissue by Light Microscopy With DFM, additional small light- scattering particles are seen alongside alveolar septa. (E–H) Same tissue area after removal of the coverslip, rinsing and drying. (E,F) Microscopic image of the dry section without coverslip, taken with a reflective light microscope. Prominent Fe-Si-NP agglomerates are still identifiable as yellow patches. (G,H) Optical profilometry analysis showing the relief structure of the dry lung section as subjected to ToF-SIMS; the topographic map reveals an amplitude of several micrometers. Arrows in (A,C,E,G) point to a particle-laden macrophage (A,C), which became lost in the course of tissue preparation. Similarly, arrows in (B,D,F,H) point to small Fe-Si-NP (seen in (B,D)), which were absent after drying. Bright-field microscopy and DFM demand that the tissue section is immersed in ringer solution, co erslipped and iewed with oil immersion lenses As ToF SIMS analysis is carried out under 5.75 µm 0 Figure 3. Light microscopic analyses of a Fe-Si-NP-laden cryo-section from rat lung. Overview (A,C,E,G) and detail (B,D,F,H) of the boxed area shown on the left. (A–D) Routine images of the coverslipped section taken with bright-field optics (A,B) and DFM (C,D); Fe-Si-NP agglomerates appear as brownish patches at alveolar septa or in macrophages. With DFM, additional small light-scattering particles are seen alongside alveolar septa. (E–H) Same tissue area after removal of the coverslip, rinsing and drying. (E,F) Microscopic image of the dry section without coverslip, taken with a reflective light microscope. Prominent Fe-Si-NP agglomerates are still identifiable as yellow patches. (G,H) Optical profilometry analysis showing the relief structure of the dry lung section as subjected to ToF-SIMS; the topographic map reveals an amplitude of several micrometers. Arrows in (A,C,E,G) point to a particle-laden macrophage (A,C), which became lost in the course of tissue preparation. Similarly, arrows in (B,D,F,H) point to small Fe-Si-NP (seen in (B,D)), which were absent after drying. 5.75 µm 0 5.75 µm 0 Figure 3. Light microscopic analyses of a Fe-Si-NP-laden cryo-section from rat lung. Overview (A,C,E,G) and detail (B,D,F,H) of the boxed area shown on the left. (A–D) Routine images of the coverslipped section taken with bright-field optics (A,B) and DFM (C,D); Fe-Si-NP agglomerates appear as brownish patches at alveolar septa or in macrophages. With DFM, additional small light- scattering particles are seen alongside alveolar septa. (E–H) Same tissue area after removal of the coverslip, rinsing and drying. (E,F) Microscopic image of the dry section without coverslip, taken with a reflective light microscope. 3 2 Inspection of Fe Si NP Laden Lung Tissue by Light Microscopy 3.2. Inspection of Fe-Si-NP-Laden Lung Tissue by Light Microscopy Prominent Fe-Si-NP agglomerates are still identifiable as yellow patches. (G,H) Optical profilometry analysis showing the relief structure of the dry lung section as subjected to ToF-SIMS; the topographic map reveals an amplitude of several micrometers. Arrows in (A,C,E,G) point to a particle-laden macrophage (A,C), which became lost in the course of tissue preparation. Similarly, arrows in (B,D,F,H) point to small Fe-Si-NP (seen in (B,D)), which were absent after drying. Bright-field microscopy and DFM demand that the tissue section is immersed in ringer solution, Figure 3. Light microscopic analyses of a Fe-Si-NP-laden cryo-section from rat lung. Overview (A,C,E,G) and detail (B,D,F,H) of the boxed area shown on the left. (A–D) Routine images of the coverslipped section taken with bright-field optics (A,B) and DFM (C,D); Fe-Si-NP agglomerates appear as brownish patches at alveolar septa or in macrophages. With DFM, additional small light-scattering particles are seen alongside alveolar septa. (E–H) Same tissue area after removal of the coverslip, rinsing and drying. (E,F) Microscopic image of the dry section without coverslip, taken with a reflective light microscope. Prominent Fe-Si-NP agglomerates are still identifiable as yellow patches. (G,H) Optical profilometry analysis showing the relief structure of the dry lung section as subjected to ToF-SIMS; the topographic map reveals an amplitude of several micrometers. Arrows in (A,C,E,G) point to a particle-laden macrophage (A,C), which became lost in the course of tissue preparation. Similarly, arrows in (B,D,F,H) point to small Fe-Si-NP (seen in (B,D)), which were absent after drying. 7 of 14 Nanomaterials 2018, 8, 571 Bright-field microscopy and DFM demand that the tissue section is immersed in ringer solution, coverslipped and viewed with oil-immersion lenses. As ToF-SIMS analysis is carried out under vacuum conditions, the coverslip has to be cautiously removed and the sections needs to be dried. Mild shearing forces and washing steps are indispensable during this procedure and may influence the signals gathered from particles in the subsequent ToF-SIMS analysis. To show these effects, reflective light microscopic images were taken at the same position of the dried section. Reflective light microscopic images have a lower optical resolution but are highly useful to visualize the structure of a section prepared for ToF-SIMS analysis. 3 2 Inspection of Fe Si NP Laden Lung Tissue by Light Microscopy 3.2. Inspection of Fe-Si-NP-Laden Lung Tissue by Light Microscopy Here, it is shown that numerous large yellow spots (up to 15 µm) previously seen in the bright-field or DFM image were still in place, whereas others structures, which were interpreted as Fe-Si-NP filled macrophages, were no longer visible (Figure 3E,F; white arrows). Similarly, smaller particles (<3 µm), which are marked by arrows in the magnified bright-field and DFM images (Figure 3B,D), were not seen on reflective light microscopy images. py g As ToF-SIMS analysis makes use of sputtering and ion bombardment, which is applied under a certain angle, the relief structure of the dried tissue section will unavoidably influence image resolution. Figure 3G,H shows the relief of the dried tissue on the indium tin oxide (ITO)-coated glass substrate, as analyzed by profilometry. The height of the formalin-fixed and afterwards dried alveolar septa on the substrate ranged 2.5–5.25 µm, which was 36–75% of the initial thickness of the cryo-section (7 µm) and may be explained by evaporation of H2O prior to immersion fixation. Although the nature of the highest peaks remains unknown, these sites do not contain agglomerated Fe-Si-NP, which were mainly found in areas ranging in height from 2–3.5 µm. In summary, the combination of several microscopic methods showed that Fe-Si-NP were distributed alongside the alveolar wall or were contained in macrophage-like structures. The successive observation by DFM and ToF-SIMS may lead to a loss of Fe-Si-NP containing structures and this has to be considered when both images are compared (see below). 3.3. ToF-SIMS Detection of Nanoparticles in Tissue—Localization However, due to the preparation steps of the lung prior to fixation (filling with cryomatrix, freezing, sectioning, and drying) and to the relief structure of the tissue (Figure 3G), a precise cellular localization of particles cannot be made by light microscopy or ToF-SIMS. A study by Shon et al. carried out on air-dried unfixed macrophages laden with dimercaptosuccinic acid-coated Fe3O4 nanoparticles succeeded in localizing particles in the cytoplasm [27]. Phosphatidylcholine components in that study were detected as specific C4H7+ ions only. Nanomaterials 2018, 7, x FOR PEER REVIEW 8 of 14 tissue (Figure 3G), a precise cellular localization of particles cannot be made by light microscopy or ToF-SIMS. A study by Shon et al. carried out on air-dried unfixed macrophages laden with dimercaptosuccinic acid-coated Fe3O4 nanoparticles succeeded in localizing particles in the cytoplasm [27]. Phosphatidylcholine components in that study were detected as specific C4H7+ ions only. Figure 4. ToF-SIMS signal distributions of organic and inorganic components in a Fe/Si core/shell nanoparticle-containing lung tissue section 30 min post intratracheal instillation of particles. (A) CH4N+ indicative of proteins to show tissue distribution and lung structure. (B) C5H15NPO4+ is the phosphocholine (PC) head group fragment showing the distribution of lipids of lung tissue and surfactant; PC signal seen in the alveolar space is interpreted as to be artificially dislocated during filling of the lungs with cryomatrix. (C) Overlay of 54Fe+ (green) and CH4N+ (red) signal showing Fe- Si-NP distribution in lung tissue. (D) 56Fe+, and (E) 54Fe+ distributions of Fe-Si-NP particles. Small and large agglomerates appear alongside the alveolar walls. (F) The overlay of 54Fe+ (D, red) and 28Si+ (G, green) reveals the colocalization of both particle-related signals. (G) ToF-SIMS signal distribution of Si species from the topmost layers as indicated by the horizontal green lines in the X-Y slice of the ToF-SIMS dataset in (H). (I) 28Si+ signal distribution integrated for the complete depth including the indium tin oxide-coated glass slide. 3.3. ToF-SIMS Detection of Nanoparticles in Tissue—Localization 3.3. ToF-SIMS Detection of Nanoparticles in Tissue—Localization In the next step, we tested the intactness of the Fe/Si core/shell in the tissue using both the Fe- and Si-signal distributions in the ToF-SIMS analysis. A sputter erosion with 1 keV O2+ ions was necessary to analyze the complete depth of the sample section. The tissue structure of the lung with all its alveolar septa, as shown in Figure 3, was properly reflected by the CH4N+ signal, which is a typical fragment of amino acids and, thus, indicates the position of fixed proteins (Figure 4A). Due to the relatively harsh sputtering conditions of 1 keV O2+ ions, only few organic molecular species were preserved. Nevertheless, the lateral distribution of the signal at m/z 184.07 could be summed up over the whole depth of the analysis. This fragment is indicative of the phosphocholine head group C5H15NPO4+ and most likely attributable to phospholipids previously located in cell membranes and/or in the pulmonary surfactant. A minor fraction of the signal was found in alveolar spaces (Figure 4B), suggesting that it was transferred onto the substrate during the preparation process. With respect to Fe, several relevant signals (besides the main isotopes 54Fe+ and 56Fe+) were identified in the spectrum of both, the isolated and the lung-incorporated Fe-Si-NP (Figure 2). Of note, further iron-related species with the same lateral distribution were found, e.g., 56FeOH+, 54FeOH+, and 56Fe2O+, proving that the signals were correctly assigned to all Fe species. In any case, the lateral signal distribution of the main Fe isotopes 54Fe+ and 56Fe+ (Figure 4D,E) was largely congruent with the distribution of Fe-Si-NP in the tissue (Figure 3C,E). This is particularly obvious for some larger areas with diameters of up to 15 µm (Figure 4D,E), but also for numerous smaller and less intense Fe patches. As most of these areas have a diameter larger than 200 nm, they most likely represent Fe-Si-NP agglomerates. Colocalization with the CH4N+ signal (Figure 4C) shows that the majority of Fe signals (green) in the range of 3–10 µm was localized alongside the alveolar septa (red), which is in accordance 8 of 14 Nanomaterials 2018, 8, 571 with the microscopic results of Figure 3 and was expected due to the short incubation time of only 30 min. Some of the smaller Fe-signals (green) were colocalized with the epithelium and might be interpreted as to be incorporated by the epithelial cell. 3.3. ToF-SIMS Detection of Nanoparticles in Tissue—Localization 300 250 200 150 100 50 0 300 200 100 0 μm CH4N+ MC: 17; TC: 7.854e+005 15 13 10 8 5 3 0 300 250 200 150 100 50 0 300 200 100 0 μm C5H15NO4P+ MC: 7; TC: 4.348e+005 6 5 4 3 2 1 0 300 250 200 150 100 50 0 300 200 100 0 μm Overlay of: CH4N+, 54Fe+ 300 250 200 150 100 50 0 300 200 100 0 μm 56Fe+ MC: 126; TC: 1.820e+006 2 ∙10 1.2 1.0 0.8 0.6 0.4 0.2 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ MC: 148; TC: 2.462e+007 2 ∙10 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 300 250 200 150 100 50 0 300 200 100 0 μm 54Fe+ MC: 12; TC: 6.369e+004 10 8 6 4 2 0 300 200 100 0 μm X-Z slice of: 28Si+ MC: 60; TC: 9.601e+005 60 50 40 30 20 10 0 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ (Scan 1-20) MC: 7; TC: 2.048e+004 6 5 4 3 2 1 0 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ (Scan 1-20) MC: 7; TC: 2.048e+004 6 5 4 3 2 1 0 Overlay of: 54Fe+, 28Si+ (Scan 1-20) Overlay of: 54Fe+, 28Si+ (Scan 1-20) Figure 4. ToF-SIMS signal distributions of organic and inorganic components in a Fe/Si core/shell nanoparticle-containing lung tissue section 30 min post intratracheal instillation of particles. (A) CH4N+ indicative of proteins to show tissue distribution and lung structure. (B) C5H15NPO4+ is the phosphocholine (PC) head group fragment showing the distribution of lipids of lung tissue and surfactant; PC signal seen in the alveolar space is interpreted as to be artificially dislocated during filling of the lungs with cryomatrix. (C) Overlay of 54Fe+ (green) and CH4N+ (red) signal showing Fe-Si-NP distribution in lung tissue. (D) 56Fe+, and (E) 54Fe+ distributions of Fe-Si-NP particles. Small and large agglomerates appear alongside the alveolar walls. (F) The overlay of 54Fe+ (D, red) and 28Si+ (G, green) reveals the colocalization of both particle-related signals. (G) ToF-SIMS signal distribution of Si species from the topmost layers as indicated by the horizontal green lines in the X-Y slice of the ToF-SIMS dataset in (H). (I) 28Si+ signal distribution integrated for the complete depth including the indium tin oxide-coated glass slide. 3.3. ToF-SIMS Detection of Nanoparticles in Tissue—Localization 300 250 200 150 100 50 0 300 200 100 0 μm CH4N+ MC: 17; TC: 7.854e+005 15 13 10 8 5 3 0 300 250 200 150 100 50 0 300 200 100 0 μm Overlay of: CH4N+, 54Fe+ 300 250 200 150 100 50 0 300 200 100 0 μm C5H15NO4P+ MC: 7; TC: 4.348e+005 6 5 4 3 2 1 0 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ (Scan 1-20) MC: 7; TC: 2.048e+004 6 5 4 3 2 1 0 Overlay of: 54Fe+, 28Si+ (Scan 1-20) Overlay of: 54Fe+, 28Si+ (Scan 1-20) 300 250 200 150 100 50 0 300 200 100 0 μm 56Fe+ MC: 126; TC: 1.820e+006 00 50 56Fe+ MC: 126; TC: 1.820e+006 28Si+ (Scan 1-20) MC: 7; TC: 2.048e+004 Overlay of: 54Fe+, 28Si+ (Scan 1-20) MC: 12; TC: 6.369e+004 300 200 100 0 μm X-Z slice of: 28Si+ MC: 60; TC: 9.601e+005 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ MC: 148; TC: 2.462e+007 2 ∙10 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 60 50 40 30 20 10 0 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ (Scan 1-20) MC: 7; TC: 2.048e+004 6 5 4 3 2 1 0 0 0 Si MC: 148; TC: 2.462e+007 Figure 4. ToF-SIMS signal distributions of organic and inorganic components in a Fe/Si core/shell nanoparticle-containing lung tissue section 30 min post intratracheal instillation of particles. (A) CH4N+ indicative of proteins to show tissue distribution and lung structure. (B) C5H15NPO4+ is the phosphocholine (PC) head group fragment showing the distribution of lipids of lung tissue and surfactant; PC signal seen in the alveolar space is interpreted as to be artificially dislocated during filling of the lungs with cryomatrix. (C) Overlay of 54Fe+ (green) and CH4N+ (red) signal showing Fe- Si-NP distribution in lung tissue. (D) 56Fe+, and (E) 54Fe+ distributions of Fe-Si-NP particles. Small and large agglomerates appear alongside the alveolar walls. (F) The overlay of 54Fe+ (D, red) and 28Si+ (G, green) reveals the colocalization of both particle-related signals. (G) ToF-SIMS signal distribution of Si species from the topmost layers as indicated by the horizontal green lines in the X-Y slice of the ToF-SIMS dataset in (H). (I) 28Si+ signal distribution integrated for the complete depth including the indium tin oxide-coated glass slide. Figure 4. 3.4. Colocalization of Fe and Si in Lung Tissue by ToF-SIMS 3.4. Colocalization of Fe and Si in Lung Tissue by ToF-SIMS With respect to the simultaneous identification of Fe and Si, isobaric interferences may occur due to the similarity of the theoretical mass of 56Fe+ (m/z 55.934939) and 28Si2+ (m/z 55.953854). This hampers a reliable distinction by mass, and only an improvement of mass resolution from the current R = 1500 to R > 3000 could solve this problem. However, the 54Fe+ signal, although relatively weak, is not influenced by any Si cluster and, therefore, allows analyzing the Fe distribution unbiased from Si signals. Thus, adopting 54Fe+ as a reference, it turned out that 56Fe+ signals virtually showed the same distribution as did 54Fe+. In addition, the 56Fe+ signal was hardly influenced by 28Si2+ from any non-particle source and showed a low background intensity in tissue-free areas (Figure 4D,G). Due to the obvious correlation of the signal distributions for the 54Fe+ and 56Fe+ isotopes, we decided to use the 56Fe+ ion to measure the distribution of Fe in lung sections. g To identify Si on the paramagnetic Fe core, any Si signal from the ITO-covered glass substrate had to be avoided. Therefore, the 28Si signal was reconstructed from only the first 20 sputter/analysis cycles (i.e., scans). The signal distribution obtained by this was highly similar to the Fe distributions (Figure 4G), indicating that the ITO layer had successfully shielded the underlying glass substrate against the analysis beam. In contrast, if the signal was summed up over all 81 cycles (compare X–Z slice in Figure 4H), the sputter cycles had eroded the ITO layer and the apparent 28Si+ distribution reflected the underlying substrate in tissue-free regions (Figure 4I). Based on selected data evaluation and an isotopic signal distribution, the presence of Si on nanoparticles could be confirmed even in the positive ion polarity upon oxygen sputtering, as opposed to earlier studies using Cs sputtering and the negative ion polarity [13]. Consequently, the identification by ToF-SIMS of pure, unlabeled SiO2 nanoparticles in tissue sections should be possible with this protocol, provided that any contamination, e.g., with the widespread polysiloxanes, can be ruled out. Another option to identify correlating distributions of Si and Fe species is the comparison of Fe isotope distributions with 30Si, as oxide clusters of this ion cannot form signals with the same mass-to-charge-ratio as the Fe isotopes in the observed mass range. 3.4. Colocalization of Fe and Si in Lung Tissue by ToF-SIMS However, as 30Si is also a part of the ITO shielded substrate, the same limitations as for 28Si apply. Of note, the reconstruction of the less abundant 30Si+ isotope ruled out that the 28Si+ signal merely originated from 56Fe+ (m/z 27.967470), which would partially interfere with the 28Si+ (m/z 27.976927), since both, 28Si and 30Si show the same signal distributions (not shown here). Based on the comparison of Figure 4D,E (for Fe) and Figure 4G (for Si), the most important result is that the 28Si+ distribution derived from the first 20 sputter/analysis cycles was properly colocalized with the Fe distributions, as is shown by the numerous yellow spots in Figure 4F, which result from red 54Fe pixels overlaid with green 28Si pixels. This demonstrates that the Si shell of the paramagnetic Fe particles is stable under in vivo conditions. 3.3. ToF-SIMS Detection of Nanoparticles in Tissue—Localization ToF-SIMS signal distributions of organic and inorganic components in a Fe/Si core/shell nanoparticle-containing lung tissue section 30 min post intratracheal instillation of particles. (A) CH4N+ indicative of proteins to show tissue distribution and lung structure. (B) C5H15NPO4+ is the phosphocholine (PC) head group fragment showing the distribution of lipids of lung tissue and surfactant; PC signal seen in the alveolar space is interpreted as to be artificially dislocated during filling of the lungs with cryomatrix. (C) Overlay of 54Fe+ (green) and CH4N+ (red) signal showing Fe-Si-NP distribution in lung tissue. (D) 56Fe+, and (E) 54Fe+ distributions of Fe-Si-NP particles. Small and large agglomerates appear alongside the alveolar walls. (F) The overlay of 54Fe+ (D, red) and 28Si+ (G, green) reveals the colocalization of both particle-related signals. (G) ToF-SIMS signal distribution of Si species from the topmost layers as indicated by the horizontal green lines in the X-Y slice of the ToF-SIMS dataset in (H). (I) 28Si+ signal distribution integrated for the complete depth including the indium tin oxide-coated glass slide. Figure 4. ToF-SIMS signal distributions of organic and inorganic components in a Fe/Si core/shell Figure 4. ToF-SIMS signal distributions of organic and inorganic components in a Fe/Si core/shell 9 of 14 9 of 14 Nanomaterials 2018, 8, 571 3.5. Correlation and Comparison of Dark-Field Microscopy and ToF-SIMS Enhanced dark-field microscopy (DFM) detects NP down to a size of 15 nm [28] and was, therefore, used as a sensitive tool to visualize the Fe-Si-NP in tissue sections prior to ToF-SIMS analysis. Overall, the ToF-SIMS 54Fe+ signal and the DFM signal showed a high degree of similarity (Figure 5A,B). Colocalization analysis of both signals using Pearson’s correlation coefficient confirmed this high degree of co-localization. As this coefficient ranges from −1 (perfect anti-correlation) to +1 (perfect correlation), a value of 0.541 confirmed that a considerable number of pixels (54Fe+ positive by ToF-SIMS) correlate with the bright spots in the DFM image (Figure 5C). Of note, this result strongly suggests that ToF-SIMS had not detected false positive signals. However, the correlation was not perfect and differences were observed for some larger dots (marked in Figure 5), most likely corresponding to particle-laden macrophages, but also for smaller particles seen in DFM. As outlined above, especially inappropriately fixed material may have become lost during preparation. 10 of 14 10 of 14 Nanomaterials 2018, 8, 571 Other reasons may also account for such differences: Fe-Si-NP might not have been detected by ToF-SIMS due to insufficient sample erosion (false negative ToF-SIMS signals). However, as the sputtering with O2+ and subsequent analysis were extended into the depth of the substrate, it appears unlikely that the soft lung tissue shielded Fe-Si-NP and prevented the accumulation of supra-threshold signal intensities in the ToF-SIMS analysis. Rather, the accessible mass (i.e., particle size and/or concentration) was too small, and a low signal did not reach the limit of detection. This appears unlikely for most of the Fe-Si-NP aggregates larger than 100 nm but may account for the 10–50 nm fraction of Fe-Si-NP, which is visible by TEM (Figure 1) and, at least in part, should be visible by DFM. Nanomaterials 2018, 7, x FOR PEER REVIEW 10 of 14 50 nm fraction of Fe-Si-NP, which is visible by TEM (Figure 1) and, at least in part, should be visible by DFM. y g p y Finally, unlike the large brown agglomerates (Figure 3A,B), small objects can scatter light as well and may have been erroneously designated as Fe-Si-NP by DFM. Hyperspectral microscopy may be used to further identify such questionable structures. y Finally, unlike the large brown agglomerates (Figure 3A,B), small objects can scatter light as well and may have been erroneously designated as Fe-Si-NP by DFM. 3.5. Correlation and Comparison of Dark-Field Microscopy and ToF-SIMS Hyperspectral microscopy may be used to further identify such questionable structures. In summary, the ion distribution measured by ToF-SIMS explains most of the DFM signals, thereby serving as a validation technique. However, DFM images contained several signals without any ToF-SIMS equivalent. These DFM signals were most likely caused by salt residues or tissue material and indicate that it is necessary to confirm the chemical identity of nanoparticles in DFM imaging studies. In summary, the ion distribution measured by ToF-SIMS explains most of the DFM signals, thereby serving as a validation technique. However, DFM images contained several signals without any ToF-SIMS equivalent. These DFM signals were most likely caused by salt residues or tissue material and indicate that it is necessary to confirm the chemical identity of nanoparticles in DFM imaging studies. Figure 5. Comparison of ToF-SIMS 54Fe+ signal distribution and dark-field microscopy (DFM) images. Image taken from Figure 3. (A) 54Fe+ ToF-SIMS signal (red); (B) DFM signal (green) taken before washing and ToF-SIMS analysis; and (C) Colocalization of (A,B). A high degree of correlation is evident (white overlay). The Pearson correlation coefficient for this image was 0.541. Arrows point to green spots (B) which were detected by DFM, but not by ToF-SIMS. 6 Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings Figure 5. Comparison of ToF-SIMS 54Fe+ signal distribution and dark-field microscopy (DFM) images. Image taken from Figure 3. (A) 54Fe+ ToF-SIMS signal (red); (B) DFM signal (green) taken before washing and ToF-SIMS analysis; and (C) Colocalization of (A,B). A high degree of correlation is evident (white overlay). The Pearson correlation coefficient for this image was 0.541. Arrows point to green spots (B) which were detected by DFM, but not by ToF-SIMS. Figure 5. Comparison of ToF-SIMS 54Fe+ signal distribution and dark-field microscopy (DFM) images. Image taken from Figure 3. (A) 54Fe+ ToF-SIMS signal (red); (B) DFM signal (green) taken before washing and ToF-SIMS analysis; and (C) Colocalization of (A,B). A high degree of correlation is evident (white overlay). The Pearson correlation coefficient for this image was 0.541. Arrows point to green spots (B) which were detected by DFM, but not by ToF-SIMS. Figure 5. Comparison of ToF-SIMS 54Fe+ signal distribution and dark-field microscopy (DFM) images. Image taken from Figure 3. (A) 54Fe+ ToF-SIMS signal (red); (B) DFM signal (green) taken before washing and ToF-SIMS analysis; and (C) Colocalization of (A,B). 3.5. Correlation and Comparison of Dark-Field Microscopy and ToF-SIMS A high degree of correlation is evident (white overlay). The Pearson correlation coefficient for this image was 0.541. Arrows point to green spots (B) which were detected by DFM, but not by ToF-SIMS. .6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings .6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Finding 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings It appears conceivable that this sequential arrangement of biomolecules and ions in the lung orchestrates the formation of the protein corona as well as agglomeration of NP. A protein corona, which can be artificially generated with different preparations of lung surfactant [11] may, therefore, differ from the native corona. To monitor the putative binding of (phosho) lipids to silica nanoparticles in the lung, we To monitor the putative binding of (phosho) lipids to silica nanoparticles in the lung, we compared the m/z 184.07 signal (representing phosphatidylcholine as the main constituent of pulmonary surfactant and cell membranes), with the distribution of Si, which forms the outer shell of the Fe-Si-NP: While the m/z 184.07 signal delineated the alveolar septal structure, and was also found on the ITO substrate at lower intensities, volumes occupied by the Si signal in the first 20 scans were completely free of PC (Figure 6, arrows). Although we are aware that the sensitivity of analytical methods can be improved, e.g., by well-adapted sputtering methods, we conclude that there is no major accumulation of phospholipids on the polar surface of Si nanoparticles. We furthermore suggest that phospholipids, as constituents of the lung surfactant, are not involved in the formation of particle agglomerates. p g (p ) p p g compared the m/z 184.07 signal (representing phosphatidylcholine as the main constituent of pulmonary surfactant and cell membranes), with the distribution of Si, which forms the outer shell of the Fe-Si-NP: While the m/z 184.07 signal delineated the alveolar septal structure, and was also found on the ITO substrate at lower intensities, volumes occupied by the Si signal in the first 20 scans were completely free of PC (Figure 6, arrows). Although we are aware that the sensitivity of analytical methods can be improved, e.g., by well-adapted sputtering methods, we conclude that there is no major accumulation of phospholipids on the polar surface of Si nanoparticles. We furthermore suggest that phospholipids, as constituents of the lung surfactant, are not involved in the formation of particle agglomerates. Figure 6. Comparison of the Si and phosphocholine distribution in a cryo-section of the rat lung 30 min post administration of Fe-Si-NP. All images contain information of the uppermost 20 scans only. (A) 28Si+; (B) C5H15NPO4+, which is the 184.07 fragment of the phosphocholine (PC) head group showing the distribution phosphatidylcholine; and (C) the overlay of (A,B). 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Finding 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings In this study, most of the nanomaterial was found closely related to the alveolar septa by both optical and mass spectrometric techniques (Figures 3 and 4G). In addition, a partial uptake into macrophages was observed despite the relatively short incubation time of 30 min. The obviously persistent macrophage activity suggests that the intratracheal instillation of H2O, which was used to In this study, most of the nanomaterial was found closely related to the alveolar septa by both optical and mass spectrometric techniques (Figures 3 and 4G). In addition, a partial uptake into macrophages was observed despite the relatively short incubation time of 30 min. The obviously 11 of 14 Nanomaterials 2018, 8, 571 persistent macrophage activity suggests that the intratracheal instillation of H2O, which was used to favor the dispersion of the polar NP, has no negative impact on the micro-milieu of the lung. Nevertheless, larger agglomerates of Fe-Si-NP with diameters of several micrometers were found in the lung. These agglomerates may have formed upon contact with the lung lining fluid and/or during lymphatic retrieval of the aqueous suspension fluid. As such, an agglomeration of nanoparticles in lung instillation studies is common [29], although, e.g., citrate-stabilized gold nanoparticles do not agglomerate under these conditions [30]. Nanomaterials 2018, 7, x FOR PEER REVIEW 11 of 14 In any case, it appears plausible that Fe-Si-NP, which had a size of less than 200 nm (see Figure gg In any case, it appears plausible that Fe-Si-NP, which had a size of less than 200 nm (see Figure 1) when they were administered to the lung, may have come into direct contact with several well organized layers of biomolecules such as the lung surfactant, the hypophase, and/or the membrane of (epithelial) cells. It appears conceivable that this sequential arrangement of biomolecules and ions in the lung orchestrates the formation of the protein corona as well as agglomeration of NP. A protein corona, which can be artificially generated with different preparations of lung surfactant [11] may, therefore, differ from the native corona. In any case, it appears plausible that Fe Si NP, which had a size of less than 200 nm (see Figure 1) when they were administered to the lung, may have come into direct contact with several well organized layers of biomolecules such as the lung surfactant, the hypophase, and/or the membrane of (epithelial) cells. 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Finding 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings (A) 28Si+; (B) C5H15NPO4+, which is the 184.07 fragment of the phosphocholine (PC) head group showing the distribution phosphatidylcholine; and (C) the overlay of (A,B). Arrows in all images point to the position of larger agglomerates of Fe-Si-NP; note that in (B) PC is not detected at the indicated sites. Figure 6. Comparison of the Si and phosphocholine distribution in a cryo section of the rat lung 30 min post administration of Fe-Si-NP. All images contain information of the uppermost 20 scans only. (A) 28Si+; (B) C5H15NPO4+, which is the 184.07 fragment of the phosphocholine (PC) head group showing the distribution phosphatidylcholine; and (C) the overlay of (A,B). Arrows in all images point to the position of larger agglomerates of Fe-Si-NP; note that in (B) PC is not detected at the indicated sites. Figure 6. Comparison of the Si and phosphocholine distribution in a cryo section of the rat lung 30 min post administration of Fe-Si-NP. All images contain information of the uppermost 20 scans only. (A) 28Si+; (B) C5H15NPO4+, which is the 184.07 fragment of the phosphocholine (PC) head group showing the distribution phosphatidylcholine; and (C) the overlay of (A,B). Arrows in all images point to the position of larger agglomerates of Fe-Si-NP; note that in (B) PC is not detected at the indicated sites. This finding seems to be in contrast to a recent investigation on lipid binding to SiO2, ZrO2, or AlOOH in vitro [11]. Using two different preparations of pulmonary surfactant, it was found that polar nanoparticles bind indeed to phospholipids, if the mixture (Curoserve®) contained surfactant protein A. However, the role of surfactant proteins for an in-situ opsonization of nanoparticles is still not known. As mentioned above, the incubation of nanoparticles in artificial or isolated pulmonary surfactant may be profoundly different from binding conditions in the lung. While in the lung the air–liquid interface carries non-polar lipid residues facing the gas phase, lipids in isolated lung surfactant may form micelles with their polar residues directed outwards. The current results provide for the first time new and more realistic insights in the formation This finding seems to be in contrast to a recent investigation on lipid binding to SiO2, ZrO2, or AlOOH in vitro [11]. Using two different preparations of pulmonary surfactant, it was found that polar nanoparticles bind indeed to phospholipids, if the mixture (Curoserve®) contained surfactant protein A. 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Finding 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings Arrows in all images point to the position of larger agglomerates of Fe-Si-NP; note that in (B) PC is not detected at the indicated sites. 300 250 200 150 100 50 0 300 200 100 0 μm Phosphatidylcholine MC: 30; TC: 2.287e+006 25 20 15 10 5 0 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ (scan 1-20) MC: 5; TC: 3.396e+004 4 3 2 1 300 250 200 150 100 50 0 300 200 100 0 μm Overlay of Phosphatidylcholine, 28Si (scan1-20) Figure 6. Comparison of the Si and phosphocholine distribution in a cryo-section of the rat lung 30 min post administration of Fe-Si-NP. All images contain information of the uppermost 20 scans only. (A) 28Si+; (B) C5H15NPO4+, which is the 184.07 fragment of the phosphocholine (PC) head group showing the distribution phosphatidylcholine; and (C) the overlay of (A,B). Arrows in all images point to the position of larger agglomerates of Fe-Si-NP; note that in (B) PC is not detected at the indicated sites. 300 250 200 150 100 50 0 300 200 100 0 μm Phosphatidylcholine MC: 30; TC: 2.287e+006 25 20 15 10 5 0 300 250 200 150 100 50 0 300 200 100 0 μm Overlay of Phosphatidylcholine, 28Si (scan1-20) 300 250 200 150 100 50 0 300 200 100 0 μm 28Si+ (scan 1-20) MC: 5; TC: 3.396e+004 Phosphatidylcholine MC: 30; TC: 2.287e+006 (scan 1 20) 5; TC: 3.396e+004 Figure 6. Comparison of the Si and phosphocholine distribution in a cryo-section of the rat lung Figure 6. Comparison of the Si and phosphocholine distribution in a cryo-section of the rat lung Figure 6. Comparison of the Si and phosphocholine distribution in a cryo-section of the rat lung 30 min post administration of Fe-Si-NP. All images contain information of the uppermost 20 scans only. (A) 28Si+; (B) C5H15NPO4+, which is the 184.07 fragment of the phosphocholine (PC) head group showing the distribution phosphatidylcholine; and (C) the overlay of (A,B). Arrows in all images point to the position of larger agglomerates of Fe-Si-NP; note that in (B) PC is not detected at the indicated sites. Figure 6. Comparison of the Si and phosphocholine distribution in a cryo-section of the rat lung 30 min post administration of Fe-Si-NP. All images contain information of the uppermost 20 scans only. 3.7. Methodical Considerations It is well known that any type of tissue preparation may have an influence on bio-imaging results. As will be described below (3.2–3.4), tissue sections analyzed by ToF-SIMS were taken from lavaged lungs, which were filled with a cryomatrix and immediately snap-frozen in liquid nitrogen. Although filling with a cryomatrix preserves the tissue architecture of the lung, the compound does not fix subcellular structures, such that a reliable distinction between cell-attached and intracellular NP cannot be made. Furthermore, sections were cut, dried onto ITO slides, stored in a frozen state, and eventually fixed with formalin on the slide. We are aware that this procedure cannot prevent a partial loss of NP, especially if they are not in contact with fixable tissue components. However, numerous particles and agglomerates proved to be firmly attached, because even the withdrawal of the coverslip prior to ToF-SIMS analysis left the majority of particles in place, as shown in Figure 5. There may also be a partial loss or wash-out of lipids, as these molecules are not fixable with formalin. However, neither ethanol nor other organic solvents were used, such that PC molecules remained highly abundant in all tissue structures. Nevertheless, there was a low background signal of PC in alveolar areas, which may be interpreted as an artificial displacement of PC. However, as levels of PC were low in the vicinity of the alveolar septa (Figure 6B), we assume that PC found within alveoli was not displaced from cellular structures. Rather, it may reflect a mixture of lung surfactant and liquid cryomatrix which dried onto the ITO carrier. Considering all these facts, we cannot completely rule out that the lack of PC on Fe-Si-NP was partially artificial, but the complete absence of PC over all Si signals (see Figure 6C) makes this interpretation highly unlikely. Finally, the lack of PC on Fe-Si-NP might also hint at an inappropriate sensitivity of the ToF-SIMS method. However, in a previous investigation on the composition of Langmuir–Blodgett films, PC and other phospholipids were successfully identified in a lung surfactant-like monolayer [32]. Thus, although some artificial displacement of particles and/or a loss of PC cannot completely be ruled out, we suggest that the absence of PC on the silica surface of the Fe-Si-NP in the lung, as measured by the highly sensitive ToF-SIMS method, is not an artifact and, therefore, of biological significance. 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Finding 3.6. Lack of Colocalization of Phophatidylcholine with Fe-Si-NP and General Impact of Findings However, the role of surfactant proteins for an in-situ opsonization of nanoparticles is still not known. As mentioned above, the incubation of nanoparticles in artificial or isolated pulmonary surfactant may be profoundly different from binding conditions in the lung. While in the lung the air–liquid interface carries non-polar lipid residues facing the gas phase, lipids in isolated lung surfactant may form micelles with their polar residues directed outwards. 12 of 14 Nanomaterials 2018, 8, 571 The current results provide, for the first time, new and more realistic insights in the formation of the lipid/protein corona around silica nanoparticles under native conditions of the lung. Findings may be representative for pulverulent silica nanomaterials, unless they are coated, e.g., by hydrophobic molecules. These findings have been achieved with high-resolution ToF-SIMS, a state-of-the-art bio-imaging method, which is universally applicable and circumvents the need to isolate particles from tissues. Future developments of the technique such as an ultra-high mass resolution detector are in sight, which may further increase the performance of the method [31]. This will allow us to obtain more detailed analyses of molecules bound to NP under in vivo conditions. References 1. Chaudhry, Q.; Scotter, M.; Blackburn, J.; Ross, B.; Boxall, A.; Castle, L.; Aitken, R.; Watkins, R. Applications and implications of nanotechnologies for the food sector. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2008, 25, 241–258. [CrossRef] [PubMed] 2. Johnston, C.J.; Driscoll, K.E.; Finkelstein, J.N.; Baggs, R.; O’Reilly, M.A.; Carter, J.; Gelein, R.; Oberdorster, G. Pulmonary chemokine and mutagenic responses in rats after subchronic inhalation of amorphous and crystalline silica. Toxicol. Sci. 2000, 56, 405–413. [CrossRef] [PubMed] 3. Arts, J.H.E.; Muijser, H.; Duistermaat, E.; Junker, K.; Kuper, C.F. Five-day inhalation toxicity study of three types of synthetic amorphous silicas in wistar rats and post-exposure evaluations for up to 3 months. Food Chem. Toxicol. 2007, 45, 1856–1867. [CrossRef] [PubMed] 4. Wiemann, M.; Sauer, U.; Vennemann, A.; Backer, S.; Keller, J.-G.; Ma-Hock, L.; Wohlleben, W.; Landsiedel, R. In vitro and in vivo short-term pulmonary toxicity of differently sized colloidal amorphous SiO2. Nanomaterials 2018, 8, 160. [CrossRef] [PubMed] 5. Landsiedel, R.; Ma-Hock, L.; Hofmann, T.; Wiemann, M.; Strauss, V.; Treumann, S.; Wohlleben, W.; Groters, S.; Wiench, K.; van Ravenzwaay, B. Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials. Part. Fibre Toxicol. 2014, 11, 16. [CrossRef] [PubMed] 6. Marzaioli, V.; Aguilar-Pimentel, J.A.; Weichenmeier, I.; Luxenhofer, G.; Wiemann, M.; Landsiedel, R.; Wohlleben, W.; Eiden, S.; Mempel, M.; Behrendt, H.; et al. Surface modifications of silica nanoparticles are crucial for their inert versus proinflammatory and immunomodulatory properties. Int. J. Nanomed. 2014, 9, 2815–2832. 7. Geiser, M.; Kreyling, W.G. Deposition and biokinetcis of inhaled nanoparticles. Part. Fibre Toxicol. 2010, 7, 2–17. [CrossRef] [PubMed] 8. Lopez-Rodriguez, E.; Gay-Jordi, G.; Mucci, A.; Lachmann, N.; Serrano-Mollar, A. Lung surfactant metabolism: Early in life, early in disease and target in cell therapy. Cell Tissue Res. 2017, 367, 721–735. [CrossRef] [PubMed] 9. Wright, J.R. Immunoregulatory functions of surfactant proteins. Nat. Rev. Immunol. 2005, 5, 58–68. [CrossRef] [PubMed] 10. Monopoli, M.P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Baldelli Bombelli, F.; Dawson, K.A. Physical−chemical aspects of protein corona: Relevance to in vitro and in vivo biological impacts of nanoparticles. J. Am. Chem. Soc. 2011, 133, 2525–2534. [CrossRef] [PubMed] 11. Wohlleben, W.; Driessen, M.D.; Raesch, S.; Schaefer, U.F.; Schulze, C.; Vacano, B.v.; Vennemann, A.; Wiemann, M.; Ruge, C.A.; Platsch, H. Influence of agglomeration and specific lung lining lipid/protein interaction on short-term inhalation toxicity. Nanotoxicology 2016, 10, 970–980. [CrossRef] [PubMed] 12. Nanomaterials 2018, 8, 571 Nanomaterials 2018, 8, 571 Author Contributions: L.V., D.B., C.E. and M.W., conceived and designed the experiments. L.V. and D.B. performed the ToF-SIMS analyses. B.H., D.B., and L.V. analyzed the ToF-SIMS data. A.V. and M.W. performed the animal experiments and contributed the tissue thin sections. L.V. and M.W. wrote the paper. Author Contributions: L.V., D.B., C.E. and M.W., conceived and designed the experiments. L.V. and D.B. performed the ToF-SIMS analyses. B.H., D.B., and L.V. analyzed the ToF-SIMS data. A.V. and M.W. performed the animal experiments and contributed the tissue thin sections. L.V. and M.W. wrote the paper. Funding: This research was funded by German Federal Ministry of Education and Research (BMBF), grant number [03X0146A and 03X0146C]. BMBF also covered the costs for Open Access publishing. Funding: This research was funded by German Federal Ministry of Education and Research (BMBF), grant number [03X0146A and 03X0146C]. BMBF also covered the costs for Open Access publishing. Acknowledgments: Authors would like to thank Christian Schechtmann for excellent technical assistance. Acknowledgments: Authors would like to thank Christian Schechtmann for excellent technical assistance. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. 4. Conclusions In this study on paramagnetic silica nanoparticles (Fe-Si-NP) administered to the lung via intratracheal instillation for 30 min, we show that particles were attached to alveolar walls and only marginally internalized by cells such as alveolar macrophages. Due to the successful colocalization of Fe and Si signals by ToF-SIMS analysis of lung cryo-sections, we conclude that the core/shell structure of the Fe-Si-NP remains intact under these conditions, such that Fe-Si-NP may now be used to study the early protein adsorption to a nanosized silica surface in the lung. Finally, the analysis of the phosphocholine head group, as a representative of phosphatidylcholine (PC), which is a major lipid of the pulmonary surfactant, revealed no augmented PC binding to Fe-Si-NP. This is an important finding as previous in vitro studies suggested that, e.g., surfactant protein A could mediate the binding of phospholipids to the polar silica surface. In this respect, ToF-SIMS proved to be a valuable technique with high spatial resolution and appropriate limits of detection, which made it possible to analyze nanoparticles together with bound organic molecules within their cellular environment. 13 of 14 13 of 14 References Kapralov, A.A.; Feng, W.H.; Amoscato, A.A.; Yanamala, N.; Balasubramanian, K.; Winnica, D.E.; Kisin, E.R.; Kotchey, G.P.; Gou, P.; Sparvero, L.J.; et al. Adsorption of surfactant lipids by single-walled carbon nanotubes in mouse lung upon pharyngeal aspiration. ACS Nano 2012, 6, 4147–4156. [CrossRef] [PubMed] 13. Veith, L.; Vennemann, A.; Breitenstein, D.; Engelhard, C.; Wiemann, M.; Hagenhoff, B. Detection of SiO2 nanoparticles in lung tissue by tof-sims imaging and fluorescence microscopy. Analyst 2017, 142, 2631–2639. [CrossRef] [PubMed] 14. Rehn, B.; Bruch, J.; Zou, T.; Hobusch, G. Recovery of rat alveolar macrophages by bronchoalveolar lavage under normal and activated conditions. Environ. Health Perspect. 1992, 97, 11–16. [CrossRef] [PubMed] 14 of 14 14 of 14 Nanomaterials 2018, 8, 571 15. Forster, B.; Van De Ville, D.; Berent, J.; Sage, D.; Unser, M. Complex wavelets for extended depth-of-field: A new method for the fusion of multichannel microscopy images. Microsc. Res. Tech. 2004, 65, 33–42. [CrossRef] [PubMed] 6. Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. Nih image to imagej: 25 years of image analysis. Nat. Met 2012, 9, 671–675. [CrossRef] [PubMed] 17. Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods 2012, 9, 676–682. [CrossRef] [PubMed] 18. Adler, J.; Parmryd, I. Quantifying colocalization by correlation: The pearson correlation coefficient is superior to the mander's overlap coefficient. Cytometry A 2010, 77, 733–742. [CrossRef] [PubMed] 19. Thevenaz, P.; Ruttimann, U.E.; Unser, M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 1998, 7, 27–41. [CrossRef] [PubMed] 20. Jasieniak, M.; Graham, D.; Kingshott, P.; Gamble, L.J.; Griesser, H.J. Surface analysis of biomaterials. In Handbook of Surface and Interface Analysis: Methods for Problem-Solving, 2nd ed.; Riviere, J.C., Myhra, S., Eds.; CRC Press: Boca Raton, FL, USA; London, UK; New York, NY, USA, 2009; pp. 536–538. 21. Sandoval, L.; Urbassek, H.M. Collision-spike sputtering of au nanoparticles. Nanoscale Res. Lett. 2015, 10, 1009. [CrossRef] [PubMed] 22. Yang, L.; Seah, M.P.; Anstis, E.H.; Gilmore, I.S.; Lee, J.L.S. Sputtering yields of gold nanoparticles by c60 ions. J. Phys. Chem. C 2012, 116, 9311–9318. [CrossRef] 23. Evennett, P.J.; Hammond, C. Microscopy Overview, 2nd ed.; Elsevier: Amsterdam, The Netherlands, 2005; pp. 32–41. 24. Davidson, M.W.; Abramowitz, M. Optical Microscopy; John Wiley & Sons, Inc.: New York, NY, USA 24. Davidson, M.W.; Abramowitz, M. References Optical Microscopy; John Wiley & Sons, Inc.: New York, NY, USA, 2002. 25. Alivisatos, A.P. Semiconductor clusters, nanocrystals, and quantum dots. Science 1996, 271, 933–937. [CrossRef] 25. Alivisatos, A.P. Semiconductor clusters, nanocrystals, and quantum dots. Science 1996, 271, [CrossRef] 26. Hu, M.; Novo, C.; Funston, A.; Wang, H.; Staleva, H.; Zou, S.; Mulvaney, P.; Xia, Y.; Hartland, G.V. Dark-field microscopy studies of single metal nanoparticles: Understanding the factors that influence the linewidth of the localized surface plasmon resonance. J. Mater. Chem. 2008, 18, 1949–1960. [CrossRef] [PubMed] 7. Shon, H.K.; Park, J.; Choi, I.; Park, H.M.; Moon, D.W.; Lee, T.G. Mass imaging of iron oxide nanoparti inside cells for in vitro cytotoxicity. J. Nanosci. Nanotechnol. 2011, 11, 638–641. [CrossRef] [PubMed] 28. Mercer, R.R.; Scabilloni, J.F.; Wang, L.; Battelli, L.A.; Antonini, J.M.; Roberts, J.R.; Qian, Y.; Sisler, J.D.; Castranova, V.; Porter, D.W.; et al. The fate of inhaled nanoparticles: Detection and measurement by enhanced dark-field microscopy. Toxicol. Pathol. 2018, 46, 28–46. [CrossRef] [PubMed] 29. Creutzenberg, O.; Bellmann, B.; Korolewitz, R.; Koch, W.; Mangelsdorf, I.; Tillmann, T.; Schaudien, D. Change in agglomeration status and toxicokinetic fate of various nanoparticles in vivo following lung exposure in rats. Inhal. Toxicol. 2012, 24, 821–830. [CrossRef] [PubMed] 30. Gosens, I.; Post, J.A.; de la Fonteyne, L.J.; Jansen, E.H.; Geus, J.W.; Cassee, F.R.; de Jong, W.H. Impact of agglomeration state of nano- and submicron sized gold particles on pulmonary inflammation. Part. Fibre Toxicol. 2010, 7, 37. [CrossRef] [PubMed] 31. Passarelli, M.K.; Pirkl, A.; Moellers, R.; Grinfeld, D.; Kollmer, F.; Havelund, R.; Newman, C.F.; Marshall, P.S.; Arlinghaus, H.; Alexander, M.R.; et al. The 3d orbisims—Label-Free metabolic imaging with subcellular lateral resolution and high mass-resolving power. Nat. Methods 2017, 14, 1175. [CrossRef] [PubMed] g g p 32. Breitenstein, D.; Batenburg, J.J.; Hagenhoff, B.; Galla, H.J. Lipid specificity of surfactant protein b studied by time-of-flight secondary ion mass spectrometry. Biophys. J. 2006, 91, 1347–1356. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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https://tuprints.ulb.tu-darmstadt.de/22526/3/applsci-1575858-supplementary.pdf
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Catchment Soil Properties Affect Metal(loid) Enrichment in Reservoir Sediments of German Low Mountain Regions
Applied sciences
2,022
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538
License: CC BY 4.0 International - Creative Commons, Attribution Supplementary material Table S1. Precautionary values according to the German Federal Soil Protection Ordinance (mg kg‐1). Texture As Cd Cr Cu Fe Hg Mn Ni Pb V Zn Sand - 0.4 30 20 - 0.1 - 15 40 - 60 Silt or Loam - 1.0 60 40 - 0.5 - 50 70 - 150 Clay - 1.5 100 60 - 1 - 70 100 - 200 1 The application on arable land requires that the metal concentrations of the applied material do not exceed 70%  of the precautionary values. Table S1. Precautionary values according to the German Federal Soil Protection Ordinance (mg kg‐1). 1 The application on arable land requires that the metal concentrations of the applied material do not exceed 70%  of the precautionary values. Table S2. Rotated component matrix of the three analyzed components (EV: Eigen value; Vt (%): percentage of  total variance; Vc (%): cumulative percentage of variance). Table S2. Rotated component matrix of the three analyzed components (EV: Eigen value; Vt (%): percentage of  total variance; Vc (%): cumulative percentage of variance). Table S2. Rotated component matrix of the three analyzed components (EV: Eigen value; Vt (%): percentage of  total variance; Vc (%): cumulative percentage of variance). total variance; Vc (%): cumulative percentage of variance). Comp. 1  2  3  Cr  .942  .156  ‐.051  Al  .905  .096  ‐.021  V  .903  .229  .026  Fe  .854  .333  ‐.173  As  .784  .215  .049  CN  ‐.536  ‐.349  .454  Zn  .478  .803  .130  Cd  .244  .762  .447  pH  .167  .756  ‐.274  Ni  .561  .727  .000  Mn  .135  .654  ‐.007  Pb  .012  .463  .328  TOC  ‐.156  ‐.030  .957  Nt  .068  .180  .925  EV  4.83  3.37  2.42  Vt (%)  34.5  24.0  17.2  Vc (%)  34.5  58.5  75.8 Table S3. Percentage of easily extractable trace metal concentrations on the aqua regia soluble concentrations of  air‐dried reservoir sediments (RS). Table S3. Percentage of easily extractable trace metal concentrations on the aqua regia soluble concentrations of  air‐dried reservoir sediments (RS). As  Cd  Cr  Fe  Mn  Ni  Pb  V  Zn  Mean  1.26  10.9  0.07  0.02  62.1  4.41  0.19  0.03  2.93  Median  0.59  7.17  0.06  0.01  71.2  3.30  0.06  0.02  0.67  SD  1.92  9.61  0.03  0.02  36.8  5.43  0.29  0.02  4.87  Min  0.20  0.88  0.02  0.00  0.73  0.28  0.01  0.01  0.13  Max  9.31  38.1  0.15  0.06  100  20.5  1.13  0.13  19.4 License: CC BY 4.0 International - Creative Commons, Attribution Table S4. Supplementary material The variability of the median values of the element‐specific enrichment factors (EF) illustrated by the  example of the Breitenbach, Obernau and Bitburg reservoirs (RS: reservoir sediment; SS: stream sediment; CS:  catchment soil). As Cd Cr Fe Mn Ni Pb V Zn Breitenbach RS SS CS 4.3 7.1 6.3 10.2 13.9 2.4 2.8 3.7 3.5 5.3 8.9 5.2 40.5 19.0 3.1 8.9 20.9 3.9 21.2 42.6 36.2 3.0 4.0 4.1 10.4 14.0 3.3 Obernau RS SS CS 3.0 3.8 3.9 8.2 2.2 2.5 3.2 3.2 3.1 4.2 4.9 4.3 27.6 7.6 4.4 7.4 6.5 3.9 6.0 4.7 6.2 3.2 2.4 3.0 6.4 3.5 2.9 Bitburg RS SS CS 2.9 4.7 2.9 2.5 1.7 1.1 2.8 3.4 2.7 3.9 5.4 4.0 3.5 6.0 5.9 4.9 5.8 4.2 8.9 3.5 2.4 3.0 2.9 2.4 4.1 3.3 2.5
https://openalex.org/W1686233141
https://zenodo.org/records/1083921/files/14948.pdf
English
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Adaptive Motion Estimator Based on Variable Block Size Scheme
Zenodo (CERN European Organization for Nuclear Research)
2,009
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5,202
World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 Authors are with Electronics and Micro-Electronics Laboratory (LAB- IT06), Faculty of Sciences of Monastir, Monastir, 5000, Tunisia. the image and the handled video. The choice of the research strategy to be used in the stage of motion estimation is International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 In order to efficiently reduce the computational cost while achieving better visual quality with low cost power, the proposed motion estimator is based on a Variable Block Size (VBS) scheme that uses only the 16x16, 16x8, 8x16 and 8x8 modes. The computational cost of a motion estimation algorithm is greatly affected by the number of candidate blocks to be computed. In order to compute the Somme of Absolute Difference (SAD), the FBMA evaluates every possible motion vector inside the search area, find the best matched block and give the highest PSNR. Other strategies are based on the so called fast block motion estimation algorithms group which includes the Three-Step Search algorithm (TSS), the Modified Log Search algorithm (LS), the Four Steep Search (FSS) and the Alternative Pixel-Decimation Search algorithm (APDS) [3, 4, 5, 6, 7, 8, 9]. These algorithms take into account a reduced group of available candidate blocks at the expense of a reduction in term of PSNR. Experimental results show that the adaptive motion estimator allows better results in term of Peak Signal to Noise Ratio (PSNR), computational cost, FPGA occupied area, and dissipated power relatively to the most popular variable block size schemes presented in the literature. Keywords—H264, Configurable Motion Estimator, Variable Block Size, PSNR, Dissipated power. Adaptive Motion Estimator Based on Variable Block Size Scheme S. Dhahri, A. Zitouni, H. Chaouch, and R. Tourki decisive in the total execution and on the video quality. One of the principal strategies to obtain an effective compression is to use a technique which exploits the space and temporal redundancy. Two main techniques are used for motion estimation: pixel recursive algorithms and block matching algorithm (BMA). The first one estimates the motion between successive frames on a pixel by pixel base, whereas the BMA estimates motion on a block by block basis. In general, due to its simple hardware realization, the block-matching approach is the most suitable. Abstract—This paper presents an adaptive motion estimator that can be dynamically reconfigured by the best algorithm depending on the variation of the video nature during the lifetime of an application under running. The 4 Step Search (4SS) and the Gradient Search (GS) algorithms are integrated in the estimator in order to be used in the case of rapid and slow video sequences respectively. The Full Search Block Matching (FSBM) algorithm has been also integrated in order to be used in the case of the video sequences which are not real time oriented. A. Four Step Search International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 munication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 The Four-Step Search (4SS) is an algorithm that builds upon the TSS, while being more center-based. The 4SS differs in that it maintains a more regular search pattern with half-stop techniques employed in the algorithm [6]. The 4SS offers a similar best-case scenario for computational complexity when compared to the TSS (17 searches). The worst-case situation for the 4SS is 27 search points compared to 33 for the TSS, which is an improvement. Experimental studies performed on multitude standard video sequences show that this algorithm is well adapted to the fast sequences by giving the better PSNR and compression rates relatively to other algorithms. International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Pu 4 SS GS Adaptive motion estimator Rapid Slow Two motion types Fig. 1 Principal of the adaptive motion estimator Rapid Fig. 2 Four Step Search paths Fig. 2 Four Step Search paths Adaptive motion estimator Two motion types Fig. 1 Principal of the adaptive motion estimator This paper is organized as follow: Section 2 presents brief overview of the Block Matching Algorithms (BMA) which are integrated in the adaptive motion estimator (4SS, GS, FS). In section 3, the architecture of the proposed motion estimator is presented. The proposed VBS scheme is described in section 4. Section 5 presents some experimental results and Section 6 concludes the paper. Fig. 2 Four Step Search paths Fig. 2 Four Step Search paths World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 split [12], and low complexity motion estimation [13], etc. ( ) ∑∑ = = + + − − = 16 1 i 16 1 j ) l j, k i( 1 tP j,i tP ) l, k ( SAD split [12], and low complexity motion estimation [13], etc. In general, since, a video application can contain many sequences that are different in terms of motion nature (slow, stationary, fast), an adaptive motion estimation scheme is very suitable to improve performances of a video Codec (Fig. 1). Where (k, l) is the location in the search window, Pt(i, j) is a pixel at (i, j) in the current frame, and Pt−1(i, j) is a pixel in the previous frame. When the value SAD(k,l) is minimum, (k,l) is the motion vector of the macro block. This paper presents the proposed adaptive motion estimator that can be dynamically configured by three algorithms (FSBM, 4SS, GS) depending on the variation of the video nature during the lifetime of a running application. This configuration is based on a recognition step that collects motion information of each block from its same block in the previous images and their neighbor’s blocks in the current image. Contrary to many solutions proposed in the literature, the proposed approach is based on an efficient technique for decomposing the motion block. In fact, a new VBS motion estimation algorithm based on 16x16, 16x8, 8x16 and 8x8 only has been proposed, which can efficiently reduce the computational cost while achieving similar or better visual quality with low cost power on different architectures. The advantages of implementing FSBM as the motion estimation algorithm include both the guaranteed optimality of the solution and the regularity of a hardware implementation. This regularity stems from the fact that consecutive motion vectors share many of the pixel values in calculation. nce Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 B. Gradient Search The block-based gradient descent search (BBGDS) algorithm has been proposed by literature [5]. Based on the observation that global minimum distribution is centralized in real video sequence, this algorithm uses a very center- based search pattern of nine checking points in each step with a step size of one. It does not restrict the number of searching steps but it stops when the minimum checking point of the current step is the central one or it reaches the search window boundary. This algorithm has advantages: very low complexity in terms of candidate to evaluate, good regularity in terms of motion vector generation and only I. INTRODUCTION IDEO communication is a rapidly evolving field for several applications which include video telephony, videoconference, remote surveillance, remote working and learning, etc. It is also a key feature for the upcoming information and communication technologies based on residential digital lines (VDSL, ADSL and ISDN) and the 3rd generation of mobile telephony system (UMTS). In this scenario, video image compression plays a fundamental role in reducing the enormous bit-rate for transmission and storage (approximately hundreds of Mbits/s for main image formats). To this objective, the ISO and the ITU-T committees have worked on several compression standards such as JPEG, MPEG (versions 1, 2, 4), H.261, H.263 and H.26L [1, 2]. V V Experimental results performed in [10] show that the full search algorithm is very greedy in computing times, but always gives the best results in compression ratio and PSNR (Fig. 1). This work shows also that in the case of fast sequences (ex. Forman) the 4SS algorithm gives better PSNR and compression rates relatively to other algorithms. In the case of slow sequences (ex. Miss America), [10] shows that the GS algorithm is more advantageous since the best block are always very close to the fetched block. By studying the sequences that contain the two types of motion (fast and slow) (ex. News and Carphone), [10] shows that the results are very close and speed can be improved by considering methods of research adapted for each type of block. Motion estimation is regarded as one of the most effective technique to reduce the flow required by a video codec. This stage is the most expensive in computing times and power consumption for the standard H264. Indeed, the method of estimation influences enormously the quality of The motion estimation for each 16x16 macro-block in the emerging H.264/AVC can be divided into 16x16, 16x8, 8x16, and 8x8 modes. If the 8x8 mode is chosen, each 8x8 block can be independently coded into 8x4, 4x8, and even 4x4 blocks. Consequently, many VBS motion estimation algorithms has been proposed in literature, such as bottom- up merge and top-down split [11], prediction by merge and scholar.waset.org/1307-6892/14948 International Scholarly and Scientific Research & Innovation 3(2) 2009 International Scholarly and Scientific Research & Innovation 3(2) 2009 218 International Scholarly and Scientific Research & Innovation 3(2) 2009 C. Full Search This algorithm defines a rectangular geometry for the image areas, consequently, the research window is limited to [- p, p] which is the area of research around the origin of the block in the reference image (Fig. 4). For an image with a size of (2p+1) × (2p+1) and for a window with a size of M*M and a block with a size of N ×N, the number of operations that are treated by the FSBM algorithm for each block is Nb = (2p+1)2×N2. The number of operations for each window is Nf = (2p+1)2×M2. Since this algorithm is the slowest in term of computing times, it is generally used for the applications with high image quality. It allows the sweeping of all the blocks of the research window. This makes it possible to reach the adequate motion vector in this window. 4 -Phase Bus Reference Memory SAD FSM FSBM FSM 4SS FSM GS Configuration Module Current Memory Fig. 5 Optimal architecture of the estimator Configuration Module Fig. 4 Method of search for the full search algorithm Fig. 5 Optimal architecture of the estimator In our realization, each algorithm is specified by its control part that is described as a Finite State Machine (FSM). The architecture is optimized in order to make the redundant blocks reusable by the three algorithms (Fig. 5). According to the algorithm to be implemented, the corresponding FSM communicates with the other blocks via a 4-Phase handshaking bus. According to the application to be treated, the estimator is configured by the suitable algorithm. This allows the use of a single estimator for various applications, which makes it possible to guarantee an effective video compression. The estimator is composed of a set of modules which are configured with the proposed VBS scheme. The calculated SAD is stored in a memory. If the scanning of the search window is finished, then the memorized SAD is compared and the minimal SAD will be detected. Then a new research can be started. The optimal SAD with its position is sent towards a buffer Fig. 4 Method of search for the full search algorithm This algorithm selects the minimum among the possible vectors inside the research window (the motion vector belongs to the area [- p, p]). This approach is inadaptable by the majority of real time applications because of the high number of operations that is performed. II. BLOCK MATCHING ALGORITHMS The FSBM algorithm is the most straightforward motion estimation operation. The process of block-matching is found in a search window of previous frames of the macro blocks most similar to the macro blocks in the current frame. The accuracy of ME depends on the matching criteria and one of the most popular criteria is the sum of absolute difference (SAD) given by: scholar.waset.org/1307-6892/14948 International Scholarly and Scientific Research & Innovation 3(2) 2009 219 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 portion of search area memory can be accessed. Experimental studies performed on multitude standard video sequences show that this algorithm is well adapted to the slow sequences by giving the better PSNR and compression rates relatively to other algorithms algorithm suffers from greatest data-processing complexity. For example, considering the research area of [- 7, +7], there are 226 calculations steps of the SAD which must be carried out for each macro block in a window. III. ADAPTIVE MOTION ESTIMATOR MODELING Fig.3 Gradient Search paths The motion estimation suggested integrates three algorithms: FSBM, 4SS and GS. The 4 modes (16x16, 16x8, 8x16 and 8x8) defined by the proposed VBS technique are integrated in this estimator. Knowing that all the motion estimators use memory to store the pixels of the block under running as well as block of reference and carry out the SAD calculation, these modules were defined only once inside the proposed architecture. Thus, each technique of motion estimation is defined only by its controller. Once configured, the selected controller communicates with the common SAD module inside architecture by using the memory common resources and ensures the estimation according to the algorithm of estimation in question. Such a strategy allows the estimator to be configured according to the nature of the application under running. Generally, the new Codec use motion estimation which integrates only one algorithm for each application [14, 15, 16, 17, 18]. For an application which handles video sequences of variable types, several estimators are normally necessary in order to guarantee an acceptable video quality for each type of sequence. nce Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/P Fig.3 Gradient Search paths memory. 6 Algorithmic description of the Full Search algorith International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 dex, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 For (k = -p; k ≤ (p- bloc_col_v+1); k++) For l = -p; l≤ (p- bloc_line_v +1); l++) req <= '1'; For (r = 0; r≤ length_v-1; r++) data_cour <= var_cour(r); data_ref <= var_ref(r); End; For v = 0; v≤length_v-1; v++) var_cour (v) <= "00000000"; End; wait until ack ='1'; motion_vec (z) <= sad_in; motion_vec2 (z, 0) <= k; motion_vec2 (z, 1) <= l; Req <= '0'; z: = z+1; wait until ack ='0'; For (m = k; m≤k+ (bloc_col_v-1) ; m++) For (n = l; n ≤ l+ (bloc_line_v-1); n++) var_cour (v) <= memory (m, n); v: =v+1; End; End; End; End; For (h = 0; h≤ nsad_v -1; h++) If (motion_vec (h) <= sad_min) then sad_min:= motion_vec (h); posit_x:= motion_vec2 (h, 0); posit_y:= motion_vec2 (h, 1); Else sad_min:= sad_min; End if; sum_sort0 <= sad_min; x_sort0 <= posit_x; y_sort0 <= posit_y; End; Fig. 6 Algorithmic description of the Full Search algorithm International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 Fig. 7 The window read/write protocol memory. The proposed motion estimator has been designed by Register Transfer Level (RTL) level by using the VHDL language. After being calculated by the chosen algorithm, the calculated SAD values are memorized in a memory in order to define for each block the starting point in the research window. All the calculated SAD values of the window are stored in a memory buffer and the minimal value is determined by trying. Then the output of the corresponding algorithm search block is the value of the minimal SAD and their positions. In other terms, the outputs are the motion vectors which will be used for the reconstruction of the image by the coder. Fig. 7 The window read/write protocol For (k = -p; k ≤ (p- bloc_col_v+1); k++) For l = -p; l≤ (p- bloc_line_v +1); l++) req <= '1'; For (r = 0; r≤ length_v-1; r++) data_cour <= var_cour(r); data_ref <= var_ref(r); End; For v = 0; v≤length_v-1; v++) var_cour (v) <= "00000000"; End; wait until ack ='1'; motion_vec (z) <= sad_in; motion_vec2 (z, 0) <= k; motion_vec2 (z, 1) <= l; Req <= '0'; z: = z+1; wait until ack ='0'; For (m = k; m≤k+ (bloc_col_v-1) ; m++) For (n = l; n ≤ l+ (bloc_line_v-1); n++) var_cour (v) <= memory (m, n); v: =v+1; End; End; End; End; For (h = 0; h≤ nsad_v -1; h++) If (motion_vec (h) <= sad_min) then sad_min:= motion_vec (h); posit_x:= motion_vec2 (h, 0); posit_y:= motion_vec2 (h, 1); Else sad_min:= sad_min; End if; sum_sort0 <= sad_min; x_sort0 <= posit_x; y_sort0 <= posit_y; End; Fig. A. Variable Block Size 1 (VBS1) Unlike previous standards, H.264/AVC adopts a tree- based decomposition to partition the macro-block (MB) into smaller sub-blocks of specified sizes. The quad-tree structure enables the possibility of a MB being coded in four different modes illustrated in Fig. 8, with partitions sizes of 16x16, 16x8, 8x16 and 8x8, while in the 8x8 partition mode, each 8x8 partition can be further split into 8x4, 4x8, and 4x4 sub-partitions. The availability of smaller ME blocks improves prediction in general, and in particular, the small blocks improve the ability of the model to handle fine motion detail and result in better subjective viewing quality because they do not produce large blocking artifacts. Fig. 8 Multiple motion estimation modes defined in H.264/AVC Fig. 8 Multiple motion estimation modes defined in H.264/AVC Fig. 8 Multiple motion estimation modes defined in H.264/AVC C. Full Search Obviously, this algorithm always finds the optimal motion vector for the given research window. It should also be obvious, that this scholar.waset.org/1307-6892/14948 International Scholarly and Scientific Research & Innovation 3(2) 2009 220 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 and reference). The setting to '0' of this signal allows the beginning of the sweeping of the window. For a new window the same operation will be repeated. An algorithmic description of the Full Search algorithm is represented by Fig. 6. memory. V. EXPERIMENTAL RESULTS To evaluate the performances of the proposed adaptive motion estimator, both software and hardware implementations have been performed. The impact of the new VBS3 motion scheme on the encoding process, along with several analyses on three CIF (352x288) sequences test with different characteristics is proposed. At the beginning, the performances evaluation of the VBS3 algorithm compared to the VBS1 and VBS2 on a SW DSP (TMS320C64) design platform are presented. The used parameters consists on a QP =38 and a search window (Horizontal: [-15, 15] and Vertical:[-15, 15]). Finally, the logic synthesis and the power estimation results of the proposed estimator with the VBS3 scheme are presented and compared with the VBS1 and VBS2 schemes. These designs target the Altera FPGA technology and performed by the ISE and the X-Power tools. ndex, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 B. Variable Block Size 2 (VBS2) Serious experiments on the test video sequences used in JVT Test Model Ad Hoc Group [11] show that there is an average of 35 per cent homogeneous area in a typical video frame, and these areas are suitable for larger size inter mode coding. Therefore, several cost calculation of small size modes can be saved. Based on this consideration, several mode decision algorithms were proposed to reduce the number of candidate modes [11, 12]. In [19], many Fast VBS Motion Estimation algorithms were tested before extracting the Zoom Motion Estimation (ZME) algorithm [19] based on 3 partitions sizes 16x16, 8x8 and 4x4 arranged as shown in Fig. 9. Fig. 6 Algorithmic description of the Full Search algorithm In the proposed architecture, the presence of a new window is indicated by a command signal (cmd_fen). This signal allows the initialization of the memory. After initialization, the algorithm begins the treatment by a signal called wr_ena (Fig. 7). The setting to '1' of the signal wr_ena makes it possible to charge a new window (current International Scholarly and Scientific Research & Innovation 3(2) 2009 scholar.waset.org/1307-6892/14948 221 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 Fig. 9 Multiple motion estimation modes defined in H.264/AVC org/Publication/14948 A. Subjective, Objective and Complexity Analysis International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Pu Fig. 9 Multiple motion estimation modes defined in H.264/AVC Table I and Table II show that the elimination of 4x8, 8x4 and 4x4 block size (performed by our VBS3 scheme) from the mode selection does not affect the video quality (PSNR and SSIM [20]). Thus, the VBS3 algorithm provides better subjective and objective quality compared to the VBS2 scheme with complexity reduction in terms of clock cycles up to 44 per cent (Table III). C. Variable Block Size 3 (VBS3) C. Variable Block Size 3 (VBS3) The proposal of the new typical based VBS scheme is to extract, using the SAD criteria, the block-size introducing the minimum computational complexity with minimum memory requirement. In this algorithm, only 16x16, 16x8, 8x16, and 8x8 block-sizes for motion estimation are considered. This approach will reduce extensively the computational complexity since the 3 other modes are eliminated, without affecting the overall visual video quality. For better understanding, the following flowchart explains in detail our proposed algorithm steps Fig. 10. Fig. 11 Number of use modes for different variable block decision algorithm International Science Index, Electronics and Communication En Fig. 10 Fast block size selection algorithm FBSA International Science Index, Electronics and Communica Fig. 11 Number of use modes for different variable block decision algorithm Fig. 11 Number of use modes for different variable block decision algorithm Fig. 10 Fast block size selection algorithm FBSA International Scholarly and Scientific Research & Innovation 3(2) 2009 scholar.waset.org/1307-6892/14948 222 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 motion estimator would be critical for inclusion in a portable or other power-conscious device. These results demonstrate the fact that from one hand, VBS3 outperforms the other variable block size techniques used in this study, such as VBS1 and VBS2 in terms of MB sizes (bit) distortion with a major loss in complexity. On the other hand, VBS3 is very similar to the other VBS predicting algorithms in terms of subjective quality with a major gain in the computational complexity and a better image quality depending on the motion characteristic of the video. In fact, the visual image quality with a major reduction in the computational effort complexity has been objectively improved. However, the penalized is performed only for the low motion sequence since the extra search will not be necessary (Fig. 11). The gain in terms of the FPGA equivalent area designed with our VBS and the VBS2 schemes is about +4,35% relatively to the FPGA equivalent area designed by the standard VBS1. Also the gain in terms of the power dissipated by our VBS scheme is about +5,17% and +1,79% relatively to the power dissipated by the VBS1 and the VBS2 schemes (Table V). TABLE V COMPARISON OF THE VBS SCHEMES IN TERMS OF FPGA EQUIVALENT AREA AND DISSIPATED POWER TABLE V COMPARISON OF THE VBS SCHEMES IN TERMS OF FPGA EQUIVALENT AREA AND DISSIPATED POWER AREA AND DISSIPATED POWER Equivalent area: VBS2 vs. VBS1 )% 100 1 VBS 2 VBS 100 ( × − +4,35% Equivalent area: VBS3. vs. VBS1 )% 100 1 VBS 3 VBS 100 ( × − +4,35% Dissipated power: VBS2 vs. VBS1 )% 100 1 VBS 2 VBS 100 ( × − +3,45% Dissipated power: VBS3. vs. VBS1 )% 100 1 VBS 3 VBS 100 ( × − +5,17% Dissipated power: VBS3. vs. VBS2 )% 100 2 VBS 3 VBS 100 ( × − +1,79% TABLE II SUBJECTIVE QUALITY PERFORMANCE (SSIM) OF BLOCK MODE SELECTION SCHEMES TABLE II SUBJECTIVE QUALITY PERFORMANCE (SSIM) OF BLOCK MODE SELECTION SCHEMES TABLE III SPEED PERFORMANCE (MILLIONS CYCLES) OF BLOCK MODE SELECTION SCHEMES B. RTL Design Results TABLE III SPEED PERFORMANCE (MILLIONS CYCLES) OF BLOCK MODE SELECTION SCHEMES VI. CONCLUSION In this paper a configurable motion estimator for the codec H264 has been presented. This estimator integrates three algorithms (FSBM, 4SS and GS) and implements a VBS scheme. Depending on the nature of the application under running this algorithm will be reconfigured dynamically by the adequate algorithm. Since, the motion estimator is responsible for nearly 70% of the power dissipated and computation cost in certain video encoders, any amelioration of the motion estimator performances would be critical for inclusion in a portable or other power- conscious device. Experimental results lead us to conclude that the proposed estimator integrating our VBS scheme allows better image quality with less computing time, less FPGA occupied area and less dissipated power relatively to the other VBS schemes. As a perspective, further design targeting ASIC technology for proposed adaptive motion estimator show that substantial improvements, in terms of encoding speed and dissipated power, can be obtained through optimizing heavily. This research work is ongoing, and the results will be presented in future publications. C. Variable Block Size 3 (VBS3) TABLE IV FPGA EQUIVALENT AREA AND POWER ESTIMATION RESULTS Used VBS Equivalent area Dissipated power (mW) VBS1 23% 58 VBS2 22% 56 VBS3 22% 55 International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 TABLE I OBJECTIVE QUALITY PERFORMANCE (PSNR) OF BLOCK MODE SELECTION SCHEMES TABLE II SUBJECTIVE QUALITY PERFORMANCE (SSIM) OF BLOCK MODE SELECTION SCHEMES TABLE III SPEED PERFORMANCE (MILLIONS CYCLES) OF BLOCK MODE SELECTION SCHEMES B. RTL Design Results In order to evaluate the occupied area and the dissipated Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/Publication/14948 TABLE I TABLE I OBJECTIVE QUALITY PERFORMANCE (PSNR) OF BLOCK MODE SELECTION SCHEMES REFERENCES [1] G. Cote, and L. Winger, Recent progress in the field of video compression, IEEE Canadian Review- Spring 2002. p p g [2] ITU-T SG15, Video Codec for Audiovisual Service at Px64 Kbits/s, In ITU-T recommendation H.261 Version 3, Mars 1993. [2] ITU-T SG15, Video Codec for Audiovisual Service at P In ITU-T recommendation H.261 Version 3, Mars 1993. [3] R. Srinivasan, and K. R. Rao, “Predictive coding based on efficient motion estimation”, IEEE Trans. on Circuits and Syst. on Video Technol., vol. 33, no. 8, August 1985, pp. 888-896. [4] M. Ghanbari, “The cross-search algorithm for motion estimation,” IEEE Trans. on Communications, vol. 38, no. 7, July 1990, pp. 950- 953. [5] L.-K. Liu et E. Feig, 'A block-based gradient descent search algorithm for block motion estimation in video coding', IEEE Trans. on Circuits and Sys. for Video Technol., Vol. 6, No 4, pp. 419- 422,1996. [6] Lai-Man Po, and Wing-Chung Ma, “A novel four-step search algorithm for fast block motion estimation,” IEEE Trans. on Circuits and Syst. for Video Technol., Vol. 6, no. 3, June 1996, pp.313 – 317. [7] C. Zhu, X. Lin, L-P. Chau, K-P. Lim, H-A. Ang and C-Y. Ong, “A novel hexagon-based search algorithm for fast block motion estimation,” IEEE International Conference on Acoustics, Speech, and Signal Processing, 2001. Vol. 3, pp.1593 – 1596. [8] C. Chun-Ho, and P. Lai-Man, “A novel cross-diamond search algorithm for fast block motion estimation,” IEEE Trans. on Circuits and Syst. for Video Technol., vol. 12, no. 12, December 2002, pp.1168-1177. International Science Index, Electronics and Communication Engineering Vol:3, No:2, 2009 waset.org/P [9] Jae Hun Lee, and Al, “Variable block size motion estimation algorithm and its hardware architecture for H.264 /AVC”, IEEE Inter. Symp. on Circuits and Sys., May 2004. [10] A. Djeffal, and Z. Baarir, “Video coding adaptive block matching “, 8th African Conference on Research in Computer Science, November 2006, pp.1-8. [11] Y.K. Tu, J.F. Yang and M.T. Sun, Fast Variable-size Block Motion Estimation Using Merging Procedure with an Adaptive Threshold, IEEE International Conference on Multimedia and Expo. Baltimore, July 2003, p.II-789-792. [12] Z. Zhou, M.T. Sun and Y.F. Hsu, Fast variable block-size motion estimation algorithms based on merge and split procedures for H.264/MPEG-4 AVC, IEEE International Symposium on Circuits and Syst., ISCAS. Vancouver, British Columbia, Canada, May 23-26, 2004.II-789-792. [13] Y. Jiang,S. Li and S. B. RTL Design Results In order to evaluate the occupied area and the dissipated power of the proposed motion estimator, the ISE and the X- Power FPGA design tools have been used. Table IV presents the logic synthesis and the power estimation results of the adaptive motion estimator configured with the FSBM algorithm and integrating the VBS1, VBS2, and the VBS3 schemes targeting the Virtex2pro FPGA technology. Experimental results show that the FPGA equivalent area and the dissipated power obtained by our VBS scheme are slightly inferior to those obtained by the VBS1 and the VBS2 schemes. But since the motion estimator is responsible for nearly 70% of the power dissipated in certain video encoders, any reduction in power in the scholar.waset.org/1307-6892/14948 International Scholarly and Scientific Research & Innovation 3(2) 2009 223 World Academy of Science, Engineering and Technology International Journal of Electronics and Communication Engineering Vol:3, No:2, 2009 scholar.waset.org/1307-6892/14948 REFERENCES Goto, A Low Complexity Variable Block Size Motion Estimation Algorithm for Video Telephony Communication, 47th IEEE International Midwest Symposium on Circuits and Syst.. July 2004, p.II-465 - II-468.II-789-792. y p [14] M. G. Xavier, “Optimizing performance of an encoder following the standard Advanced Video Coding for a vector machine,” Master Memory, Faculty of Sciences libre University of Bruxcelle, 2006. [15] S. Yalcin, H.F. Ates, I.Hamzaoglu, “A high performance hardware architecture for an SAD reuse based hierarchical motion estimation algorithm for H.264 video coding”, Proc. Inter. Conf. on Field Prog. Logic and App., Tampere, Finland, Aug 2005. [16] P. Brault, “Motion Estimation and image segmentation”, Thesis Memory, Faculty of Sciences of Orsay, November 2005. [17] LIU Hao, ZHANG Wen-jun, CAI Jun, “A fast block-matching algorithm based on variable shape search”, Journal of Zhejiang University Science A, Mars 2005. [18] Tiago Miguel Braga da Silva Dias, “High-Performance VLSI Motion Estimation Processors: Data Reuse and Sub-Pixel Accuracy”, Master Memoir, Univ. de Tec. de Lisboa, Inst. Sup. Tec., Sep. 2004. [19] M.A. Ben Ayed,A. Samet and N. Masmoudi, Toward an Optimal Block Motion Estimation Algorithm for H.264/AVC, International Journal of Image and Graphics (IJIG). 2006. [20] Z. Wang,A.C. Bovik,H.R. Sheikh and E.P. Simoncelli, Image quality assessment: From error visibility to structural similarity, IEEE Trans. on Image Processing. vol. 13, no. 4, 2004. 224 International Scholarly and Scientific Research & Innovation 3(2) 2009 scholar.waset.org/1307-6892/14948
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https://www.frontiersin.org/articles/10.3389/fvets.2021.688656/pdf
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ORIGINAL RESEARCH published: 09 September 2021 doi: 10.3389/fvets.2021.688656 ORIGINAL RESEARCH published: 09 September 2021 doi: 10.3389/fvets.2021.688656 Stefan Deleuze 1*†, Fany Brotcorne 2†, Roland Polet 2, Gede Soma 3, Goulven Rigaux 4, Gwennan Giraud 2, Fanny Cloutier 2, Pascal Poncin 5, Nengah Wandia 3 and Marie-Claude Huynen 2 Stefan Deleuze 1*†, Fany Brotcorne 2†, Roland Polet 2, Gede Soma 3, Goulven Rigaux 4, Gwennan Giraud 2, Fanny Cloutier 2, Pascal Poncin 5, Nengah Wandia 3 and Marie-Claude Huynen 2 Edited by: Reviewed by: Paolo Martelli, Ocean Park, Hong Kong, SAR China Joanne Paul-Murphy, University of California, Davis, United States Worldwide, primates, and humans increasingly share habitats and often enter in conflict when primates thrive in human-dominated environments, calling for special management measures. Reproductive control is increasingly used to manage population growth but very few monitoring data are available. Therefore, the efficiency and implications of such programs require a careful examination. In the context of a contraception program in wild female long-tailed macaques in Ubud, Bali, conducted over four successive campaigns between 2017 and 2019, including 140 females (i.e., 41.9% of the reproductive females of the population in 2019), modifications of an endoscopic tubectomy procedure, a permanent sterilization method, clinical evaluation of this method, and the post-operative monitoring results of the neutered females after release are described. This surgical approach was applicable for pregnant females: 28.6% of the treated females were pregnant at the time of the surgery. The procedure used a single lateral port to reach and cauterize both oviducts in non-pregnant as well as in early to mid-term pregnant females. Pregnant females nearer to term required a second lateral port to access both oviducts masked by the size of the gravid uterus. Moreover, bipolar thermocauterization was utilized successfully without resection to realize the tubectomy. The average duration of the laparoscopic surgery was 14 min for non-pregnant females and 22 min for pregnant females. Animals were released 3 h 22 min in average following their capture. This short holding time, recommended for free-ranging primates, was made possible by the minimal invasiveness of the sterilization approach. A laparoscopic post-operative evaluation conducted on two patients during the following campaign confirmed that the *Correspondence: Stefan Deleuze s.deleuze@uliege.be *Correspondence: Stefan Deleuze s.deleuze@uliege.be †These authors share first authorship Specialty section: This article was submitted to Zoological Medicine, a section of the journal Frontiers in Veterinary Science Received: 31 March 2021 Accepted: 09 August 2021 Published: 09 September 2021 Specialty section: This article was submitted to Zoological Medicine, a section of the journal Frontiers in Veterinary Science Received: 31 March 2021 Accepted: 09 August 2021 Published: 09 September 2021 1 Research Unit FARAH, Equine and Companion Animal Reproduction Pathologies Clinic, Veterinary Medicine Faculty, University of Liège, Liège, Belgium, 2 Research Unit SPHERES, Department of Biology, Ecology and Evolution, Sciences Faculty, University of Liège, Liège, Belgium, 3 Primate Research Center, Veterinary Medicine Faculty, Udayana University, Denpasar, Indonesia, 4 Pan Anima, Tournai, Belgium, 5 Research Unit FOCUS, Department of Biology, Ecology and Evolution, University of Liège, Liège, Belgium Edited by: Kathryn Christine Gamble, Lincoln Park Zoo, United States INTRODUCTION urges conducting systematic studies during and in the aftermath of the fertility control interventions to monitor the impacts, efficiency, and adequacy of the methods adopted with primates (10, 19). As for laparoscopic tubectomy, clinical intra-, and post- operative evaluations have been described in a small number of rhesus (17) and Formosan macaques (M. cyclopis) (20) in captive settings, and more recently in a long term program in field conditions in free-ranging rhesus cross long-tailed macaques (14). However, the application of laparoscopic tubectomy to M. fascicularis in free-ranging settings, its clinical evaluation and possible modification to optimize the technique as well as a long-term post-surgery monitoring remain lacking and need to be documented. Reasons for selecting this technique with urban long-tailed macaques are manyfold but most of all, the species’ reproductive biology [i.e., a multimale/multifemale mating system where a female is likely to mate with multiple males; (21)] and social dynamics [i.e., a female philopatry system of resident matrilineal groups wherein demographic recruitment is a function of births and male migrations; (22)], naturally orient the choice toward female-based sterilization. Worldwide, a growing proportion of primate populations of various species thrive in human-modified habitats (1). As a consequence of the rapid land-use change, these populations are increasingly forced to cohabit with humans (2). The macaques (Macaca sp.) in particular share one of the widest spatial overlaps with humans, and sometimes proliferate, even in urban environments (3). This is the case for the long-tailed macaque (Macaca fascicularis) which increasingly gravitates toward human environments throughout south-east Asia, becoming sometimes locally overabundant within human-dominated lands (4). Uncontrolled increase of populations in urban or peri- urban settings often causes conflicts between macaques and people, threatens public health, multiplies nuisance problems for people, and has in turn significant impacts for macaques (2, 5). Regarding sanitary risk, non-human primates present a high potential for zoonotic pathogen exchange with humans by their close phylogenetic relationship (6), especially when they expand their range in anthropogenic landscape and increase in abundance (7). The risk of infectious disease outbreaks increases in human-macaque interface zones with intensified inter-species contacts, with implications for both public health and primate conservation (8). The recent COVID-19 pandemic events have recalled for a pressing need to monitor these populations at the interface with humans (9). Citation: Deleuze S, Brotcorne F, Polet R, Soma G, Rigaux G, Giraud G, Cloutier F, Poncin P, Wandia N and Huynen M-C (2021) Tubectomy of Pregnant and Non-pregnant Female Balinese Macaques (Macaca Fascicularis) With Post-operative Monitoring. Front. Vet. Sci. 8:688656. doi: 10.3389/fvets.2021.688656 September 2021 | Volume 8 | Article 688656 Frontiers in Veterinary Science | www.frontiersin.org Endoscopic Tubectomy in Long-Tailed Macaques Deleuze et al. oviducts were definitely disrupted and no longer patent. Moreover, no new pregnancies in sterilized females were recorded during the 3-year observation period. The survival rate of the treated females 6 months after sterilization was high (96.3%) with no major post-operative complications clinically recorded. Among females that were pregnant during surgery, 81.1% were confirmed to experience term delivery. This study demonstrates the safety and efficiency of endoscopic tubectomy, even for pregnant females, as a mean of wild macaques’ population control. Keywords: bipolar thermocautery, laparoscopy, monkey, population control, salpingectomy, sterilization, wildlife Keywords: bipolar thermocautery, laparoscopy, monkey, population control, salpingectomy, sterilization, wildlife INTRODUCTION Managing these escalating conflicts has to consider altogether macaque population sustainability, public health implications, as well as cultural and economic concerns of neighboring local communities (10). The first objective of this study is to document the modification of the endoscopic tubectomy procedure used in the long-tailed macaque, to assess its applicability with gravid females, and to provide a clinical evaluation of the method and its potential associated complications. The second objective is to assess the suitability of this sterilization technique in a free-ranging setting via a long-term post-op monitoring of the contracepted females after release as regards to survival and births. In several places in Asia, measures have been carried on to limit the expansion of monkey overpopulation associated with human-macaque conflicts (3). Contraception represents an ethical alternative to culling and translocation and is increasingly used in free-ranging populations. Over the years, various approaches have been used including male castration (11), progestin-based contraceptive treatment administered to females (12), programs combining immune-contraception on females and neutering chemical vasectomy on males (13), or endoscopic tubectomy as a permanent sterilization method for females. The latter technique has since been used either alone (14) or combined with vasectomies (15–17) on a large-scale on rhesus macaques (Macaca mulatta) in different sites in India and Hong Kong. Frontiers in Veterinary Science | www.frontiersin.org Keywords: bipolar thermocautery, laparoscopy, monkey, population control, salpingectomy, sterilization, wildlife Study Population and Local Issue Study Population and Local Issue Despite the limited size of available habitat (∼21 ha in 2018), the population of macaques in the Ubud Monkey Forest dramatically grew over 30 years, with a 10% mean annual growth rate (24). In 2017, the population counted 754 individuals split into seven social groups (C, X, S, N, H, T, L) and kept increasing up to 1,059 individuals in 2019, reaching the highest population density (50 individuals/ha) reported for that species in the semi-wild (25). Reasons for this constant growth and high density include the combined effects of forest loss and limitation to emigration imposed by the recent development of human barriers (26), the absence of natural predators, the cultural protection the macaques granted in the temple forest site, and the regular large-scale food provisioning of the monkeys which otherwise would not find enough resources in the forest to sustain themselves (27). Therefore, the site is now overcrowded and this situation imposes an array of detrimental consequences and costs to the macaques, including growing social tensions, increased aggressions and fading social group cohesiveness (28). Concurrently, the stakeholders of the sanctuary report increasing complaints from the neighborhood about macaque nuisances as well as aggressive interactions between macaques and visitors of the site. The capture protocol complies with the ethical recommendations of Jolly et al. (35) for capturing free-ranging habituated primates. The capture-release process was conducted by the trained local staffof the Ubud Monkey Forest. Four giant trapping cages (5 mL × 2.4 mW × 2 mH) located within the groups’ home range were baited daily with food since 2016 to habituate the monkeys to use the cage (Supplementary Figure 1). The cages were equipped with a trapdoor that had to be manually maneuvered by an operator at distance. On capture days, once an acceptable number of reproductive females had entered the cage, the trap door was closed and another operator slid a movable partition on the back side of the cage to lead the monkeys into smaller squeeze cages for anesthesia (Supplementary Figure 2). Darting using blowpipe was occasionally used to increase numbers of females and reach a minimum of 8–10 surgeries per day. The first dose administrated was based on the visual assessment of the age class. Based on previously collected data, estimated bodyweights of adult and subadult females were 5.5 and 3.5 kg, respectively. Study Site h h This research was conducted in the Ubud Monkey Forest (Mandala Wisata Wenara Wana), a forest sanctuary located in Ubud in south-central Bali (8◦31′S-155◦15′E), Indonesia. It is a highly frequented tourist site, visited by more than one million of tourists per year, generating a plethora of commercial activities and economic incomes (23). The site consists of a Hindu temple complex within a 21 ha of secondary mixed forest enclosed in an urban landscape (Figure 1). This sanctuary shelters both a long-term residential and free-ranging population of long-tailed macaques. The village committee has managed this site since 1980 and provisions the macaques on a daily basis with sweet potatoes and various fruits and vegetables. Although wildlife fertility control has gained influence over the past decades (18), experimental data are critically lacking in this field when it comes to free-ranging primates. This situation September 2021 | Volume 8 | Article 688656 Frontiers in Veterinary Science | www.frontiersin.org 2 Endoscopic Tubectomy in Long-Tailed Macaques Deleuze et al. females were sterilized. A demographical monitoring and further projections are in progress to check the model suitability and calibrate the needed adjustment campaigns (31). FIGURE 1 | The study site and location of the four trapping cages and the surgery room (modified from www.monkeyforestubud.com). Frontiers in Veterinary Science | www.frontiersin.org Study Animals Over successive sterilization campaigns between 2017 and 2019, 140 reproductive females underwent an intervention for tubal cauterization. Only reproductive females (identified by the presence of nipples development and sexual skin swelling) were selected for surgery. These reproductive females were either full adults or subadults, identified on a combination of morphological characteristics including dental eruption patterns (32) and development stage of sexual organs (33, 34). Subadults (2.5– 3.5 years) are nulliparous females characterized by buttonlike nipples and 24–28 permanent teeth. Adults are primiparous or multiparous females with elongated nipples and 28–32 permanent teeth. Out of 140 females, 129 (92.1%) were adult and 11 (7.9%) were subadults. Characteristics of the treated animals are documented in Supplementary Table 1. FIGURE 1 | The study site and location of the four trapping cages and the surgery room (modified from www.monkeyforestubud.com). Study Population and Local Issue Animals were induced using intramuscular injection of 5 mg/kg of ketamine (Ketamine 10% R⃝, Kepro, Holland) and 1.33 mg/kg of xylazine (Xyla R⃝, Interchemie, Holland). Once induced all animals were weighed to allow adjusted dosage of all drugs administered thereafter. For animals that underwent surgery, anesthesia was then maintained with intermittent intravenous boluses of 1% propofol (Recofol R⃝, 1 mg/kg, Primex, Switzerland). Vital parameters, respiratory and heart rate, pulse oximetry (with the probe placed either on the cheek, tongue, or tip of the index finger) and rectal temperature were monitored all along the procedure. Given that the capture method used a food-baiting strategy, the animals were not fasted before their anesthesia. After anesthesia, monkeys were equipped with a pediatric bracelet marked with an individual code for ID tracking and were transported to an air-conditioned operation building located inside the sanctuary (Figure 1). In the aforementioned conditions, the question of population control has become crucial and a sterilization program has been initiated in 2017 as a means of controlling the density of monkeys in an ethical way. This first required the careful estimation of the magnitude of the sterilization needed. A stochastic matrix population model (29) based on a 10-year census dataset of the population was implemented to estimate the sterilization proportion of reproductive females required to achieve population stability (i.e., a null population growth). Simulations suggested that 47% of the reproductive females in 2017 (i.e., 135 individuals) had to be sterilized in order to reach population stability (30). By 2019, 41.9% of the reproductive September 2021 | Volume 8 | Article 688656 3 Endoscopic Tubectomy in Long-Tailed Macaques Deleuze et al. Marking and Release A permanent double marking for sterilized females was used with a microchip transponder inserted in the left antebrachium and a tattoo on their chest to permit remote identification (Supplementary Figure 4). After marking, the identifying pediatric bracelet was removed, a reversal, atipamezole hydrochloride (Antimedin R⃝, 0.02 mg/kg, Dong Bang Co., Korea) was administrated intramuscularly (using a volume consistent with that of the previously administered xylazine) and animals were positioned in lateral recumbency in individual holding cages for recovery (Supplementary Figure 5). Animals were monitored for signs of pain during the recovery period. When they showed clear signs of full recovery from anesthesia (i.e., strong grip and climbing reflex, visual fixation, and following reflexes), individuals were transported in their holding cage to the capture site and released in their group. Post-operative Monitoring p g This study has been conducted over 3 years (2017–2020) during four successive sterilization campaigns (16 days in total: 3 days in July 2017 and July 2018, 5 days in February 2019 and August 2019). In parallel with the campaigns, a long-term monitoring of the contracepted females was conducted. Those females were monitored over 27 months split in several field observation periods between 2017 and 2020 (i.e., July 2017– September 2018, February 2019–September 2019, and December 2019–Mars 2020). Within 6 months after their surgery, the general body condition of contracepted females in the field, as well as any injury on sutures or elsewhere, disabilities, or cues indicating major unhealthy conditions were assessed. For the females pregnant at the time of the surgery, information about the pregnancy process and births was collected. Delivery failure in contracepted pregnant females was defined as abortion and perinatal mortality (36), based on clinical observation of (non- )occurrence of births after the intervention. Finally, survival of the sterilized females was defined as individuals still present at least 6 months after surgery. Clinical Exam and Preparation cavity were carefully evaluated for any sign of bleeding or complication. The telescope and cannulae were removed and the abdomen was deflated by gentle massage. The two abdominal incisions were closed using 3–0 polyglecaprone (Monocryl R⃝, Ethicon, USA) in simple interrupted pattern. An intradermal suture with the same material was used for the skin incisions (Supplementary Figure 3). A general clinical exam was conducted to care for potential injuries, remove food material from the oral cavity and cheek pouches to reduce the risk of aspiration pneumonia, and assess the position and size of the uterus that enlarged cranially with pregnancy for subjective staging via abdominal palpation (early stage defined as <1.5 months, mid-term as 1.5–3 months, and advanced stage as >3 months). Females selected for surgery were catheterized in the cephalic vein for fluidotherapy with saline perfusion (NaCl 0.9%) and received a dose of antibiotic (Betamox LA R⃝, amoxicillin, 15 mg/kg, I.M., Norbrook, United Kingdom), non-steroidal anti-inflammatory (Tolfedine R⃝CS, 4 mg/kg, subcutaneous, Vétoquinol, France), and eye lubricant (Visine R⃝, Johnson & Johnson) to protect the cornea from desiccation. The abdomen was shaved from the xiphoid appendix to the pubis and as far as the lower line of the flank. Patients were placed in dorsal recumbency with the pelvis placed on a cushion to slightly elevate it and allow movement of the abdominal organs cranially in the abdominal cavity (Trendelenburg position). The surgical site was scrubbed with antiseptic soap and 70% alcohol and draped. Anesthesia charts including time of drug administration, duration of surgery from incision to suture, and from induction to release, as well as clinical remarks were recorded. Surgery The tubectomy procedure routinely consisted in a laparoscopic two-5 mm-ports approach. Briefly, a 15 mm skin incision was performed with a scalpel blade ∼1–2 cm cranially to the umbilicus to allow placement of a Veress needle (150 mm, CovidienTM, USA), taking care to avoid the underlying organs (e.g., the gravid uterus) or any blood vessel. The abdomen was then insufflated with CO2 to a pressure of 10–12 mmHg (Insufflator, WISAP R⃝, Germany) until a satisfactory abdominal distension was achieved. The Veress needle was then removed and a 5 mm trocar was inserted through the same skin incision. The rigid telescope (Hopkins R⃝straight 0◦, 5 mm, 29 cm, Karl Storz, Germany), connected to an endoscopic camera (Sopro- Comeg 368 HD, Germany) and light source (LED Nova 150, Karl Storz, Germany) was introduced in the abdomen via the cannula. The abdominal cavity was evaluated for topography of the reproductive organs, gravidity and presence of adhesions. The site for the insertion of a second 5 mm trocar was localized in the left lower quadrant, just cranially to the oviduct. After a skin incision of ∼5 mm, the second trocar was inserted under visual endoscopic control, guided by transillumination thus easily avoiding large blood vessels. The ampulla of the left oviduct was easily recognized as flexuous, and paler than the rest of the organ. Using a bipolar Kelly grasping forceps (RoBi R⃝38151, Karl Storz, Germany) connected to a thermo-cauterization unit (Autocon R⃝II 80, Karl Storz, Germany), it was grasped and coagulated until blanching of the entire oviductal segment was observed. A second cautery was performed proximally to the first. The ampulla of the right side was accessed through the same port and cauterized in a similar way (Supplementary Video 1), except for pregnant females nearer to term for which a third access in the right flank was performed to reach the right oviduct (Supplementary Video 2). The sites of thermo- cauterization were checked for adequate cauterization of the sections of the oviduct as they were not resected and were left in place. The reproductive organs and the rest of the abdominal Complications The major complication encountered during the surgery was caused by the extent of fibrous adhesions, that significantly interfered with the exploration of the abdominal cavity. These adhesions were recorded in 24/140 (17.1%) of the treated females (Supplementary Table 1) and were mostly located in the caudal abdomen. A biopsy, obtained from a lesion appearing as a small mass located at the surface of the uterus of the patient (X=) with such adhesions and for which the laparoscopy was converted to a laparotomy, confirmed a diagnosis of endometriosis. Five cases of herniation were recorded, usually associated with dense adhesions. For exhaustive data, see Supplementary Table 1. Intraoperative Evaluation Success of Tubectomy and Pregnancy Stage-Related Feasibility proportion (%) of age class (adult, subadult), pregnancy stage at the time of the intervention, pregnancy outcome, female survival at least 6 months following surgery, and intraoperative complications. Parameter Category Occurrence % Age Adult 129 92.1 Subadult 11 7.9 Pregnancy stage Not gravid 100 71.4 Early 11 7.9 Mid-term 10 7.1 Advanced 19 13.6 Pregnancy outcome Delivery 30 83.3 Abortion 6 16.7 Unknown 4 10 Survival (6 month) Yes 131 96.3 No 5 3.7 Intraoperative complication Tubectomy failure 4 2.9 Adhesions 24 17.1 For exhaustive data, see Supplementary Table 1. y Among the 140 treated females, 136 (97.1%) were successfully tubectomized. Forty of these females (28.6%) were at various stages of pregnancy (early stage: 7.9%, mid-term: 7.1%, advanced stage: 13.6%). Among these 40 pregnant females, 3 (7.5%) could not be successfully sterilized due to various intraoperative complications. All three were at an advanced stage of pregnancy at the time of surgery. In two cases (C6, H8), the size of the uterus was such that the oviducts could not be adequately visualized and accessed, and the procedure was interrupted to avoid prolonged manipulations and anesthesia. The third case (S14) was a female at an advanced stage of gestation that died during the surgery for undetermined cause. In a fourth case, that was tattooed with a mark “X=,” a non-pregnant female presented abundant adhesions in the uterine zone, making it impossible to access the ovaries despite a conversion to an open laparotomy approach. The mean surgery duration was 14 ± 3 min for non-pregnant females and 22 ± 18 min for pregnant females. The overall whole capture-release process lasted in average 3 h 22 min, and ranged from 1 h 11 min to 5 h 52 min. Post-operative Clinical Evaluation The potential post-operative complications by direct observations of the released females within days after the surgery were monitored (Supplementary Figure 4). No issues apart from dehiscence of the skin suture, due to intensive scratching by the monkey, seen in 8 cases (5.7%) without major infection cues or systemic impact, no major complication and no pain-associated behavior associated with surgery in non-human primates (37, 38) have been observed after release. To monitor the efficacy of the laparoscopic tubectomy approach, a laparoscopic post-operative assessment was made on two patients (L3, T4) randomly re-captured 1 or 2 years after their intervention. Capture, anesthesia, analgesia and laparoscopy procedures were the same as previously described. Both laparoscopic post-operative evaluations confirmed that the oviducts were definitely disrupted and no longer patent. The ovaries looked normal and were clearly no longer connected to the uterus as part of the oviduct was missing. There was no sign of local reaction or adhesions on the surgical site (Supplementary Video 3). Long-Term Field Monitoring The 3-year monitoring has revealed a 100% reliability rate of the sterilization method since no new birth has been recorded so far in females which underwent surgery, and were not pregnant at that time. 96.3% of the sterilized females survived at least 6 months after the surgery and 91.9% until 3 years after surgery. This survival rate is similar to the annual survival rate of all reproductive females of this population (92.3%) calculated via a matrix population model based on 10-year life table dataset (30). Most bodies of deceased sterilized females could not be found but their behavior and condition before their disappearance suggested causes unrelated to surgery (e.g., falls from trees, human-related hazards such as electric shock in poles). Finally, the pregnancy outcome for 36 of the 40 gestating females that underwent surgery was confirmed: delivery failure (including abortion and perinatal mortality) was observed in 6 (16.7%) of these females. Relevant results are summarized in Table 1. Intraoperative Evaluation Success of Tubectomy and Pregnancy Stage-Related TABLE 1 | Data summary of the treated females (N = 140) with frequency and proportion (%) of age class (adult, subadult), pregnancy stage at the time of the intervention, pregnancy outcome, female survival at least 6 months following surgery, and intraoperative complications. Frontiers in Veterinary Science | www.frontiersin.org RESULTS Over the four sterilization campaigns, 140 long-tailed female macaques (129 adult and 11 subadult females) have been treated and monitored (Supplementary Table 1). The average weight of these females was 5.2 kg (N = 140). Adult females weighed more (5.3 kg, N = 129) than subadults (4.0 kg, N = 11). Over the four sterilization campaigns, 140 long-tailed female macaques (129 adult and 11 subadult females) have been treated and monitored (Supplementary Table 1). The average weight of these females was 5.2 kg (N = 140). Adult females weighed more (5.3 kg, N = 129) than subadults (4.0 kg, N = 11). September 2021 | Volume 8 | Article 688656 Frontiers in Veterinary Science | www.frontiersin.org 4 Endoscopic Tubectomy in Long-Tailed Macaques Deleuze et al. DISCUSSION Contraception is increasingly used as a tool for population control of primates living in anthropogenic habitats in Asia. Despite an increasing interest in using this method, their implications and suitability remain poorly understood (39). Very few systematic studies have indeed evaluated the outputs of such measures, which constitutes a major research gap calling for robust assessments (40). This study aimed to September 2021 | Volume 8 | Article 688656 Frontiers in Veterinary Science | www.frontiersin.org 5 Endoscopic Tubectomy in Long-Tailed Macaques Deleuze et al. provide an evaluation of the laparoscopic tubectomy by bipolar thermocautery as a mean of fertility control in free-ranging long- tailed macaques. It also aimed at adjusting the procedure for pregnant patients and provide a clinical evaluation of the method and its potential associated complications. supports the unlikelihood of recanalization and reversal with this technique in long-tailed macaques as supported by the 100% efficacy of the method (absence of recorded pregnancy or birth) for all the patients that survived over the 3-year- period monitoring we report. These results confirm that a bipolar thermocautery alone, without further resection of the fallopian tubes is a safe and simpler approach to achieve an effective hemostasis and permanent disruption of the oviducts. Previous studies have shown various undesirable side effects associated with castration, ovariectomy or contraceptive agents in primates (41–45). Conversely, while impeding fecundation, vasectomy and tubectomy keep intact the steroid hormonal functions underpinning normal sexual activity (20, 46). Preservation of the gonadal hormones represents the best option when sterilizing wild female macaques as it allows them to keep normal sexual and social behaviors in their group. It has also been shown to have beneficial effects on bone metabolism both in women (47) and monkeys (48). Laparoscopic tubectomy is a minimally-invasive intervention highly recommended as a substitute for conventional ovariectomy for permanent contraception of macaques as it does not induce observable hormonal alterations (49). It is associated with a lower incidence of intra- and post-operative complications (17) and significantly reduces the surgical and post-operative recovery times in primates (50). This fact is of paramount importance with social primates since prolonged separation of individuals is likely to jeopardize their reintroduction to social group. Common adverse consequences of prolonged isolation are fighting, increased aggressions toward the reintroduced animals and socio-behavioral changes including loss of the social status after reintroduction (51). The maximum holding period recommended is 24 h (52). DISCUSSION In this study, all animals were reintroduced to their group within 6 h after capture, as reported on large cohorts (14), and the release process monitoring showed no substantial fights during release. Previous reports of laparoscopic tubectomy in macaques accessed each ovary from its own side (17, 20, 50) resulting in a three-port technique. More recently, both ovaries were accessed from the same side but maintained a three-port approach (14). Although, the original plan had been to access each ovary from its own side, it became immediately obvious that the first port in the left flank could easily be used to access the right oviduct as well with the bipolar forceps, at least for non-pregnant and early to mid-term pregnant females. The two-port approach was therefore preferred as easier to perform and avoiding the opening and closing of the third access, thus reducing the duration of the procedure. p Capture is a keypoint of free-ranging animal sterilization programs. Although less stressful for the animals, and giving access to higher number of them (35), trapping in cages does not allow selection of the captured animals. Individuals that may not be contracepted have to be released with reduced chances to be trapped again in a short interval. These include females with palpable pregnancies which were not usually considered suitable candidates for surgery (14). In order to avoid having to release animals without neutering them, the technique was adjusted to allow surgery on pregnant females. The placement of the first trocar was adjusted based on gestational age and the relative position of the gravid uterus evaluated by palpation as commonly described in humans (55). While the two-port approach proved to grant easy access to both oviducts in early to mid-term pregnancies, in females nearer to their term the size of the gravid uterus precluded the adequate visualization of the contralateral oviduct. For those cases, following cautery of the left oviduct, patients were slightly tilted to the left and a third access in the right flank is performed as described above. This position is routinely recommended in human laparoscopy (55, 56) and it has been shown that it is unnecessary to place the patient in a strict lateral decubitus position (57). Not only does it allow better visualization of the right oviduct but it also reduces aortocaval compression. Frontiers in Veterinary Science | www.frontiersin.org September 2021 | Volume 8 | Article 688656 DISCUSSION In humans, one of the major concerns with laparoscopy on pregnant patients was originally the effect of the CO2 pneumoperitoneum on the fetal physiology. However, it has been shown that no substantial adverse effects are observed when the duration of the procedure is limited (under 60 min) and the intraabdominal pressure is kept under 10–12 mmHg (58, 59). While the insufflator is set at 12 mmHg as default in this approach for non-pregnant monkeys, the intraabdominal pressure was limited as possible to 10 mmHg in advanced pregnancies as long as a sufficient visualization of the oviducts can be achieved. Accidental puncture of the gravid uterus with the Veress needle is considered the most serious complication in pregnant women (58, 60). This complication has not been observed in any of the 40 pregnant females that underwent surgery during the campaigns. The technique we describe here is adapted from the endoscopic salpingectomy in wild macaques first demonstrated by P. Martelli in a DVD entitled “Endoscopic Tubectomy in Macaques” for a German company selling endoscopic equipment (Karl Storz-Endoskope) in 2009. Endoscopic salpingectomy has since widely been used for neutering campaigns in Formosan (20), rhesus (14, 17) macaques and quite recently, on a chimpanzee (Pan troglodytes) (53). Here the choice of cautery, a two-port-approach, the inclusion of pregnant females and the complications as well as the modifications to the original technique are discussed. While all previous reports combined coagulation of the oviducts followed by resection using endoscopic scissors of a section of each tube, that was removed from the abdominal cavity through the cannula (17, 20, 50, 53), this study opted for thermocautery only. The oviduct is grasped and cauterized twice but no section is resected and removed. The severed tissues are left to aseptically necrotize in situ, which was considered safe and sufficient to ensure sterilization of the animal as it has been shown to be as safe and effective as partial salpingectomy in women (54). Although that information is limited to two patients that underwent a second laparoscopy for control purposes, the anatomical disruption of the portion of the oviduct that had been severed by thermocautery and the consequent loss of connection between the ovary and the uterus was confirmed visually. This September 2021 | Volume 8 | Article 688656 6 Endoscopic Tubectomy in Long-Tailed Macaques Deleuze et al. DISCUSSION Laparoscopic tubal cautery during pregnancy was also safe and feasible. However, the procedure feasibility was limited by the gestational stage: females near term (≥4 months) could not easily be treated because the size of the uterus reduced the access to oviducts. Provided that this limitation is considered, this new insight is of great interest for sterilization of animals living in free-ranging settings where captures are very challenging. That is particularly true for non-seasonal breeding species, where no optimal timing of capture can be identified because of year-round distribution of births. Captures can therefore be optimized if most of the gestating females can be included and undergo tubectomy. Post-operatively, 8 cases of partial wound dehiscence were observed which did not impact the general condition of the animals and was self-limited. These delayed wound healing came unsurprisingly as grooming behavior, wound, or stitching material picking are common in macaques (66). However, this complication has not been mentioned in other studies (17) and more particularly in a large scale study (14) where the skin was closed using surgical glue rather than absorbable suturing material, which should be considered to further improve our technique. No other complications were recorded. Systematic post-operative observations are not feasible with animals living in a semi-wild habitat, and although no pain scoring observations were performed, females that were seen in the days following surgery showed no apparent signs of pain and displayed normal behavior of feeding and grooming. These observations support the fact that, laparoscopic tubectomy being a minor surgical procedure, one single administration of NSAIDs for analgesia was sufficient to allow swift release of the animals back to their social groups. Although this duration may need to be reduced for smaller animals (<2 kg) because of their propensity to hypoglycemia, it is usually considered advisable to withhold food for 12–16 h and water for 2 h before induction of anesthesia of non-human primates (67). This reduces the risk of gastrointestinal bloat and aspiration pneumonia as macaques may vomit on induction of anesthesia. Fasting the animals—or even administration of metoclopramide 30–60 min before induction which is also sometimes recommended (67)—was unfortunately not possible under the conditions of this study. However, like others (14), no such complications were observed during our sterilization campaigns and thorough emptying of the cheek pouches was considered sufficient to prevent aspiration pneumonia. DISCUSSION involved fibrous adhesions, especially between the omentum and the uterus, liver, spleen, and urinary bladder (64). The same paper also describes cases where one or both ovaries could not be identified and were suspected to be incorporated or effaced by the fibrous adhesions. Although, it would be hazardous to conclude that all cases with intra-abdominal adhesions would necessarily be due to endometriosis, this is exactly what was observed in the patient [“(X=)” where endometriosis was confirmed. In non-human primates, decidualization of endometriotic tissue most often occurs under the influence of exogenous progestins or during pregnancy (65). In this study, none of the 24 animals with such lesions received exogenous progestins and 16 were not pregnant, supporting the hypothesis of that endometriotic tissue may decidualize under the influence of elevated endogenous progesterone (64). It may be suspected that the size of the patients, allowing a good transabdominal palpation and localization of the uterus, permitted readjustment of the site of insertion more cranially to avoid the uterine fundus. It is worth noticing that while all the previous reports based on the initial description of the technique place the first trocar caudal to the umbilicus (14, 17, 20, 50, 53), the first access is located just cranially to the umbilicus in this study. This may contribute to an easier access to the contralateral oviduct even in pregnant patients and limit conflicts between instruments inside the abdominal cavity. y As laparoscopic oviduct cautery does not induce observable hormonal alterations (49), it is therefore not expected to interfere with pregnancy maintenance. By comparison, delivery failure was observed in 16.7% females. This proportion of abortion in gravid sterilized females was similar to or even lower than the one reported for long-tailed macaques in captivity (23.7%) (61). Out of a very large cohort, where pregnant females were normally excluded, 9 cases of tubectomy on females suspected of being pregnant based on the presence of mildly rounded congested and enlarged uteri, among which 3 completed a gestation successfully have been reported. However, as these findings were incidental and pregnancy had not been confirmed, the authors preferred not to draw conclusions (14). These results establish that the laparoscopic tubectomy did not interfere with gestation and is therefore a safe and reliable sterilization technique even for advanced pregnant female long-tailed macaques. Laparoscopic procedures for various surgical interventions during pregnancy have been considered effective and safe in women and macaques (62). Frontiers in Veterinary Science | www.frontiersin.org DISCUSSION Complications of endoscopic salpingectomy, including difficult entry into the abdominal cavity using the Veress needle, intraoperative hemorrhage and inadvertent cauterization of the uterus have been reported in Formosan macaques (20). As in other reports (14), very few intraoperative complications were observed during this study’s campaigns. The main intraoperative limitation was due to the presence of adhesions, observed in 24/140 (17%) of the operated females out of which 16 were not pregnant. In one case, these adhesions were such that access to the ovaries could not be achieved even after conversion to a standard laparotomy. A biopsy obtained from this patient confirmed the presumptive diagnosis of endometriosis associated with these cases of adhesions mainly located in the caudal abdomen. The incidence of spontaneous endometriosis in a breeding colony of Macaca fascicularis has been reported to be 28.7% (63) in aging females. Lesions of endometriosis have recently been characterized in long-tailed macaques and In this study, the mean surgery duration of the laparoscopic tubectomy was 14 ± 3 min for non-pregnant females and 22 ± 18 min for pregnant females. This is longer than the 3–10 min reported in other papers (14, 17) that included non-pregnant females only. Possible explanations for this difference may be found in the degree of experience of the operator but also in at least two technical aspects. First, females with pre-existing severe adhesions that sometimes seriously interfered with the access to the oviducts were included and thus prolonged the duration of the procedure. Second, 5 mm trocars instead of 3 mm September 2021 | Volume 8 | Article 688656 Frontiers in Veterinary Science | www.frontiersin.org 7 Endoscopic Tubectomy in Long-Tailed Macaques Deleuze et al. of NSAID for analgesia and a holding period as short as possible). This research has been approved by the Animal Ethics Commission of Udayana University (No. 282/KE-PH/I/2017) and has been granted with permission of the Provincial Office of Conservation in Bali (BKSDA, No. KT49/BKSDA BL-1/KK/2017; Capture license: #29/PPSP/XII/2017). Moreover, this research was conducted under research permission from the Indonesian Ministry of Research and Technology (No. 186/SIP/FRP/E5/Dit.KI/VII/2017, No. 46/SIP/FRP/E5/Dit.KI/II/ 2018, No. 10/E5/E5.4/ SIP.EXT/2019, No. 83/E5/E5.4/SIP.EXT/2019). Universal precautions (gloves, masks, and overshoes/caps specifically for surgery) against hazards and zoonotic pathogen transmission were carefully taken during the whole process from capture, clinical exam, surgery and postoperative procedures. ones were used, which led to suture the abdominal wall rather than leaving it unsutured. ACKNOWLEDGMENTS We thank the Indonesian Ministry of Research and Technology, the Ubud Monkey Forest Management Committee, Mr. Nyoman Buana and Mr. Wayan Buda for support and permission to conduct this research in Indonesia. We are very grateful to the Ubud Monkey Forest staff, the field assistants (from ULiege, UCL, and UNUD) and the senior colleagues from Udayana University for their assistance with the sterilization campaigns and data collection. We also thank Dr. Nicolas Schtickzelle for his help in matrix population modelling, Dr. Paolo Martelli and Mrs. Femke den Haas for fruitful advices and logistic support regarding the sterilization campaigns. ETHICS STATEMENT The whole protocol of this study followed the ethical recommendations for animal welfare (36). All procedures have been chosen in order to cause the least stress, pain and discomfort to the monkeys (e.g., collective trapping allowing to achieve a maximal number of captures while causing the least stress and discomfort to the monkeys, limiting handling when animals are not sedated, minimally invasive endoscopic tubectomy procedure, administration DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. AUTHOR CONTRIBUTIONS FB, SD, and M-CH: conceived and designed the study. SD, RP, and GS: undertook the laparoscopic tubectomy and collected clinical data. SD, GR, M-CH, FB, NW, GS, GG, and FC: veterinary care and logistics during the sterilization campaigns. GG, FB, and FC: data collection for field monitoring. FB: data analyses. NW and PP: facilitation to fieldwork. SD and FB: wrote the manuscript. M-CH: provided in-depth editorial advice. All authors reviewed and edited the manuscript and approved the submitted version. Our follow-up over a 3-year period on patients’ clinical evolution, pregnancy outcome, and later infertility demonstrates the safety and efficiency of the modifications brought to the procedure. Moreover, as controlling effects of fertility control on wild animal life is of paramount importance, a long-term monitoring of this population is still ongoing and further focuses on the potential impact of reproduction suppression on behavior and social dynamics (68). Effects of reproduction suppression on the health, welfare, social behavior, and ranging patterns of primates are often only superficially considered. This influence needs to be examined to further complete the data on the technique and its impacts beyond the population control aspects. FUNDING This study was carried out with the financial support of the Padangtegal village leadership committee, the Belgian National Fund for Scientific Research, the Erasmus + International Credit Mobility Programme, the University of Liège, and Wallonie-Bruxelles International. This study was carried out with the financial support of the Padangtegal village leadership committee, the Belgian National Fund for Scientific Research, the Erasmus + International Credit Mobility Programme, the University of Liège, and Wallonie-Bruxelles International. DISCUSSION Finally, to limit wound picking, the skin was apposed with an intradermal suture instead of surgical glue which is obviously faster. Laparoscopic vessel sealant tools, not yet available in 3 mm, were shown not necessary and that standard bipolar forceps, that are available both in 3- and 5- millimeter size, were sufficient to efficiently sterilize the patients. Smaller size instruments should therefore probably be preferred for surgeries on animals the size of a macaque. As a conclusion, laparoscopic oviduct cautery with standard bipolar forceps can be adjusted to safely sterilize barren and pregnant long-tailed macaques even at late stages of pregnancy. Use of a single lateral port is feasible in non-gestating and early to mid-term pregnant females, while females nearer to their term are best operated using a three-port entry. The idea that including pregnant females and patients with some degree of adhesions will optimize the trapping efforts and the overall success of population control programs. The absence of significant complications with the technique, which allows release of free-ranging animals very shortly after procedure and avoids complications related to prolonged isolation from the group. 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Laparoscopy in the second and third trimesters of pregnancy for abdominal surgical emergencies. J Gynecol Obstet Hum Reprod. (2017) 46:417–22. doi: 10.1016/j.jogoh.2017.03.008 57. Moreno-Sanz C, Pascual-Pedreño A, Picazo-Yeste JS, Seoane-Gonzalez JB. Laparoscopic appendectomy during pregnancy: between personal September 2021 | Volume 8 | Article 688656 Frontiers in Veterinary Science | www.frontiersin.org 10 10
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Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30
Frontiers in bioengineering and biotechnology
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ORIGINAL RESEARCH published: 22 October 2018 doi: 10.3389/fbioe.2018.00151 Gustavo Pagotto Borin 1,2, Marcelo Falsarella Carazzolle 3, Renato Augusto Corrêa dos Santos 4, Diego Mauricio Riaño-Pachón 5 and Juliana Velasco de Castro Oliveira 1,2* 1 Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil, 2 Programa de Pós-Graduação em Genética e Biologia Molecular, Instituto de Biologia, Universidade de Campinas (UNICAMP), Campinas, Brazil, 3 Laboratório de Genômica e Expressão (LGE), Departamento de Genética, Evolução, Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil, 4 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, Brazil, 5 Centro de Energia Nuclear na Agricultura, Universidade de São Paulo (USP), Piracicaba, Brazil Edited by: Roberto Silva, Universidade de São Paulo, Brazil The biomass-degrading fungus Trichoderma reesei has been considered a model for cellulose degradation, and it is the primary source of the industrial enzymatic cocktails used in second-generation (2G) ethanol production. However, although various studies and advances have been conducted to understand the cellulolytic system and the transcriptional regulation of T. reesei, the whole set of genes related to lignocellulose degradation has not been completely elucidated. In this study, we inferred a weighted gene co-expression network analysis based on the transcriptome dataset of the T. reesei RUT-C30 strain aiming to identify new target genes involved in sugarcane bagasse breakdown. In total, ∼70% of all the differentially expressed genes were found in 28 highly connected gene modules. Several cellulases, sugar transporters, and hypothetical proteins coding genes upregulated in bagasse were grouped into the same modules. Among them, a single module contained the most representative core of cellulolytic enzymes (cellobiohydrolase, endoglucanase, β-glucosidase, and lytic polysaccharide monooxygenase). In addition, functional analysis using Gene Ontology (GO) revealed various classes of hydrolytic activity, cellulase activity, carbohydrate binding and cation:sugar symporter activity enriched in these modules. Several modules also showed GO enrichment for transcription factor activity, indicating the presence of transcriptional regulators along with the genes involved in cellulose breakdown and sugar transport as well as other genes encoding proteins with unknown functions. Highly connected genes (hubs) were also identified within each module, such as predicted transcription factors and genes encoding hypothetical proteins. In addition, various hubs contained at least one DNA binding site for the master activator Xyr1 according to our in silico analysis. The prediction of Xyr1 binding sites and the co-expression with genes encoding carbohydrate active enzymes and sugar transporters suggest a putative role Reviewed by: Kentaro Inokuma, Kobe University, Japan Andrei Steindorff, Joint Genome Institute (JGI), United States *Correspondence: Juliana Velasco de Castro Oliveira juliana.velasco@ctbe.cnpem.br Specialty section: This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Bioengineering and Biotechnology Specialty section: This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Bioengineering and Biotechnology Received: 10 July 2018 Accepted: 03 October 2018 Published: 22 October 2018 Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30 Gustavo Pagotto Borin 1,2, Marcelo Falsarella Carazzolle 3, Renato Augusto Corrêa dos Santos 4, Diego Mauricio Riaño-Pachón 5 and Juliana Velasco de Castro Oliveira 1,2* INTRODUCTION These genes represent interesting new targets, because several were found either activated or repressed in culture media having complex carbon sources (Häkkinen et al., 2012; Borin et al., 2017; Daly et al., 2017; Horta et al., 2018). In this context, the gene co-expression network analysis has become a valuable bioinformatic toolkit for data integration and the identification of new candidate genes related to a biological process of interest (Gonzalez-Valbuena and Treviño, 2017). Only a few studies have inferred gene networks from the transcriptome of T. reesei. T. reesei RUT-C30 is a well-known industrial strain able to abundantly produce and secrete cellulases, and it has been used as a genetic background to develop other industrial strains. RUT-C30 was isolated following three rounds of mutagenesis and screening from the ancestral QM6a strain, and its hypercellulolytic phenotype is attributed in part to the truncation of cre1, the main player of carbon catabolic repression. Since then, RUT-C30 strain has been the target of numerous studies on the conversion of biomass into biofuels and other high-value products (Marx et al., 2013; Mello-De-Sousa et al., 2014; Druzhinina and Kubicek, 2017). 2G ethanol technology primarily consists of three major steps: pretreatment, enzymatic hydrolysis and sugar fermentation, being pretreatment and enzymatic hydrolysis the major limitations for the economic feasibility of 2G ethanol. In this context, a cost-effective process is required to make this biofuel more attractive and competitive (Bornscheuer et al., 2014; Gupta and Verma, 2015). Trichoderma reesei is the primary fungal source of the industrial cellulases present in enzymatic cocktails for lignocellulose degradation and the production of 2G ethanol. Due to its remarkable capacity to produce and secrete enzymes that are active on carbohydrates, T. reesei has been used as a microbial factory for the breakdown of lignocellulose and a host for heterologous expression (Schmoll et al., 2016). These enzymes and other accessory proteins are collectively named as Carbohydrate-Active enZymes (CAZymes) (Lombard et al., 2014) and their genes are under control of several transcriptional regulators, which are activated or repressed according to sugar (xyr1 and cre1, for example) and nitrogen (areA) availability, pH alteration (pacC), light (env1), and other factors (Stricker et al., 2006; Rassinger et al., 2018; Schmoll, 2018a). In this context, some of them have been target of studies of genetic manipulation involving deletion and overexpression of transcriptional regulators in order to enhance the cellulolytic phenotype of T. INTRODUCTION In the last few years, several advances have been achieved in different research fields and contributed to a better understanding of the physiology and key features behind the T. reesei hypercellulolytic capacity. Such advances include the development of new tools for genetic manipulation (Derntl et al., 2015; Liu et al., 2015), transcriptomic and proteomic studies using lignocellulosic residues (Dos Santos Castro et al., 2014; Borin et al., 2015, 2017; Daly et al., 2017; Ellilä et al., 2017; Cologna et al., 2018), the discovery of new transcription factors (TFs) and regulatory elements (Derntl et al., 2016, 2017; Stappler et al., 2017; Zheng et al., 2017b; Benocci et al., 2018), promoter characterization (Zheng et al., 2017a; Kiesenhofer et al., 2018) and structural studies of cellulases (Li et al., 2015; Bodenheimer and Meilleur, 2016; Eibinger et al., 2016; Ma et al., 2017; Borisova et al., 2018). Over the previous years, the development of new sustainable alternatives to fossil fuels has become critical to mitigate greenhouse gas emissions and to avoid the exhaustion of natural sources. Among the biofuels, second generation (2G) ethanol has emerged as one of the most promising substitutes for gasoline since it can be produced from lignocellulosic feedstocks, including agroindustrial residues and municipal waste (Lynd et al., 2017). The United States and Brazil are the world’s largest 1G ethanol producers and are responsible for 57 and 27% of its global production, respectively (Renewable Fuels Association, 2017). In Brazil, the 1G ethanol production system is well established and is based on sugarcane milling and sugar-rich juice fermentation with generation of bagasse and straw as byproducts. Currently bagasse is used to produce steam and energy in the sugar-ethanol biorefineries, and the straw is left on the soil to prevent erosion and enhance the organic carbon content (da Rosa, 2013; Pereira et al., 2015; Lisboa et al., 2017). These residues have already been used as feedstocks for 2G ethanol in Brazilian industrial plants (GranBio and Raízen), and they have a high potential to be explored more thoroughly in an integrated process with 1G ethanol technology (Junqueira et al., 2017; Lynd et al., 2017). However, despite all of the studies conducted and the knowledge acquired, the biotechnological potential of T. reesei has not been completely explored since various genes identified by different “omic” approaches have still not been characterized. Citation: Borin GP, Carazzolle MF, dos Santos RAC, Riaño-Pachón DM and Oliveira JVdC (2018) Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30. Front. Bioeng. Biotechnol. 6:151. doi: 10.3389/fbioe.2018.00151 October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Borin et al. Gene Co-expression Network of Trichoderma reesei of these hubs in bagasse cell wall deconstruction. Our results demonstrate a vast range of new promising targets that merit additional studies to improve the cellulolytic potential of T. reesei strains and to decrease the production costs of 2G ethanol. Keywords: Trichoderma reesei, sugarcane bagasse, 2G ethanol, enzymatic cocktail, gene co-expression network, Xyr1-binding site Fungal Strain and Culture Conditions Fungal Strain and Culture Conditions Gene co-expression network analysis was performed based on a RNA-Seq dataset from a previous study conducted by our research group (Borin et al., 2017). Briefly, T. reesei RUT- C30 spores were first cultivated in potato dextrose agar for 7–10 days at 29◦C, and harvested in sterile distilled water. The spore suspensions were inoculated to a final concentration of 1 × 106 spores per 30 mL of basic culture medium (BCM) (pH 5.5) composed of 0.05% yeast extract (w/v), 50 mL/L salt solution (6 g/L NaNO3, 1.5 g/L KH2PO4, 0.5 g/L KCl, and 0.5 g/L MgSO4), 200 µL/L trace elements (10 g/L ethylenediaminetetraacetic acid, 4.4 g/L ZnSO4·7H2O, 1.0 g/L MnCl2·4H2O, 0.32 g/L CoCl2·6H2O, 0.315 g/L CuSO4·5H2O, 0.22 g/L (NH4)6Mo7O24·4H2O), 1.47 g/L CaCl2·2H2O, and 1 g/L FeSO4·7H2O), with 1% fructose (w/v) as the carbon source at 29◦C, 200 rpm for 48 h. The pre-grown mycelia were transferred to fresh BCM (without yeast extract) with 0.5% bagasse (w/v) as the carbon source for 6, 12, and 24 h, and to 1% fructose (w/v) for 24 h. Fructose was used as control condition in the RNA- Seq experiment as it is an inert sugar, which neither induces nor suppresses overall expression of lignocellulolytic enzymes (Amore et al., 2013). The cultures were grown under continuous light exposure, as it influences positively cellulase gene expression (Schmoll, 2018b). Mycelia were harvested by filtration, washed with sterile water and immediately ground into powder in liquid nitrogen. Frozen material was then used for the RNA extraction. The MCODE (v1.4.2) plugin (Bader and Hogue, 2003) of Cytoscape was used to identify subclusters of genes densely connected within each module with the parameters set to default (degree cutoff= 2, node score cutoff= 0.2, K-core = 2, maximum depth = 100, haircut method). Genes with high connectivity within the modules, i.e., nodes with degree values higher than 90% of the entire degree distribution, were considered to be hubs (Liang et al., 2012; Bi et al., 2015). The individual betweenness centrality of the nodes generated by Cytoscape was also compared to the corresponding degree value in order to confirm the centrality of the hubs. Hub genes tend to demonstrate a positive correlation between these two parameters (Potapov et al., 2005; Lee, 2006; Li et al., 2014). INTRODUCTION reesei (Meng et al., 2018; Rassinger et al., 2018; Zhang et al., 2018). Previously, Borin et al. (2017) investigated the transcriptome of T. reesei RUT-C30 grown on steam-exploded sugarcane bagasse (referred to as “bagasse” from this point on) in a time course of 6, 12, and 24 h as well as on fructose after 24 h. Interestingly, a set of cellulase, hemicellulase, TF, and sugar transporter coding genes were activated in bagasse along with genes encoding hypothetical or uncharacterized proteins. Based on the assumption that co-expressed genes tend to share similar expression patterns and that they could be co-regulated by the same elements, such as the carbon source and pH (van Dam et al., 2017), this study attempts to identify new Frontiers in Bioengineering and Biotechnology | www.frontiersin.org October 2018 | Volume 6 | Article 151 2 Gene Co-expression Network of Trichoderma reesei Borin et al. Portal (9,852 gene models) (Le Crom et al., 2009; Nordberg et al., 2014) using TopHat2 (Kim et al., 2013). The mapped reads were counted with the featureCounts function from the Rsubread v1.12.6 package (Liao et al., 2014). Low abundance genes were filtered out, keeping only genes with a cpm ≥1 in at least three samples. RPKM values were calculated following TMM normalization using the edgeR package v3.12.1 (Bioconductor) (Robinson et al., 2010) within the R environment (R Core Team, 2015). Only differentially expressed genes (DEGs) with two-fold change cutoff(SEB 6, 12 or 24 h vs. fructose 24 h) were considered, i.e., log2-fold change ≥1 (upregulated) or ≤- 1 (downregulated). The identification of the genes from T. reesei was based on RUT-C30 strain retrieved from JGI database. genes related to the T. reesei lignocellulose degradation response using a Weighted Correlation Network Analysis (WGCNA) (Langfelder and Horvath, 2008). WGCNA estimates the co- expression similarity between the genes and constructs a weighted correlation matrix following a scale-free topology. In scale-free networks, a few nodes (genes) have a high degree (links), while most nodes have a small number of interactions (edges). These highly connected genes (hubs) play a central role in the network stability against perturbations, and they are very important in diverse cellular processes (Han et al., 2004; Luscombe et al., 2004). INTRODUCTION In addition, an in silico prediction of the DNA binding sites for Xyr1, the master activator of cellulases (Stricker et al., 2006), was performed using the promoters to find genes of unknown function that could be regulated by this TF. To our knowledge, this is the first report of the use of a network approach combined with regulatory motif analyses to reveal new genes of biotechnological interest in T. reesei RUT-C30. In this study, an extensive number of genes were found to be co-expressed in bagasse, and they could be the target of new studies to evaluate their role in lignocellulose degradation. Gene co-expression network analysis was then performed using the RPKM values and the WGCNA package v1.51 (Langfelder and Horvath, 2008). Briefly, a softpower β was chosen using the function pickSoftThreshold to fit the signed network to a scale-free topology. Next, an adjacency matrix was generated as follows: adj = (0.5 ∗(1+cor))β, where adj, cor and β are adjacency, pairwise Pearson correlation and softpower value, respectively. Topological Overlap Matrix (TOM) was used as an input in the function hclust (“average” method) to construct a hierarchical clustering tree (dendrogram). TOM is a measure that quantifies the topological similarity between the genes within a network, i.e., it evaluates whether two or more nodes share links within the network and groups them into the same module (Ravasz et al., 2002; Langfelder, 2013). A threshold of 0.15 (correlation > 85%) was chosen to merge similar modules, and only modules having at least 30 genes were kept. Network visualization and analyses for the highly connected genes (absolute Pearson correlation > 0.8, adj = 0.064) were carried out in Cytoscape v3.3.0. The entire R script used in the WGCNA analysis is available in Data Sheet 1. Fungal Strain and Culture Conditions Biological process and molecular function annotation from the Gene Ontology (GO) consortium was obtained from the JGI database (https://genome.jgi.doe.gov/TrireRUTC30_1/ TrireRUTC30_1.home.html) and used for the enrichment analysis using Cytoscape’s plugin BiNGO v3.0.3 (Maere et al., 2005) configured to perform hypergeometric test and adjust p- values for multiple testing using the Benjamini & Hochberg’s false discovery rate (FDR) method (p ≤0.05). To completely annotate the function of the T. reesei proteins, KEGG and KOG annotations were retrieved from the JGI database. In addition, manually curated annotation of the QM6a strain based Gene Co-expression Network Analysis Gene Co-expression Network Analysis The reads obtained by Borin et al. (2017) (BioProject accession PRJNA350272) were size-filtered (minimum of 40 bp) and selected by quality (Q > 20) using AlienTrimmer software (Criscuolo and Brisse, 2013). The filtered reads were mapped to the T. reesei RUT-C30 v1.0 genome available in the JGI Genome October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 Gene Co-expression Network of Trichoderma reesei Borin et al. with secretion signal peptides were gathered in a few modules. Most of the upregulated genes of these functional classes were found in four modules: coral1, darkorange, black, and darkred (Figure 1A, Table S1), while most of the downregulated genes were in additional four modules: brown, darkolivegreen4, grey60, and lightcyan1 (Figure 1B, Table S1). The first four modules will be designated the “up set” and the latter four the “down set.” These different classes of DEGs are of major importance to the identification of new targets related to bagasse deconstruction and other processes associated, as they cover enzymes for the lignocellulose breakdown, transcriptional regulators of the gene expression, transport of inducer-acting molecules and other unknown players. Taken together, these modules represented more than half of all the DEGs in the T. reesei transcriptome. For this reason, further analyses focused only on them. on the trichoCODE pipeline (Druzhinina et al., 2016) was also transferred to the RUT-C30 strain using clusters of 1:1 orthologs generated by applying OrthoMCL pipeline (Li et al., 2003). Identification of Subclusters and Go Enrichment reesei network and gathered a significant share of the CAZyme and predicted sugar transporter genes (Figure 1A). Therefore, it is not surprising that the enrichment of these GO terms were identified for these modules and shows that our analyses were directed toward the identification of the genes of unknown function, which were co-expressed with known players in lignocellulose degradation and/or sugar transport. Prediction of the Xyr1-Binding Sites The promoter region of the T. reesei RUT-C30 genes was searched for Xyr1-binding sites (XBS) according to a modified pipeline developed by Silva-Rocha et al. (2014). The 1.5 kb sequences immediately upstream from the start codon ATG of 22 cellulase genes regulated directly by Xyr1 (Castro et al., 2014) were retrieved from the JGI database according to an in-house Biopython script available online (https://github.com/ SantosRAC/UNICAMP_RACSMaster/tree/master/GFFTools). For motif discovery, these sequences were used as input in the MEME program (Bailey et al., 2009) using the following parameters: (i) zero or one occurrence per sequence at the forward and reverse strand, (ii) a minimum of 10 motifs, and (iii) a minimum and maximum motif width of 6 and 10, respectively. The frequency matrix of the motif most similar to the Xyr1 consensus sequence 5 ′-GGC(A/T)3-3 ′ (Rauscher et al., 2006) was chosen to be sought in the promoter of the network genes using the matrix-scan tool from the RSAT server (p-value ≤ 1.00E-04) (http://rsat-tagc.univ-mrs.fr/rsat/matrix-scan_form. cgi). KOG annotation was used to identify the functional groups of genes that have the predicted XBS, and a hypergeometric test was applied to enrich the statistically significant KOG groups (p-value ≤1.00E-03) using KOG annotation for all the genes as background. P-values were adjusted for multiple testing corrections using Benjamini & Hochberg’s false discovery rate (FDR) method (p ≤0.05). Identification of Subclusters and Go Enrichment Enrichment Subclusters of co-expressed genes were classified using the MCODE algorithm to find protein coding genes acting together during the process of lignocellulose deconstruction from bagasse. Each of the modules previously identified was partitioned, and a few subclusters were formed for each module from the up and down sets (Table S2). Next, a GO enrichment was conducted with the genes within each subcluster. Only subclusters having at least 10 genes were considered for GO enrichment (Table S3). Overall, subclusters of coral1, black, darkorange, and darkred modules presented 26 GO terms enriched, including cellulose binding (GO:0030248) and hydrolase activity (GO:0004553, 0016798), transferase activity (GO:0016758, 0016740), carbohydrate transport (GO:0008643) and regulation of transcription (GO:0006355), and transcription factor activity (GO:0003700), respectively (Table S3). As already described, these modules had the largest number of upregulated genes of the whole T. reesei network and gathered a significant share of the CAZyme and predicted sugar transporter genes (Figure 1A). Therefore, it is not surprising that the enrichment of these GO terms were identified for these modules and shows that our analyses were directed toward the identification of the genes of unknown function, which were co-expressed with known players in lignocellulose degradation and/or sugar transport. Subclusters of co-expressed genes were classified using the MCODE algorithm to find protein coding genes acting together during the process of lignocellulose deconstruction from bagasse. Each of the modules previously identified was partitioned, and a few subclusters were formed for each module from the up and down sets (Table S2). Next, a GO enrichment was conducted with the genes within each subcluster. Only subclusters having at least 10 genes were considered for GO enrichment (Table S3). during the process of lignocellulose deconstruction from bagasse. Each of the modules previously identified was partitioned, and a few subclusters were formed for each module from the up and down sets (Table S2). Next, a GO enrichment was conducted with the genes within each subcluster. Only subclusters having at least 10 genes were considered for GO enrichment (Table S3). g ( ) Overall, subclusters of coral1, black, darkorange, and darkred modules presented 26 GO terms enriched, including cellulose binding (GO:0030248) and hydrolase activity (GO:0004553, 0016798), transferase activity (GO:0016758, 0016740), carbohydrate transport (GO:0008643) and regulation of transcription (GO:0006355), and transcription factor activity (GO:0003700), respectively (Table S3). As already described, these modules had the largest number of upregulated genes of the whole T. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Construction of a Weighted Gene Co-expression Network Recently, the transcriptome of T. reesei RUT-C30 grown on bagasse after a time course of 6, 12 and 24 h as well as on fructose after 24 h was investigated (Borin et al., 2017). Several DEGs were identified, including CAZymes, genes encoding sugar transporters and uncharacterized TFs and proteins. Using this dataset, a gene co-expression network was inferred to identify new targets related to the cell wall degradation of bagasse. g g g p Alternatively, the down set demonstrated only 15 GO terms enriched, such as threonine-type endopeptidase activity (GO:0004298) (module brown), carbohydrate biosynthetic process (GO:0016051) (darkolivegreen4), DNA conformation change (GO:0071103) and electron carrier activity (GO:0009055) (grey60), and anion binding (GO:0043168) (lightcyan1) (Table S3). The difference between the GO terms enriched in the up and down sets could be explained by the carbon sources used in the culture media. Bagasse is a complex recalcitrant structure that must be broken down by the combined action of CAZymes. The sugars released in this process are transported to the intracellular environment, where they are utilized in carbohydrate catabolism to produce energy. These sugars, including cellobiose and xylose, can also act as inducers of cellulase and hemicellulase expression (Mach-Aigner et al., From 9,852 genes, 8,402 were kept for further analyses after gene expression filtering and normalization. The data were imported into the WGCNA package, and a softpower β of 26 (R² = 0.85) was chosen to fit the scale independence to a scale-free topology and taking into account the connectivity between the genes based on their expression (Data Sheet 2A). Genes having similar expression patterns were grouped into modules, and highly connected modules were merged (Data Sheet 2B). In total, 28 different modules were formed with the genes highly co-expressed (Pearson correlation > 0.8). The DEGs identified and annotated by Borin et al. (2017) were sought within each module generated using the WGCNA package, and ∼70% of all of the up and downregulated genes were identified in the T. reesei network (Table 1, Table S1). Genes encoding CAZymes, TFs, sugar transporters and uncharacterized proteins October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 4 Gene Co-expression Network of Trichoderma reesei Borin et al. TABLE 1 | Modules of co-expressed genes in the T. reesei network and number of DEGs found within each module. Construction of a Weighted Gene Co-expression Network Set Module Nodes Edges Up (% total)8 Down (% total) 8 Down Brown 1676 1149157 – 672 (44.8) Up Coral1 1173 482242 386 (26.2) – Up Black 875 335415 157 (10.6) 2 (0.1) Up Darkorange 683 156692 270 (18.3) – Down Darkolivegreen4 615 136748 – 144 (9.6) Up Darkred 607 124171 170 (11.5) – Coral2 375 45234 16 (1.1) 10 (0.7) Down Grey60 370 53283 – 130 (8.7) Down Lightcyan1 332 35644 – 27 (1.8) Brown2 291 28624 – 6 (0.4) Navajowhite2 159 8570 5 (0.3) 8 (0.5) Bisque4 135 6022 10 (0.7) – Royalblue 117 4724 – 22 (1.5) Darkgreen 116 4704 – 1 (0.1) Yellowgreen 91 895 – – Plum1 85 1537 2 (0.1) 3 (0.2) Ivory 79 1641 – – Brown4 72 743 6 (0.4) – Darkslateblue 71 1258 – – Thistle2 66 1552 – 4 (0.3) Lavenderblush3 62 1464 – 1 (0.1) Darkseagreen4 59 1464 – – Orangered3 52 1143 4 (0.3) – Lightsteelblue 49 579 2 (0.1) 3 (0.2) Lightcoral 48 470 3 (0.2) – Firebrick4 45 548 – – Blue2 43 378 3 (0.2) – Darkviolet 41 671 2 (0.1) – Total 8387 2585573 1036 (70.1) 1033 (68.9) Genes were up or downregulated in at least one time point of T. reesei grown on sugarcane bagasse. Nodes and edges represent genes and pairwise interactions, respectively. 8 The percentage was calculated based on the total number of up (1475) and downregulated (1500) genes expressed in the transcriptome of T. reesei (Borin et al., 2017). co-expressed genes in the T. reesei network and number of DEGs found within each module. Genes were up or downregulated in at least one time point of T. reesei grown on sugarcane bagasse. Nodes and edges represent genes and pairwise interactions, respectively. 8 The percentage was calculated based on the total number of up (1475) and downregulated (1500) genes expressed in the transcriptome of T. reesei (Borin et al., 2017). amino acid transporters; CAZymes; TFs; proteins of chromatin remodeling; enzymes from carbohydrate, amino acid, lipid and nucleotide metabolism; chaperones and hypothetical proteins without annotated function (Table S5). 2010; Zhou et al., 2012; Zhang et al., 2013). In contrast, the disaccharide fructose is a readily assimilable sugar that does not require cellulase enzymes to be utilized by the fungus. Therefore, it is evident that T. reesei adapts its metabolism according to the available carbon source. Construction of a Weighted Gene Co-expression Network For simplification purposes, only a few hubs are shown in Table 2. In the up set, 3 CAZymes, 9 sugar transporters, 6 TFs and 5 genes encoding unknown proteins with a predicted secretion signal peptide were found to be hub genes. A few TFs and other proteins having a putative regulatory function in gene transcription were identified as hub genes, and most were only characterized in their corresponding fungal orthologs, including the PRO1 Zn2Cys6 transcriptional regulator (jgi|136533, module darkorange) (Masloffet al., 1999), the PRO41 protein (jgi|8730, module coral1) (Nowrousian et al., 2007) and the AMA1 activator (jgi|114362, module coral1) (Diamond et al., 2009) (Table 2). Interestingly, two genes encoding methyltransferases (jgi|79832, module darkred; jgi|72465, module coral1) and one gene encoding a GCN5-acetyltransferase (jgi|133861, module coral1) were also identified as hub genes. Among the hub genes encoding proteins of unknown function with signal peptides, the Hub Genes Identification Important genes for a biological process tend to be central in a gene co-expression network and share a high number of co-expressed neighbors having a lower degree (Villa-Vialaneix et al., 2013). In this study, hubs were defined as the genes at the top of the degree distribution and those that demonstrated a positive correlation between the degree and betweenness centrality (Table S4). In total, 321 and 294 hubs were identified in the up and down sets, respectively (Table S5). Among the 321 hubs, 129 (40%) were upregulated in bagasse in at least one time point (6, 12 or 24 h), and 191 (65%) out of 294 genes were downregulated in the down set. Various genes encoding proteins that have different functions were found to be hubs in all eight modules investigated, including sugar, ion, and October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 Gene Co-expression Network of Trichoderma reesei Borin et al. Borin et al. FIGURE 1 | DEGs identified in the modules of the T. reesei RUT-C30 network encoding CAZyme, sugar transporter, transcription factor and unknown proteins with signal peptide. Most of the upregulated (A) and downregulated (B) genes in sugarcane bagasse were found in a few modules. work encoding CAZyme, sugar transporter, transcription factor and unknown proteins with i b f d i f d l FIGURE 1 | DEGs identified in the modules of the T. reesei RUT-C30 network encoding CAZyme, sugar transporter, transcription factor and unknown proteins with signal peptide. Most of the upregulated (A) and downregulated (B) genes in sugarcane bagasse were found in a few modules. (Rauscher et al., 2006) and the motif used in the study of Silva- Rocha et al. (2014). As a result, only one putative XBS of 10 nucleotides resembling the Xyr1 motifs was chosen for further analyses (Figure 2). The frequency matrix of the motif chosen was then retrieved from the MEME server and used as a model in the identification of XBS predicted within the promoter of the genes from the up and down sets. The complete list of the motifs predicted in the promoter of the 22 CAZymes and the frequency matrix is available in Table S6. (Rauscher et al., 2006) and the motif used in the study of Silva- Rocha et al. (2014). Hub Genes Identification As a result, only one putative XBS of 10 nucleotides resembling the Xyr1 motifs was chosen for further analyses (Figure 2). The frequency matrix of the motif chosen was then retrieved from the MEME server and used as a model in the identification of XBS predicted within the promoter of the genes from the up and down sets. The complete list of the motifs predicted in the promoter of the 22 CAZymes and the frequency matrix is available in Table S6. genes jgi| 124417, 128655 were co-expressed in the module coral1 and darkorange, respectively, and were upregulated in bagasse. The trichoCODE annotation indicated that their orthologous genes in T. reesei QM6a encode SSCRPs, and therefore they could be acting in the extracellular environment (Table 2, Table S5). However, the true evidence that they are pivotal to T. reesei response when grown on bagasse still needs to be evaluated. Similar to the up set, various hub genes encoding different proteins were found in the down set. In total, 7 CAZymes, 5 sugar transporters, 8 TFs and 4 proteins of unknown functions that have predicted secretion signal peptide were found to be downregulated in bagasse. In addition to the identification of the hub genes that have a putative regulatory role in the gene expression, such as TFs, chromatin remodeling and signal transduction proteins, several genes encoding unknown proteins significantly downregulated were also found, especially in the brown module (Table 2, Table S5). To validate the motifs found in silico, they were compared with the motifs already characterized in the promoters of the CAZyme genes (cbh1 and xyn1) of other T. reesei strains. Cellobiohydrolase 1 (Cbh1/Cel7a) is one the most produced and secreted enzymes from T. reesei (Kiesenhofer et al., 2018), and endo-β-1,4-xylanase 1 (Xyn1) is a hemicellulase with important role in xylan deconstruction (Liu et al., 2017). The comparison of the motifs showed that our pipeline could predict XBS that had been previously described and characterized (Figures S3, S4) (Rauscher et al., 2006; Furukawa et al., 2009; Ries et al., 2014; Kiesenhofer et al., 2018), similarly to the prediction of Silva-Rocha et al. (2014). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Prediction of the Xyr1-Binding Sites in the Promoter Region Next, the predicted XBS found in the promoter of the genes from the up and down sets were investigated (Table S7). In total, 245 upregulated and 210 downregulated genes had at least one XBS predicted in the promoter region. This represented 24 and 22% of the entire set of DEGs present in the up and down sets, respectively. Considering only the number of DEGs in each module, coral1 (45.5%) and grey60 (40.4%) were the modules having the largest percentage of genes with predicted XBS (Table S1). Previously, Castro et al. (2014) showed that 22 genes encoding cellulases and hemicellulases of the T. reesei QM9414 strain are regulated directly by the master activator Xyr1 (jgi|98788). This TF is essential for the induction of cellulase and hemicellulase genes, and it has been the target of various studies aiming to improve the hypercellulolytic phenotype of T. reesei using genetic manipulation (Wang et al., 2013; Lv et al., 2015; Zhang et al., 2017). To predict the presence of a specific DNA binding motif for the RUT-C30 strain, the promoter regions (1.5 kb immediately upstream the start codon ATG) of the orthologous genes between the RUT-C30 and QM9414 strains were used to seek motifs that were similar to the Xyr1 consensus sequence 5′-GGC(A/T)3-3′ KOG functional annotation of the genes having predicted XBS from up and down sets was also investigated (Figure 3). The hypergeometric test and multiple testing correction showed that the KOG classes metabolism, carbohydrate transport and metabolism, and information storage and processing were October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 Gene Co-expression Network of Trichoderma reesei Borin et al. TABLE 2 | Hub genes found in the up and down sets that were differently expressed in sugarcane bagasse (Borin et al., 2017). Prediction of the Xyr1-Binding Sites in the Promoter Region In the down set, 33 (17.3%) hub genes had XBS predicted in this study, including two CAZymes (chitin synthase and glucan endo- 1,3-β-glucosidase), one ABC transporter family protein, one putative nonribosomal peptide synthase (NRPS), one putative SWI-SNF chromatin-remodeling complex protein and several uncharacterized proteins (Table S5). FIGURE 2 | Putative XBS predicted in the promoter of 22 genes encoding cellulases and hemicellulases and chosen to be sought in the gene promoter regions of T. reesei. Only this XBS was found to resemble the Xyr1 consensus sequence 5 ′-GGC(A/T)3-3 ′ and the motif used by Silva-Rocha et al. (2014). Prediction of the Xyr1-Binding Sites in the Promoter Region RUT-C30 ID1 QM6a ID2 Description3 Module Degree log2FC4 B6h B12h B24h Up sets 23456 122048 Sec61 beta subunit of ER translocase Black 859 – – 1.09 103979 81884 Translocon-associated protein (TRAP) Black 859 – – 1.06 133861 111236 GCN5-related N-acetyltransferase Coral1 1106 3.29 3.18 4.12 8730 57737 PRO41 protein Coral1 1086 2.03 1.27 2.69 114362 67408 AMA1 activator Coral1 1097 – – 1.11 139402 121107 Zn2Cys6 transcriptional regulator Coral1 1104 – – 1.38 95791 120698 C2H2 transcriptional regulator PacC Darkorange 652 2.28 2.15 1.94 136533 76590 Zn2Cys6 transcriptional regulator Pro1 Darkorange 650 2.01 2.00 2.09 79832 5366 S-adenosyl-L-methionine-dependent Methyltransferase Darkred 558 1.93 1.71 – 38522 103158 Zn2Cys6 transcriptional regulator Darkred 572 2.12 1.99 – Down sets 98900 78688 Heat shock factor-type DNA-binding domain-containing protein Brown 1642 −1.69 −1.53 −2.29 140865 106171 HET protein Brown 1640 −4.10 −2.82 −4.99 124027 122943 SWI-SNF chromatin-remodeling complex protein Brown 1639 – −1.10 −1.28 77229 106259 Zn2Cys6 transcriptional regulator brown 1643 −2.18 −1.86 −2.86 139518 124228 GT2 chitin synthase Darkolivegreen4 572 – – −1.28 86800 66606 Zn2Cys6 transcriptional regulator Darkolivegreen4 577 – – −1.11 139776 21255 bHLH transcriptional regulator Grey60 347 −1.12 −1.05 – 131957 53893 Hypothetical protein Grey60 353 −2.17 −2.79 −1.55 105289 82667 ThrB Homoserine kinase Lightcyan1 307 −1.22 −1.17 – 96918 120953 Unknown secreted protein Lightcyan1 310 −3.29 −3.04 – 1Trichoderma reesei RUT C30 v1.0 database from JGI was used to recover the T. reesei proteins ID; 2QM6a ortholog genes; 3Functional annotation according to KEGG, KOG and Druzhinina et al.’s work (2016); 4Log2 fold change (FC), B: sugarcane bagasse. Red and blue colors indicate gene expression of genes up and downregulated in sugarcane bagasse, respectively. Major Facilitator Superfamily (MFS) and one putative calcium transporter, one Ras GTPase, one putative SWI-SNF chromatin- remodeling complex protein, one polyketide synthase (PKS) and various hypothetical proteins with unknown function (Table S5). In the down set, 33 (17.3%) hub genes had XBS predicted in this study, including two CAZymes (chitin synthase and glucan endo- 1,3-β-glucosidase), one ABC transporter family protein, one putative nonribosomal peptide synthase (NRPS), one putative SWI-SNF chromatin-remodeling complex protein and several uncharacterized proteins (Table S5). Major Facilitator Superfamily (MFS) and one putative calcium transporter, one Ras GTPase, one putative SWI-SNF chromatin- remodeling complex protein, one polyketide synthase (PKS) and various hypothetical proteins with unknown function (Table S5). Xyr1 and Co-expressed Genes in the Coral1 Module DISCUSSION transcriptional regulator, one protein with an ankyrin repeat, and several other genes still not characterized (Figure 4B, Table S8). cbh2), endoglucanases (egl1, egl2, egl3, and egl5), β-glucosidases (bgl1 and bgl2), endo-β-1,4-xylanases (xyn2, xyn3, xyn4, and xyn5), and monooxygenases from the AA9 family (cel61a and cel61b) (Figure 4A, Table S8). Xyr1 and Co-expressed Genes in the Coral1 Module As already described, Xyr1 is critical for the activation of cellulases and hemicellulases, and it was expressed in bagasse more highly than fructose during the entire time course (Log2FC: 6 h: 0.48; 12 h: 0.99; 24 h: 1.65). Along with the other 385 upregulated genes, xyr1 was grouped into the coral1 module which had the largest number of CAZyme genes upregulated in bagasse (Figure 1A). Thus, it is worth investigating the genes that were co-expressed with this activator, since they could be involved in the fungal response to the lignocellulosic biomass. FIGURE 2 | Putative XBS predicted in the promoter of 22 genes encoding cellulases and hemicellulases and chosen to be sought in the gene promoter regions of T. reesei. Only this XBS was found to resemble the Xyr1 consensus sequence 5 ′-GGC(A/T)3-3 ′ and the motif used by Silva-Rocha et al. (2014). statistically significant in the up set, while secondary metabolites biosynthesis, transport and catabolism was the only class enriched in the down set (Figure 3). This difference stresses the diversity of genes encoding proteins with several functions that could be modulated by the activator Xyr1, in addition to other elements. From the pairwise edges of the coral1 module, 858 neighbor nodes to Xyr1 were retrieved. A total of 338 out of 858 genes were upregulated in bagasse, and only 115 had at least one XBS in the promoter. Among these genes, 35 CAZymes were found to be co-expressed with xyr1, including cellobiohydrolases (cbh1 and y y Analyzing the hub genes of the up set, 30 genes (23.3%) demonstrated at least one XBS in their promoter, including one Zn2Cys6 transcriptional regulator, transporters from the October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 7 Borin et al. Gene Co-expression Network of Trichoderma reesei FIGURE 3 | KOG function classification of the DEGs identified in the up and down sets that had at least one predicted XBS. KOG classes significantly enriched (p-value ≤1.00E-03) are shown with an asterisk (*). FIGURE 3 | KOG function classification of the DEGs identified in the up and down sets that had at least one predicted XBS. KOG classes significantly enriched (p-value ≤1.00E-03) are shown with an asterisk (*). transcriptional regulator, one protein with an ankyrin repeat, and several other genes still not characterized (Figure 4B, Table S8). DISCUSSION The list of hubs included 14 genes encoding one MFS transporter, one Zn2Cys6 October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 Gene Co-expression Network of Trichoderma reesei Borin et al. FIGURE 4 | (A) Upregulated genes encoding proteins of different functions being co-expressed with the activator xyr1 in the coral1 module. (B) Hub genes identified in the same module having at least one predicted XBS in the promoter region. The nodes are identified with the T. reesei RUT-C30 ID retrieved from JGI database. The complete list of co-expressed genes with xyr1 is shown in Table S8. FIGURE 4 | (A) Upregulated genes encoding proteins of different functions being co-expressed with the activator xyr1 in the coral1 module. (B) Hub genes identified in the same module having at least one predicted XBS in the promoter region. The nodes are identified with the T. reesei RUT-C30 ID retrieved from JGI database. The complete list of co-expressed genes with xyr1 is shown in Table S8. et al., 2018), clarifying the importance of these putative sugar transporters in the induction of CAZymes and lignocellulose degradation. were chosen for further analysis due to the largest number of up and downregulated genes encoding CAZymes, TFs, sugar transporters and other genes of unknown function that were co-expressed (Figure 1). g Distinct GO terms were enriched in the down set, such as threonine-type endopeptidase activity (GO:0004298) and carbohydrate biosynthetic process (GO:0016051) (Table S3). The latter was enriched in the darkolivegreen4, and interestingly, this GO term consisted of seven enzymes possibly involved in fungal cell wall biosynthesis, including three glycosyl transferases, one α-amylase, one rhamnose reductase, one pyruvate carboxylase, and one glucose 6-phosphate isomerase (GPI) (Table S3). Fungal cell wall is primarily composed of β-1,3-glucan, β-1,6- glucan, mixed β-1,3-/ β-1,4-glucan, α-1,3-glucan, chitin, and glycoproteins. They are organized in a complex backbone, and it is thought that glycosyl hydrolases and glycosyl transferases are fundamentally important in cell wall biosynthesis (Free, 2013). Genes encoding enzymes from the gluconeogenesis pathway, such as pyruvate carboxylase and GPI, could also provide hexose phosphates as building blocks for the cell wall biosynthesis (Ene et al., 2012). Considering that fructose is a simpler sugar than the bagasse lignocellulose, the fungus must be utilizing this readily assimilable carbon source to grow, and consequently it has to remodel its cell wall. DISCUSSION Interestingly, swo1 (jgi|104220) and cip1 (jgi| 121449) were found as neighbor nodes to xyr1, and they were under strong activation in bagasse during the entire time course (6, 12, and 24 h) (Table S8). Swo1 acts synergistically with xylanases to remove the hemicellulosic fraction of the lignocellulose (Gourlay et al., 2013), and Cip1 appears to be important to the hydrolysis of the lignocellulosic biomass (Lehmann et al., 2016). Both proteins have a carbohydrate-binding domain that belongs to family 1 (CBM1), and one XBS was predicted in each gene promoter. Therefore, they play a role in bagasse deconstruction and are transcriptionally regulated by Xyr1, as reported previously (Reithner et al., 2014; Ma et al., 2016). In addition, 14 putative sugar transporters, three Zn2Cys6 transcriptional regulators, one putative GCN5-acetyltransferase, one methyltransferase and several other genes were also co-expressed with xyr1 and had XBS in their promoters (Figure 4A, Table S8). The construction of a gene co-expression network allows us to identify clusters of genes that have a similar expression pattern and to assess the biological information that is relevant to a specific phenotype. Hitherto there were only a few studies investigating gene co-expression network in T. reesei. Dos Santos Castro et al. (2014), for instance, performed RNA-Seq of T. reesei QM9414 strain grown on sophorose, cellulose and glucose, and their analysis revealed specific differentially expressed genes in sophorose and cellulose. More recently, Horta et al. (2018) examined the transcriptome and exoproteome of T. reesei, T. harzianum, and T. atroviride grown on cellulose and glucose. Based on their co-expression network, they found a set of 80 genes shared between the three Trichoderma species that could represent a common cellulose degradation system. However, no research has been reported exploring the gene co-expression network of T. reesei RUT-C30 grown on sugarcane bagasse. In this study, the recently published transcriptome of T. reesei RUT-C30 grown on bagasse after 6, 12 and 24 h was used to determine the modules of co-expressed genes. As a result, 28 modules of co-expressed genes were formed, and only eight Finally, hub genes that were neighbor nodes to the master activator Xyr1 (represented in the coral1 module) and that had XBS in their promoter were also identified. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org DISCUSSION Although most of those genes were downregulated and highly co-expressed, the engagement of these genes in this process remains elusive. p g After the MCODE clustering, the GO enrichment showed that several terms related to lignocellulose breakdown, including polysaccharide (GO:0030247) and cellulose binding (GO:0030248), and hydrolase activities (GO:0004553 and GO:0016798), were enriched in the subcluster 1 of the coral1 module (Table S3) where the master activator Xyr1 was also found. The presence of 50 upregulated CAZyme genes encoding cellulases, hemicellulases and oxidative enzymes in the coral1 module (Table S1), along with the co-expression of xyr1, allows us to hypothesize that soon after xyr1 is expressed in bagasse, its protein product regulates the transcription of the (hemi)cellulolytic genes. In addition, 36 out of the CAZyme 50 genes demonstrated at least one XBS in their promoter, which is consistent with previous studies (Castro et al., 2014; Silva-Rocha et al., 2014) and highlights the ability of our approach to identify the Xyr1 target genes. In addition to coral1, the darkorange module had the carbohydrate transport (GO:0008643) term enriched (Table S3), revealing that several genes encoding transporters were grouped in this module. Most of them belonged to the MFS family, and its members are thought to transport a vast array of small molecules, such as sugars, inorganic ions, siderophores and amino acids (Yan, 2015; Quistgaard et al., 2016). Some studies have also shown that MFS transporters can still mediate the induction of cellulases in T. reesei (Zhang et al., 2013; Huang et al., 2015; Nogueira Hub genes were also sought out within the main modules, and a large number of new targets that were co-expressed were discovered (Table 2, Table S5). For example, the Pro1 coding gene (jgi|136533) was found as a hub node within the October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 9 Gene Co-expression Network of Trichoderma reesei Borin et al. identity: 33%) encodes a meiosis-specific activator related to spore morphogenesis (Okaz et al., 2012; Schmoll et al., 2016). One gene encoding the developmental regulatory protein WetA (jgi|9281) was found in the same module being upregulated during the entire time course. In Fusarium graminearum, the putative ortholog WetA (I1S0E2.2; identity: 43%) is necessary for conidiogenesis and conidial maturation, and the wetA mutants produced conidia that were more sensitive to oxidative and heat stress (Son et al., 2014). Intriguingly, Wu et al. DISCUSSION (2017) suggested that the Aspergillus flavus ortholog WetA (EED47149.1; identity: 57%) could be a global player in the regulation of conidial development, acting during the fungal cell wall biogenesis and regulating the secondary metabolic pathways. In addition, the putative TF (jgi|98900) was one of the hub genes of the brown module, and its corresponding orthologs encode the heat shock transcription factor 1 (Hsf1) (Schmoll et al., 2016), the master stress response regulator in eukaryotes (Zheng et al., 2016) and the activator of virulence in the pathogen Candida albicans (Nicholls et al., 2011) (Table 2, Table S5). Another four and seven putative TF coding genes were also found to be hubs in the up and down set, respectively (Table S5). The search for their orthologous genes in other ascomycetes revealed that none had been characterized, and therefore, their function remains to be elucidated. The relevant role of the characterized ortholog hub genes identified in this study highlights the central position of these nodes in the up and down sets and support the necessity of investigating their putative regulatory influence on the T. reesei response in bagasse. darkorange module, and it was upregulated in the three time points analyzed (6, 12, and 24 h). Pro1 is a member of the Zn2Cys6 transcription factors, and several orthologs had already been characterized in ascomycetes. In Neurospora crassa, the ortholog Adv-1 (NCU07392; identity: 66%) regulates cell-to-cell fusion and sexual development (Chinnici et al., 2014; Dekhang et al., 2017), and in Sordaria macrospora (CAB52588.2; identity: 66%), Pro1 has a pivotal role in various different processes, including the regulation of genes involved in cell wall integrity, the NADH oxidase pathway and pheromone signaling (Masloff et al., 2002; Steffens et al., 2016). Steffens et al. (2016) demonstrated that, in addition to Adv-1, N. crassa requires the pH response transcription regulator PacC to activate its female development. Intriguingly, its ortholog gene pac1 (jgi|95791) in T. reesei RUT-C30 was also identified to be a hub node (Table 2, Table S5). pac1 was upregulated in bagasse and co-expressed with the pro1 gene in the same module. He et al. (2014) functionally characterized pac1 in T. reesei, and they observed increased cellulase transcription and production in the 1pac1 mutants at neutral pH. They also noted that the pac1 deletion impaired fungal growth and development. DISCUSSION In total, the results point to possible crosstalk between Pro1, Pac1 and other genes involved in an intricate regulatory network. As already described, two putative methyltransferase coding genes (jgi|79832, module darkred; jgi|72465, module coral1) and a GCN5-acetyltransferase coding gene (jgi|133861, module coral1) were also identified as hubs in the up set (Table S5). These classes of enzymes are thought to be responsible for DNA methylation and histone modification, respectively, and epigenetically regulate various important cellular processes, such as the silencing of transposable elements and the activation of gene transcription (Xin et al., 2013; Su et al., 2016; Lyko, 2017). Intriguingly, recent studies have suggested that methyltransferases are also implicated in lignin degradation in the basidiomycete Phanerochaete chrysosporium, broadening the functions of these enzymes (Korripally et al., 2015; Thanh Mai Pham and Kim, 2016; Kameshwar and Qin, 2017). In addition, five genes encoding unknown proteins that have secretion peptide signal were considered to be hubs in the up set (Table S5). Among them, two (jgi|124417, 128655) had their QM6a orthologs annotated as annotated as small secreted cysteine-rich proteins (SSCRPs) using trichoCODE. In general, SSCRPs are related to interactions between the microorganisms and the environment, including biocontrol, the induction of plant resistance and adhesion (Shcherbakova et al., 2015; Qi et al., 2016). The presence of a predicted secretion signal peptide allows us to hypothesize that these proteins could be secreted to support the bagasse-fungal adhesion. In total, the identification of these hub genes shows that they could be acting as regulatory players or accessory proteins in the bagasse degradation response. Finally, other genes encoding proteins of different functions were also identified as hub nodes in the up and down sets (Table S5). In the up set, three CAZymes (jgi|97768, 75420, 128705), two SSCRPs (jgi|124417, 128655), the translocon- associated protein (TRAP) (jgi|103979) and the Sec61 beta subunit (jgi|23456) from the endoplasmic reticulum (ER) translocon, ion and amino acid transporters, and various unknown proteins were found. In the down set, other hubs were discovered, including seven CAZymes (jgi|24326, 104519, 103899, 124897, 97721, 139518, 104242), six proteases (jgi|90298, 135507, 75405, 82006, 138263, 87936), a SWI-SNF chromatin-remodeling complex protein (jgi|124027) and unknown proteins (Table 2, Table S5). This vast array of hub genes demonstrates that the great diversity of genes is central in the co-expressed modules. However, most of them are still not characterized, and efforts are required to elucidate their function in fungal physiology. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org DISCUSSION The prediction of XBS based on the promoter of cellulase and hemicellulase coding genes was used in this study to verify the genes possibly regulated by Xyr1 in the presence of a complex lignocellulosic biomass. Xyr1 is the key transcriptional regulator of the CAZymes involved in cell wall deconstruction, and it is regulated by the carbon catabolite repressor Cre1, which is truncated and confers a hypercellulolytic phenotype in the RUT- C30 strain (Ilmén et al., 1996; Silva-Rocha et al., 2014). To validate our pipeline of regulatory motif predictions, the XBS predicted in the cbh1 and xyn1 promoters were compared with those from other studies. Other putative regulators were also identified as hub nodes in the up and down sets, and some of them were not characterized in T. reesei, only in its homologs (Table S5). For example, the gene encoding the Ama1 activator (jgi|114362) was found in the coral1 module having an XBS in the promoter, and its putative ortholog in Saccharomyces cerevisiae (YGR225W; October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 10 Gene Co-expression Network of Trichoderma reesei Borin et al. According to Ries et al. (2014), there are two XBS in the positions −733 and −320 in relation to the start codon ATG of the cbh1 gene (Data Sheet S3). The −733 motif (5′-TTTGCC- 3′) was predicted by the pipeline of Silva-Rocha et al. (2014). However, it was not predicted in this study. Alternatively, we identified four XBS in the promoter region of cbh1 at positions −508, −748, −771, and −1376 that were not identified in the study by Silva-Rocha et al. (2014). This was likely to be due to the differences between the strains and pipelines. Using a labeled Xyr155−195 probe, Furukawa et al. (2009) found that two of these predicted sequences (positions−508 and−748) showed strong binding to the probe. In addition, Kiesenhofer et al. (2018) replaced the promoter of the glycine oxidase (goxA) reporter gene with the cbh1 promoter containing deletions in three different regions and observed a significant decrease in the GoxA activity when the mutant strains were grown on different carbon sources, including lactose, carboxymethylcellulose (CMC) and pretreated wheat straw. Two of these regions spanned the XBS predicted in this study at positions −748 and −771 (Data Sheet S3). DISCUSSION To verify the putative targets of Xyr1, the direct co-expressed neighbors of the xyr1 node were retrieved from module1, and we searched for the XBS in the gene promoters (Table S8). From a total of 115 Xyr1-partner genes having a predicted XBS, almost half encode CAZymes and putative sugar transporters. Three Zn2Cys6 transcription factors (jgi|139402, 77124, 141251) were also found, and one (jgi|141251) demonstrated 55 and 72% identity to the NirA ortholog in F. fujikuroi (KLO85312.1) and A. nidulans (AN0098), respectively (Table S8). NirA is a nitrate- specific transcription factor that modulates nitrogen metabolite repression (NMR), and this TF accumulates in the nucleus in the presence of nitrate or nitrite and a low concentration of assimilable nitrogen sources, such as ammonium. The nitrogen metabolism regulator AreA interacts physically with NirA, and the complex formed activates the genes for nitrate assimilation (Gallmetzer et al., 2015; Pfannmüller et al., 2017). The T. reesei ortholog areA (jgi|140814) was not differentially expressed in bagasse, and therefore it is curious to note the upregulation of the T. reesei ortholog nirA in this carbon source and its co-expression with xyr1. In addition to cbh1, the XBS predicted in the xyn1 promoter were also compared with the ones previously reported (Rauscher et al., 2006; Furukawa et al., 2009; Kiesenhofer et al., 2018). Two XBS were predicted at positions −417 and −621 being the first one identified and characterized in other studies (Data Sheet S4). For example, Rauscher et al. (2006) investigated a 217-bp region (−321 to −538) inside the xyn1 promoter and discovered that Xyr1 was able to bind to two sequences at positions −404 and −420. Point mutations in each of these two motifs caused a substantial decrease in the reporter activity of the gene glucose oxidase and showed that they are critical for the transcriptional activation of xyn1 in the presence of xylan, one of the primary sugar inducers of hemicellulases (Rauscher et al., 2006). Some years later, Furukawa et al. (2009) analyzed three XBS (−80, −420 and −887) in the xyn1 promoter and discovered that only the XBS at position −420 showed strong binding to the Xyr1 probe. Finally, Kiesenhofer et al. (2018) recently demonstrated that the deletion of these XBS at positions −404 and −420 abolished the GoxA activity under control of the xyn1 promoter even in the presence of 0.5 mM xylose (Data Sheet S4). DISCUSSION In summary, the previous studies indicate that some of the XBS predicted in this study could be functional in T. reesei RUT-C30 and therefore, are important for the transcriptional regulation of CAZyme genes and probable additional genes. In addition to the cbh1 and xyn1, the promoter of the cbh2 gene also had two XBS predicted in this study that were shared with its homologs in other T. reesei strains (data not shown). Three genes (jgi|126063, 73493, 92240) encoding putative transporters of non-sugar solutes were co-expressed with xyr1 and had XBS in their promoters (Table S8). The gene jgi|73493 encodes a putative calcium transporter with 10 transmembrane domains, and it was found as a hub in the coral1 module (Table S8). It has already been reported that metal ions, such as Ca2+ and Mn2+, have a positive effect on the mycelial growth of T. reesei and cellulase production, and this molecular signaling mechanism is mediated by the Mn2+ transporters TPHO84-1 and TPHO82-2, a Ca2+/ Mn2+ ATPase (TPMR1) and the components of the Ca2+/calmodulin signal transduction, including the TF Crz1 (Chen et al., 2016, 2018). Therefore, it is worth investigating the role of this putative calcium transporter in the induction of the genes responsive to lignocellulose degradation, since it could transport cations that activate gene expression. In addition to the non-sugar transporters, 15 putative sugar transporter coding genes were co-expressed with xyr1 and demonstrated at least one XBS predicted at their promoter region (Table S7). One of them was considered to be a hub gene encoding a protein annotated as allantoate permease (jgi|93149), but its participation in the fungal response toward the biomass deconstruction remains unclear. Most of these putative transporters are members of the MFS superfamily, and therefore, could transport a variety of solutes, including sugar and amino acids. Sloothaak et al. (2016) developed a Hidden Markov Model (HMM) to identify new xylose transporters in T. reesei and A. niger. Several candidates were identified, and one (RUT-C30: jgi| 7811; QM6a ortholog: jgi| 106330) was found upregulated and co-expressed with xyr1 in this study (Table S7). Unexpectedly, this putative xylose transporter coding gene had one XBS predicted in the promoter and showed an increasing expression profile in bagasse. The prediction of an XBS in the promoter region of this gene suggests that it could be regulated by Xyr1 and could be involved in the xylose assimilation after the hemicellulose breakdown of bagasse. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org REFERENCES Bornscheuer, U., Buchholz, K., and Seibel, J. (2014). Enzymatic degradation of (ligno)cellulose. Angew. Chemie Int. Ed. 53, 10876–10893. doi: 10.1002/anie.201309953 Amore, A., Giacobbe, S., and Faraco, V. (2013). Regulation of cellulase and hemicellulase gene expression in fungi. Curr. Genomics 14, 230–249. doi: 10.2174/1389202911314040002 Castro, L. D. S., Antoniêto, A. C. C., Pedersoli, W. R., Silva-Rocha, R., Persinoti, G. F., and Silva, R. N. (2014). Expression pattern of cellulolytic and xylanolytic genes regulated by transcriptional factors XYR1 and CRE1 are affected by carbon source in Trichoderma reesei. Gene Expr. Patterns 14, 88–95. doi: 10.1016/j.gep.2014.01.003 Bader, G. D., and Hogue, C. W. (2003). 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A., Moreira, L. R., et al. (2018). Exploring Trichoderma and Aspergillus secretomes: proteomics approaches for the identification of enzymes of biotechnological interest. Enzyme Microb. Technol. 109, 1–10. DISCUSSION Based on the assumption that Xyr1 is essential to the induction of the CAZymes and sugar transporters, it was not surprising that genes related to carbohydrate transport and metabolism, such as CAZymes and putative sugar transporters, were the most abundant upregulated genes that had XBS in the KOG analyses. Alternatively, the XBS were enriched in the genes of secondary metabolites biosynthesis, transport, and catabolism class in the down set (Figure 3), which suggests that Xyr1 participates in the regulation of secondary metabolism. October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 11 Gene Co-expression Network of Trichoderma reesei Borin et al. ACKNOWLEDGMENTS We would like to thank the Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) and the Centro Nacional de Pesquisa em Energia e Materiais (CNPEM) for the computational facility. FUNDING This study received financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP processes n 2014/15799-7, 2014/11766-7, 2015/08222-8, and 2017/18987- 7), from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq process number 141574/2015-1), and also in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES). AUTHOR CONTRIBUTIONS GB performed the analyses, and RdS carried out the data processing. DR-P and MC supervised the study and performed the analyses. JO supervised the study and planned the analyses. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2018.00151/full#supplementary-material CONCLUSION All the authors wrote the draft and approved its final version. The T. reesei gene co-expression network analysis grouped several differentially expressed genes into modules based on their expression patterns in steam-exploded sugarcane bagasse. A large number of interesting genes encoding CAZymes, putative sugar and ion transporters, as well as TFs and proteins with a putative regulatory role were highly co-expressed within some modules. The prediction of XBS in the promoters confirmed the influence of Xyr1 in the CAZyme coding genes regulation and enabled the identification of new putative targets of this master regulator. Hub nodes were also found within the modules, and many of them had not been characterized. 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Frontiers in Bioengineering and Biotechnology | www.frontiersin.org October 2018 | Volume 6 | Article 151 REFERENCES doi: 10.1093/bib/bbw139 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Villa-Vialaneix, N., Liaubet, L., Laurent, T., Cherel, P., Gamot, A., and SanCristobal, M. (2013). The structure of a gene co-expression network reveals biological functions underlying eQTLs. PLoS ONE 8:e60045. doi: 10.1371/journal.pone.0060045 The handling editor declared a shared affiliation, though no other collaboration, with several of the authors, RS and DR, at the time of the review. Copyright © 2018 Borin, Carazzolle, dos Santos, Riaño-Pachón and Oliveira. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Wang, S., Liu, G., Wang, J., Yu, J., Huang, B., and Xing, M. (2013). Enhancing cellulase production in Trichoderma reesei RUT C30 through combined manipulation of activating and repressing genes. J. Ind. Microbiol. Biotechnol. 40, 633–641. doi: 10.1007/s10295-013-1253-y Wu, M.-Y., Mead, M. E., Kim, S.-C., Rokas, A., and Yu, J.-H. (2017). WetA bridges cellular and chemical development in Aspergillus flavus. PLoS ONE 12:e0179571. doi: 10.1371/journal.pone.0179571 October 2018 | Volume 6 | Article 151 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 15
https://openalex.org/W2141769581
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Recent Progress in Osteocyte Research
Endocrinology and metabolism
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11879695 Published Version doi:10.3803/EnM.2013.28.4.255 doi:10.3803/EnM.2013.28.4.255 Citation Divieti Pajevic, Paola. 2013. “Recent Progress in Osteocyte Research.” Endocrinology and Metabolism 28 (4): 255-261. doi:10.3803/EnM.2013.28.4.255. http://dx.doi.org/10.3803/ EnM.2013.28.4.255. INTRODUCTION blasts that during the process of matrix mineralization remain entrapped in the mineralizing matrix that they are actively synthesizing. They reside both in the mineralized matrix and in the newly formed osteoid. Earlier work of Gaillard et al. [1] and Rutishauser and Majno [2], described two stages into the life of an osteocyte; an early stage (young osteocyte) in which the cell is smaller in size, reside into the osteoid and do not express alkaline phosphatase and a late stage (mature osteo- cyte) in which the larger cell re-express alkaline phosphatase and is deeply embedded in the mineralized bone. The larger cell will then degenerate and leave an empty lacuna [1]. This classification has been recently revised to include an addition- al stage of differentiation: according to their spatial localiza- tion and gene expression, these cells are now divided into os- teoid, mineralizing, and the mature osteocytes [3]. The oste- oid, or nascent osteocyte is characterized by a relative proxim- Our understanding of the function of osteocytes has expanded dramatically over the last decade primarily due to the identifi- cation of osteocytes specific markers, such as dentin matrix protein 1 (DMP1) and SOST/sclerostin, that has allowed, for the first time a closer look at the biology of these cells. Osteo- cytes, the cells deeply entrapped into the mineralized matrix, have emerged as key regulators not only of skeletal and min- eral homeostasis, but also hematopoiesis. This review will summarize novel findings in osteocyte biology and future ave- nues of research. Recent Progress in Osteocyte Research Paola Divieti Pajevic Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA The last decade has seen an exponential increase in our understanding of osteocytes function and biology. These cells, once con- sidered inert by-standers trapped into the mineralized bone, has now risen to be key regulators of skeletal metabolism, mineral homeostasis, and hematopoiesis. As tools and techniques to study osteocytes improved and expanded, it has become evident that there is more to these cells than initially thought. Osteocytes are now recognized not only as the key responders to mechanical forces but also as orchestrators of bone remodeling and mineral homeostasis. These cells are the primary source of several impor- tant proteins, such as sclerostin and fibroblast growth factor 23, that are currently target as novel therapies for bone loss (as the case for antisclerostin antibodies) or phosphate disorders. Better understanding of the intricate cellular and molecular mecha- nisms that govern osteocyte biology will open new avenue of research and ultimately indentify novel therapeutics to treat bone and mineral disorders. This review summarizes novel findings and discusses future avenues of research. Keywords: Osteocytes; Sclerostin; Mineral homeostasis; Bone homeostasis Corresponding author: Paola Divieti Pajevic Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, 50 Blossom Street, Boston, MA 02114, USA Tel: +1-617-726-6184, Fax: +1-617-726-7543, E-mail: divieti@helix.mgh. harvard.edu Review Article Endocrinol Metab 2013;28:255-261 http://dx.doi.org/10.3803/EnM.2013.28.4.255 pISSN 2093-596X · eISSN 2093-5978 Endocrinol Metab 2013;28:255-261 http://dx.doi.org/10.3803/EnM.2013.28.4.255 pISSN 2093-596X · eISSN 2093-5978 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Keywords: Osteocytes; Sclerostin; Mineral homeostasis; Bone homeostasis THE OSTEOCYTES Primary cilia have been described as important mechanosen- sor and mice lacking Kif3a or ITF88 (polaris), both key com- ponents of the cilia, in bone cells, display skeletal abnormali- ties. Targeted ablation of Kif3a from osteoblasts, and conse- quently from osteocytes, using the Col1α 2.3 promoter, affects skeletal anabolic responses to cyclic axial compression, sug- gesting that primary cilia are needed for proper skeletal mech- anosensory [14,15]. Similarly, mice with in which PDK1 is ablated in osteocytes, using the 10kb-DMP1 promoter have impaired anabolic response to mechanical loading, further supporting the importance of cilia in skeletal mechanosensa- tion [16,17]. Marotti et al. [7] and Palumbo et al. [8,9] have extensively described the morphological changes that accompany the transformation from a motile osteoblast into an entombed os- teocyte. They demonstrate, by histological analysis of new- born rabbit bones, that the formation of osteocyte cytoplasmic processes is asynchronous and asymmetrical and precede the mineralization of the organic matrix. Moreover, osteocytes are evolutionary highly conserved and the organized structure of these cells within a mineralized matrix is present in bone spec- imens from Tyrannosaurus rex, dating back more than 80 mil- lion of years ago, clearly indicating an important role for these cells in skeletal metabolism [10]. Osteocytes communicate with each other and with cells at the endosteal and periosteal surface through an extensive and intricate system of canaliculi. These cells are also in close proximity of capillary and vessels, raising the hypothesis that osteocytes might function as an endocrine organ and directly secrete proteins, such as FGF-23, Phex, or sclerostin into the circulation [11]. Moreover, the osteocytic network, with its extensive system, is an ideal structure to sense mechanical loading and control mineral homeostasis. It has been postulat- ed that osteocytes can send signals for both bone resorption and formation and thus orchestrate a proper cycle of remodel- ing, as discussed in details below. Osteocytes responses to load, both in vivo and in vitro, include secretion of prostaglandin E2 and nitric oxide and the expression of several mechanosensitive transcripts such as DMP1, MEPE, and Phex [18,19]. Another osteocytic gene highly regulated by mechanical forces is SOST. SOST/sclerostin in suppressed dur- ing load whereas it is increased during hindlimb unloading [20]. Moreover mice lacking SOST are resistant to disuse induced bone loss and antisclerostin antibodies can prevent bone loss as- sociated with hindlimb unloading [21]. THE OSTEOCYTES Osteocytes are postmitotic, terminally differentiated osteo- Copyright © 2013 Korean Endocrine Society Corresponding author: Paola Divieti Pajevic This is an Open Access article distributed under the terms of the Creative Com­ mons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribu­ tion, and reproduction in any medium, provided the original work is properly cited. 255 255 www.e-enm.org Pajevic PD Pajevic PD ity to the endosteal (and possibly periostal) surface and the ex- pression of transcripts such as E11/gp38/podoplain [4], matrix extracellular phosphoglycoprotein (MEPE) and phosphate- regulating gene with homologies to endopeptidases on the X chromosome (Phex). This cell, opposite to the more mature osteocyte, is negative for sclerostin and fibroblast growth fac- tor (FGF)-23 expression but does express DMP1, as elegantly demonstrated by Kalajzic et al. [5] in a recent review. It is not clear, however, if osteoid, mineralizing and mature osteocytes exert different biological functions or if they all work as mechanosensor and skeletal regulators. Indeed recent studies suggest that cortical and trabecular osteocytes might have dis- tinct roles with the former being the sensor of load while the latter being the controller of bone metabolism. Windahl et al. [6], for example, reported that estrogen receptor-α ablation in osteocytes differentially affects trabecular and cortical bone compartments in males mice. for over 30 years that osteocytes are mechanosensor of bone. They are indeed the cells capable of sensing mechanical forces applied to the skeleton and transform these forces into biologi- cal stimuli. It is now widely accepted that the cell perceives forces in the form of shear stress created by the flow of fluid inside the lacuna-canalicular network. It has been proposed that the flow derives from both the compression induced by loading and the extravascular pressure. Efforts to physically measure and quantify this flow are currently ongoing and re- cent reports suggest that osteocytes are subjected to forces in the order of 5 Pa [12]. Although the theory that osteocytes are the mechanosensor of bone has been around for decades, the definite demonstration of this theory comes from work of Tat- sumi et al. [13]. Using a mouse model of targeted osteocytes ablation, they demonstrated resistance to disuse induces bone loss highlighting the central role that osteocytes play in mech- anosensation. How an osteocyte can sense the fluid flow in the lacuno-canalicular network is still not completely understood. THE OSTEOCYTES This demonstrates that osteocytes are key orchestrators not only of skeletal responses to loading but also to unloading, as occurs during paralysis, pro- longed bed rest or space flight. Frost [22] postulated that osteo- cytes are the mechanostat of bone, ascribing to these cells the role of driving skeletal adaptation to mechanical forces. Bone adapts its form to mechanical demands trough a mechanisms known as bone modeling. During modeling, bone resorption, OSTEOCYTE FUNCTIONS: MECHANOTRANSDUCTION What are the functions of an osteocyte? It has been recognized Copyright © 2013 Korean Endocrine Society www.e-enm.org Recent Progress in Osteocytes driven by osteoclasts, and bone formation, osteoblasts-mediated, occurs on different surfaces of the skeleton such as the bone un- dergoes reshaping to adapt to different loading conditions. Bone remodeling, on the other hand, is the continuous and spatially coupled, resorption and formation of the skeleton to preserve functional integrity. According to Frost [22], the mechanostat distinguishes between bone modeling (shape change) and re- modeling (replacement only). Examples of bone adaptation to loading are evident in professional athletes, where the high load applied to the skeleton results in stronger and bigger bones, whereas in astronauts or in paralyzed patients there is severe bone loss due to unloading. As described above, compelling evi- dences ascribe to sclerostin an important role in this disuse in- duced bone loss. duced periosteal circumference and have a blunted response to mechanical forces. When IGF-1 conditional KO mice were subjected to mechanical loading, there was a significant reduc- tion in osteoblastic bone formation response, indicating that IGF-1 is an important determinant of both bone size and bone strength [30]. Pioneering studies on osteocytes involving histological analysis of bone specimens raised the possibility that osteo- cytes could reabsorb bone and directly contribute to mineral- ion homeostasis. The finding that in various diseases (renal osteodystrophy, hyperparathyroidism, and immobilization) the size of the osteocytic lacunae was increased led to the hypoth- esis that these cells were indeed capable of modifying the perilacunar mineralized matrix. This theory was quickly aban- doned when isolated osteocytes failed to reabsorb bone is a classical osteoclast pit-forming assay and few decades later, the concept that osteocytes can indeed reabsorb their perilacu- nal matrix has been revisited. The current osteocytic osteolysis concept, as proposed by Cullinane [31], suggests that osteo- cytes do not remove substantial amount of bone (as osteo- clast), but rather modify the matrix minerals in the perilacunar areas. This perilacunal remodeling is evident during continu- ous infusion with PTH [32], treatment with prednisone [33] or during lactation [34]. As described above, osteocytes express receptor for PTH, a known regulator of calcium homeostasis, and they are the ideal candidates for systemic homeostasis regulation. Mice lacking the PTH1R specifically in osteocytes have indeed an impaired calcium homeostasis when subjected to a low calcium diet, indicating these cells also control calci- um homeostasis [35]. OSTEOCYTE FUNCTIONS: MECHANOTRANSDUCTION Lastly, when the vitamin D receptor is ablated from osteocytes, the animals are unable to mobilize calcium from bone under a vitamin D stimulus, indicating that vitamin D signaling in osteocytes is critical for moving calci- um from skeletal stores to the blood [36]. In light to recent re- port indicating that osteocytes are the major source of RANKL and therefore bone remodeling [26,27], we can speculate that the calcemic effect of vitamin D on bone is via osteocyte-de- rived RANKL although evidences for this pathway are still missing. BONE AND MINERAL HOMEOSTASIS Osteocytes are not only the sensor of mechanical forces, they are also master controller of bone and mineral homeostasis. These cells are, postnatally, the main source sclerostin and re- ceptor activation of nuclear factor-κB ligand (RANKL). Sclerostin, the product of the gene SOST, has emerged as a powerful inhibitor of bone formation. The protein binds to LRP5 and the related LRP6 and four receptors and block the canonical Wnt-ßcatenin signaling pathway. Lack (or mutation) of sclerostin induces sclerosteosis whereas its over expression causes severe osteopenia (for review on Wnts) [23]. Osteo- cytes are also the main source of FGF-23, a key regulator, to- gether with parathyroid hormone (PTH) of phosphate homeo- stasis [24,25]. Thus, it is not surprising that genetic manipula- tion of osteocytes induced both skeletal and mineral defects. Mice lacking RANKL in osteocytes have increased bone min- eral density and reduced bone remodeling [26,27], as do mice lacking the receptor of PTH (PTH1R). Mice lacking Atg7, an autophagy gene, from osteocytes, display similar skeletal phe- notype as aging mice, indicating that these cells are key regu- lator of skeletal metabolism [28]. Both DMP1-null mice and individuals with inactivating mutation of this gene have rickets and osteomalacia and are hypophostatemic as a consequence of high levels of FGF-23 [29]. On the contrary, mice lacking FGF-23 are hyperphosphatemic, osteopenic, and dye prema- turely. It has been reported that osteocytes are also the major source of insulin-like growth factor (IGF)-1 and IGF-1 pro- duced by these cells is required for proper anabolic responses to mechanical loading. Deletion of IGF-1 in osteocytes, (using the DMP1-Cre promoter) impairs skeletal growth, have re- www.e-enm.org  2 www.e-enm.org  257 HEMATOPOIESIS Hematopoiesis, the process that continuously gives rise blood cells, is tightly regulated not only by intrinsic factors but also by extrinsic clues derived from various cells within the bone marrow microenvironment. The bone marrow is indeed the pri- Copyright © 2013 Korean Endocrine Society www.e-enm.org  257 www.e-enm.org  257 Pajevic PD Pajevic PD mary site for hematopoiesis that is highly orchestrated by inter- actions between the hematopoietic stem cells (HSCs) and their niches on the endosteal bone surface. The HSC niche consists of cells from the osteolineage, sinusoidal endothelial cells, mesenchymal stromal and stem cells, sympathetic neurons, and the extracellular matrix [37,38]. In these niches, the HSC un- dergo self-renewal and/or differentiate into hematopoietic lin- eage with cues from the niche-supporting cells, which provide structural as well as signaling support for the hematopoietic microenvironment. Osteoblasts have been shown as critical regulators of HSC maintenance, proliferation, and maturation [39,40] via various cell-surface proteins such as Notch1 ligand Jagged1 and via secreted cytokines such as CXCL12. Osteo- blasts also provide a niche for B-lymphopoiesis in part via in- terleukin-7 expression through Gsα-signaling [41]. Hypoxic osteoblasts are also a major producer of erythropoietin through which they regulate erythropoiesis [42]. Moreover, osteoclasts has been shown to be potential HSC regulators through degra- dation of endosteal components and promotion of mobilization of HSCs [43]. In contrast, the role of osteocytes in HSC and progenitor regulation, and hence hematopoiesis, has remained completely unexplored despite the fact that these cells com- prise 90% to 95% of all bone cells in the adult skeleton and they produce an extensive canalicular network that reaches the endosteal bone surface, a preferred site for the HSC niche. Os- teoblasts and osteocytes express several G-protein coupled re- ceptor (GPCR) and signaling trough these receptor s has been shown to control the niche. Interestingly constitutive activation of the PTH/PTHrP receptor in osteoblasts increased the num- bers of HSCs [39] whereas its expression in osteocytes does not [44], suggesting that cells early in the osteoblast lineage are critical for maintaining an intact HSC niche. To examine the role of osteocytes and GPCR signaling in regulation of hema- topoiesis we recently generated mice lacking Gsα in osteocytes (Ocy-GsαKO). Surprisingly, ablation of Gsα from these cells in- duces a marked neutrophilia, thrombocytopenia and spleno- megaly. CONFLICTS OF INTEREST No potential conflict of interest relevant to this article was re- ported. FUTURE DIRECTIONS It is evident that there is a need for further investigations aimed at better understand osteocytes function. Several ques- tion remain unanswered and areas in need of better under- standing are numerous. For example, which are the molecular cues that signal an osteoblast to become an osteocyte? How can osteocytes sense mechanical forces applied to the skeleton and what is the role of primary cilia? What control the in- creased in SOST/sclerostin induced by unloading? How can an osteocyte sense phosphate? What is the role of osteocytes in hematopoiesis? These are many more other unanswered questions will require better tools and in vitro models to study and analyze these cells and advance our understanding of their complex functions even further. HEMATOPOIESIS Ocy-GsαKO mice displayed a profound myeloprolifera- tive phenotype characterized by a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood and marked [45]. Using a novel ex vivo coculture system using osteocyte- enriched bone explants we identified granulocyte colony-stim- ulating factor as the principal cytokine regulating granulopoie- sis in these mice Interestingly PTH1R signaling in osteocytes SOST/sclerostin, a Wnt inhibitor and suppressor of osteoblast proliferation and functions, was increased significantly in these mice. Treatment with antisclerostin antibody partially restored the number of osteoblasts in Ocy-GsαKO mice without rescuing the bone marrow abnormalities demonstrating that the hemato- poietic abnormalities present in these mice are independent on osteoblasts or increased SOST/sclerostin expression [45]. On the other hand, SOST/sclerostin can directly control the fate of B-cells, as recently reported [46]. Cain et al. [46] showed that SOST-/- mice, despite the high bone mass and increased num- ber of osteoblasts, have no differences in the frequency or ab- solute number of HSCs, common lymphoid progenitors, com- mon myeloid/megakaryocyte erythroid progenitors, or granu- locyte/monocyte progenitors, confirming the findings in mice overexpressing the constitutive PTH/PTHrP receptor in osteo- cytes [44]. Interestingly, in these mice, B cells are significantly reduced in both their frequency and cell number in the bone marrow and the reduction was a consequence of increased apoptosis due to a reduction in Cxcl12 expression in the stro- mal cells. www.e-enm.org REFERENCES 13. Tatsumi S, Ishii K, Amizuka N, Li M, Kobayashi T, Kohno K, Ito M, Takeshita S, Ikeda K. Targeted ablation of osteo- cytes induces osteoporosis with defective mechanotrans- duction. Cell Metab 2007;5:464-75. 1. Gaillard PJ, Talmage RV, Budy AM, te Leiden R; Rice Uni- versity. 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Adv Exp Med Biol 2012; 728:41-64. 25. Bergwitz C, Jüppner H. Regulation of phosphate homeosta- sis by PTH, vitamin D, and FGF23. Annu Rev Med 2010; 61:91-104. 36. Lieben L, Masuyama R, Torrekens S, Van Looveren R, Schrooten J, Baatsen P, Lafage-Proust MH, Dresselaers T, Feng JQ, Bonewald LF, Meyer MB, Pike JW, Bouillon R, Carmeliet G. Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization. J Clin Invest 2012;122: 1803-15. 26. Nakashima T, Hayashi M, Fukunaga T, Kurata K, Oh-Hora M, Feng JQ, Bonewald LF, Kodama T, Wutz A, Wagner EF, Penninger JM, Takayanagi H. Evidence for osteocyte regulation of bone homeostasis through RANKL expres- sion. Nat Med 2011;17:1231-4. 27. Xiong J, Onal M, Jilka RL, Weinstein RS, Manolagas SC, O’Brien CA. Matrix-embedded cells control osteoclast for- mation. Nat Med 2011;17:1235-41. 37. Krause DS, Scadden DT. Deconstructing the complexity of a microenvironmental niche. Cell 2012;149:16-7. 38. Krause DS, Scadden DT, Preffer FI. The hematopoietic stem cell niche: home for friend and foe? Cytometry B Clin Cytom 2013;84:7-20. 28. Onal M, Piemontese M, Xiong J, Wang Y, Han L, Ye S, Komatsu M, Selig M, Weinstein RS, Zhao H, Jilka RL, Al- meida M, Manolagas SC, O’Brien CA. Suppression of au- tophagy in osteocytes mimics skeletal aging. J Biol Chem 2013;288:17432-40. 39. Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP, Schipani E, Divieti P, Bring- hurst FR, Milner LA, Kronenberg HM, Scadden DT. Os- teoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841-6. 29. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, Yu X, Rauch F, Davis SI, Zhang S, Rios H, Drezner MK, Quarles LD, Bonewald LF, White KE. Loss of DMP1 causes rick- ets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310-5. 40. Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, Ross J, Haug J, Johnson T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Li L. Pajevic PD REFERENCES J Bone Miner Res 2003;18:807-17. JM, Koskela A, Tuukkanen J, Divieti Pajevic P, Feng JQ, 19. Harris SE, Gluhak-Heinrich J, Harris MA, Yang W, Bone- wald LF, Riha D, Rowe PS, Robling AG, Turner CH, Feng JQ, McKee MD, Nicollela D. DMP1 and MEPE expression are elevated in osteocytes after mechanical loading in vivo: theoretical role in controlling mineral quality in the perila- cunar matrix. J Musculoskelet Neuronal Interact 2007; 7:313-5. 7. Marotti G, Ferretti M, Remaggi F, Palumbo C. Quantita- tive evaluation on osteocyte canalicular density in human secondary osteons. Bone 1995;16:125-8. 8. Palumbo C, Palazzini S, Marotti G. Morphological study of intercellular junctions during osteocyte differentiation. Bone 1990;11:401-6. 20. 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Copyright © 2013 Korean Endocrine Society www.e-enm.org Recent Progress in Osteocytes Gsα-dependent signaling. Blood 2013;121:930-9. Gsα-dependent signaling. Blood 2013;121:930-9. cells. Blood 2012;119:2489-99. 46. Cain CJ, Rueda R, McLelland B, Collette NM, Loots GG, Manilay JO. Absence of sclerostin adversely affects B-cell survival. J Bone Miner Res 2012;27:1451-61. 45. Fulzele K, Krause DS, Panaroni C, Saini V, Barry KJ, Liu X, Lotinun S, Baron R, Bonewald L, Feng JQ, Chen M, Wein- stein LS, Wu JY, Kronenberg HM, Scadden DT, Divieti Pa- jevic P. Myelopoiesis is regulated by osteocytes through www.e-enm.org  261 Copyright © 2013 Korean Endocrine Society
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Outcomes in patients with lacrimal gland carcinoma treated with definitive radiotherapy or eye-sparing surgery followed by adjuvant radiotherapy
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RESEARCH Open Access Abstract Background: The optimal treatment for lacrimal gland cancer remains unclear. Eye-preserving surgery, as opposed to exenteration, followed by adjuvant radiotherapy (RT), has recently been reported to deliver satisfactory outcomes, but evidence is sparse. The aim of the present study was to evaluate outcomes in patients with lacrimal gland cancer treated at two tertiary medical centers. Methods: We retrospectively examined data from patients with lacrimal gland cancer who had received eye- preserving surgical treatment followed by adjuvant RT with or without chemotherapy, or (if the tumor was inoperable) needle biopsy with definitive RT with or without chemotherapy. Baseline clinical and pathological characteristics were considered. Outcomes of interest included post-treatment complications, overall survival (OS), locoregional progression-free survival (LPFS), and distant metastasis-free survival (DMFS). Results: Eighteen patients were included. Two-year OS, LPFS, and DMFS rates were 69.0, 76.7, and 71.4%, respectively. Patients with early-stage (T1–T2) lacrimal gland cancer had significantly better outcomes than those with advanced-stage disease (T3–T4). Two-year OS, LPFS, and DMFS rates were each 100% in patients with disease stages T1–T2, and 37.5, 50, and 37.5%, respectively, in those with disease stages T3–T4 (P < 0.05). Orbital complications were well tolerated. Conclusions: Eye-sparing surgery with adjuvant RT can achieve satisfactory results in patients with T1–T2 lacrimal gland carcinoma. Disease stage T3 and above was associated with poor outcomes even with post-operative RT, likely due to distant metastasis. Adding neoadjuvant chemotherapy or adjuvant chemotherapy to current treatment strategies might be a suitable choice for this group of patients. Keywords: Lacrimal gland carcinoma, Orbital tumors, Radiotherapy, Orbital exenteration * Correspondence: ocean@ntut.edu.tw †Yun-Hsuan Lin and Shih-Ming Huang contributed equally to this work. 1Department of Electro-Optical Engineering, National Taipei University of Technology, 1, Sec. 3, Chung-Hsiao E. Rd, Taipei 10608, Taiwan, ROC Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. RESEARCH Open Access Outcomes in patients with lacrimal gland carcinoma treated with definitive radiotherapy or eye-sparing surgery followed by adjuvant radiotherapy Yun-Hsuan Lin1,2,3†, Shih-Ming Huang3,4,5†, Wing-Keen Yap5, Ju-Wen Yang2,3, Ling Yeung2,3, Din-Li Tsan4, Joseph Tung-Chieh Chang5 and Lung-Chien Chen1* Lin et al. Radiation Oncology (2020) 15:156 https://doi.org/10.1186/s13014-020-01601-8 RESEARCH Open Access Outcomes in patients with lacrimal gland carcinoma treated with definitive radiotherapy or eye-sparing surgery followed by adjuvant radiotherapy Yun-Hsuan Lin1,2,3†, Shih-Ming Huang3,4,5†, Wing-Keen Yap5, Ju-Wen Yang2,3, Ling Yeung2,3, Din-Li Tsan4, Joseph Tung-Chieh Chang5 and Lung-Chien Chen1* Lin et al. Radiation Oncology (2020) 15:156 https://doi.org/10.1186/s13014-020-01601-8 Lin et al. Radiation Oncology (2020) 15:156 https://doi.org/10.1186/s13014-020-01601-8 Outcomes in patients with lacrimal gland carcinoma treated with definitive radiotherapy or eye-sparing surgery followed by adjuvant radiotherapy Yun-Hsuan Lin1,2,3†, Shih-Ming Huang3,4,5†, Wing-Keen Yap5, Ju-Wen Yang2,3, Ling Yeung2,3, Din-Li Tsan4, Joseph Tung-Chieh Chang5 and Lung-Chien Chen1* © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 10 Page 2 of 10 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Radiation Oncology Precis Eye-sparing surgery with adjuvant radiotherapy can achieve satisfactory results in patients with T1–2 lacri- mal gland carcinoma. Disease stage T3 and above was associated with poor outcomes even with post-operative RT, likely due to distant metastasis. This retrospective study included patients with lacrimal gland cancer who underwent either eye-preserving surgi- cal treatment followed by adjuvant RT with or without chemotherapy, or (in cases involving an inoperable tumor), needle biopsy followed by definitive RT with or without chemotherapy. Patients diagnosed with recur- rent tumors at their first visit, and patients diagnosed with lacrimal duct cancer or other type of orbital cancer, were excluded. Disease stage was reviewed by a radiolo- gist and ophthalmologist based on the American Joint Committee on Cancer (AJCC) staging system, eighth edition. All patient records included detailed patho- logical reports with data on cell type, margin status, perineural invasion (PNI), and lymphovascular invasion (LVSI), if the tumor had undergone resection. Treatment Radiotherapy was recommended for patients at high risk of recurrence, including those with an advanced stage lacrimal gland cancer and/or multiple unfavorable pathological factors such as bony structure invasion, positive surgical margins, LVSI, or PNI. RT was deliv- ered in the form of three-dimensional conformal RT, intensity modulated RT, volumetric modulated arc ther- apy, or proton beam therapy. The RT field included the tumor bed and the residual gross tumor volume. The median prescribed dose for adjuvant RT was 50–60 Gy and 66–70 Gy for the definitive setting, with 1.8–2 Gy daily fractions administered over 5–7 weeks. Whether or not chemotherapy was given was at the attending physi- cian’s discretion. The regimens used were platinum- based chemotherapy. Lacrimal gland carcinomas are associated with poor local control and significant morbidity and mortality rates [9]. To date, no guidelines on standard treatment for lacrimal gland carcinomas have been developed due to the rarity of these diseases, which are conventionally treated with orbital exenteration followed by radio- therapy (RT) [5, 10]. However, locoregional recur- rence, distant relapse, and cancer-related mortality risks are high after orbital exenteration [5, 11, 12]. Thus, eye-preserving surgery followed by adjuvant RT has recently gained popularity, but the optimal ap- proaches to surgery and RT in these patients remain subject to debate [5, 9, 13, 14]. Statistical analyses ll l ( In this study, we examined clinical outcomes associ- ated with either eye-preserving surgery followed by adjuvant RT, or needle biopsy followed by definitive RT, in a cohort of 18 patients with lacrimal gland carcinoma. Clinical outcomes such as local control, survival, and ocular complications were considered. The clinical and pathological factors that affected the outcomes were also examined. Overall survival (OS) was defined as the time from the date of surgical treatment or biopsy until death or the last follow-up appointment. Local progression-free sur- vival (LPFS) was defined as the time between the date of surgical treatment or biopsy and the detection of local tumor progression. Data on the first site(s) of distant metastasis were collected, and distant metastasis-free survival (DMFS) was determined. The Kaplan–Meier method was used to estimate the OS, LPFS, and DMFS rates, while the statistical significance of between-group differences in clinical characteristics (including sex, age, RT dose, cell types, and pathological features) and OS, LPFS, DMFS was determined using the log-rank test. All statistical analyses were performed using SPSS v. 23.0 (IBM Corp., New York, NY; formerly SPSS Inc., Chicago, IL). All P-values were two-sided, and a P-value < 0.05 was considered statistically significant. Introduction Lacrimal gland tumors are among the rarest types of head and neck cancers, accounting for approximately 10% of all orbital tumors [1]. Previous studies have esti- mated annual incidence for these tumors at 0.19–1 per 1,000,000 people [1–3]. Epithelial lesions, including be- nign pleomorphic adenoma and malignant tumors, ac- count for 20–30% of all lacrimal gland tumors. Malignant epithelial tumors include adenoid cystic car- cinoma (ACC) (60%), carcinoma ex pleomorphic aden- oma or adenocarcinoma (20–30%), and mucoepidermoid carcinoma (5%) [1, 4, 5]. In contrast, non-epithelial le- sions, such as inflammation, lymphoid tumors, plasma- cytoma, histiocytoma, lipoma, and hemangioma account for 70–80% of all lacrimal gland tumors [2, 4, 6–8]. Survival analysis For the entire cohort, 2-year OS, LPFS, and DMFS rates were 69.0, 76.7, and 71.4%, respectively (Fig. 2). Asso- ciations between clinicopathological factors and 2-year OS, LPFS, and DMFS rates are shown in Table 3. Pa- tients with early-stage (T1–T2) lacrimal cancer achieved significantly better 2-year OS, LPFS, and DMFS than those with advanced-stage disease (T3– T4). Two-year OS was 100% in patients with disease stage T1–T2, and 37.5% in those with disease stage T3–T4. Moreover, 2-year LPFS was 100% in patients with disease stage T1–T2 and 50% in those with dis- ease stage T3–T4. Finally, 2-year DMFS was 100% in patients with disease stage T1–T2 and 37.5% in those with disease stage T3–T4 (Fig. 3). Other factors, in- cluding sex, age (< 54 yrs. vs. ≥54 yrs), RT dose (< 66 vs. ≥66 Gy), cell types (ACC vs. others), margin (posi- tive vs. negative), PNI (positive vs. negative), and LVSI (positive vs. negative) had no significant impact on OS, LPFS, or DMFS. Materials and methods This study protocol was reviewed and approved by the institutional review board of Chang Gung Me- morial Hospital (Reference No.: 202000138B0). All data were stored in the hospital database and ex- tracted for research. The participants’ informed con- sent requirement was waived due to the retrospective nature of this study. Page 3 of 10 Page 3 of 10 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Radiation Oncology Post-therapy surveillance RT was 6000 cGy (range 3600–7000 cGy) in 1.8–2 Gy per fraction, and the definitive RT dose was 6000 cGy/25 fractions, 7200 cGy/36 fractions, and 7200 cGy/36 fractions for three patients, respectively. Follow-up in the form of radio–oncologic and ophthal- mic clinic appointments was arranged every 3 months during the first 2 years, every 4–6 months during the third and fourth year, and every 6–12 months thereafter. Imaging examination was performed at specific intervals: chest radiography every 3 months, and computed tom- ography or orbital magnetic resonance imaging every 3– 6 months, until symptoms indicating recurrence were noted. Ocular complication evaluation Follow-up in the form of ophthalmic clinic appoint- ments was arranged every 3 months during the first 2 years and every 6–12 months thereafter. Detailed ocular examination, including visual acuity, intraocular pres- sure, slit lamp examination, and fundus examination was performed at every ophthalmologic visit. Medical or sur- gical treatment was administered if an ocular complica- tion was noted. Patient characteristics During 2000–2018, a total of 18 patients from the Lin- kou and Keelung branches of Chang Gung Memorial Hospital were included in the present study. The median follow-up time was 39 months (range 5–161 months) for the patients who were alive at the end of the study period, and 26 months (range 5–161 months) for the whole cohort. The patients’ demographic and clinical characteristics are shown in Table 1 and Table 2. Fifteen patients (83.3%) underwent eye-sparing surgery and the remaining patients had needle biopsy alone due to inop- erable tumor invading brain tissue or extending to the infratemporal fossa. ACC was found in nine, carcinoma ex pleomorphic adenoma in seven, and poorly differenti- ated adenocarcinoma in two patients. In five patients (27.8%), negative margins were achieved during surgery. Six patients (33.3%) had pathologically confirmed LVSI and eight patients (44.4%) had pathologically confirmed PNI. Patients who underwent eye-sparing surgery also received adjuvant RT or concurrent platinum-based che- moradiotherapy (CCRT) due to the presence of risk fac- tors, such as positive surgical margin, confirmed LVSI or PNI, or initial advanced tumor size and invasion status. Three patients who underwent needle biopsy received definitive RT or CCRT, also platinum-based. Ocular toxicity analysis In our study subjects, one patient was lost follow up at the ophthalmic clinic 3 months after surgery and three patients 6 months after surgery. During their follow-up period, no ocular complications were reported. In the remaining 14 patients who were followed up by the oph- thalmologist for more than 12 months, the administered treatment was well tolerated. Treatment-related acute and late ocular toxicity effects are shown in Table 4. Ocular toxicities, including punctate keratitis, devel- oped in five patients (27.78%) 1.14–7.9 months after radiotherapy (RT total/fraction dose: 5000–7000/200 cGy). Cataract developed in four patients (22.22%) and occurred 2.13–112.7 months after the RT (RT total/fraction dose: 6000–6600/200 cGy). Dry eye de- veloped in two patients (11.11%) and occurred 6.29– 11.05 months after the RT (RT total/fraction dose: 6600/200 cGy). Blepharitits, which developed in two patients (11.11%), occurred 2.06–8.00 months after the treatment (RT total/fraction dose: 6600/200 cGy). Other complications, including trichiasis, acute con- junctivitis, corneal epithelial defect, filamentary kera- titis, radiation retinopathy, and vitreous hemorrhage were each reported in one patient (5.56%). The pa- tient with radiation retinopathy received an intravitre- ous injection of 1.25 mg Avastin (bevacizumab) for retinal neovascularization. Regarding RT technique, 2 patients received three- dimensional conformal radiotherapy (3DCRT), 14 re- ceived intensity-modulated radiotherapy (IMRT), one received proton beam therapy alone, and one received combined proton–photon RT. The dosimetry com- parison between photon plan and proton plan is pre- sented in Fig. 1. The median RT dose for adjuvant Page 4 of 10 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Ocular toxicity analysis Radiation Oncology Table 1 Baseline characteristics of patients with lacrimal gland carcinoma Total (n = 18) Patient number Percent Median age (years) 54 (20–85) Sex Male 8 44.4 Female 10 55.6 T stage 1 1 5.6 2 9 50.0 3 3 16.7 4 5 27.7 Types of surgery Excision 15 83.3 Needle biopsy 3 16.7 RT treatment Adjuvant RT alone 13 72.2 Adjuvant CCRT 2 11.1 Definitive RT 2 11.1 Definitive CCRT 1 5.6 RT type 3DCRT 2 11.1 IMRT 14 77.8 Proton 2 11.1 RT dose < 6600 cGy 9 50.0 ≥6600 cGy 9 50.0 Cell type Adenoid cystic carcinoma 9 50.0 Carcinoma ex pleomorphic adenoma 7 38.9 Poorly differentiated adenocarcinoma 2 11.1 Margin positive 13 72.2 negative 5 27.8 LVSI Positive 6 33.3 Negative 12 66.7 PNI Positive 8 44.4 Negative 10 55.6 RT radiotherapy, CCRT concurrent chemoradiotherapy, 3DCRT 3-dimensional conformal radiotherapy, IMRT intensity-modulated radiotherapy, cGy centigray, lymphovascular invasion, PNI perineural invasion. ncurrent chemoradiotherapy, 3DCRT 3-dimensional conformal radiotherapy, IMRT intensity-modulated radiotherapy, cGy centigray, LVSI PNI perineural invasion. RT radiotherapy, CCRT concurrent chemoradiotherapy, 3DCRT 3-dimensional conformal radiotherapy, IMRT intensity-modulated radiotherapy, cGy centigray, LVSI lymphovascular invasion, PNI perineural invasion. Discussion treatment followed by adjuvant radiotherapy with or with- out chemotherapy. In the present cohort, 2-year OS, LPFS, and DMFS rates were 69.0, 76.7, and 71.4%, respectively. These outcomes were inferior to those reported in previ- ously, which was probably due to differences in patient This study reported outcomes in patients with lacrimal gland carcinoma who underwent either needle biopsy followed by definitive radiotherapy with or without chemo- therapy due to inoperable tumor, or eye-preserving surgical Page 5 of 10 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Discussion Page 6 of 10 (2020) 15:156 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Radiation Oncology Fig. 1 Patient number 7, cT2bN0M0 lacrimal gland cancer, pre-surgery T1-MRI and the photon or proton beam dosimetry of adjuvant radiotherapy. Total RT dose was 70 Gy in 33 fractions. Dose constraints for organs at risk: Brain stem: Dmax < 54 Gy. Left eye and left optic nerve: Dmax ≤59.5Gy (85% dose). Right eye and right optic nerve: Dmax ≤50 Gy. Optic chiasm: Dmax < 54 Gy. Right len: Dmax ≤18Gy. No ocular complication was noted during our study period. a. Axial view of the tumor. b. Sagittal view of the tumor. c. Photon beam dosimetry. 1d. Proton beam dosimetry Fig. 1 Patient number 7, cT2bN0M0 lacrimal gland cancer, pre-surgery T1-MRI and the photon or proton beam dosimetry of adjuvant radiotherapy. Total RT dose was 70 Gy in 33 fractions. Dose constraints for organs at risk: Brain stem: Dmax < 54 Gy. Left eye and left optic nerve: Dmax ≤59.5Gy (85% dose). Right eye and right optic nerve: Dmax ≤50 Gy. Optic chiasm: Dmax < 54 Gy. Right len: Dmax ≤18Gy. No ocular complication was noted during our study period. a. Axial view of the tumor. b. Sagittal view of the tumor. c. Photon beam dosimetry. 1d. Proton beam dosimetry carcinoma (three patients with T3 [16.7%], and five patients with T4 [27.7%]). In addition, the majority of patients were at high risk of locoregional or distant recurrence, with a positive surgical margin in 72.2% of the patients, and confirmed LVSI and PNI in 33.3 and 44.4% of patients, respectively. The differences in patient baseline characteristics may explain the rela- tively lower OS, LPFS, and DMFS rates reported in our study. characteristics. Esmaeli et al. [15] studied the eye-sparing approach to treating lacrimal gland carcinoma in 11 pa- tients during 2007–2014. All 11 patients were disease-free at the last follow-up visit after the eye-sparing surgery, with a median follow-up time of 33 months (range, 14–64 months). A significant proportion of patients recruited by Esmaeli et al. had early-stage disease (one patient with T1, six patients with T2, one with T3, and three with T4). In addition, Han et al. Discussion Radiation Oncology Table 2 Detailed patient, tumor, treatment characteristics and outcomes Patient Age Gender Cell type AJCC stage laterality Surgery type Margin PNI LVSI Adjuvant treatment Post treatment status Time to local progression (months) Time to distant metastasis (months) Metastasis site Total follow up time (months) Final status 1 45 Female CEPA cT1aN0M0 L Excision + – – RT alone CR – – 161 alive 2 20 Female ACC cT2aN0M0 L excision – + – RT alone CR – – 28 dead 3 58 Female ACC cT2aN0M0 L excision + – – RT alone CR – – 20 alive 4 55 Male CEPA cT2aN0M0 R excision + – + RT alone CR – – 39 alive 5 77 Male CEPA cT2aN0M0 R excision + – – RT alone CR – – 26 alive 6 53 Female CEPA cT2aN0M0 L excision – + + RT alone (proton) CR – – 5 alive 7 45 Male ACC cT2bN0M0 L excision + – – CCRT (proton + photon) CR – – 14 alive 8 35 Male ACC cT2cN0M0 L excision + + + RT alone CR – – 158 alive 9 43 Female ACC cT2cN0M0 R excision + + – RT alone CR – 26 mediastinal LNs, liver, bone, lungs 26 dead with disease 10 53 Female ACC cT2cN0M0 R excision + + + RT alone CR – – 40 alive 11 85 Male ACC cT3cN0M0 L needle biopsy – – – RT alone PR 10 34 lungs 43 alive with disease 12 62 Male CEPA cT3cN0M0 L excision + – + RT alone CR 50 51 lungs 92 dead with disease 13 80 Male CEPA cT3cN0M0 R needle biopsy – – – RT alone CR – – 17 dead 14 36 Female ACC cT4bN0M0 L excision + + – RT alone CR 10 6 liver 16 dead with disease 15 30 Female ACC cT4bN0M0 L excision + + – RT alone CR 5 10 bone and lungs 28 dead with disease 16 58 Female CEPA cT4bN0M0 R excision + + + RT alone CR 6 5 parotid, neck nodes, lungs 12 dead with disease 17 60 Male PD adeno cT4cN0M0 L needle biopsy – – – CCRT PR – 5 bone 20 dead with disease 18 71 Female PD adeno cT4cN0M0 R excision + – – CCRT CR – 5 liver and bone 15 dead with disease AJCC the American Joint Committee on Cancer, PNI perineural invasion, LVSI lymphovascular invasion, ACC adenoid cystic carcinoma, CEPA carcinoma ex pleomorphic adenoma, PD adeno poorly differentiated adenocarcinoma, L left, R right, RT radiotherapy, CCRT concurrent chemoradiotherapy, CR=, LN lymph node. Discussion [16] reported outcomes following eye-sparing surgery and adjuvant radiother- apy in 10 patients with adenoid cystic carcinoma of the lacrimal gland, treated during 1998–2012 (one pa- tient with T1, seven with T2, and two with T3 stage disease), at a median follow-up time of 89.5 months (range 37–217 months). Reported OS was 90%, with a single patient who died 58 months after surgery. Moreover, DMFS was 100%, while local recurrence occurred in one patient. In our series, almost half of the patients had advanced-stage lacrimal gland The present study is among a small number that have examined factors affecting outcomes in patients with lacrimal gland carcinoma, which is an aim that is difficult to achieve due to the rarity of this disease. In a previous report by Woo et al., the only tumor characteristic associated with outcome was T stage. In the same study, adjuvant radiotherapy was strongly associated with a better 5-year recurrence-free sur- vival rate [17]. Friedrich and Bleckmann also found that lower T stage was associated with a better Lin et al. Radiation Oncology (2020) 15 Page 7 of 10 Lin et al. Radiation Oncology Fig. 2 The overall survival (OS), local progression-free survival (LPFS), and distant metastasis-free survival (DMFS) graphs of the whole cohort. For the entire cohort, 2-year OS, LPFS, and DMFS rates were 69.0, 76.7, and 71.4%, respectively Fig. 2 The overall survival (OS), local progression-free survival (LPFS), and distant metastasis-free survival (DMFS) graphs of the whole cohort. For the entire cohort, 2-year OS, LPFS, and DMFS rates were 69.0, 76.7, and 71.4%, respectively Fig. 2 The overall survival (OS), local progression-free survival (LPFS), and distant metastasis- the entire cohort, 2-year OS, LPFS, and DMFS rates were 69.0, 76.7, and 71.4%, respectively outcomes even when treated with surgical resection followed by adjuvant RT, which occurred most likely due to distant metastasis. Adding neoadjuvant chemo- therapy or adjuvant chemotherapy to current treat- ment strategies might be a suitable choice for this group of patients to improve the control of distant disease. prognosis [18]. Among factors reported as negatively affecting patient outcomes were older age, basaloid or solid pattern ACC, perineural invasion, and the pres- ence of a macroscopic tumor on imaging scans before RT. [12, 19–22] In the present study, among patients who had received adjuvant radiotherapy, T stage and tumor size emerged as the most important factors that determined patient outcome. LPFS local progression free survival, DMFS distant-metastasis free survival, RT radiotherapy, ACC adenoid cystic carcinoma, PNI perineu ular invasion g Y year, OS overall survival, LPFS local progression free survival, DMFS distant-metastasis free survival, RT radiotherapy, ACC adenoid cy invasion, LVSI lymphovascular invasion Log rank test Y year, OS overall survival, LPFS local progression free survival, DMFS distant-metastasis free survival, RT radiotherapy, ACC adenoid cystic carcinoma, PNI perineural invasion, LVSI lymphovascular invasion Discussion Specifically, patients with tumor at stage T1–2 treated with eye-sparing surgery followed by adjuvant radiotherapy achieved 100% 2-year LPFS, DMFS, and OS rates. However, tumor stage T3 and higher was associated with poor Proton therapy or even heavy ion therapy applied to lacrimal gland cancer is considered promising for re- ducing low dose delivered outside the treated fields [3]. We have observed better dosimetry associated with the proton plan compared to the photon plan. Table 3 Two-year overall survival, local progression-free survival, and distant-metastasis free survival rates for patients with lacrimal gland carcinoma, including factors affecting survival 2-Y OS (%) P-value* 2-Y LPFS (%) P-value* 2-Y DMFS (%) P-value* Sex (male vs. female) 87.5 vs. 67.5 0.533 87.5 vs. 67.5 0.533 87.5 vs. 57.1 0.353 Age (< 54 vs. ≧54) 85.7 vs. 55.6 0.529 76.2 vs. 77.8 0.621 75.0 vs. 66.7 0.331 T stage (T1–2 vs. T3–4) 100.0 vs. 37.5 0.013 100.0 vs.50.0 0.006 100.0 vs. 37.5 0.002 RT dose (< 66 vs. ≧66 Gy) 87.5 vs. 50.8 0.114 63.5 vs. 88.9 0.464 75.0 vs. 66.7 0.344 Cell types (ACC vs. others) 87.5 vs. 50.0 0.517 66.7 vs. 87.5 0.581 77.8 vs. 66.7 0.914 Margin (positive vs. negative) 75.5 vs. 50.0 0.43 76.9 vs. 75.0 0.944 69.2 vs. 80.0 0.76 PNI (positive vs. negative) 66.7 vs. 71.4 0.438 58.3 vs. 90.0 0.264 58.3 vs. 80.0 0.583 LVSI (positive vs. negative) 80.0 vs. 75.0 0.879 80.0 vs. 75.0 0.879 83.3 vs. 66.7 0.292 *Log-rank test Y year, OS overall survival, LPFS local progression free survival, DMFS distant-metastasis free survival, RT radiotherapy, ACC adenoid cystic carcinoma, PNI perineural invasion, LVSI lymphovascular invasion survival, local progression-free survival, and distant-metastasis free survival rates for patients with lacrimal ng factors affecting survival Lin et al. Radiation Oncology (2020) 15:156 Page 8 of 10 Lin et al. Radiation Oncology Fig. 3 Kaplan-Meier survival curves showing overall survival (a), local progression free survival (b), and distant-metastasis free survival (c) in patients with lacrimal gland carcinoma according to T category as determined using the eighth edition of the American Joint Committee on Cancer staging system. * Log-rank test. Discussion (a) The 2-year OS rates were 100 and 37.5% in T1–2 and T3–4 groups (p = 0.013), with (b) the 2-year DMFS rates were 100 and 37.5% (p = 0.002), and (c) the 2-year LPFS rates of 100 and 50%, respectively (p = 0.006) At a high dose (> 70% dose), proton beam dosimetry is equivalent to photon therapy; however, low-dose distribution is better for proton therapy, whereby the 20% isodose line can spare the opposite eye. Ensuring low dose spread to the nearby healthy tissue is critical in certain cases. Lower dose delivered to the regional organ at risk can reduce the likelihood of secondary malignancy and long-term complications, and there- fore should be considered for patients with early-stage lacrimal gland cancer who are likely to achieve good local control and long-term survival after the treatment. Several ocular toxicities were recorded among our pa- tients. These complications were expected as the radi- ation doses used were mostly above the dose that is well tolerated by ocular structures other than the sclera. Sen- sitivity to radiation of the ocular structures has been shown to vary [23–25]. The lens has been reported as the most radiosensitive ocular structure, with a mini- mum cataractogenic dose of 5.5 Gy. The lacrimal gland, cornea, and conjunctiva can each tolerate up to 50 Gy of radiation. The retina tolerates doses > 55 Gy. The sclera is the most resistant ocular structure and is believed to tolerate doses > 1000 Gy. The majority of ocular compli- cations reported in the present study were effectively managed by ophthalmologists. Only one patient received a punctal plug due to intolerable dry eye and another patient required intravitreous injection of Avastin (beva- cizumab) at 1.25 mg due to radiation-associated retinop- athy with retinal neovascularization. Therefore, we recommend that all patients who have received RT for lacrimal gland carcinoma visit an ophthalmologic clinic regularly. This study has some limitations. First, this was a retro- spective study based on medical records, which makes selection bias inevitable. Moreover, some of the patients did not attend the ophthalmologic clinic as recom- mended, making it likely that the rate of ocular compli- cations was underestimated. Second, due to the rarity of lacrimal gland cancer, the small sample size meant that the analysis was unlikely to yield statistically significant results. Fig. 3 Kaplan-Meier survival curves showing overall survival (a), local progression free survival (b), and distant-metastasis free survival (c) in patients with lacrimal gland carcinoma according to T category as determined using the eighth edition of the American Joint Committee on Cancer staging system. * Log-rank test. (a) The 2-year OS rates were 100 and 37.5% in T1–2 and T3–4 groups (p = 0.013), with (b) the 2-year DMFS rates were 100 and 37.5% (p = 0.002), and (c) the 2-year LPFS rates of 100 and 50%, respectively (p = 0.006) Acknowledgements Not applicable. 3. Hayashi K, Koto M, Ikawa H, Ogawa K, Kamada T. Efficacy and safety of carbon-ion radiotherapy for lacrimal gland carcinomas with extraorbital extension: a retrospective cohort study. Oncotarget. 2018;9(16):12932–40. extension: a retrospective cohort study. Oncotarget. 2018;9(16):12932–40. 4. Ni C, Kuo PK, Dryja TP. Histopathological classification of 272 primary epithelial tumors of the lacrimal gland. Chin Med J. 1992;105(6):481–5. 5. Wright JE, Rose GE, Garner A. Primary malignant neoplasms of the lacrimal gland. Br J Ophthalmol. 1992;76(7):401–7. Ethics approval and consent to participate 10. Henderson JW, Neault RW. En bloc removal of intrinsic neoplasms of the lacrimal gland. Am J Ophthalmol. 1976;82(6):905–9. This study protocol was reviewed and approved by the institutional review board of Chang Gung Memorial Hospital (Reference No.: 202000138B0). This study protocol was reviewed and approved by the institutional review board of Chang Gung Memorial Hospital (Reference No.: 202000138B0). 11. Esmaeli B, Golio D, Kies M, DeMonte F. Surgical management of locally advanced adenoid cystic carcinoma of the lacrimal gland. Ophthalmic Plast Reconstr Surg. 2006;22(5):366–70. Authors’ contributions d y 4. Ni C, Kuo PK, Dryja TP. Histopathological classification of 272 primary epithelial tumors of the lacrimal gland. Chin Med J. 1992;105(6):481–5. 4. Ni C, Kuo PK, Dryja TP. Histopathological classification of 272 primary epithelial tumors of the lacrimal gland. Chin Med J. 1992;105(6):481–5. 5. Wright JE, Rose GE, Garner A. Primary malignant neoplasms of the lacrimal y y epithelial tumors of the lacrimal gland. Chin Med J. 1992;105(6):481–5. 5. Wright JE, Rose GE, Garner A. Primary malignant neoplasms of the lacrimal gland. Br J Ophthalmol. 1992;76(7):401–7. SMH, YHL and LCC conceived the study; SMH and YHL collected data; SMH analyzed and interpreted data; YHL, SMH and WKY wrote manuscript; LCC provided statistical analyses; JWY, LY, DLT, and JTCC provided critical review and revision of manuscript; All authors reviewed the results and approved the final version of the manuscript. p g 5. Wright JE, Rose GE, Garner A. Primary malignant neoplasms of the lacrimal gland. Br J Ophthalmol. 1992;76(7):401–7. 6. Alkatan HM, Al-Harkan DH, Al-Mutlaq M, Maktabi A, Elkhamary SM. Epithelial lacrimal gland tumors: a comprehensive clinicopathologic review of 26 lesions with radiologic correlation. Saudi J Ophthalmol. 2014;28(1):49–57. 6. Alkatan HM, Al-Harkan DH, Al-Mutlaq M, Maktabi A, Elkhamary SM. Epithelial lacrimal gland tumors: a comprehensive clinicopathologic review of 26 lesions with radiologic correlation. Saudi J Ophthalmol. 2014;28(1):49–57. 7. Gao Y, Moonis G, Cunnane ME, Eisenberg RL. Lacrimal gland masses. AJR Am J Roentgenol 2013 201(3) W371 81 lesions with radiologic correlation. Saudi J Ophthalmol. 2014;28(1):49 57. 7. Gao Y, Moonis G, Cunnane ME, Eisenberg RL. Lacrimal gland masses. AJR Am J Roentgenol. 2013;201(3):W371–81. Funding 7. Gao Y, Moonis G, Cunnane ME, Eisenbe Am J Roentgenol. 2013;201(3):W371–81 Funding The authors state that this work has not received any funding. The authors state that this work has not received any funding. 8. Zeng J, Shi JT, Li B, Sun XL, An YZ, Li LQ, et al. Epithelial tumors of the lacrimal gland in the Chinese: a clinicopathologic study of 298 patients. Graefes Arch Clin Exp Ophthalmol. 2010;248(9):1345–9. Discussion Finally, the patients receiving proton treatment were only followed for 1–2 years, as our hospital began proton beam therapy in 2017. A longer follow-up period Lin et al. Radiation Oncology (2020) 15:156 Page 9 of 10 Lin et al. Radiation Oncology Table 4 Frequency of ocular complications after radiotherapy reported in the current study and the corresponding radiotherapy dose Complications Patient number Percentage (%) Time of occurrence (m) RT total dose (cGy) RT fraction dose (cGy) Punctate keratitis 5 27.78 1.14–7.9 5000–7000 200 Cataract 4 22.22 2.13–112.7 6000–6600 200 Dry eye 2 11.11 6.49–11.05 6600 200 Blepharitis 2 11.11 2.06–8.00 6000 200 Trichiasis 1 5.56 16.36 6000 200 Acute conjunctivitis 1 5.56 0.68 5000 250 Corneal epithelial defect 1 5.56 23.38 7200 200 Filamentary keratitis 1 5.56 6.65 7000 200 Radiation retinopathy 1 5.56 74.39 6600 200 Vitreous hemorrhage 1 5.56 10.92 6840 180 RT radiotherapy, cGy centigray Table 4 Frequency of ocular complications after radiotherapy reported in the current study and the corresponding radiotherapy d Table 4 Frequency of ocular complications after radiotherapy reported in the current study and the corresponding radiotherapy Keelung, Taiwan. 5Department of Radiation Oncology, Chang Gung Memorial Hospital, Linkou, Taiwan. is required to evaluate the effects of proton therapy on patient survival. is required to evaluate the effects of proton therapy on patient survival. In conclusion, eye-sparing surgery with adjuvant RT can achieve satisfactory results in patients with T1–2 lacrimal gland carcinoma. Disease stage T3 and above was associated with poor outcomes even with post- operative RT, probably due to distant metastasis. Adding neoadjuvant or adjuvant chemotherapy to current treat- ment strategies may be a suitable choice for this group of patients. Received: 1 May 2020 Accepted: 16 June 2020 Received: 1 May 2020 Accepted: 16 June 2020 Consent for publication All data were stored in the hospital database and extracted for research. The participants’ informed consent requirement was waived due to the retrospective nature of this study. 12. Esmaeli B, Ahmadi MA, Youssef A, Diba R, Amato M, Myers JN, et al. Outcomes in patients with adenoid cystic carcinoma of the lacrimal gland. Ophthalmic Plast Reconstr Surg. 2004;20(1):22–6. Competing interests 13. Pommier P, Liebsch NJ, Deschler DG, Lin DT, McIntyre JF, Barker FG 2nd, et al. Proton beam radiation therapy for skull base adenoid cystic carcinoma. Arch Otolaryngol Head Neck Surg. 2006;132(11):1242–9. 13. Pommier P, Liebsch NJ, Deschler DG, Lin DT, McIntyre JF, Barker FG 2nd, et al. Proton beam radiation therapy for skull base adenoid cystic carcinoma. Arch Otolaryngol Head Neck Surg. 2006;132(11):1242–9. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Availability of data and materials The datasets used during the current study are available from the corresponding author on reasonable request. The datasets used during the current study are available from the corresponding author on reasonable request. 9. Woo KI, Yeom A, Esmaeli B. Management of Lacrimal Gland Carcinoma: lessons from the literature in the past 40 years. Ophthalmic Plast Reconstr Surg. 2016;32(1):1–10. References 1. Shields JA, Shields CL, Epstein JA, Scartozzi R, Eagle RC Jr. Review: primary epithelial malignancies of the lacrimal gland: the 2003 Ramon L. font lecture. Ophthalmic Plast Reconstr Surg. 2004;20(1):10–21. 2. von Holstein SL, Therkildsen MH, Prause JU, Stenman G, Siersma VD, Heegaard S. Lacrimal gland lesions in Denmark between 1974 and 2007. Acta Ophthalmol. 2013;91(4):349–54. Author details 1 Thariat J, Racadot S, Pointreau Y, Boisselier P, Grange JD, Graff P, et al. Intensity- modulated radiotherapy of head and neck cancers: dose effects on the ocular, orbital and eyelid structures. Cancer Radiother. 2016;20(6–7):467–74. 24. Marchand V, Dendale R. Normal tissue tolerance to external beam radiation therapy: eye structures. Cancer Radiother. 2010;14(4–5):277–83. 25. Brady LW. Ocular complications of high-dose radiotherapy. Oncology (Williston Park). 1996;10(7):981–2 4. Author details 1 14. Meldrum ML, Tse DT, Benedetto P. Neoadjuvant intracarotid chemotherapy for treatment of advanced adenocystic carcinoma of the lacrimal gland. Arch Ophthalmol. 1998;116(3):315–21. 14. Meldrum ML, Tse DT, Benedetto P. Neoadjuvant intracarotid chemotherapy for treatment of advanced adenocystic carcinoma of the lacrimal gland. Arch Ophthalmol. 1998;116(3):315–21. Author details 1Department of Electro-Optical Engineering, National Taipei University of Technology, 1, Sec. 3, Chung-Hsiao E. Rd, Taipei 10608, Taiwan, ROC. 2Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung, Taiwan. 3College of Medicine, Chang Gung University, Taoyuan, Taiwan. 4Department of Radiation Oncology, Chang Gung Memorial Hospital, 15. Esmaeli B, Yin VT, Hanna EY, Kies MS, William WN Jr, Bell D, et al. Eye-sparing multidisciplinary approach for the management of lacrimal gland carcinoma. Head Neck. 2016;38(8):1258–62. 15. Esmaeli B, Yin VT, Hanna EY, Kies MS, William WN Jr, Bell D, et al. Eye-sparing multidisciplinary approach for the management of lacrimal gland carcinoma. Head Neck. 2016;38(8):1258–62. Page 10 of 10 Lin et al. Radiation Oncology (2020) 15:156 Lin et al. Radiation Oncology (2020) 15:156 16. Han J, Kim YD, Woo KI, Sobti D. Long-term outcomes of eye-sparing surgery for adenoid cystic carcinoma of lacrimal gland. Ophthalmic Plast Reconstr Surg. 2018;34(1):74–8. 17. Woo KI, Sagiv O, Han J, Frank SJ, Kim YD, Esmaeli B. Eye-preserving surgery followed by adjuvant radiotherapy for lacrimal gland carcinoma: outcomes in 37 patients. Ophthalmic Plast Reconstr Surg. 2018;34(6):570–4. 18. Friedrich RE, Bleckmann V. Adenoid cystic carcinoma of salivary and lacrimal gland origin: localization, classification, clinical pathological correlation, treatment results and long-term follow-up control in 84 patients. Anticancer Res. 2003;23(2A):931–40. 19. Ni C, Cheng SC, Dryja TP, Cheng TY. Lacrimal gland tumors: a clinicopathological analysis of 160 cases. Int Ophthalmol Clin. 1982;22(1):99–120. 20. Tellado MV, McLean IW, Specht CS, Varga J. Adenoid cystic carcinomas of the lacrimal gland in childhood and adolescence. Ophthalmology. 1997; 104(10):1622–5. 21. Weis E, Rootman J, Joly TJ, Berean KW, Al-Katan HM, Pasternak S, et al. Epithelial lacrimal gland tumors: pathologic classification and current understanding. Arch Ophthalmol. 2009;127(8):1016–28. 22. Chawla B, Kashyap S, Sen S, Bajaj MS, Pushker N, Gupta K, et al. Clinicopathologic review of epithelial tumors of the lacrimal gland. Ophthalmic Plast Reconstr Surg. 2013;29(6):440–5. 23. Thariat J, Racadot S, Pointreau Y, Boisselier P, Grange JD, Graff P, et al. Intensity- modulated radiotherapy of head and neck cancers: dose effects on the ocular, orbital and eyelid structures. Cancer Radiother. 2016;20(6–7):467–74. 23. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Leaky pipeline, gender bias, self-selection or all three? A quantitative analysis of gender balance at an international palliative care research conference
BMJ supportive & palliative care
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Citation for published version (APA): Sleeman, K. E., Koffman, J., & Higginson, I. J. (2019). Leaky pipeline, gender bias, self-selection or all three? A quantitative analysis of gender balance at an international palliative care research conference. BMJ Supportive and Palliative Care, 9(2), 146-148. https://doi.org/10.1136/bmjspcare-2016-001211 Citation for published version (APA): Sleeman, K. E., Koffman, J., & Higginson, I. J. (2019). Leaky pipeline, gender bias, self-selection or all three? A quantitative analysis of gender balance at an international palliative care research conference. BMJ Supportive and Palliative Care, 9(2), 146-148. https://doi.org/10.1136/bmjspcare-2016-001211 Citing this paper Pl h C t g t s pape Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA): Sleeman, K. E., Koffman, J., & Higginson, I. J. (2019). Leaky pipeline, gender bias, self-selection or all three? A quantitative analysis of gender balance at an international palliative care research conference. BMJ Supportive and Palliative Care, 9(2), 146-148. https://doi.org/10.1136/bmjspcare-2016-001211 ABSTRACT Department of Palliative Care, Policy and Rehabilitation, King’s College London—Cicely Saunders Institute, London, UK The gender balance in academia is the subject of considerable debate.1–3 The progress of women through academia has been described as a ‘leaky pipeline’, where there is attrition of women at each step of the career ladder. Initiatives to redress this are gaining momentum, for example the UK Athena SWAN Charter which was established in 2005 to encour- age and recognise commitment to advan- cing the careers of women in science, technology, engineering, maths and medi- cine (STEMM). Objectives The ‘leaky pipeline’ in academia is a clearly described phenomenon, but has not been examined in palliative care. We analysed the gender balance of speakers at the 9th World Research Congress of the European Association of Palliative Care (EAPC) to test the null hypothesis that there is no difference in the proportion of women and men with senior academic visibility in palliative care conference programmes. Correspondence to Dr Katherine E Sleeman, Department of Palliative Care, Policy and Rehabilitation, King’s College London—Cicely Saunders Institute, London, UK; katherine.sleeman@kcl.ac.uk Correspondence to Dr Katherine E Sleeman, Department of Palliative Care, Policy and Rehabilitation, King’s College London—Cicely Saunders Institute, London, UK; katherine.sleeman@kcl.ac.uk Received 28 July 2016 Revised 22 November 2016 Accepted 26 January 2017 Received 28 July 2016 Revised 22 November 2016 Accepted 26 January 2017 Methods The final programme of the 2016 EAPC World Congress was examined, and the gender of each speaker was recorded. Presentations were assessed using a three-tier hierarchy of senior academic visibility: Free Communication sessions, Themed sessions and invited Plenaries (low to high). As there was only one Invited Plenary at EAPC 2016, we examined the gender balance at EAPC Plenaries from 2012 to 2016. Visibility, for example through confer- ence talks and plenaries, is an important dimension of gender equality. A paucity of women conference speakers has been demonstrated in several academic disci- plines.4 However, it is unclear if the same phenomenon exists in palliative care, a specialty where women traditionally make up the majority of the workforce.5 Results Overall, the majority of speakers at EAPC 2016 (96/130, 73.8%) were women. The proportion of women was highest in the Free Communication sessions (84/107, 78.5%). In the Themed sessions, women made up just over half of speakers (12/22, 54.5%). In 2016, there was 1 invited Plenary speaker, a man. 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Download date: 24. Oct. 2024 Short report To cite: Sleeman KE, Koffman J, Higginson IJ. BMJ Supportive & Palliative Care Published Online First: [please include Day Month Year] doi:10.1136/bmjspcare-2016- 001211 Katherine E Sleeman, Jonathan Koffman, Irene J Higginson Katherine E Sleeman, Jonathan Koffman, Irene J Higginson Received 28 July 2016 Revised 22 November 2016 Accepted 26 January 2017 ABSTRACT From 2012 to 2016, just 6 of 23 invited Plenary speakers at EAPC conferences have been women (26.1%) (χ2=25.4, p<0.001). In June 2016, 1200 delegates attended the 9th World Research Congress of the European Association of Palliative Care (EAPC) in Dublin, making it one of the largest research conferences in palliative care globally. The programme featured 17 Free Communication sessions where researchers were chosen on the quality of anonymous (and gender-blind) abstracts to present their work in 8 min talks. In addition, there were seven Themed ses- sions in which speakers were invited on the basis of their level of authority on a specific subject to give 20 min talks. The most prestigious speaking opportunity is the invited Plenary where high-profile Conclusions These data reject our null hypothesis and suggest that there is attrition of women along the academic pipeline in palliative care. Other factors such as self-selection (that women decline invitations to give talks) and unconscious gender bias need further exploration, as well as actions to address the imbalance. To cite: Sleeman KE, Koffman J, Higginson IJ. BMJ Supportive & Palliative Care Published Online First: [please include Day Month Year] doi:10.1136/bmjspcare-2016- 001211 1 Sleeman KE, et al. BMJ Supportive & Palliative Care 2017;0:1–3. doi:10.1136/bmjspcare-2016-001211 Short report academics are invited to give longer talks (30 min) on the main conference stage. gender balance of invited Plenary speakers for a 5-year period (figure 1). At EAPC 2015, there were eight invited Plenary speakers (including a debate), of whom two were women (25%). At EAPC 2014, there were two Plenary speakers, both men. At EAPC 2013, there were nine Plenary speakers, including three women (33.3%). At EAPC 2012, one of three Plenary speakers was a woman (33.3%). Taking 5 years together, 6 of 23 invited Plenary speakers at EAPC conferences have been women (26.1%) (χ2=25.4, p<0.001). We aimed to analyse the gender balance of speakers at the 2016 EAPC World Congress to test the null hypothesis that there is no attrition of women in senior academic positions in palliative care, using the three-tiered hierarchy of Free Communication ses- sions, Themed sessions and Plenaries as a proxy for seniority. DISCUSSION P lli i Palliative care is a profession where women make up the majority of the workforce,5 and this is reflected in the overall gender balance of those giving talks at EAPC 2016. However, we identified that the propor- tion of women is lower for invited talks compared to those chosen anonymously from abstracts: whereas around three quarters of speakers in the Free Communication sessions were women, this proportion reduced to roughly half for the Themed sessions, and to only a quarter for the invited Plenaries (using pooled data from 2012 to 2016). Given the large number of women working in pal- liative care, it is of concern that the most prestigious invited Plenaries are so infrequently given by women, and it is noteworthy that two EAPC conferences over the past 5 years have included no female Plenary speakers at all (2014 and 2016). These data suggest that there is attrition of women along the ‘academic pipeline’ in palliative care. However, the relative con- tribution of other potential factors such as self- selection (that women decline invitations to give talks) and unconscious gender bias (that women are not invited to give talks) is unclear. In addition, palliative care is multiprofessional, with gender unequally repre- sented within each of the professions. The potential contribution of discipline should also be explored. METHODS The final programme of the 2016 EAPC World Congress was examined, and the gender of each speaker was recorded. Speaker gender was determined either by direct observation of their talk or by infer- ence from their first name. In eight cases, Google was used for more information. Where there was a substi- tution of speaker at the last minute, the gender of the speaker originally planned was used. At the 2016 EAPC, five top scoring abstracts were presented as 15 min Plenaries on the main stage. Since the decision to feature these presentations was made on the basis of an anonymous abstract, these were analysed with the Free Communication sessions. Since there was only one invited Plenary speaker at 2016 EAPC, we examined the gender balance of invited EAPC Plenary speakers over 5 years (2012–2016) by searching through relevant scientific programmes online. χ2 tests were used to assess differences. We did not analyse Meet the Expert sessions or Poster Discussion sessions. Sleeman KE, et al. BMJ Supportive & Palliative Care 2017;0:1–3. doi:10.1136/bmjspcare-2016-001211 RESULTS At EAPC 2016, there were a total of 17 Free Communication sessions comprising 102 talks. An additional five top scoring abstracts were presented on the main stage. There were seven Themed sessions which included 22 speakers. There was a single invited Plenary, the Ventafridda Lecture. Given that there was a single Plenary speaker in 2016, we examined the gender balance of Plenary speakers over 5 years. This was based on the assump- tion that variation from conference to conference is likely to outweigh any temporal trends. The propor- tion of female Plenary speakers was one-third or fewer for each of the 5 years examined. There was no indication that the proportion of female Plenary speakers improved over time. Indeed, the 2012 and 2013 conferences had the greatest proportion of female Plenary speakers (one-third). y Overall, the majority of speakers at EAPC 2016 were women. The proportion of women was highest in the Free Communication sessions. In the Themed sessions, women made up just over half of speakers. The Ventafridda Lecture was given by a man (table 1). g y Since there was only one invited Plenary speaker at EAPC 2016, we examined previous EAPC conference programmes to obtain a more representative view of Table 1 Men and women giving talks at EAPC 2016, according to session type Men (%) Women (%) Invited plenary 1 (100) 0 (0) Themed session 10 (45.5) 12 (54.5) Free communication 23 (21.5) 84* (78.5) Total 34 (26.2) 96 (73.8) *Includes five top scoring abstracts presented on the main stage. Table 1 Men and women giving talks at EAPC 2016, according to session type Speaking at academic conferences is important for career advancement. Such opportunities not only facilitate networking and collaboration, but are used as a marker of quality by promotions panels.6 Strategies to improve the proportion of women invited to speak, particularly those invited to give Plenaries, should be explored. These include consider- ation of the gender balance on scientific organising 2 Sleeman KE, et al. BMJ Supportive & Palliative Care 2017;0:1–3. doi:10.1136/bmjspcare-2016-001211 Figure 1 Number of men and women giving invited Plenaries, 2012–2016. Short report Short report Figure 1 Number of men and women giving invited Plenaries, 2012–2016. Competing interests IJH was Chair of the Scientific Organising Committee of the 2015 EAPC World Congress in Copenhagen and gave an invited Plenary at the 2012 EAPC Research Congress in Trondheim. RESULTS KES, JK and IJH are members of the King’s College London Department of Palliative Care Athena SWAN Self-Assessment Team. committees, as the proportion of women on conven- ing panels has been shown to be correlated with the proportion of women speakers.4 6 The issue of self- selection is important: previous studies have shown that women are more likely than men to decline invi- tations to speak at conferences,7 and less likely than men to put themselves forward for talks.8 These issues should be explored and addressed, for example through routine collection and analysis of the gender of all authors who submit abstracts, not only for EAPC Congresses but for all major palliative care con- ferences. Scientific organising committees should scru- tinise information on invitations, acceptances and refusals for high-profile speaking positions. Where women decline invitations to speak, the reasons for this should be explored, and steps to increase future acceptance rates identified. These initiatives will help us to understand the relative contributions of lack of assertiveness and lack of recognition. In addition, rou- tinely collecting feedback from delegates on the issue of gender equality might identify hidden issues. Last, we suggest that explicit acknowledgement of low visi- bility of female speakers by conference organisers may itself act as an intervention to redress the balance. committees, as the proportion of women on conven- ing panels has been shown to be correlated with the proportion of women speakers.4 6 The issue of self- selection is important: previous studies have shown that women are more likely than men to decline invi- tations to speak at conferences,7 and less likely than men to put themselves forward for talks.8 These issues should be explored and addressed, for example through routine collection and analysis of the gender of all authors who submit abstracts, not only for EAPC Congresses but for all major palliative care con- ferences. Scientific organising committees should scru- tinise information on invitations, acceptances and refusals for high-profile speaking positions. Where women decline invitations to speak, the reasons for this should be explored, and steps to increase future acceptance rates identified. These initiatives will help us to understand the relative contributions of lack of assertiveness and lack of recognition. In addition, rou- tinely collecting feedback from delegates on the issue of gender equality might identify hidden issues. RESULTS Last, we suggest that explicit acknowledgement of low visi- bility of female speakers by conference organisers may itself act as an intervention to redress the balance. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. Open Access This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/4.0/ REFERENCES 1 Moss-Racusin CA, Dovidio JF, Brescoll VL, et al. Science faculty’s subtle gender biases favor male students. Proc Natl Acad Sci USA 2012;109:16474–9. 2 Edmunds LD, Ovseiko PV, Shepperd S, et al. Why do women choose or reject careers in academic medicine? A narrative review of empirical evidence. Lancet 2016;388:2948–58. 3 Handley IM, Brown ER, Moss-Racusin CA, et al. Quality of evidence revealing subtle gender biases in science is in the eye of the beholder. Proc Natl Acad Sci USA 2015;112:13201–6. Failure of promotion on merit is deleterious for aca- demia and ultimately for patient care. Whether these data represent a leaky pipeline, gender bias, self- selection or all three, it is essential that as a specialty we take this issue seriously. We hope that the data pre- sented here provide a starting point for more in-depth consideration of this issue and appropriate strategies to tackle it. 4 Isbell LA, Young TP, Harcourt AH. Stag parties linger: continued gender bias in a female-rich scientific discipline. PLoS ONE 2012;7:e49682. 5 NCPC Specialist Palliative Care (SPC) Workforce Survey 2013. http://www.ncpc.org.uk/sites/default/files/NCPC_SPC_Workforce_ Survey_2013.pdf (accessed 6 February 2017) 6 Casadevall A. Achieving speaker gender equity at the American Society for Microbiology General Meeting. mBio 2015;6:e01146. 7 Schroeder J, Dugdale HL, Radersma R, et al. Fewer invited talks by women in evolutionary biology symposia. J Evol Biol 2013;26:2063–9. 7 Schroeder J, Dugdale HL, Radersma R, et al. Fewer invited talks by women in evolutionary biology symposia. J Evol Biol 2013;26:2063–9. Contributors KES conceived and designed this study, performed the analysis and wrote the manuscript, with help from IJH and JK throughout. 8 Jones TM, Fanson KV, Lanfear R, et al. Gender differences in conference presentations: a consequence of self-selection? PeerJ 2014;2:e627. Funding KES is funded by an NIHR Clinician Scientist Fellowship and has received funding from Cicely Saunders International. No specific funding was received for this study. 3 Sleeman KE, et al. BMJ Supportive & Palliative Care 2017;0:1–3. doi:10.1136/bmjspcare-2016-001211
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Fabrication of a Double Core–Shell Particle-Based Magnetic Nanocomposite for Effective Adsorption-Controlled Release of Drugs
Polymers
2,022
cc-by
12,976
Article Fabrication of a Double Core–Shell Particle-Based Magnetic Nanocomposite for Effective Adsorption-Controlled Release of Drugs Manzoor Hussain 1, Touseef Rehan 2, Khang Wen Goh 3, Sayyed Ibrahim Shah 4,*, Abbas Khan 1 , Long Chiau Ming 5,* and Nasrullah Shah 1,* 1 Department of Chemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan; 1 Department of Chemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan; manzoor14ics@gmail.com (M.H.); abbas053@gmail.com (A.K.) 1 Department of Chemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan; manzoor14ics@gmail.com (M.H.); abbas053@gmail.com (A.K.) 2 Department of Food and Nutrition, Shaheed Benazir Bhutto Women University, Peshawar 25000, Pakistan; touseefnasr@gmail.com 2 Department of Food and Nutrition, Shaheed Benazir Bhutto Women University, Peshawar 25000, Pakistan; touseefnasr@gmail.com 3 Faculty of Data Science and Information Technologies, INTI International University, Nilai 78100, Malaysia khangwen.goh@newinti.edu.my 3 Faculty of Data Science and Information Technologies, INTI International University, Nilai 78100, Malaysia; khangwen.goh@newinti.edu.my g g y 4 Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan 4 Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan 5 PAP Rashidah Sa’adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Gadong BE1410, Brunei Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan 5 PAP Rashidah Sa’adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Gadong BE1410, Brunei * Correspondence: ibrahimshah@awkum.edu.pk (S.I.S.); long.ming@ubd.edu.bn (L.C.M.); nasrullah@awkum.edu.pk (N.S.) Abstract: There has been very limited work on the control loading and release of the drugs aprepitant and sofosbuvir. These drugs need a significant material for the control of their loading and release phenomenon that can supply the drug at its target site. Magnetic nanoparticles have characteristics that enable them to be applied in biomedical fields and, more specifically, as a drug delivery system when they are incorporated with a biocompatible polymer. The coating with magnetic nanoparticles is performed to increase efficiency and reduce side effects. In this regard, attempts are made to search for suitable materials retaining biocompatibility and magnetic behavior. In the present study, silica-coated iron oxide nanoparticles were incorporated with core–shell particles made of poly(2- acrylamido-2-methylpropane sulfonic acid)@butyl methacrylate to produce a magnetic composite material (MCM-PA@B) through the free radical polymerization method. The as-prepared compos- ite materials were characterized through Fourier-transform infrared (FTIR)spectroscopy, scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), energy-dispersive X-Ray Analysis (EDX), and thermogravimetric analysis (TGA), and were further investigated for the loading and release of the drugs aprepitant and sofosbuvir. Citation: Hussain, M.; Rehan, T.; Goh, K.W.; Shah, S.I.; Khan, A.; Ming, L.C.; Shah, N. Fabrication of a Double Core–Shell Particle-Based Magnetic Nanocomposite for Effective Adsorption-Controlled Release of Drugs. Polymers 2022, 14, 2681. https://doi.org/10.3390/ polym14132681 Academic Editors: Faisal Raza, Muhammad Ovais and Carmine Coluccini Received: 21 May 2022 Accepted: 27 June 2022 Published: 30 June 2022 Citation: Hussain, M.; Rehan, T.; Goh, K.W.; Shah, S.I.; Khan, A.; Ming, L.C.; Shah, N. Fabrication of a Double Core–Shell Particle-Based Magnetic Nanocomposite for Effective Adsorption-Controlled Release of Drugs. Polymers 2022, 14, 2681. https://doi.org/10.3390/ polym14132681 Academic Editors: Faisal Raza, Muhammad Ovais and Carmine Coluccini Received: 21 May 2022 Accepted: 27 June 2022 Published: 30 June 2022 Citation: Hussain, M.; Rehan, T.; Goh, K.W.; Shah, S.I.; Khan, A.; Ming, L.C.; Shah, N. Fabrication of a Double Core–Shell Particle-Based Magnetic Nanocomposite for Effective Adsorption-Controlled Release of Drugs. Polymers 2022, 14, 2681. https://doi.org/10.3390/ polym14132681 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Keywords: magnetic core–shell particles; composite material; thermogravimteric analysis; controlled release; adsorption kinetic model Article Fabrication of a Double Core–Shell Particle-Based Magnetic Nanocomposite for Effective Adsorption-Controlled Release of Drugs The maximum loading capacity of 305.76 mg/g for aprepitant and 307 mg/g for sofosbuvir was obtained at pH 4. Various adsorption kinetic models and isotherms were applied on the loading of both drugs. From all of the results obtained, it was found that MCM-PA@B can retain the drug for more than 24 h and release it slowly, due to which it can be applied for the controlled loading and targeted release of the drugs. polymers polymers 1. Introduction Among the list of essential drugs, it has been included in basic health systems by the World Health Organization. By combining it with the NS5A inhibitor ledipasvir, sofosbuvir is used to treat genotypes 1, 2, 3, 4, 5, and 6, as well as hepatitis C infections [15–17]. Aprepitant and sofosbuvir have little water solubility, resulting in erratic bioavailability. This is because the active substance present in the drugs should be in dissolved form in aqueous form in the gastrointestinal tract, such that it crosses the luminal wall to make its therapeutic effects more significant. Moreover, higher solubility allows the active form of the drug to easily enter the blood and reach its target site to show its therapeutic effects. The loading and release of these drugs into the carrier molecules is also of great concern due to limited data available for them. Copolymers of 2-acrylamido-2-methyl propane sulfonic acid (AMPS) with other poly- mers have been synthesized and applied for several purposes. Molecularly imprinted polymer of N-maleoyl chitosan with AMPS have been prepared and applied for the recog- nition and delivery of bovine serum albumin [18]. A copolymer of AMPS with acrylamide has been applied for adsorption of cadmium from aqueous solution and for the loading and release of 5- fluorouracil, as well as the antiviral drug indinavir sulfate [19–21]. An- other copolymer of AMPS with polyvinyl alcohol and acrylamide has been applied for the controlled loading and release of ibuprofen [22]. Hydrogel prepared from chitosan and poly(AMPS) has been applied for the removal of methylene blue, acid red dye, Cd(II), and Cr(II) from wastewater [23]. Hydrogel prepared from poly(AMPS) and pectine has been applied for the controlled loading and release of captopril [24]. Different types of nanomaterials prepared from poly(α-L-glutamic acid) have been prepared and applied for the loading and controlled release of various types of drugs [25]. In one of the recent studies, hollow polymeric nanosphere (HPN)-supported imidazolium-based ionic liquids were shown to be efficient materials against multidrug-resistant bacteria, due to which they can also be applied in the biomedical field [26]. The fabrication of magnetic core–shell composites for drug loading and controlled release is of great value due to their efficiency and ability of controlled targeted drug delivery. The controlled loading and release of curcumin has also been demonstrated by the application of core–shell particles comprising chitosan with magnetic iron oxide nanoparti- cles (IONPs) and gold nanoparticles [27]. 1. Introduction Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Magnetic nanoparticles have recently been synthesized and applied in a large number of fields, including loading and controlled release of drugs, separation of cells, enhancing magnetic resonance imaging, the treatment of cancer, and many more [1]. Nanoparticles based on iron oxide are chemically stable, easily diffusible, less toxic, and biocompatible, due to which they are highly significant [2]. The use of bare iron oxide nanoparticles has problems of low accumulation, low separation yield by magnets, and the possibility that their atoms may oxidize, decreasing the properties of dispersion and magnetism [3]. The https://www.mdpi.com/journal/polymers Polymers 2022, 14, 2681. https://doi.org/10.3390/polym14132681 Polymers 2022, 14, 2681 2 of 20 property of having a high surface-to-volume ratio of iron oxide nanoparticles provides room for the adsorption of plasma proteins, followed by the activation of clearance mechanisms before they reach the target site. The time for the circulation of nanoparticles and decrease in the risk of adsorption of plasma proteins is also enhanced by the modification of the surface with some biocompatible polymers with a hydrophilic nature [4,5]. Iron oxide nanoparticles are mostly encapsulated by the silica, because it is inert and has ease of functionalization, and after encapsulation these nanoparticles become water-compatible [6,7]. The use of bare magnetic nanoparticles is usually prohibited for the application of drug delivery, because of the compromised biocompatibility and they do not possess various functionalities facilitating the coating of polymers on their surfaces. For this reason, they are coated with silica so that their surfaces are ready for coating and enhanced interaction with drugs [8–10]. y g g Aprepitant is an orally active neurokinin receptor antagonist used for chemotherapy- induced nausea and vomiting (CINV) [11]. It is the first FDA-approved drug that is used to treat CINV that works for more than 24 h after chemotherapy treatment [12]. It has a very low affinity for serotonin (5-HT3), corticosteroid, and dopamine receptors. Various drugs have been developed to prevent acute and delayed emesis, but aprepitant has been approved for PONV and nausea treatment during cancer therapy [13]. Sofosbuvir is a drug that is used in combination with other drugs in order to treat hepatitis C [14]. 2.1. Materials Sodium hydride 60%, magnesium turnings, and potassium persulfate were pur- chased from Daejung, S. Korea. Dimethylformamide (DMF), 4-vinylbenzyl chloride (VBC), 2-acrylamido-2-methylpropane sulfonic acid (AMPS), 4,4′-azobis(4-cyanovaleric acid) (ABCA), dimethyl sulfoxide (DMSO), butyl methacrylate (BMA), bromobenzene, and iodine were purchased from Sigma-Aldrich, Schnelldorf, Germany. Pyrroles and carbon disulfide were purchased from Daejung, Korea, diethyl ether from Sigma-Aldrich, Schnelldorf, Germany, magnesium sulfate and petroleum ether were from Fisher Scien- tific, Chicago, IL, USA, and tetrahydrofuran (THF) was purchased from Daejung, Korea. Silica-gel, FeCl2.4H2O, and FeCl3.6H2O were purchased from UniChem USA. n-Hexane was purchased from Sigma-Aldrich, Germany. Aprepitant and sofosbuvir were donated by Ferozsons pharmaceutical company, Nowshera, Pakistan. 2.2. Methods 2.2.1. Preparation of Poly(2-acrylamido-2-methylpropane sulfonic acid) (poly(AMPS)) 1. Introduction The core-shell structure made of IONPs@Au has been applied as an anticancer agent [28]. Iron oxide magnetic nanoparticles have been com- bined with large number of polymers for vast applications, e.g.,polylactide-co-glycolide combined with iron oxide nanoparticles has been applied for the encapsulation of both hydrophilic and hydrophobic drugs [29]. AMPS incorporated with pectin followed by the loading of IONPs has been applied for the controlled release of diclofenac sodium [30]. Polymers 2022, 14, 2681 3 of 20 Poly-(sodium-2-acrylamido-2-methyl propane sulfonic acid-co-styrene)/magnetite have been used for the purpose to inhibit corrosion from steel [31]. Poly-(sodium-2-acrylamido-2-methyl propane sulfonic acid-co-styrene)/magnetite have been used for the purpose to inhibit corrosion from steel [31]. p p Generally, there is a large number of drug delivery systems used, including liposomes, graphene, gold, carbon nanotubes, and magnetic nanoparticles [32–36]. These aforemen- tioned materials have little ability to deliver the drug in a controlled way. Bare magnetic nanoparticles have limitations to their application, due to which they are incorporated in some polymeric materials to enhance their applicability. Current research is focused on site-specific drug delivery systems, by releasing the drug in response to an external trigger, i.e., physiological pH and temperature [37–39]. In order to overcome the problem of controlled drug delivery, we here focus on the synthesis of a novel drug delivery system based on the combination of magnetic nanoparticles (silica-coated IONPs) and core–shell particles (poly(2-acrylamido-2-methylpropane sulfonic acid)@butyl methacrylate). The as-prepared magnetic composite material (MCM-PA@B) was thoroughly characterized by various analytical techniques, and was applied for loading and controlled release using aprepitant and sofosbuvir as model drugs. 2.2.2. Preparation of Core–Shell Latex of Poly(AMPS)@BMA (PA@B) 2.2.2. Preparation of Core–Shell Latex of Poly(AMPS)@BMA (PA@B) The fabricated Poly(AMPS) (1 g) was taken and dissolved in DI water in a reactor, and then BMA (25 mL) was added, followed by the addition of K2S2O8 (1 g in 15 mL of DI water). An overhead condenser was installed over the flask, and the mixture was stirred at 60 ◦C for 24 h in a water bath at 900 rpm using a magnetic stirrer. A milky, white-colored suspension of CS was obtained, which was transferred to a reagent bottle and stored for further use. 2.2.3. Synthesis of Silica-Coated IONPs (IONPs@SiO2) The synthesis of silica-coated IONPs was carried out in two consecutive steps: In the first step, a co-precipitation method was used to synthesize the magnetic IONPs, as reported by Veisi et al. [41]. Fe2+ and Fe3+ were mixed in 40 mL of double-DI water at a molar concentration of 0.15 mol and 0.3 mol, respectively. Sodium hydroxide solution (2 M) was added dropwise to keep the pH above 10, and temperature was maintained at 80 ◦C for 6 h. Then, the solution was cooled and filtered, followed by washing three times with distilled water. The black-colored particles were then dried in an oven to remove moisture, and then stored in an inert environment. In the second step, the synthesized IONPs were coated with SiO2 using theStöber method [10]. From the synthesized IONPs, 1.6 g was taken in a flask. Then, ethanol (80 mL), deionized water (16 mL), 10% NaOH solution (16 mL), and TEOS (5 mL) were added to it, followed by sonication for 1 h at 30 ◦C. Blackish grey-colored particles were produced, which were further separated by using an external magnet. These synthesized silica-coated magnetic nanoparticles were further washed with distilled water and ethanol, followed by drying at 80 ◦C in an oven for six hours. 2.2.1. Preparation of Poly(2-acrylamido-2-methylpropane sulfonic acid) (poly(AMPS)) The as-prepared poly(AMPS) revealed the following NMR data: g 1H NMR (400 MHz (D2O), ppm): 7.6 2H, (br s, N-pyrrole–H), 7.1 (br m, –Ar–), 6.3 (2H, br s, N-pyrrole–H), 3.3 (2H, br, m, C-CH2-C), 2.1 (C-CHC-C) 1.4 (6H, br m, C-CH3) 13C NMR (400 Mhz (D2O) ppm): 58.1 (RCH2SO3H), 52 (RCHS), 38.6 (RCH2), 26.6 (RCH3) g 1H NMR (400 MHz (D2O), ppm): 7.6 2H, (br s, N-pyrrole–H), 7.1 (br m, –Ar–), 6.3 (2H, br s, N-pyrrole–H), 3.3 (2H, br, m, C-CH2-C), 2.1 (C-CHC-C) 1.4 (6H, br m, C-CH3) 13C NMR (400 Mhz (D2O) ppm): 58.1 (RCH2SO3H), 52 (RCHS), 38.6 (RCH2), 26.6 (RCH3) 2.2.1. Preparation of Poly(2-acrylamido-2-methylpropane sulfonic acid) (poly(AMPS)) First of all, the RAFT agent 4-vinylbenzyl pyrrole carbodithioate (VP) was synthesized as reported in our previous study [40]. Briefly, 160 mL of DMF was taken, in which sodium hydride (6.03 g) was dissolved to produce a grey-colored suspension. Pyrrole solution was prepared by dissolving 10.02 g in DMF to make 20 mL of solution, which was further added to the mixture, forming a yellow-colored product. The temperature of this mixture was dropped to 0 ◦C, and CS2 solution already prepared from 9.01 mL in 20 mL of DMF was added to the mixture through continuous stirring for 30 min. Then, 4-VBC (22.2 g) solution in 20 mL of DMF was added with continuous stirring to produce a dark-red-colored mixture that was stirred overnight at room temperature (25 ◦C). This product was separated from its mixture by using a separating funnel that contained a mixture of deionized water and diethyl ether (1:1) and was further dried with 20 g of solid MgSO4. The product was filtered, followed by vacuum distillation, and then, using silica gel as stationary phase, the product was separated through the column. The obtained product was again vacuum-distilled, producing a yellow-colored product thatwas kept in a nitrogen-purged environment at −18 ◦C. For the preparation of poly(AMPS), 1 g of the RAFT agent 4-VP was added to 25 mL of DMSO in a two-neck flask reactor. Then, 25 g of AMPS monomer was added to it, followed by the addition of 0.5 g of 4,4′-azobis(4-cyanovaleric acid) (ABCA) initiator. The flask containing the mixture was made oxygen-free by purging with N2 several times. Then, the mixture was heated to 60 ◦C for 12 h. After heating, the mixture converted to brown-colored poly(AMPS). The obtained product was stored at −5 ◦C in a nitrogen atmosphere, and was Polymers 2022, 14, 2681 4 of 20 used further for the synthesis of core–shells. The as-prepared poly(AMPS) revealed the following NMR data: used further for the synthesis of core–shells. 2.2.4. Fabrication of Magnetic Composite Material (MCM-PA@B) Next, IONPs@SiO2wastaken in different amounts (0.1 g to 0.5 g) and dispersed in deionized (DI) water by sonication. PA@B was added to it in different amounts (1 mL to 5 mL) and sonicated for 15 min. Then, 4,4′-azobis(4-cyanovaleric acid)(0.015 g) in 1.5 mL of DI water was added to it and again sonicated for 15 min, followed by the addition of IONPs@SiO2 suspension with continuous sonication for 10 min (Table 1). The as-prepared mixture was then stirred for 2 h under nitrogen-purged conditions at 60 ◦C toproduce the composite materials. The synthesized magnetic composite material (MCM-PA@B) was then transferred to a beaker and kept in the oven until dried and converted to a black-colored material. Table 1. Different ratios of IONPs@SiO2 and PA@B in the synthesis of MCM-PA@B. S. No. IONPs@SiO2:PA@B IONPs@SiO2 (g) PA@B (mL) ABCA (mL) 1 IONPs@SiO21:PA@B1 0.1 1 2 2 IONPs@SiO21:PA@B3 0.1 3 2 3 IONPs@SiO21:PA@B5 0.1 5 2 4 IONPs@SiO23:PA@B5 0.3 5 2 5 IONPs@SiO25:PA@B5 0.5 5 2 Table 1. Different ratios of IONPs@SiO2 and PA@B in the synthesis of MCM-PA@B. 2.2.6. Application of MCM-PA@B for Drug Loading and Release Studies The as-prepared composite materials were further applied for the loading and release of the drugs aprepitant and sofosbuvir. A stock solution of eachdrug (100 ppm) was prepared in 100 mL of methanol through sonication at room temperature. From the stock solution, five separate sample solutions were further prepared through dilution with a concentration of 20 ppm. The composite material was added to each of the sample solutions at neutral pH and STP. All of the drug solutions containing the composite were shaken overnight. From each of the samples, aliquots were taken out after a certain interval of time and checked through the UV spectrophotometer to find the loading amount of the drug. To optimize the conditions for maximum loading of the drugs, both of the drugs were tested for their loading at different pH and drug concentrations. For pH optimization, drug solutions were prepared, and their pH was set to2 by the addition of Britton–Robinson buffer, after which 0.05 g of composite was added. This solution was shaken for 2 h, and its absorbance was checked using the UV spectrophotometer. A similar procedure was followed for pH 4, 6, 8, and 10. For optimization of the drug concentration used, drug solutions from 20 ppm to 100 ppm were prepared, and their pH was set to 4 by adding buffer, followed by the addition of 0.05 g of composite materials. These solutions were shaken for 2 h and then their absorbance was checked. Pseudo-2nd-Order Kinetic Model Pseudo-2nd-Order Kinetic Model A second-order kinetic equation was used for the interpretation of the data (Equation (3)): t/qt = t/qe + 1/k2 qe2 (3) (3) where k2 shows the rate constant for the second order, while qe and qt show the amount of loaded drug at equilibrium and at any time. 2.2.5. Characterization of the Synthesized MCM-PA@B FTIR Spectrometer (Varian 640-IR, USA) was used to obtain the FTIR spectra of the prepared MCM-PA@B in the wavenumber region of 4000–400 cm−1. A thermogravimetric analyzer (TGA-50H Shimadzu, Kyoto, Japan) was used to study the thermal stability of Polymers 2022, 14, 2681 5 of 20 5 of 20 the prepared composite materials. The machine was run from 25 to 600 ◦C ata heating rate of 30 ◦C/min. An X-ray diffractometer (D-2 Phraser, Bruker, Denver, CO, USA) was used to analyze the XRD patterns by applying a scan rate of 20◦/min, and a Cu Kα radiation source was used. A field-emission scanning electron microscope (SEM) (model JSM5910, JEOL. Kyoto, Japan,) was used with an acceleration voltage of 30 KV to analyze the surface morphology of the composite materials. A Thermo Electron Co., USA, Helios β UV–Vis Spectrometer (Lambda 25, Perkin Elmer, Waltham, MA, USA) was used to determine the UV–Vis absorption spectra of the samples. 2.2.7. Kinetic Study of Adsorption Pseudo-1st-Order Kinetic Model Drugs’ loading data were obtained and, using a pseudo-1st-order kinetic model, were used to interpret the obtained results (Equations (1) and (2)): Log (qe −qt)= log qe −k1t/2.303 (1) qe or qt = (Ci −Ce)V/m (2) Log (qe −qt)= log qe −k1t/2.303 (1) (1) qe or qt = (Ci −Ce)V/m (2) (2) where qt is the amount of drug loaded at any time, while qe shows the amount of loaded drug at equilibrium time, k1 is the constant for the first order, Ce and Ci are the drug’s concentration at equilibrium and initial use, respectively, V is the volume of the drug solution, and m is the mass of the composite materials (0.1 g). Intra-Particle Diffusion Kinetic Model The data were also interpreted using an intra-particle diffusion model (Equation (4)) qt = Kintt1⁄2 + C (4) (4) Polymers 2022, 14, 2681 6 of 20 where C is the intercept and is associated withthe width of the border layer i.e., the higher the value of C, the further is its boundary layer outcome. where C is the intercept and is associated withthe width of the border layer i.e., the higher the value of C, the further is its boundary layer outcome. 2.2.8. Equilibrium Studies 3.1. Scanning Electron Microscope Study of MCM-PA@B 3.1. Scanning Electron Microscope Study of MCM-PA@B The morphological, internal, and topographical structure of the as-prepared MCM- PA@B was evaluated by SEM analysis. It can be seen in Figure 1A,B that the IONPs and IONPs@SiO2 had a small particle size; however, the incorporation of silica onto the IONPs converted them into rough and heterogeneous surface particles [42]. The incorporation of IONPs@SiO2 into the PA@B to produce MCM-PA@B can be seen in Figure 1C–G. The IONPs@SiO21: PA@B1 sample contained an equal ratio of both IONPs@SiO2 and PA@B, due to which its morphological structure did not look uniform. However, as the IONPs@SiO2 were incorporated with a higher amount of PA@B, uniformity in the structure enhanced and made it more recognizable, as shown in Figure 1D–F. Moreover, the core–shell polymer of PA@B was coated over the silica-coated IONPs to form double core–shell nanocompos- ites. The surface polymer molecules covered the nanoparticles from all sides and, hence, prevented their agglomeration, due to which these double core–shell nanocomposites were distinguishable and clear. The similar composition of PA@B, with changes only to its quantity, did not cause significant changes in the morphology of the nanocomposites [41]. As the ratio of IONPs@SiO2 increased, the surface again shifted towards non-uniformity. From the SEM results, it can be seen that using the IONPs@SiO2 and PA@B latex at ratios of 1:3, 1:5, and 3:5 gave us composite materials with similar morphological structures. The magnetic behavior of the synthesized magnetic nanoparticles and MCM-PA@B is presented in Figure 2. 2.2.8. Equilibrium Studies Langmuir and Freundlich isotherms were used to determine the equilibrium study of the drug incorporated into the synthesized composite materials. The Langmuir equation shows monolayer attachment of the drug with the composite materials by providing many sites for linking the drug with the composite materials. The Langmuir equation in presented in Equation (5): Ce/qe = 1/KL + aL Ce/KL (5) (5) where KL shows the equilibrium adsorption constant, while qe is the monolayer loading capacity of the drug. p y g The Freundlich isotherm is given in Equation (6): Log qe = log KF +1/n log Ce (6) (6) The values for the constants (KF and N) of Freundlich and Langmuir (Q and KL) as well as the regression coefficient R2 were calculated. 3.3. Thermogravimetric Analysis 3.3. Thermogravimetric Analysis By looking into the spectra of TGA analysis given in Figure 4, we can observe two degradation stages for IONPs and IONPs@SiO2. In the first stage, the weight loss of IONPs at 125 °C is due to the loss of water molecules attached tothe surface of the IONPs, while in the second stage the weight loss at 170 °C and 360 °C is due to the transformation of crystal from Fe3O4 to γ-Fe2O3 caused by heating,and only a 5% decrease in the weight of the composite occurs [47]. In the case of IONPs@SiO2, in the first stage, 2% weight loss occurs up to 230 °C due to the loss of any remaining water and volatile molecules, and then the weight loss decreases by 12% due to the conversion of Fe3O4 to γ-Fe2O3 at 360 °C, followed by no significant weight loss, indicating a higher degree of thermal stability [48]. All of the composites of CS and IONPs@SiO2 contain different amounts of poly(AMPS) and IONPs@SiO2. The graph shows the TGA of all of these composites, and indicates that each of the composite has three degradation stages. For the composites IONPs@SiO21:PA@B1, IONPs@SiO21:PA@B3, IONPs@SiO21:PA@B5; the first degradation stage occurs below 150 °C, showing a 10% loss of water and volatile molecules contents [46], followed by degradation up to 300 °C, showing the loss of sulfonate groups of the polymers up to 30%, and then 85% weight loss occurs at 450 °C due to the degradation of the polymer on the IONPs@SiO2. Above 450 °C, there is no significant change in the weight, confirming the presence of IONPs, as these particles have high thermal stability. Moreover, for the composites IONPs@SiO23:PA@B5 and IONPs@SiO25:PA@B5, both have high amounts of IONPs as compared to previous composites, so they have relatively high thermal stability, and a maximum of 70% weight loss occurs at 450 °C, followed by no significant decrease in the weight of the samples. As IONPs@SiO25:PA@B5 contains more IONPs@SiO2, it gives more thermal stability to the composite, due to which its graph is slightly above that of the IONPs@SiO23:PA@B5 [31,46]. From the above discussion it can By looking into the spectra of TGA analysis given in Figure 4, we can observe two degradation stages for IONPs and IONPs@SiO2. 3.2. FTIR Study of the Composite The synthesis of IONPs is confirmed by the presence of peak at 565 cm−1, which is due to the presence of stretching of Fe–O bonds in Fe2O3 in IONPs [43], while the band at 1084 confirms the silica coating on the IONPs to produce Fe2O3@SiO2 [44]. The bands at 1353 cm−1 are due to the presence of the sulfonate group of the poly(AMPS) [45].The peaks at 2872 and 1452 cm−1 are due to CH and CH2 as well as C-N stretching vibrations [46]. The broad peak at 3100–3500 cm−1 indicates OH stretching and the presence of the NH group. The peak at 941 cm−1 is due to S–O–C symmetrical stretching vibrations of the poly(AMPS) shell, while that at 1735 cm–1 assures the presence of a CO group, and that at 1249 cm−1 shows the presence of a BMA core [20] (Figure 3). 7 of 20 Polymers 2022, 14, 2681 Polymers 2022, 14, x Figure 1. SEM images of IONPs (A), IONPs@SiO2 (B), MCM-PA@B (C–G), IONPs@SiO21:PA@B1 (C), IONPs@SiO21:PA@B3 (D), IONPs@SiO21:PA@B5 (E), IONPs@SiO23:PA@B5 (F), and IONPs@SiO25:PA@B5 (G). Figure 1. SEM images of IONPs (A), IONPs@SiO2 (B), MCM-PA@B (C–G), IONPs@SiO21:PA@ (C), IONPs@SiO21:PA@B3 (D), IONPs@SiO21:PA@B5 (E), IONPs@SiO23:PA@B5 (F), a IONPs@SiO25:PA@B5 (G). rs 2022, 14, x 8 of 22 Figure 1. SEM images of IONPs (A), IONPs@SiO2 (B), MCM-PA@B (C–G), IONPs@SiO21:PA@B1 (C), IONPs@SiO21:PA@B3 (D), IONPs@SiO21:PA@B5 (E), IONPs@SiO23:PA@B5 (F), and IONPs@SiO25:PA@B5 (G). Figure 1. SEM images of IONPs (A), IONPs@SiO2 (B), MCM-PA@B (C–G), IONPs@SiO21:PA@B1 (C), IONPs@SiO21:PA@B3 (D), IONPs@SiO21:PA@B5 (E), IONPs@SiO23:PA@B5 (F), and IONPs@SiO25:PA@B5 (G). 8 of 22 Figure 1. SEM images of IONPs (A), IONPs@SiO2 (B), MCM-PA@B (C–G), IONPs@SiO21:PA@B1 (C), IONPs@SiO21:PA@B3 (D), IONPs@SiO21:PA@B5 (E), IONPs@SiO23:PA@B5 (F), and IONPs@SiO25:PA@B5 (G). Figure 1. SEM images of IONPs (A), IONPs@SiO2 (B), MCM-PA@B (C–G), IONPs@SiO21:PA@B1 (C), IONPs@SiO21:PA@B3 (D), IONPs@SiO21:PA@B5 (E), IONPs@SiO23:PA@B5 (F), and IONPs@SiO25:PA@B5 (G). 8 of 22 Figure 2. Magnetic behavior of the synthesized magnetic nanoparticles (A,B) and MCM-PA@B (C,D). Figure 2. Magnetic behavior of the synthesized magnetic nanoparticles (A,B) and MCM-PA@B (C,D). Figure 2. Magnetic behavior of the synthesized magnetic nanoparticles (A,B) and MCM-PA@B (C,D). Figure 2. Magnetic behavior of the synthesized magnetic nanoparticles (A,B) and MCM-PA@B (C,D). 8 of 20 9 of 22 Polymers 2022, 14, 2681 Polymers 2022, 14, x Figure 3. FTIR spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Figure 3. 3.2. FTIR Study of the Composite FTIR spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Figure 3. FTIR spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Figure 3. FTIR spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. 3.3. Thermogravimetric Analysis 3.3. Thermogravimetric Analysis 0 100 200 300 400 500 600 Weight loss (%) 0 20 40 60 80 100 120 IONPs IONPs@SiO2 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 composite materials with high concentrations of IONPs@SiO2 are more thermally stable than those with low amounts of IONPs@SiO2. composite materials with high concentrations of IONPs@SiO2 are more thermally stable than those with low amounts of IONPs@SiO2. Temperature 0 100 200 300 400 500 600 Weight loss (%) 0 20 40 60 80 100 120 IONPs IONPs@SiO2 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Figure 4. Thermogravimetric analysis of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Figure 4. Thermogravimetric analysis of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Weight loss (%) 0 Figure 4. Thermogravimetric analysis of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@ with different ratios of IONPs@SiO2 and PA@B. Figure 4. Thermogravimetric analysis of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. 3.4. XRD Analysis 3.4. XRD Analysis The XRD analysis results of the synthesized composite materials are given in Figure 5. The data from the graph show us that the IONPs have some sharp peaks that confirm their crystalline structure. Moreover, the crystalline form of the nanoparticles is confirmed by the sharp peaks at 30.99°, 36.3°, 44.1°, 58°, and 63.6° [46]. The graph of the IONPs@SiO2 also shows similar peaks that ensures its crystalline nature, with no loss of any of the en- tity. This shows that after coating of of silica on the IONPs, the crystalline structure of the nanoparticles was not disturbed, and showed the peaks in the similar range. By incorpo- ration of the core–shell latex with IONPs@SiO2, the intensity of these peaks decreased and became very low, ensuring their combination. However, due to the presence of the IONPs@SiO2, the peaks are still present with low intensity, confirming the presence of both the PA@B and IONPs@SiO2. The XRD analysis results of the synthesized composite materials are given in Figure 5. The data from the graph show us that the IONPs have some sharp peaks that confirm their crystalline structure. Moreover, the crystalline form of the nanoparticles is confirmed by the sharp peaks at 30.99◦, 36.3◦, 44.1◦, 58◦, and 63.6◦[46]. The graph of the IONPs@SiO2 also shows similar peaks that ensures its crystalline nature, with no loss of any of the entity. This shows that after coating of of silica on the IONPs, the crystalline structure of the nanoparti- cles was not disturbed, and showed the peaks in the similar range. By incorporation of the core–shell latex with IONPs@SiO2, the intensity of these peaks decreased and became very low, ensuring their combination. However, due to the presence of the IONPs@SiO2, the peaks are still present with low intensity, confirming the presence of both the PA@B and IONPs@SiO2. 3.5. Zero-Point Charge (pHzpc) on the Composite 3.3. Thermogravimetric Analysis 3.3. Thermogravimetric Analysis In the first stage, the weight loss of IONPs at 125 ◦C is due to the loss of water molecules attached to the surface of the IONPs, while in the second stage the weight loss at 170 ◦C and 360 ◦C is due to the transformation of crystal from Fe3O4 to γ-Fe2O3 caused by heating, and only a 5% decrease in the weight of the composite occurs [47]. In the case of IONPs@SiO2, in the first stage, 2% weight loss occurs up to 230 ◦C due to the loss of any remaining water and volatile molecules, and then the weight loss decreases by 12% due to the conversion of Fe3O4 to γ-Fe2O3 at 360 ◦C, followed by no significant weight loss, indicating a higher degree of thermal stability [48]. All of the composites of CS and IONPs@SiO2 contain different amounts of poly(AMPS) and IONPs@SiO2. The graph shows the TGA of all of these composites, and indicates that each of the composite has three degradation stages. For the composites IONPs@SiO21:PA@B1, IONPs@SiO21:PA@B3, IONPs@SiO21:PA@B5; the first degradation stage occurs below 150 ◦C, showing a 10% loss of water and volatile molecules contents [46], followed by degradation up to 300 ◦C, showing the loss of sulfonate groups of the polymers up to 30%, and then 85% weight loss occurs at 450 ◦C due to the degradation of the polymer on the IONPs@SiO2. Above 450 ◦C, there is no significant change in the weight, confirming the presence of IONPs, as these particles have high thermal stability. Moreover, for the composites IONPs@SiO23:PA@B5 and IONPs@SiO25:PA@B5, both have high amounts of IONPs as compared to previous composites, so they have relatively high thermal stability, and a maximum of 70% weight loss occurs at 450 ◦C, followed by no significant decrease in the weight of the samples. As IONPs@SiO25:PA@B5 contains more IONPs@SiO2, it gives more thermal stability to the composite, due to which its graph is slightly above that of the IONPs@SiO23:PA@B5 [31,46]. From the above discussion it can be concluded that Polymers 2022, 14, 2681 9 of 20 re more 9 of 20 re more composite materials with high concentrations of IONPs@SiO2 are more thermally sta than those with low amounts of IONPs@SiO2. 3.5. Zero-Point Charge (pHzpc) on the Composite The PA@B contains a sulfonate group from the poly(AMPS) shell, which is incorpo- rated with the IONPs@SiO2. Due to the presence of these acidic sulfonate groups, the pHzpc of the composite material was expected to be in the acidic range, as confirmed by the results obtained, with a value of 2.5. Below the pHzpc value, the composite has positive charge, while increasing above this value it has negative charge (Figure 6). 10 of 20 10 of 20 Polymers 2022, 14, 2681 Figure 5. XRD spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. 3.5. Zero-Point Charge (pHzpc) on the Composite The PA@B contains a sulfonate group from the poly(AMPS) shell, which is incorpo- rated with the IONPs@SiO2. Due to the presence of these acidic sulfonate groups, the pHzpc of the composite material was expected to be in the acidic range, as confirmed by the results obtained, with a value of 2.5. Below the pHzpc value, the composite has posi- tive charge, while increasing above this value it has negative charge (Figure 6). Figure 5. XRD spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Polymers 2022, 14, x 12 of 22 Figure 6. pHzpc of the fabricated composite materials (MCM-PA@B). 3.6. EDX Analysis Figure 7 shows the EDX analysis of the synthesized IONPs. The incorporation of sil- i ith th IONP i fi d b th f it k i Fi 7 M th pH 0 2 4 6 8 10 12 14 ΔpH -1 0 1 2 3 4 5 6 7 Figure 6. pHzpc of the fabricated composite materials (MCM-PA@B). Figure 5. XRD spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Figure 5. XRD spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. olymers 2022, 14, x 12 of 22 Polymers 2022, 14, x 12 of 22 Figure 5. XRD spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. Figure 5. XRD spectra of pure IONPs, IONPs@SiO2, and the fabricated MCM-PA@B with different ratios of IONPs@SiO2 and PA@B. 3.5. Zero-Point Charge (pHzpc) on the Composite The PA@B contains a sulfonate group from the poly(AMPS) shell, which is i rated with the IONPs@SiO2. 3.5. Zero-Point Charge (pHzpc) on the Composite Due to the presence of these acidic sulfonate gro pHzpc of the composite material was expected to be in the acidic range, as confir the results obtained, with a value of 2.5. Below the pHzpc value, the composite h tive charge, while increasing above this value it has negative charge (Figure 6). Figure 6. pHzpc of the fabricated composite materials (MCM-PA@B). 3 6 EDX A l i pH 0 2 4 6 8 10 12 14 ΔpH -1 0 1 2 3 4 5 6 7 Figure 6. pHzpc of the fabricated composite materials (MCM-PA@B). 3.5. Zero-Point Charge (pHzpc) on the Composite The PA@B contains a sulfonate group from the poly(AMPS) shell, which is rated with the IONPs@SiO2. Due to the presence of these acidic sulfonate gr pHzpc of the composite material was expected to be in the acidic range, as con the results obtained, with a value of 2.5. Below the pHzpc value, the composite tive charge, while increasing above this value it has negative charge (Figure 6). pH 0 2 4 6 8 10 12 14 ΔpH -1 0 1 2 3 4 5 6 7 pH Figure 6. pHzpc of the fabricated composite materials (MCM-PA@B). Figure 6. pHzpc of the fabricated composite materials (MCM-PA@B). 11 of 20 Polymers 2022, 14, 2681 11 of 20 3.6. EDX Analysis 3.6. EDX Analysis y Figure 7 shows the EDX analysis of the synthesized IONPs. The incorporation of silica with the IONPs is confirmed by the presence of its peaks in Figure 7a. Moreover, the presence of the peaks for carbon and sulfur also shows the presence of the core–shell latex linked with IONPs@SiO2. The presence of peaks for Fe confirms the magnetic nature of all of the prepared MCM-PA@B (Figure 7). 13 of 22 Figure 7 shows the EDX analysis of the synthesized IONPs. The incorporation of silica with the IONPs is confirmed by the presence of its peaks in Figure 7a. Moreover, the presence of the peaks for carbon and sulfur also shows the presence of the core–shell latex linked with IONPs@SiO2. The presence of peaks for Fe confirms the magnetic nature of all of the prepared MCM-PA@B (Figure 7). 13 of 22 Figure 7 shows the EDX analysis of the synthesized IONPs. The incorporation of silica with the IONPs is confirmed by the presence of its peaks in Figure 7a. Moreover, the presence of the peaks for carbon and sulfur also shows the presence of the core–shell latex linked with IONPs@SiO2. The presence of peaks for Fe confirms the magnetic nature of all of the prepared MCM-PA@B (Figure 7). 13 of 22 linked with IONPs@SiO2. The presence of peaks for Fe confirms the magnetic nature of a of the prepared MCM-PA@B (Figure 7). Figure 7. EDX analysis of the IONPs@SiO21:PA@B1 (a), IONPs@SiO21:PA@B3 ( IONPs@SiO21:PA@B5 (c), IONPs@SiO23:PA@B5 (d), and IONPs@SiO25:PA@B5 (e). Figure 7. EDX analysis of the IONPs@SiO21:PA@B1 (a), IONPs@SiO21:PA@B3 (b IONPs@SiO21:PA@B5 (c), IONPs@SiO23:PA@B5 (d), and IONPs@SiO25:PA@B5 (e). Figure 7. EDX analysis of the IONPs@SiO21:PA@B1 (a), IONPs@SiO21:PA@B3 (b IONPs@SiO21:PA@B5 (c), IONPs@SiO23:PA@B5 (d), and IONPs@SiO25:PA@B5 (e). Figure 7. EDX analysis of the IONPs@SiO21:PA@B1 (a), IONPs@SiO21:PA@B3 (b) IONPs@SiO21:PA@B5 (c), IONPs@SiO23:PA@B5 (d), and IONPs@SiO25:PA@B5 (e). 3.7. Drug Loading and Release Study 3.7. Drug Loading and Release Study 3.7. Drug Loading and Release Study 3.7. Drug Loading and Release Study The loading of the drugs aprepitant and sofosbuvir at various times, pH values, and concentrations is given in Figures 8–10, respectively. It can be observed from the figures that the MCM-PA@B with a lower ratio of PA@B has lower loading, while it was found to be higher in PA@B in the MCM-PA@B. This might be due to the presence of higher attach- ment sites (functional groups) to which the drug(s) can attach, ultimately leading to higher drug loading. Moreover, as the amounts of IONPs@SiO2 are increased, there is no observ- able increase in the loading capacity of the drugs, which might be due to the incorporation of IONPs@SiO2 with the core–shell latex molecules. As shown in the figure, maximum loading of the drugs occurs at a quantity of 229 mg/g for aprepitant and 203 mg/g for sofosbuvir at a time of 3 h. After 3 h, there was no increase in the loading capacity of the drug, showing that all of the attachment sites were occupied and, hence, equilibration had occurred The loading of the drugs aprepitant and sofosbuvir at various times, pH values, and concentrations is given in Figures 8–10, respectively. It can be observed from the figures that the MCM-PA@B with a lower ratio of PA@B has lower loading, while it was found to be higher in PA@B in the MCM-PA@B. This might be due to the presence of higher attachment sites (functional groups) to which the drug(s) can attach, ultimately leading to higher drug loading. Moreover, as the amounts of IONPs@SiO2 are increased, there is no observable increase in the loading capacity of the drugs, which might be due to the incorporation of IONPs@SiO2 with the core–shell latex molecules. As shown in the figure, maximum loading of the drugs occurs at a quantity of 229 mg/g for aprepitant and 203 mg/g for sofosbuvir at a time of 3 h. After 3 h, there was no increase in the loading capacity of the drug, showing that all of the attachment sites were occupied and, hence, equilibration had occurred. 3.7. Drug Loading and Release Study 3.7. Drug Loading and Release Study 12 of 20 12 of 20 Polymers 2022, 14, 2681 in Time (min) 0 50 100 150 200 250 300 Aprepitant drug loaded (mg/g) 0 50 100 150 200 250 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 A Time (min) 0 50 100 150 200 250 300 Sofosbuvir drug Loaded (mg/g) 0 50 100 150 200 250 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 B Figure 8. Loading of drugs into the fabricated MCM-PA@B at different time points: aprepitant (A); sofosbuvir (B). Time (min) 0 50 100 150 200 250 300 Aprepitant drug loaded (mg/g) 0 50 100 150 200 250 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 A Time (min) 0 50 100 150 200 250 300 Sofosbuvir drug Loaded (mg/g) 0 50 100 150 200 250 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 B Figure 8. Loading of drugs into the fabricated MCM-PA@B at different time points: aprepitant (A); sofosbuvir (B). As previously shown in Figure 4, the synthesized composite materials have a pHzpc of 2.5. Above this pH, MCM-PA@B has positive charge, while below this pH it has negative charge. According to Bedi et al. [49], aprepitant has a pKa value of 9.7, while Sofosbuvir has a pKa value of 9.3 [50]. These pKa values show that both drugs are in an anionic form below pH 9. Maximum loading of the drugs was found to be 307 mg/g for aprepitant and 306 mg/g for sofosbuvir at pH 4 due to the presence of opposite charges on the drugs and composite materials. The loading capacity decreases as the pH is increased from 4 to 10, due to conversion of the drugs to a cationic form, which causes repulsion at higher pH. The lesser amount of drug loading is attributed to the free space available for the accommodation of drug molecules. The loading of both drugs was also studied for different concentrations of the drugs. From Figure 10 (upper), it can be observed that the loading of aprepitant reached a max- imum of 490 mg/g for composite materials with a lower quantity of core–shells and 501 mg/g for composites with higher quantities of PA@B. The increase in the loading of the drug occurred until 80 ppm, and then there was no significant change in the loading amount of the drug. 3.7. Drug Loading and Release Study 3.7. Drug Loading and Release Study The loading of sofosbuvir increased to maximum values of 515 mg/g for composite materials with lower quantities of core–shells and 525 mg/g for composites Polymers 2022, 14, 2681 13 of 20 i (A) 13 of 20 i (A) with higher quantities of core–shell latex. The loading of sofosbuvir into the composite materials is given in Figure 10 (lower). with higher quantities of core shell latex. The loading of sofosbuvir into materials is given in Figure 10 (lower). pH 0 2 4 6 8 10 12 Aprepitant drug loaded (mg/g) 0 50 100 150 200 250 300 350 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 materials is given in Figure 10 (lower). pH 0 2 4 6 8 10 12 Aprepitant drug loaded (mg/g) 0 50 100 150 200 250 300 350 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 pH 0 2 4 6 8 10 12 Sofosbuvir drug Loaded (mg/g) 50 100 150 200 250 300 350 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Figure 9. Loading of drugs into the fabricated MCM-PA@B at different pH values:aprepita per); sofosbuvir (lower). Figure 9. Loading of drugs into the fabricated MCM-PA@B at different pH values: aprepitant (u sofosbuvir (lower). pH 0 2 4 6 8 10 12 Sofosbuvir drug Loaded (mg/g) 50 100 150 200 250 300 350 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 pH Figure 9. Loading of drugs into the fabricated MCM-PA@B at different pH values:aprepitant (up- per); sofosbuvir (lower). Figure 9. Loading of drugs into the fabricated MCM-PA@B at different pH values: aprepitant (upper); sofosbuvir (lower). 14 of 20 14 of 20 Polymers 2022, 14, 2681 Concentration (ppm) 0 20 40 60 80 100 120 Aprpitant drug Loaded (mg/g) 0 100 200 300 400 500 600 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Concentration (ppm) 0 20 40 60 80 100 120 Sofosbuvir Loaded (mg/g) 0 100 200 300 400 500 600 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Figure 10. Loading of drugs into the fabricated MCM-PA@B at different concentrations: (upper); sofosbuvir (lower). Figure 10. Loading of drugs into the fabricated MCM-PA@B at different concentrations: a (upper); sofosbuvir (lower). The release of the drugs aprepitant and sofosbuvir at various time intervals in Figure 11. 3.7. Drug Loading and Release Study 3.7. Drug Loading and Release Study C t ti ( ) 0 20 40 60 80 100 120 Aprpitant drug Loaded (mg/g) 0 100 200 300 400 500 600 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Concentration (ppm) 0 20 40 60 80 100 120 Sofosbuvir Loaded (mg/g) 0 100 200 300 400 500 600 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Figure 10. Loading of drugs into the fabricated MCM-PA@B at different concentrations:aprepitant (upper); sofosbuvir (lower). Figure 10. Loading of drugs into the fabricated MCM-PA@B at different concentrations: aprepitant (upper); sofosbuvir (lower). The release of the drugs aprepitant and sofosbuvir at various time intervals is shown in Figure 11. The release of the drugs aprepitant and sofosbuvir at various time intervals is shown in Figure 11. 15 of 20 Polymers 2022, 14, 2681 Time (hr) 0 5 10 15 20 25 30 Drug Release(%) 0 10 20 30 40 50 60 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Time (h) 0 5 10 15 20 25 30 Drug Release(%) 0 10 20 30 40 50 60 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 igure 11. Controlled release study of drugs from the fabricated MCM-PA@B:aprepitant (upper); ofosbuvir (lower). Figure 11. Controlled release study of drugs from the fabricated MCM-PA@B: aprepitant (upper); sofosbuvir (lower). Time (hr) 0 5 10 15 20 25 30 Drug Release(%) 0 10 20 30 40 50 60 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 Time (h) 0 5 10 15 20 25 30 Drug Release(%) 0 10 20 30 40 50 60 IONPs@SiO21:PA@B1 IONPs@SiO21:PA@B3 IONPs@SiO21:PA@B5 IONPs@SiO23:PA@B5 IONPs@SiO25:PA@B5 1. Controlled release study of drugs from the fabricated MCM-PA@B:aprepitant (upper); ir (lower). Figure 11. Controlled release study of drugs from the fabricated MCM-PA@B: aprepitant (upper); sofosbuvir (lower). orption Kinetics Models 3.8. Adsorption Kinetics Models orption Kinetics Models 3.8. Adsorption Kinetics Models arious adsorption kinetics models were applied tothe loading of both drugs onto ricated MCM-PA@B. Table 2A,B show the data of the kinetic model applied. As we that first-order kinetics is concentration-dependent, first-order kinetics explains e incorporation of drugs into the MCM-PA@B is directly dependent on the amount drug incorporated and its saturation concentration. From the table, we can see that maximum values of 0.92 and 0.94 for MCM-PA@B, informing us that this first-order model is not enough to explain the loading of both drugs. Various adsorption kinetics models were applied tothe loading of both drugs onto the fabricated MCM-PA@B. Table 2A,B show the data of the kinetic model applied. As we know that first-order kinetics is concentration-dependent, first-order kinetics explains that the incorporation of drugs into the MCM-PA@B is directly dependent on the amount of the drug incorporated and its saturation concentration. From the table, we can see that R2 has maximum values of 0.92 and 0.94 for MCM-PA@B, informing us that this first-order kinetic model is not enough to explain the loading of both drugs. Polymers 2022, 14, 2681 16 of 20 16 of 20 Table 2. (A). Kinetic models for loading of aprepitant into the fabricated IONPs@SiO2-PA@B com- posite materials. (B). Kinetic models for the loading of sofosbuvir into the IONPs@SiO2-PA@B composite materials. orption Kinetics Models 3.8. Adsorption Kinetics Models (A) IONPs@SiO21: PA@B1 IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25: PA@B5 qe (exp) mg/g 193.82 193.903 194.02 194.02 194.06 Pseudo-first-order kinetic model K1(min−1) −0.035 −0.18 −0.042 −0.015 −0.042 qe (mg/g) 8.18 9.79 9.86 4.56 9.98 R2 0.92 0.91 0.91 0.92 0.91 Pseudo-second-order kinetic model K2(min−1) 0.001 0.001 0.001 0.001 0.001 qe (mg/g) 197.08 197.08 197.08 197.08 197.08 R2 1 1 1 1 1 Intra-particle diffusion model Kint 0.336 0.338 0.342 0.342 0.345 C 189.18 189.25 189.33 189.34 189.32 R2 0.901 0.898 0.897 0.893 0.898 (B) IONPs@SiO21: PA@B1 IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25: PA@B5 qe (exp) mg/g 194.29 194.43 194.53 194.57 194.57 Pseudo-first-order kinetic model K1(min−1) −0.027 −0.026 −0.025 −0.027 −0.029 qe (mg/g) 7.05 6.92 6.73 7.24 7.57 R2 0.93 0.94 0.94 0.93 0.93 Pseudo-second-order kinetic model K2(min−1) 0.001 0.001 0.001 0.001 0.001 qe (mg/g) 196.07 196.07 196.07 196.07 196.07 R2 1 1 1 1 1 Intra-particle diffusion model Kint 0.366 0.366 0.366 0.366 0.370 C 187.37 187.37 189.37 189.41 189.42 R2 0.895 0.899 0.902 0.898 0.893 The second-order kinetics equation is dependent on the loading equilibrium capacity, which assumes that the rate of loading over the sites of attachment is proportional to the square of unavailable sites for attachment. The rate of loading is related to the concentration of the activated sites on the surface of MCM-PA@B. According to Table 2A,B, second-order kinetics for loading of both drugs fits well due to its higher R2 value of 1. This high R2 value shows that in the loading of both of the drugs, the rate-controlling step could be chemisorption. The formation of a link between the hydrogel and the drug takes place through exchange of electrons, which is chemisorption [51]. g g p The diffusion process plays a very crucial role in the loading of drugs into the com- posite materials. According to Table 2A,B, the obtained data show good correlation for the second-order kinetics model, but cannot explain the drug diffusion mechanism. The values obtained initially for the loading of drugs can be used to evaluate the intra-particle diffusion model of kinetics. Inside the active sites of MCM-PA@B, the loading of both of the drugs may be controlled by mass transfer through pores, liquid-phase external mass transfer, or both [52]. 3.9. Application of Equilibrium Isotherms on the Loading of Drugs osite materials. (B). Equilibrium isotherms and their values for the loadin Ps@SiO2 PA@B composite materials e interaction between the drugs and composite materials was eva therms. The loading of drugs over the uniform surface of the com Langmuir and Freundlich equilibrium isotherms were used to evaluate the loading of drugs into the fabricated composite materials. The data for equilibrium isotherms are given in Table 3A,B. The interaction between the drugs and composite materials was evaluated through these isotherms. The loading of drugs over the uniform surface of the composite materials through monolayer incorporation is shown by the Langmuir adsorption isotherm. Both of the drugs follow Langmuir isotherms, for which the R2 value is 1, showing us that the loading of both drugs is favorable, due to the presence of many numbers of active sites as well as the uniform surface of the composite materials. The data for the Freundlich isotherms for both of the drugs show that the loading of the drugs does not follow them satisfactorily, with n values of less than 1 as compared to the Langmuir isotherms [54]. s@SiO2 PA@B composite materials. 1: IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25 : PA@B5 −25 −25.31 −25.31 −25.38 216.27 215.97 216.02 215.87 0.99 0.99 0.99 0.99 −250 −256.41 −256.41 −256.41 −1.35 −1.38 −1.38 −1.38 h monolayer incorporation is shown by the Langmuir adsorption ugs follow Langmuir isotherms, for which the R2 value is 1, show- of both drugs is favorable, due to the presence of many numbers s the uniform surface of the composite materials. The data for the or both of the drugs show that the loading of the drugs does not ly, with n values of less than 1 as compared to the Langmuir iso- m isotherms and their values for the loading of aprepitant on the osite materials. (B). Equilibrium isotherms and their values for the loading Table 3. (A). Equilibrium isotherms and their values for the loading of aprepitant on the IONPs@SiO2- PA@B composite materials. (B). Equilibrium isotherms and their values for the loading of sofosbuvir on the IONPs@SiO2-PA@B composite materials. 185.18 185.18 185.18 185.18 1 1 1 1 Ps@SiO2 PA@B composite materials. orption Kinetics Models 3.8. Adsorption Kinetics Models The loading of drugs over the uniform surface of the com 3.9. Application of Equilibrium Isotherms on the Loading of Drugs osite materials. (B). Equilibrium isotherms and their values for the loadin Ps@SiO2 PA@B composite materials e interaction between the drugs and composite materials was eva therms. The loading of drugs over the uniform surface of the com 1: IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25 PA@B5 (A) IONPs@SiO21: PA@B1 IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25: PA@B5 N −24.69 −25 −25.31 −25.31 −25.38 Kf 216.57 216.27 215.97 216.02 215.87 R2 0.99 0.99 0.99 0.99 0.99 KL (L/g) −243.9 −250 −256.41 −256.41 −256.41 AL (L/mol) −1.317 −1.35 −1.38 −1.38 −1.38 IONPs@SiO21: PA@B1 IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25 : PA@B5 N −25.83 −26.24 −26.52 −26.73 −26.80 Kf 215.42 215.07 214.78 214.58 214.53 R2 0.99 0.99 0.99 0.99 0.99 KL (L/g) −270.27 −277.77 −277.77 −285.71 −285.71 AL (L/mol) −1.45 −1.5 −1.5 −1.54 −1.54 Qͦ 185.18 185.18 185.18 185.18 185.18 R2 1 1 1 1 1 185.18 185.18 185.18 185.18 185.18 R2 1 1 1 1 1 (B) IONPs@SiO21: PA@B1 IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25: PA@B5 N −25.83 −26.24 −26.52 −26.73 −26.80 Kf 215.42 215.07 214.78 214.58 214.53 R2 0.99 0.99 0.99 0.99 0.99 KL (L/g) −270.27 −277.77 −277.77 −285.71 −285.71 AL (L/mol) −1.45 −1.5 −1.5 −1.54 −1.54 N −24.69 −25 −25.31 −25.31 −25.38 Kf 216.57 216.27 215.97 216.02 215.87 R2 0.99 0.99 0.99 0.99 0.99 KL (L/g) −243.9 −250 −256.41 −256.41 −256.41 AL (L/mol) −1.317 −1.35 −1.38 −1.38 −1.38 Qͦ 185.18 185.18 185.18 185.18 185.18 R2 1 1 1 1 1 (B) IONPs@SiO21: PA@B1 IONPs@SiO21: PA@B3 IONPs@SiO21: PA@B5 IONPs@SiO23: PA@B5 IONPs@SiO25 : PA@B5 N −25.83 −26.24 −26.52 −26.73 −26.80 Kf 215.42 215.07 214.78 214.58 214.53 R2 0.99 0.99 0.99 0.99 0.99 KL (L/g) −270.27 −277.77 −277.77 −285.71 −285.71 AL (L/mol) −1.45 −1.5 −1.5 −1.54 −1.54 Qͦ 185.18 185.18 185.18 185.18 185.18 R2 1 1 1 1 1 185.18 185.18 185.18 185.18 185.18 R2 1 1 1 1 1 (A) N orption Kinetics Models 3.8. Adsorption Kinetics Models The rate-determining step of the loading of drugs may be pore Polymers 2022, 14, 2681 3 9 Application (mg/g) R2 17 of 20 17 of 20 diffusion or film diffusion. However, we can conclude from the data in Table 2A,B that the intra-particle diffusion step was not involved in the loading of either drug. The results of the intra-particle diffusion models shows that three steps are involved in the loading of drugs: In the initial step, drug molecules are loaded at quicker rate from the solution, with active sites of composite materials on their surface [53]. During the second stage, the drug molecules go inside the space present in composite materials at a quicker rate. In the third stage, the drug molecules diffuse inside the composite materials through very tiny pores—where the diffusion takes place very slowly—and reach their maximum quantity, after which no further loading takes place. From all of these results obtained for the loading of both of the drugs we can conclude that this kinetic model is not the rate-controlling step for the loading of both of the drugs, for which this model does not pass through the origin. ated composite materials. The data for equilibrium isotherms are e interaction between the drugs and composite materials was eval- therms. The loading of drugs over the uniform surface of the com- h monolayer incorporation is shown by the Langmuir adsorption ugs follow Langmuir isotherms, for which the R2 value is 1, show- of both drugs is favorable, due to the presence of many numbers the uniform surface of the composite materials. The data for the or both of the drugs show that the loading of the drugs does not ly, with n values of less than 1 as compared to the Langmuir iso- m isotherms and their values for the loading of aprepitant on the 196.07 196.07 196.07 196.07 1 1 1 1 Intra-particle diffusion model 0.366 0.366 0.366 0.370 187.37 189.37 189.41 189.42 0.899 0.902 0.898 0.893 brium Isotherms on the Loading of Drugs undlich equilibrium isotherms were used to evaluate the loading ated composite materials. The data for equilibrium isotherms are i i b h d d i i l l 3.9. Application of Equilibrium Isotherms on the Loading of Drugs site materials. (B). Equilibrium isotherms and their values for the loadin @SiO2 PA@B composite materials interaction between the drugs and composite materials was eval herms. 4. Conclusions In this study, IONPs@SiO2 and CS were successfully synthesized and incorporated with one another to produce composite materials. The synthesis of these composite ma- terials was confirmed by the FTIR, TGA, XRD, and SEM analysis techniques. From the TGA, it was found that the composite materials with high ratios of IONPs@SiO2 were more Polymers 2022, 14, 2681 18 of 20 18 of 20 thermally stable. All of the synthesized composite materials were further applied for the loading and release of aprepitant and sofosbuvir. Maximum loading was found at pH 4 and at a time of 2 h; moreover, this loading amount increased with the increase in the concentration of the drugs. These loaded composite materials were further investigated for their release at pH 7.4 and 37 ◦C. The release of the drugs reached a maximum of 50% after 24 h. From all of the data obtained, we can conclude that increasing the amount of CS and IONPs@SiO2 in the composite materials can enhance the loading efficiency of both drugs, while slow release of the drugs occurs in these composite materials. All of our results show that the composite materials of IONPs@SiO2 with CS can be used as efficient drug delivery systems. Author Contributions: Conceptualization, M.H. and N.S.; methodology, L.C.M. and T.R.; software S.I.S. and A.K.; validation, S.I.S. and N.S.; formal analysis, K.W.G., L.C.M. and M.H.; investigation, N.S.; resources, K.W.G. and L.C.M.; data curation, L.C.M.; writing—original draft preparation, M.H. and N.S.; writing—review and editing, L.C.M., N.S., S.I.S. and A.K.; visualization, K.W.G. and S.I.S.; supervision, N.S.; project administration, N.S.; funding acquisition, N.S., K.W.G. and L.C.M. All authors have read and agreed to the published version of the manuscript. Funding: This research work was fully supported by the HEC Pakistan under the NRPU project number 20-5201, and partially supported by another project number NRPU-20-3868 awarded to Nasrullah Shah. Acknowledgments: The authors are indebted to the R&D section of the Ferozsons Pharmaceutical Industry, Nowshera, Pakistan for providing the standard drugs for use in the present study. Acknowledgments: The authors are indebted to the R&D section of the Ferozsons Pharmaceutical Industry, Nowshera, Pakistan for providing the standard drugs for use in the present study. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Shah, S.A.; Majeed, A.; Rashid, K.; Awan, S.U. PEG-coated folic acid-modified superparamagnetic MnFe2O4 nanoparticles for hyperthermia therapy and drug delivery. Mater. Chem. Phys. 2013, 138, 703–708. [CrossRef] yp py g y y 2. Shah, S.A.; Hashmi, M.; Alam, S. Effect of aligning magnetic field on the magnetic and calorimetric pr bi ti l i f th h th i t t t f M t S i E C 2011 31 1010 101 2. 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Design Approaches to Improve Organic Solar Cells
Journal of technology innovations in renewable energy
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Journal of Technology Innovations in Renewable Energy, 2014, 3, 63-71 Journal of Technology Innovations in Renewable Energy, 2014, 3, 63-71 63 Design Approaches to Improve Organic Solar Cells Fahmi Fariq Muhammad* t of Physics, Faculty of Science and Health, Koya University, Koya, Kurdistan Region, Iraq Abstract: Organic solar cells (OSC)s are valuable economical and environmental friendly devices capable to generate electricity from sunlight. This is because of their simple fabrication process and minimal material usage in comparison to the inorganic solar cells. OSCs have not been widely commercialized yet because of their relatively low power conversion efficiency and stability problem. It is expected that these obstacles will be surmounted in a foreseeable future upon rigorous research studies performed in the field. This paper is devoted to reviewing the design related strategies that can be taken to enhance OSC performance. These strategies can be accomplished through modulating the architecture of the devices and by considering the alignment of the molecular energy levels between their active layer components. Keywords: Organic solar cell, Nanostructure material, OSC performance, OSC efficiency, OSC improvemen Keywords: Organic solar cell, Nanostructure material, OSC performance, OSC effi *Address correspondence to this author at the Department of Physics, Faculty of Science and Health, Koya University, Koya, Kurdistan Region, Iraq; Tel: + 964-7701935331; E-mail: fahmi982@gmail.com 1. INTRODUCTION conjugated polymers, fullerenes (C60 and its derivatives), and carbon nanotubes, CNTs [11-17]. However, due to their low efficiencies of about 9% [18, 19] and stability problem [20], they are still far from being commercialized widely, while at least 10% of stable power conversion efficiency is required for practical applications [21, 22]. The unsolved issues of low efficiency, and short life time, necessitate rigorous research studies to be performed covering from materials analysis to the devices fabrication and characterization. Nevertheless, relatively little information is known about the exact correlation between the devices parameters and their materials performance, still some of the OSCs parameters are not fully understood [23, 24]. Figure 1 shows a prototype structure of OSC device. Indium-tin-oxide (ITO) is normally utilized as first electrode. The most commonly used second electrode is one of these metals; aluminum, calcium, magnesium, or gold. The continuous demand for energy and the limiting supply of its today’s main sources (petroleum, natural gas, and coal) with their detrimental long term effects on the environment necessitates a rapid development into the clean and renewable energy sources. The only sustainable source that can supply electric power with its capability of protecting our environment from being polluted is thought to be solar energy [1-3]. Solar energy can be exploited to generate electricity by means of solar cell devices in a technology known as photovoltaic, PV technology. The most widely used material in PV fabrication is crystalline silicon, c-Si representing over 90% of the global commercial PV module production in its various forms [4]. Manufacturing of inorganic based SC is expensive due to strong requirements to the high purity of this material and the techniques used in the devices fabrication [5]. Besides, emissions to the environment are mainly occurred from using fossil-fuel based energy in producing the materials for inorganic solar cells [6]. Figure 1: Prototype structure of organic solar cells. Over the last two decades great efforts have been made to develop the low cost and ambient temperature processing solar cells based on organic and polymeric materials [7-10]. Despite easy fabrication of these devices, they are able to show extra important features, such as flexibility, light weight, and degradability for recycling purposes. OSC is a green technology, which is basically made from those materials that carbon atoms are participating in their chemical structures. 2. BULK HETEROJUNCTION STRUCTURES The idea behind bilayer heterojunction, which consists of two layers of D-A, is to use two materials with different electron affinities, LUMOs and ionization potentials, HOMOs [26]. By this, favorable exciton dissociation is obtained; the electron will be accepted by the material with the higher electron affinity while the hole by the material with the lower ionization potential [27]. The main drawback of this concept resides in the rather short diffusion length of excitons (5–15 nm) [28]. Indeed, only those excitons that are created within a distance from the D-A interface shorter than their diffusion length may contribute to the photocurrent generation [25]. This limits the photocurrent and hence the overall performance of bilayer organic solar cells. To overcome this limitation, the surface area of the D-A interface needs to be increased. This can be achieved by creating a mixture of donor and acceptor materials with a nanoscale phase separation resulting in a three dimensional interpenetrating network called the bulk heterojunction like shown in Figure 2. Bulk heterojunction concept suggests the goal of achieving a larger interfacial area between the electron and hole transporting materials. The photocurrent achieved by bulk heterojunction devices are up to several milliamperes per square centimeter, improving drastically the efficiencies over that of bilayer strucutres [24, 29]. It has been proved that solution-processed bulk heterojunction structures with D-A blends sandwiched between the anode and cathode are the One major obstacle in front of OSCs is how to make excitons dissociate effectively into free charge carriers. Another challenge, which is common in both of organic and inorganic solar cells, is the full collection of photogenerated charge carriers by the correspondence electrodes (negative and positive electrodes). In the organic materials, especially disordered ones, carrier mobility is several orders of magnitude smaller than that in crystalline inorganic semiconductors. This imposes restriction on the maximum thickness of organic photovoltaic devices and makes them to have very thin active layers (in nanometers scale). Furthermore, D-A organic components suffer from an imbalance transport of electron and hole carriers. Accumulation of less mobile charge carriers in the active layer will hamper charge collection at the electrodes and thereby drastically reduce the solar cell efficiency. Another problem is the difficulties in an efficient harvest of excitons. Because of a large binding energy, intrinsic dissociation of excitons into free carriers is virtually impossible. 1. INTRODUCTION OSCs are seen to be the most plausible candidates with substantial future prospects consisting of Figure 1: Prototype structure of organic solar cells. It is clear that upon the absorption of light by OSCs, excitons (bound electron-hole) are formed in the donor- acceptor (D-A) active layer, followed by the exciton diffusion and dissociation, which occurs at the D-A interfaces via an ultra-fast charge transfer. via E-ISSN: 1929-6002/14 © 2014 Lifescience Global © 2014 Lifescience Global Journal of Technology Innovations in Renewable Energy, 2014, Vol. 3, No. 2 Fahmi Fariq Muhammad 64 Subsequently, the separated free electrons and holes transport through individual percolating pathways, and then they are extracted by the corresponding electrodes. Hence, one might conclude that the device performance can be approached by improving factors contributing in the processes of light absorption, exciton diffusion/dissociation, charge transport, and charge collection. bottlenecks and hence to produce efficient OSC fabrication. The next sections will provide an interesting overview of these approaches. 2. BULK HETEROJUNCTION STRUCTURES Due to this, diffusion of excitons towards either charge transfer centers or D-A interfaces is a prerequisite for charge photogeneration [25]. Therefore, different practical approaches are needed to be undertaken to overcome these Figure 2: Bulk heterojunction structure for OSCs. Figure 2: Bulk heterojunction structure for OSCs. Journal of Technology Innovations in Renewable Energy, 2014, Vol. 3, No. 2 Design Approaches to Improve Organic Solar Cells 65 most promising alternative to realize large-scale solar cell production [30, 31]. most promising alternative to realize large-scale solar cell production [30, 31]. the charge transport plays a crucial role in the device performance. As a result, the optimized thickness for most of OSCs is less than 100 nm [13]. Apart from bulk heterojunction structure that was discussed previously, several materials and device structures have been developed to obtain high short circuit current density, Jsc. The concept of tri-layer organic p-i-n junction [34], in which i-layer is a co-deposited layer of two different organic semiconductors is another approach, as shown in Figure 3a. Co-deposited layers have a vast number of heteromolecular (donor–acceptor) contacts acting as efficient photocarrier generation sites [28]. Additionally, tandem structure as an effective approach to enhance the light harvesting by means of stacking multiple cells with complementary absorption spectra has also been proposed (see Figure 3b). The limits to power conversion efficiency and photovoltage can be breached through fabrication of tandem solar cells [35- 37]. A tandem solar cell consists of two stacked solar cells made from materials with different optical gaps. Initially, light is absorbed by the higher-gap cell hence the lower energy photons pass through the higher gap device and then photons are absorbed by the second cell. There is a conductive layer connecting the two cells accordingly, which works as a site for charge recombination. However, in terms of device fabrication, there are difficult tasks such as optimizing the layers thickness, selection of suitable materials, and recombination sites that have to be treated with a great caution. Figure 3b shows a representative tandem solar cell with two stacked cell structure having a gold metal, Au layer as the conductive layer between the two stacked cells to provide the recombination site. The bulk heterojunction device is similar to the bilayer device with respect to the D-A concept, but it exhibits a vastly increased interfacial area dispersed throughout the bulk. 2. BULK HETEROJUNCTION STRUCTURES The bulk heterojunction requires percolated pathways for both phases throughout the volume, i.e., a bicontinuous and interpenetrating network. Therefore, the nanoscale morphology in the bulk D-A active layer is more complicated to be controlled well in terms of the phase separation between the donor and acceptor regions and hence possibly the leakage current persists due to the insufficient contact of the acceptor phase with the cathode electrode and donor phase with the anode electrode [32]. The bulk heterojunction devices used today consist, in general, of a poly(4-tyrenesulfonate) (PEDOT-PSS) covered ITO substrate, coated with a single photoactive bulk heterojunction layer (e.g., polymer–fullerene mixtures) closed by a low work function cathode such as Al [33]. 3. MULTILAYER STRUCTURES It is known that each organic material has a unique optical band gap and a broad absorption spectrum of sunlight is not easy to be achieved by a single organic layer. Therefore, multiple layers and stacked tandem cell structure, in which each layer absorbs a different light wavelength, have been proposed to mostly resolve the limited absorption problem. Organic materials have higher absorption coefficient than that of inorganic materials. Therefore, about 300 nm film is thick enough to absorb the most incident light [13]. However, the thickness is ultimately limited by the short exciton diffusion length and low charge carrier mobility [17] since the balance between the light absorption and 5. DOUBLE CABLE POLYMER It was understood that the control of nanoscale morphology in bulk heterojunction structure was not easy. The degree of the phase separation between D-A and domain size depend on solvent choice, speed of evaporation, materials solubility, miscibility of the donor and acceptor components, annealing temperature, etc. It was understood that the control of nanoscale morphology in bulk heterojunction structure was not easy. The degree of the phase separation between D-A and domain size depend on solvent choice, speed of evaporation, materials solubility, miscibility of the donor and acceptor components, annealing temperature, etc. One strategy towards increasing the D-A phase control is to covalently link donor and acceptor molecules to get some sort of polymers called the double cable polymers. Researchers [41-43] have synthesized a pendant fullerene moieties and polythiophene backbone with covalently bound tetracyanoan- thraquino-dimethane, TCAQ moieties (donor–acceptor double-cable polymer), respectively, aiming at utilizing these structures in organic solar cells. Even though the synthesized soluble cable polymers have shown some promising results, the fully optimized double-cable polymers are still not achieved. As the complexity of the designed systems increase, the more critical it becomes to optimize design parameters. The realization of effective double cable polymers will bring the D-A heterojunction at a molecular level. Figure 6a shows a schematic representation of this system, while Figure 6b shows an alternative approach to double cables polymer, known as block copolymers, consisting of donor and acceptor blocks linked side by side together. In general, block copolymers [44, 45] are well recognized for phase separation and ordered domains formation, similar to those of the double cable polymers. Stalmach et al. [46] synthesized a block copolymer consisting of an electron acceptor block and Figure 4: An OSC device incorporating exciton blocking layer of Alq3 between the cathode electrode and acceptor material. Figure 4: An OSC device incorporating exciton blocking layer of Alq3 between the cathode electrode and acceptor material. Similar to the cathode interface, atoms from the anode electrode can react with the organic material. Indium atoms from indium tin oxide, ITO electrode were found to diffuse into the organic layer, where it acts as trapping site for the charge carriers [27]. One strategy that is used to minimize indium and oxygen diffusion is to put an interfacial hole-transporting layer, such as poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfo- nic) acid, PEDOT-PSS between ITO and the active material. 4. EXCITON BLOCKING LAYER The active layer of bulk heterojunction structure is sandwiched between the anode and cathode Figure 3: The structure of (a) multilayer organic p-i-n solar cells and (b) organic tandem solar cells. Figure 3: The structure of (a) multilayer organic p-i-n solar cells and (b) organic tandem solar cells. Journal of Technology Innovations in Renewable Energy, 2014, Vol. 3, No. 2 66 Fahmi Fariq Muhammad nm) between the active layer and anode electrode, the efficiency of the device was pronouncedly increased [24]. electrodes, in which both donor and acceptor materials are in direct contact with the electrodes. So it is possible for the acceptor material to transfer electrons to the hole-collecting anode and for the donor to transfer holes to the electron-collecting cathode, thereby producing a large leakage current and lowering the performance of the device. In order to tackle these problems, interfacial buffer layers can be inserted between the active layer and electrodes to enhance the collection of the photogenerated charges and to reduce the leakage current [31]. In a study in which an exciton blocking layer, EBL of bathocuproine, BCP was incorporated, photovoltaic properties of devices based on pentacene/PCBM were investigated [38]. It was seen that a thin layer of BCP has improved the EQE and charge carrier collection in the devices. More recently, tris (8-hydroxyquinolinate) aluminium, Alq3 was also used between the cathode electrode and acceptor materials [39, 40] (see Figure 4). It was observed that this has led to increase in both efficiency and stability of the OSC devices. Figure 5: An OSC device incorporating exciton blocking layer of PEDOT:PSS between the anode electrode and acceptor material. 6. ENERGY BAND ALIGNMENT The discovery of conjugated polymers stimulated the research field of organic electronics, thereby developing a variety of organic based devices such as, solar cells, light-emitting diodes, field-effect transistors, and memory devices. The emergence of the fields can be tracked back to the mid-1970 when Shirakawa reportedly prepared the first polymer (polyacetylene) by accident, subsequently Heeger and MacDiarmid discovered that the polymer would undergo an increase in conductivity by 12 orders [47]. The most important functionality of the organic materials is the large polarizability of their extended  -conjugated electron systems formed by the delocalization of the pz orbitals of the carbon atoms [14]. Due to this basic functionality, upon the absorption of sun light, these materials can show the photo-induced charge carriers and transport properties by hopping process along their conjugated backbone. Hence, most of the organic materials have attracted considerable attention to be exploited in the fabrication of electronic and optoelectronic devices [48]. In particular, two types of materials are usually selected as photovoltaic active layers in the fabrication of OSCs. The first layer must be conductive to holes. This is referred to as donor, while the second layer is conductive to electrons and known as acceptor. Regardless of whether the device is produced in a bilayer or bulk heterojunction structure, it is of great importance the HOMO and LUMO energy levels of the D-A system are matched well to facilitate efficient Step 3: (D  A)*  (D +  A )* (polarization of excitation: partial charge transfer) Step 3: (D  A)*  (D +  A )* (polarization of excitation: partial charge transfer) Step 3: (D  A)*  (D +  A )* (polarization of excitation: partial charge transfer) Step 4: (D +  A )*  (D•+  A•) (ion radical pair formation) Step 4: (D +  A )*  (D•+  A•) (ion radical pair formation) Step 5: (D•+  A•) D•+ + A• (complete charge separation) Table 1 shows some representative donors and acceptors including their HOMO and LUMO energy levels [50, 51] with their molecular structures. Fullerenes are considered the best electron acceptors so far. Step 1: D + A  D* + A (photoexcitation of D) Step 1: D + A  D* + A (photoexcitation of D) Step 1: D + A  D* + A (photoexcitation of D) Step 2: D* + A  (D  A)* (excitation delocalized between D and A) Step 2: D* + A  (D  A)* (excitation delocalized between D and A) Step 2: D* + A  (D  A)* (excitation delocalized between D and A) 5. DOUBLE CABLE POLYMER This layer serves to smooth out the uneven surface of ITO and provides larger injection of holes into the anode electrode. Figure 5 shows a device, in which after insertion of a thin PEDOT:PSS layer (35 Journal of Technology Innovations in Renewable Energy, 2014, Vol. 3, No. 2 6 Design Approaches to Improve Organic Solar Cells 67 Figure 6: Shows (a) schematic representation of a realistic double cable polymer and (b) self-assembled layered structure of di- block copolymers. Figure 6: Shows (a) schematic representation of a realistic double cable polymer and (b) self-assembled layered structure of di- block copolymers. exciton generation, dissociation and charge transport properties. The basic process of the photo-induced charge transfer between a donor, D molecule and an acceptor, A molecule can be described as follows [49]: an electron donor block by means of atom-transfer radical addition, aiming to enhance the photovoltaic efficiency of the PPV-C60 system (PPV = poly(p- phenylenevinylene)). Since the solubility of such complicated structures is very limited, the practical handling for device fabrication is cumbersome [27]. 6. ENERGY BAND ALIGNMENT This is because of (i) ultrafast (~50 fs) photoinduced charge transfer that is happened between the donors and fullerenes, (ii) fullerenes exhibit high mobility, for example, C60 has shown field effect electron mobility of up to 1 cm2 V-1 s-1 and (iii) fullerenes show a better phase segregation in the blend films [13, 49]. Among the organic donor materials concerned, sexithiophene, 6T shows the highest mobility when they are used as hole transporting layers [52, 53]. By improving the solubility of sexithiophenes through the addition of hexyl side chains to  - sexithiophene (6T) [54], it was possible to synthesize  , -dihexyl-sexithiophene, DH6T semiconductor, which was characterized by reasonable field-effect mobility reaching as high as 0.1 cm2/V.s [55, 56]. Journal of Technology Innovations in Renewable Energy, 2014, Vol. 3, No. 2 Fahmi Fariq Muhammad 68 gy gy, , , q Table 1: Examples of Donor and Acceptor Organic Materials Including their Basic Structural and Energy Levels Information Nomenclature Molecular structure HOMO (eV) LUMO (eV) Poly [2-methoxy-5-(2’-ethyl-hexyloxy)- 1,4-phenylene vinylene] (a) O O n MEH-PPV 5.2 2.8 Poly (3-hexylthiophene) (a) S n P3HT 4.8 2.7 Donors Poly[2,6-(4,4-bis-(2-ethylhexyl)-4H- cyclopenta[2,1-b;3,4-b']dithiophene)- alt-4,7-(2,1,3-benzothiadiazole)] (b) S S N N S n PCPDTBT 4.9 3.5 ,-dihexyl-sexithiophene (c) S S S S S S C6H13 C6H13 DH6T 5.2 2.9 [6,6]-phenyl-C61 butyric acid methyl ester (PCBM) (b) OMe O PCBM, C61 6.0 3.9 [6,6]-phenyl-C70 butyric acid methyl ester (PCBM) (b) OMe O PCBM, C70 6.1 4.3 Tris (8-hydroxyquinolinate) aluminium (d) N O N O N O Al Alq3 6.3 3.4 Acceptors Tris (8-hydroxyquinolinate) gallium (d) N O N O N O Ga Gaq3 5.8 3.0 (a)[57], (b)[37], (c)[51], (d)[50]. Benefited by the high carrier mobility of DH6T and PCBM, and their appropriate energy band alignment various architectures of organic solar cells based on DH6T/PCBM have been investigated [24]. Interestingly, if a ternary bulk heterjunction structure is considered, then the right choice of materials based on their energy able 1: Examples of Donor and Acceptor Organic Materials Including their Basic Structural and Energy Levels Information O O n MEH-PPV S n P3HT ,-dihexyl-sexithiophene (c) OMe O PCBM, C61 OMe O PCBM, C70 N O N O N O Al Alq3 N O N O N O Ga Gaq3 (a)[57], (b)[37], (c)[51], (d)[50]. DH6T/PCBM have been investigated [24]. 7. OUTLINE http://dx.doi.org/10.1016/j.jpowsour.2009.12.082 The simple and low cost fabrication process of organic solar cells makes them attractive candidates for generation of electricity. The performance enhancement of these devices is of prior request by the researchers in the field. This can be accomplished upon rigorous research studies performed through materials analysis to the devices fabrication and characterization, thereby realizing valuable strategies to improve the performance of these devices. The architecture of the active layer plays a vital role in defining the overall efficiency of OSCs. In this way, various approaches can be utilized to modulate the device components including heterojunction and multilayer structures, insertion of excitons blocking layers, designing the double cable or di-block copolymers along with the optimum selection for the HOMO and LUMO energy levels between the cells components. [3] Kalogirou SA. Solar Energy Engineering, Processes and Systems. First ed. London WC1X 8RR, UK: Elsevier Inc.; 2009. [4] Foster R, Ghassemi M, Cota A. Energy and the Environment. Ghassemi A, editor. Boca Raton, FL 33487-2742: Taylor and Francis Group, LLC; 2010. Skompska M. Hybrid conjugated polymer/semiconductor photovoltaic cells. 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Photoluminescence quenching in oligothiophene single crystal. Synthetic Metals 1998; 95(2): 143-8. [46] [46] Stalmach U, de Boer B, Videlot C, van Hutten PF, Hadziioannou G. Semiconducting Diblock Copolymers Synthesized by Means of Controlled Radical Polymerization Techniques. Journal of the American Chemical Society 2000; 122(23): 5464-72. http://dx.doi.org/10.1021/ja000160a [55] [55] Iosip MD, Destri S, Pasini M, Porzio W, Pernstich KP, Batlogg B. New dithieno[3,2-b: 2',3'-d]thiophene oligomers as promising materials for organic field-effect transistor applications. Synthetic Metals 2004; 146(3): 251-7. http://dx.doi.org/10.1016/j.synthmet.2004.08.004 http://dx.doi.org/10.1021/ja000160a [47] Masi JV, editor. Polymers: conductors, insulators, and active devices. Electrical Insulation Conference and Electrical Manufacturing & Coil Winding Technology Conference, 2003 Proceedings; 2003 23-25 Sept 2003. [56] [56] Murphy AR, Fréchet JMJ, Chang P, Lee J, Subramanian V. Organic Thin Film Transistors from a Soluble Oligothiophene Derivative Containing Thermally Removable Solubilizing Groups. Journal of the American Chemical Society 2004; 126(6): 1596-7. http://dx.doi.org/10.1021/ja039529x [48] Shirota Y. Organic materials for electronic and optoelectronic devices. Journal of Materials Chemistry 2000; 10(1): 1-25. http://dx.doi.org/10.1039/a908130e [57] [57] Yamanari T, Taima T, Sakai J, Saito K. Origin of the open- circuit voltage of organic thin-film solar cells based on conjugated polymers. Solar Energy Materials and Solar Cells 2009; 93(6-7): 759-61. http://dx doi org/10 1016/j solmat 2008 09 022 [49] Koeppe R, Sariciftci NS. Photoinduced charge and energy transfer involving fullerene derivatives. Photochemical & Photobiological Sciences 2006; 5(12): 1122-31. http://dx.doi.org/10.1039/b612933c [50] Muhammad FF, Abdul Hapip AI, Sulaiman K. Study of optoelectronic energy bands and molecular energy levels of Published on 30-05-2014 Received on 23-04-2014 Received on 23-04-2014 Accepted on 01-05-2014 DOI: http://dx.doi.org/10.6000/1929-6002.2014.03.02.4 DOI: http://dx.doi.org/10.6000/1929-6002.2014.03.02.4
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https://www.e3s-conferences.org/articles/e3sconf/pdf/2018/13/e3sconf_icemee2018_02028.pdf
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Experimental study on flame propagation characteristics of Hydrogen premixed gas in gas pipeline
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1 Introduction concentration, enough oxygen and ignition source. It is a process of chemical reaction flow. The typical process of premixed flame propagation in the constrained space includes the ignition, the laminar combustion, the fire perturbation, the transformation from laminar to turbulent combustion and the final detonation. At the beginning of the reaction, the laminar flame is unstable. The fold appears caused by the instability of the thermal diffusion perturbation. The vortex generated to increase the disturbance of the flame front and to prompt the turbulent combustion, when the explosion space is constrained or there are obstacles. Then, if the flame continues to accelerate, the explosion occurs and even transforms to detonation. Figure 1 gives a sketch of the propagation process of the reaction. Flammable gas refers to the gas that can cause combustion or explosion and release a large amount of energy under the ignition energy excitation within the environment full of combustion improver. The flammable gases play a very important role in the fields of petrochemical industry, metallurgical industry and gas production and supply industry, such as methane and hydrogen. By far, hydrogen is the cleanest high-energy gas fuel, and also is the main industrial material. However, hydrogen is more explosive and more powerful than conventional gas fuels due to the lower ignition energy, the wider explosion limitation and the larger laminar burning and diffusion velocity. The hydrogen explosion accidents occur frequently. The research on the combustion and flame propagation characteristics of hydrogen premixed system has a great practical significance. The propagation of gas explosion wave, the kinetic theory of laminar flame and the constant flame theory were discussed in the early 1950s [1-2]. The propagations of gas explosion wave in the closed system were also focused as follow [3-6]. But the investigations about the hydrogen premixed system are not enough. It has very important practical significance and is the key of whether hydrogen can be used as a conventional energy. Fig. 1. A sketch of the propagation process of the reaction. The following combustion chemical reaction occurs, when premixed hydrogen is ignited. Fig. 1. A sketch of the propagation process of the reaction. Fig. 1. A sketch of the propagation process of the reaction. The following combustion chemical reaction occurs, when premixed hydrogen is ignited. The following combustion chemical reaction occurs, when premixed hydrogen is ignited. Experimental study on flame propagation characteristics of Hydrogen premixed gas in gas pipeline 1Liaoning Shihua University, Fushun, Liaoning 113001, China Abstract. Hydrogen is the cleanest high-energy gas fuel, and also is the main industrial material. However, hydrogen is more explosive and more powerful than conventional gas fuels, which restricts its application. In particular, the expansion of premixed combustion under a strong constraint is more complicated, the reaction spreads faster. The flame propagation characteristics of premixed hydrogen/air were investigated by experiment. The mechanism of reaction acceleration is discussed, and then the speed of the flame propagation and the reaction pressure were tested and analysed. * Corresponding author: danzhuma@163.com 1 Introduction 2 2 2 2 2 H +0.5O +1.88N H O+1.88N → In this paper, the deflagrating characteristic of premixed hydrogen was concerned, based on the theory of flame dynamics and pressure change. The speed of flame propagation and the pressure fluctuation were studied and analysed. In this paper, four different hydrogen concentrations were considered for test, they are 13.5%, 18%, 24%, 28.5% and 38.5%. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/20183802028 https://doi.org/10.1051/e3sconf/20183802028 E3S Web of Conferences 38, 02028 (2018) ICEMEE 2018 2 Hydrogen premixed combustion and its mechanism in the constrained space The experimental setup is shown as in figure 2, which consists of an automatically gas supply system, a high explosive pressure test system, a high-speed photography system, a high-voltage ignition system and a pressure relief channel. The simulated pipe used in the The combustion and explosion of premixed gas needs the conditions, such as the suitable flammable gas E3S Web of Conferences 38, 02028 (2018) ICEMEE 2018 https://doi.org/10.1051/e3sconf/20183802028 experiment was made by polymathic methacrylate tube. The inner diameter of the tube was 108 mm, with a uniform length of 2 m. Two relief channels are set on the side of the pipeline to ensure the safety. The ignitions were charged by high voltage spark discharge. The reaction pressures and the flame propagation were recorded by the pressure sensors and high-speed camera. with the pipe wall, the acceleration is terminated into the plane flame phase, the propagation speed of the flame front is beginning to decline. Then the flame forward reverses, gradually forming the tulip flame, which increases the surface area of the flame, the flame forward speed increases again. It can be seen from figure 4 that the tulip flame appears at 2 H 18% X = . But, at the higher Fig. 2. Schematic of the experimental setup. Fig. 2. Schematic of the experimental setup. hydrogen concentration, the premixed flame is in the accelerated propagation period, there is no feature of tulip flame in the length of the experimental pipeline. hydrogen concentration, the premixed flame is in the accelerated propagation period, there is no feature of tulip flame in the length of the experimental pipeline. Fig. 4. The flame propagation speed curves of pipelines wit different hydrogen concentration. Fig. 2. Schematic of the experimental setup. 4.1 The speed of flame propagation of premixed hydrogen in the pipeline The reaction wave fronts were recorded by the high- speed camera, and then the speed of the flame propagation could be calculated by the image processing method. The position of flame front is calculated and the speed of propagation is the ratio between the distance and the time difference between two positions, as shown in figure 3. In this paper, the speed of flame propagation under different hydrogen volume fraction is calculated by using the above method. y g Fig. 3. The sketch of the flame propagation in the pipeline. y g Fig. 3. The sketch of the flame propagation in the pipeline. Fig. 3. The sketch of the flame propagation in the pipeline. The effect of different hydrogen volume fractions on the speed of flame propagation was discussed. The results are shown in figure. 4. The propagation speed of the flame in the pipe increased with the flame front propelling, and then decreased after the peak for 2 H 13.5% X = and 2 H 18% X = . The speed of flame propagation decreased with the irregular flame front. But the decrease of the speed was not tested in the higher hydrogen content. Fig. 4. The flame propagation speed curves of pipelines with different hydrogen concentration. References gas washes out of the pipe. At the same time, the gas in the pipeline continues to participate in the reaction and consumed, the pressure drops in the pipe, resulting the negative pressure. The negative pressure in the tube causes the ejected gas to be sucked back into the reaction, causing the pressure rise again, thus causing the pressure oscillation. With the constant consumption of unburned gas in the tube, the absolute value of the pressure oscillation decreases and finally returns to the initial value. 1. W. Bartknecht, Explosions: Course, Prevention, Protection (Berlin, Springer, 1981) 2. D. Bjerketvedt, J. R. Bakke, K. Wingerden, Gas Explosion Handbook (Journal of Hazardous Materials, 1997) 3. K. K. Kuo, Principles of Combustion (Wiley- Interscience, Switzerland, 2005) 4. Mustafa Ilbas, Serhat Karyeyen, Ismail Ozdemir, Int. J Hydrogen energy, E 26, 1 (2015) The results of the pressure test show that the pressure propagation of hydrogen-air in the pipeline is divided into three stages. (1) After the input of the ignition energy, the premixed gas is ignited, the hydrogen-air continue to react, the pressure in the pipe continuously accumulates to reach the first crest value. (2) Due to the high temperature of the premixed combustion, when the water vapor which generated by the reaction contact the lower temperature environment to condense into droplets. This process causes the negative pressure in the pipe. And then, the unreacted hydrogen-air are sucked back to the pipe to join in the reaction, the pressure goes up again. This causes the pressure shock in the pipeline. (3) The pressure amplitude reduces to the initial value with the complete reaction of the premixed gas. The peak pressure and the intensity of the pressure oscillation increase with the increasing of hydrogen content, while the time of peak pressure decrease. 5. Xianzhong Hu, Qingbo Yu, Nan Sun, Qin Qin, Int. J Hydrogen energy, E 39, 9527 (2014) 6. Hongfa Huo, Xingjian Wang, Vigor Yang, Combustion and Flame, E 161, 3040 (2014) p p Fig. 5. Typical reaction pressure curve. p p Fig. 5. Typical reaction pressure curve. Fig. 5. Typical reaction pressure curve. 4.2 The reaction pressure The peak speed of flame propagation and the location of the peak are all related to the hydrogen concentration. The higher the concentration of hydrogen, the more violent the reaction is, the faster the flame spread. The propagation speed of the premixed gas in the restricted pipeline is closely related to the shape of the flame front. In the early stage, the flame is not restrained by the wall, it is semi-spherical and rapidly evolves into a fingertip flame, the surface area of the flame is accelerated by exponential growth. When the flame comes into contact The reaction pressure was recorded by the pressure sensor, which was lie in the front of the pipe. The typical curves were shown in figure 5. As shown in the figure, the reaction pressure of premixed hydrogen-air has obvious pressure oscillation and appears negative pressure. At the beginning of the reaction, the pressure increases rapidly with the reaction progresses. Then the film on the other side of the pipeline breaks, the pipe becomes to a semi-enclosed space, part of the premixed 2 2 https://doi.org/10.1051/e3sconf/20183802028 E3S Web of Conferences 38, 02028 (2018) ICEMEE 2018 5 Summary The combustion characteristics of premixed hydrogen is the foundation of its safe application as a high-energy fuel. The experiment results show that the peak speed of flame propagation and the location of the peak depend on the concentration of hydrogen. The higher hydrogen concentration, the more violent the reaction is, and the faster the flame spread. The reaction pressure develops through three stages: the pressure rises to the first peak; the pressure continues to oscillate and the pressure reduces to the initial value. Acknowledgements This work was financially supported by the Scientific Research Project of the Department of Education of Liaoning Province (L2015303) and the Breeding program of Liaoning Shihua University (2016[177]-19). 3 3
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Lactobacillus Dominate in the Intestine of Atlantic Salmon Fed Dietary Probiotics
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Lactobacillus Dominate in the Intestine of Atlantic Salmon Fed Dietary Probiotics Shruti Gupta 1, Adriána Fe ˇckaninová 2, Jep Lokesh 1, Jana Koš ˇcová 3, Mette Sørensen 1, Jorge Fernandes 1 and Viswanath Kiron 1* Shruti Gupta 1, Adriána Fe ˇckaninová 2, Jep Lokesh 1, Jana Koš ˇcová 3, Mette Sørensen 1, Jorge Fernandes 1 and Viswanath Kiron 1* 1 Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway, 2 Department of Food Hygiene and Technology, University of Veterinary Medicine and Pharmacy in Košice, Košice, Slovakia, 3 Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy in Košice, Košice, Slovakia Probiotics, the live microbial strains incorporated as dietary supplements, are known to provide health benefits to the host. These live microbes manipulate the gut microbial community by suppressing the growth of certain intestinal microbes while enhancing the establishment of some others. Lactic acid bacteria (LAB) have been widely studied as probiotics; in this study we have elucidated the effects of two fish-derived LAB types (RII and RIII) on the distal intestinal microbial communities of Atlantic salmon (Salmo salar). We employed high-throughput 16S rRNA gene amplicon sequencing to investigate the bacterial communities in the distal intestinal content and mucus of Atlantic salmon fed diets coated with the LABs or that did not have microbes included in it. Our results show that the supplementation of the microbes shifts the intestinal microbial profile differentially. LAB supplementation did not cause any significant alterations in the alpha diversity of the intestinal content bacteria but RIII feeding increased the bacterial diversity in the intestinal mucus of the fish. Beta diversity analysis revealed significant differences between the bacterial compositions of the control and LAB-fed groups. Lactobacillus was the dominant genus in LAB-fed fish. A few members of the phyla Tenericutes, Proteobacteria, Actinobacteria, and Spirochaetes were also found to be abundant in the LAB-fed groups. Furthermore, the bacterial association network analysis showed that the co-occurrence pattern of bacteria of the three study groups were different. Dietary probiotics can modulate the composition and interaction of the intestinal microbiota of Atlantic salmon Edited by: Konstantinos Papadimitriou, Agricultural University of Athens, Greece Reviewed by: Atte Von Wright, University of Eastern Finland, Finland Carmen Wacher, National Autonomous University of Mexico, Mexico *Correspondence: Viswanath Kiron kiron.viswanath@nord.no Specialty section: This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology Received: 06 October 2018 Accepted: 14 December 2018 Published: 11 January 2019 Keywords: fish, Salmo salar, feed additive, probiotics, intestinal bacteria, Lactobacillus, microbiota, amplicon sequencing ORIGINAL RESEARCH published: 11 January 2019 doi: 10.3389/fmicb.2018.03247 Test Probiotics, Feed Type, and Design Test Probiotics, Feed Type, and Design Two species of Lactobacillus (RII and RIII) that were previously isolated from the intestinal content of farmed healthy juveniles of rainbow trout (commercial fish farm–Rybárstvo PoŽehy s.r.o., Slovak Republic) were employed in this study. Antimicrobial susceptibility of the microorganisms was assessed based on the “Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance” provided by the European Food Safety Authority. Sensitivity or intrinsic resistance of the isolated organisms to a recommended set of antibiotics make them safe for use as probiotics in aquaculture. Both RII and RIII showed antagonistic activity against salmonid pathogens Aeromonas salmonicida subsp. salmonicida CCM 1307 and Yersinia ruckeri CCM 6093 (Feˇckaninová, 2017). Furthermore, high level of tolerance to different pH, bile, temperature, and high growth properties of the two species were confirmed through in vitro studies (Feˇckaninová, 2017). The test probiotics were coated on commercial salmon feeds. Briefly, a pure culture of probiotic bacteria that were grown on de Man, Rogosa and Sharpe agar (MRS) plates (HiMedia, Mumbai, India) for 48 h were inoculated into 1,000 ml of MRS broth and incubated for 18 h at 37◦C. The culture was centrifuged at 4,500 rpm for 20 min at 4◦C in a cooling centrifuge (Universal 320 R, Hettich, Germany). The resulting cell pellets were washed twice and resuspended in 30 ml of 0.9% (w/v) sterile saline. The feed (batches of 1,800 g) was thoroughly coated with the bacterial suspensions (Spirit Supreme, Skretting AS, Norway) using a vacuum coater (Rotating Vacuum Coater F-6-RVC, Forberg International AS, Norway). The bacterial counts on feeds were ∼108 cells.g−1 (RII/RIII), as determined by spread plating on MRS agar plates and incubating for 48 h at 37◦C. The control feeds were coated with 0.9% of sterile saline alone. The coated feeds were stored at 4◦C until they were offered to Atlantic salmon. Lactic acid bacteria (LAB) maintain intestinal health by producing lactic acid that can be utilized by short-chain fatty acids (SCFAs)-producing microorganisms. SCFAs (particularly acetate, propionate and butyrate) contribute to host health maintenance; for example, butyrate is used as energy source by the intestinal epithelial cells and also have anti-inflammatory effects on the host cells (Louis et al., 2014). Test Probiotics, Feed Type, and Design LAB that is generally found in the GIT of endothermic animals have been extensively investigated and their benefits have been reviewed by many researchers (Pavan et al., 2003; Masood et al., 2011; Yang et al., 2015; Karamese et al., 2016). The importance of fish gut- dwelling LAB in aquaculture has been described in other reviews (Ringø and Gatesoupe, 1998; Gatesoupe, 2008). Lactobacillus that colonize the intestinal regions of fish are able to evoke immune responses and impart protection against diseases (He et al., 2017). Feeding diets supplemented with beneficial bacteria such as LAB is being considered as an alternative approach to control diseases in farmed fish (Martínez Cruz et al., 2012; Feˇckaninová et al., 2017; Rodriguez-Nogales et al., 2017). Not many studies in fish have employed high-throughput sequencing techniques to understand the changes in bacterial communities following LAB feeding. In this study, we examined the ability of Lactobacillus to modulate the distal intestinal microbiota of Atlantic salmon, a farmed salmonid fish. In addition, we describe the differences in the topology of co-occurrence networks associated with the intestinal bacteria of Atlantic salmon offered feeds with and without Lactobacillus. Citation: Gupta S, Fe ˇckaninová A, Lokesh J, Koš ˇcová J, Sørensen M, Fernandes J and Kiron V (2019) Lactobacillus Dominate in the Intestine of Atlantic Salmon Fed Dietary Probiotics. Front. Microbiol. 9:3247. doi: 10.3389/fmicb.2018.03247 The ecological community of microorganisms that reside (Marchesi and Ravel, 2015) in the gastrointestinal tract (GIT) of an organism is referred to as the gut microbiota (Lozupone et al., 2012). The GIT of a healthy human harbors a dense (Kelsen and Wu, 2012; Marchesi et al., 2016) and diverse population (Lozupone et al., 2012) of commensal microorganisms, which offer many benefits to the host, including immune homeostasis and health maintenance (Sommer and Bäckhed, 2013). These commensal gut bacteria are also known to aid in amino-acid production The ecological community of microorganisms that reside (Marchesi and Ravel, 2015) in the gastrointestinal tract (GIT) of an organism is referred to as the gut microbiota (Lozupone et al., 2012). The GIT of a healthy human harbors a dense (Kelsen and Wu, 2012; Marchesi et al., 2016) and diverse population (Lozupone et al., 2012) of commensal microorganisms, which offer many benefits to the host, including immune homeostasis and health maintenance (Sommer and Bäckhed, 2013). These commensal gut bacteria are also known to aid in amino-acid production January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. MATERIALS AND METHODS (Lin et al., 2017), nutrient metabolism and absorption (Morowitz et al., 2011; Semova et al., 2012), vitamin and bioactive metabolite’ synthesis (Cummings and Macfarlane, 1997; LeBlanc et al., 2013), and pathogen displacement (Kamada et al., 2013). An imbalance in the gastrointestinal microbial composition can lead to immune-mediated diseases (Petersen and Round, 2014). A healthy gut bacterial assembly is essential for the well-being of the host organisms including fish, the microbiome of which is shaped by environment- and host-related factors (Wong and Rawls, 2012; Eichmiller et al., 2016; Lokesh et al., 2018). Ethics Statements This study was approved by the Norwegian Animal Research Authority, FDU (Forsøksdyrutvalget ID-7898). Fish handling and sampling procedures were in compliance with the description in LOVDATA. The rearing water was treated with UV rays to remove substances that could be harmful to the fish. Optimum values for water salinity, oxygen and nitrogen concentration were maintained in the rearing tanks. The temperature of the fish rearing hall was kept stable during the entire feeding experiment. Probiotics are “living bacteria,” and when they are administered as supplements in the right amount they can confer health benefits to humans (FAO and WHO, 2006), by targeting, among others intestinal health through stimulation of intestinal epithelial cell proliferation and differentiation, fortification of intestinal barrier and immunomodulation (Gareau et al., 2010; Thomas and Versalovic, 2010; Hemarajata and Versalovic, 2013). Probiotics also have both direct and indirect effects on the intestinal microbial composition and diversity, and global host metabolic functions (Scott et al., 2015). These live bacteria produce antimicrobial compounds that suppress the growth of other microorganisms and compete for their receptors and binding sites (Spinler et al., 2008; O’Shea et al., 2012); thus altering the gut microbiota (Collado et al., 2007). Members of the genera Lactobacillus and Bifidobacterium are the most commonly used probiotic organisms for humans (O’Toole and Cooney, 2008). Frontiers in Microbiology | www.frontiersin.org Collection of the Intestinal, Tank Biofilm, and Rearing Water Samples First, the fish were euthanized using 160 mg/L of MS222 tricaine methanesulfonate (Argent Chemical Laboratories, Redmond, WA, USA). Thereafter, the body surface of the fish was swiped with 70% ethanol. The fish were then dissected to aseptically remove the GIT from the abdominal cavity. The distal intestinal (DI) region was separated from the GIT and the content and surface mucus samples from the DI were collected (n = 18 for each group; 6 fish/tank) using sterile forceps and sterile glass slides, respectively. In addition to these fish samples, we collected environmental samples: water from the main inlet to the rearing hall (inlet water, n = 1), water from the rearing tanks (n = 3) and biofilm from the walls of the rearing tanks (n = 3). From the 3 tanks of each group, one liter of rearing water was filtered using 0.2 µm pore-size filters (Pall Corporation, Hampshire, United Kingdom) and the filter paper was stored at −80◦C. The biofilm samples were scraped from the walls of the 3 tanks of each group. The fish and biofilm samples were collected in cryotubes, snap-frozen in liquid nitrogen and stored at −80◦C. The sample abbreviations reported in this article are: (i) fish samples–Control distal intestine content (CDC), RII distal intestine content (RIIDC), RIII distal intestine content (RIIIDC), Control distal intestine mucus (CDM), RII distal intestine mucus (RIIDM), RIII distal intestine mucus (RIIIDM); (ii) environmental samples– Control tank water (CW), RII tank water (RIIW), RIII tank water (RIIIW), inlet water (IW), Control tank biofilm (CB), RII tank biofilm (RIIB), RIII tank biofilm (RIIIB). 16S rRNA Gene Sequence Data Processing Sequence data quality check, processing and analyses: The sequence quality of the raw reads generated from the Illumina MiSeq machine was checked using FastQC (Andrews, 2010). The forward reads (R1) corresponding to V3 region were employed for subsequent analyses because they were of better quality than the reverse reads (R2) corresponding to V4 region [Phred quality score (Q) ≤15]. Sequence processing was performed using the UPARSE (USEARCH version 9.2.64) software by Edgar (2013); this step included quality filtering and operational taxonomic units OTU clustering. FastQ files were used as the input file for the UPARSE pipeline. The raw reads were truncated to 240 bp and quality-filtered. The reads were truncated to remove the low- quality base pairs at the 3 ′-end and to make all samples of same sequence length. Collection of the Intestinal, Tank Biofilm, and Rearing Water Samples Furthermore, chimeric sequences were removed using the UCHIME algorithm (Edgar et al., 2011). The quality- filtered sequences were clustered into OTUs at 97% sequence similarity level. For taxonomy prediction, we employed the 16S rRNA Ribosomal Database Project (RDP) training set with species names v16. This RDP training set was used as a reference database because the large 16S databases like SILVA, Greengenes, or the full RDP database may give unreliable annotations of short 16S rRNA tags (Edgar, 2018). Taxonomic ranks were assigned to the OTUs using the SINTAX algorithm (Edgar, 2016) using a bootstrap cutoffvalue of 0.5. Afterwards, OTUs with a confidence score <1 at the domain level and the OTUs belonging to the phyla Cyanobacteria and Chlorophyta were removed to exclude the plant-related sequences from the microbiota analysis. After Experimental Fish, Feeding Regime, and Environmental Parameters The water flow rate, temperature, salinity and O2 levels in the tanks were 800 L/h, 6.7–7.1◦C, 33 ppt, >85% saturation measured at the outlet, respectively. A photoperiod of 24:0 LD was maintained throughout the feeding trial. Experimental Fish, Feeding Regime, and Environmental Parameters Atlantic salmon of average weight 522 ± 68 g were maintained in 800 L tanks in a flow-through seawater system, earlier described in Sørensen et al. (2017). A 20-day feeding trial was conducted at the research station, Nord University, Bodø, Norway. Three groups of fish (n = 45 fish/tank; 3 replicate tanks per group) were offered feeds with (RII ∼108 cells.g−1-RII; RIII ∼108 cells.g−1- RIII) or without probiotics (Control—C). The fish were fed ad January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 2 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. Biosystems, Woburn, USA), 1.5 µl of each forward and reverse primer (at a final concentration of 100 nM), 3.5 µl of DNAse and nuclease free water (Merck, Darmstadt, Germany) and 6 µl of DNA template and/ or 6 µl of negative PCR control. The thermocycling conditions included initial denaturation at 95◦C for 5 min, followed by 35 cycles of denaturation at 98◦C for 30 s, annealing at 58◦C for 30 s, extension at 72◦C for 45 s, and the final extension performed at 72◦C for 2 min. After performing the PCR, the resulting amplicon triplicates were pooled and visualized on 1.2% (w/v) agarose gel stained with SYBR R⃝Safe (Thermo Fisher Scientific, Rockford IL, USA), and the amplicon size was compared to a 1 kb DNA ladder (Thermo Fisher Scientific, Inc.). No amplification was observed in the negative PCR control. Only the amplicons (∼550 bp) with clear visible bands were selected, purified using the ZR-96 ZymocleanTM Gel DNA Recovery Kit (Zymo Research) and eluted in 15 µl of elution buffer. The eluted amplicon library (sequencing library) was quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). After quantification, each amplicon library was normalized to an equimolar concentration (3 nM) and validated on the TapeStation (Agilent Biosystems, Santa Clara, USA), prior to sequencing. The normalized library pool was further diluted to 12 pM, spiked with equimolar 10% Phix control and then paired-end sequencing was performed using the 600 cycle v3 sequencing kit on the Illumina MiSeq Desktop sequencer (Illumina, San Diego, CA, United States) in 2 runs with inter-run calibrators to reduce eventual differences between sequencing runs. libitum; the feeds were dispensed two times a day, between 08.00–09.00 and 14.00–15.00, using automatic feeders (Arvo- Teck, Huutokoski, Finland). Frontiers in Microbiology | www.frontiersin.org DNA Extraction and PCR Amplification of Bacterial 16S rRNA Gene for Illumina MiSeq Amplicon Sequencing Genomic DNA was extracted from the content, mucus and biofilm samples using the Quick-DNATM Fecal/Soil Microbe 96 kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. Metagenomic DNA Isolation kit for water (Epicenter Biotechnologies, Madison, WI, USA) was employed to extract the genomic DNA from the water samples. The quality of the extracted DNA was checked on 1.2% (w/v) agarose gel. Qubit 3.0 fluorometer (Life Technologies, Carlsbad, USA) was employed to quantify the concentration of DNA. To describe the changes in the intestinal bacteria under the influence of LAB, we amplified the V3–V4 region of the bacterial 16S rRNA gene employing a dual-index sequencing strategy described by Kozich et al. (2013). The PCR reactions were carried out in triplicates, each reaction (25 µl) volume contained 12.5 µl of Kapa HiFi Hot Start PCR Ready Mix (KAPA January 2019 | Volume 9 | Article 3247 3 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. constructing the OTU table, the counts were rarefied to the lowest number of sequences per sample to get an even sampling depth to facilitate comparisons between the treatment groups. The OTU count data was divided into 4 sets based on the sample type, namely the DI content, DI mucus, tank water and tank biofilm samples. The downstream analyses were performed separately on these 4 sets. Furthermore, to ensure that we employ content and mucus data from the same fish, only 14 fish from each group were considered for the downstream analyses. In total 103 samples were used for the downstream analyses, including the tank water and biofilm samples. The raw 16S rRNA gene sequence data from this study has been deposited in the European Nucleotide Archive (ENA) under the accession number ERP110004. interaction networks to be sparse (Kurtz et al., 2015). In this study, we employed the neighborhood selection (MB) method on the sequenced 16S rRNA gene (V3 region) data of both DI content and mucus samples to understand the community organization. We explored the co-occurrence networks to uncover the probable biological interactions occurring within the microbial communities. We used the top 200 OTUs for network construction, since it is advised to avoid extremely rare OTUs or OTUs with a large number of zeros (Banerjee et al., 2018). The co-occurrence microbial networks were constructed and analyzed using the functions of the R package “igraph” v1.2.1 and customized ggplot2 commands. Statistical Analysis of the Bacterial 16S rRNA Gene Amplicon Data The differences in the DI bacterial communities of the LAB- fed fish compared to the control fish are explained based on the following diversity metrics: overall microbial richness (i.e., counts of individual OTUs), effective number of OTUs (counts of common and dominant OTUs), taxonomic composition, relative abundances of the bacterial taxa. Furthermore, we present the significant and relevant bacterial communities of the DI microbiota. We also describe the topology of the networks of the bacterial communities in the 3 fish groups. Statistical analysis was also performed in RStudio v3.5.0. Kruskal-Wallis test followed by Dunn’s test was employed to detect differences in alpha diversity, and we report statistically significant differences at p < 0.05 and statistical trends at p ≤0.15. Betadisper was used to check the assumption of heterogeneity in dispersions; after that Adonis (PERMANOVA) followed by pairwise comparisons was employed (999 permutations) to understand the significant dissimilarities of the communities. “ANCOM” v1.1–3 (Mandal et al., 2015) was used to detect the differentially abundant OTUs in the treatment groups, and “Boruta” v5.3.0 R package (Kursa and Rudnicki, 2010) was employed to find the relevant OTUs that caused the differences in the intestinal bacteria of the three fish groups. Differences in the Microbial Diversity and Composition of the Intestinal and Environmental Microbiota LAB feeding did not affect the species richness of the bacterial community in the DI content (Figure 2A). However, this was not the case for bacteria in the DI mucus; the species richness was found to be higher in the mucus of the RIII-fed group (p = 0.004 for RII vs. RIII and p = 0.071 for RIII vs. C) (Figure 3A). We observed differences in the effective number of common and dominant OTUs in the mucus of LAB-fed groups, (p = 0.109 and p = 0.146 for RII vs. RIII; Figures 2B, 3B and Figures 2C, 3C). Comparison of the Faith’s phylogenetic diversity (PD) of the DI content did not reveal any significant differences (Figure 2D). RESULTS We analyzed the V3 region amplicons of the 16S rRNA gene that was sequenced on our high-throughput sequencing platform. A total of 28,747,884 high-quality reads were clustered into 1,823 OTUs at 97% identity threshold. These reads were rarified based on sample-size to 12,855 reads/sample; this allowed us to assess most of the underlying microbial diversity (Supplementary Figures 1A,B). The feeding design, sample processing and sequencing, and analyses are shown in Figure 1. DNA Extraction and PCR Amplification of Bacterial 16S rRNA Gene for Illumina MiSeq Amplicon Sequencing A network consists of a set of vertices (commonly called as nodes) and set of edges. The degree of a node is the number of connections it has with the other nodes in the network. Betweenness estimates the number of shortest paths that pass through the nodes in the network and assortativity coefficient quantifies the extent of the selectively connected labeled pair of nodes (Kolaczyk and Gábor, 2014). We compared the topology of the networks of the content and mucus samples separately by analyzing the node degrees and betweenness of the control and LAB-fed groups using Kruskal- Wallis test followed by Dunn’s test. Analyses of microbial diversity and composition: R codes were executed in RStudio v3.5.0 (RStudio Team, 2016) and the functions of the R packages “iNEXT” v2.0.12 (Hsieh et al., 2016), “phyloseq” v1.22.3 (McMurdie and Holmes, 2013) and “ggplot2” v2.2.1 (Wickham, 2016) were used to make the rarefaction curves for the species richness, to calculate and visualize diversity indices, and to prepare the abundance plots. Another R package called “microbiome” v1.0.2 (Lahti et al., 2017) was used to make core and rare microbiota (relative abundance of core taxa) plots. Alpha diversities were calculated based on the formula suggested by Jost (2006); for Shannon diversity (effective number of common OTUs) and Simpson diversity (effective number of most abundant OTUs). Beta diversity was examined by conducting weighted UniFrac distance metric (for fish samples)-based PCoA and double principal coordinates analysis (DPCoA, for water and biofilm samples) (Fukuyama et al., 2012). Microbial Network Construction and Comparison of Topology g We used “SPIEC-EASI” v0.1.4 R package (SParse InversE Covariance Estimation for Ecological Association Inference) for generating the single-domain bacterial network. SPIEC-EASI is a statistical method that assumes the underlying microbial January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 4 Gupta et al. Lactobacilli Modulate Salmon Intestinal Microbiota FIGURE 1 | Illustration of the study design, sampling and sequence analyses. Although we did not observe any significant differences between the bacterial communities of the tank biofilm and the intestinal mucus bacteria of the LAB-fed fish (Supplementary Figures 3B–D,F–G), the biofilm and mucus bacteria of the control group were different (Supplementary Figure 3E, F statistic = 16.29, R2 = 0.520, p = 0.003). For the DI mucus, differences were observed between the PD associated with the three fish groups (p = 0.004 for RII vs. RIII and p = 0.079 for RIII vs. C; Figure 3D). It is noteworthy that the median alpha diversities of RII lies below the corresponding values of C although we did not detect a trend or statistically significant difference between the feed groups. PCoA based on the weighted UniFrac distance matrix revealed the beta diversity of the bacterial communities; the differences between the control and LAB-fed groups were statistically significant (Figure 4A: F statistic = 9.215, R2 = 0.320, p < 0.001; and Figure 4B: F statistic = 3.114, R2 = 0.137, p < 0.002). Frontiers in Microbiology | www.frontiersin.org Intestinal Bacterial Composition Under the Influence of LAB fujisawaense), and Sphingomonas were also the shared core taxa in the content (Figure 7A). In the DI mucus, the genera that had higher abundance in the RIII-fed fish viz. Brevinema and Pelomonas (B. andersonii, P. saccharophila) were observed among the core bacterial members. Photobacterium, Ralstonia, Aquabacterium, Bradyrizhobium, Methylobacterium, Phyllobacterium, (P. phosphoreum, R. pickettii, A. parvum, B. jicamae, M. fujisawaense, P. myrsinacearum), Sphingomonas, and Mycoplasma were also the shared core taxa of the intestinal mucus (Figure 7B). At the genus level, Lactobacilli (Lactobacillus fermentum and Lactobacillus paraplantarum) were found to be the most dominant bacteria in the content and mucus of LAB- fed fish (Figure 5B, and Supplementary Figures 4B–D) and Mycoplasma was also found to be dominant in the DI mucus of LAB-fed fish (Figure 6B). The DPCoA indicated differences in the core members of the LAB-fed and the control group (content: F-statistic: 3.879, R2 = 0.165, p = 0.004; mucus: F-statistic: 5.844, R2 = 0.219, p = 0.001; Supplementary Figures 6A,B). Intestinal Bacterial Composition Under the Influence of LAB Bacteria belonging to 23 phyla were present in the DI content and mucus (Figures 5A, 6A). Firmicutes, Proteobacteria, Spirochaetes, Tenericutes, and Actinobacteria were found to be dominant in the intestine of the three study groups (Supplementary Figures 4A,C). Firmicutes were found to be more abundant than the rest, in both the content and mucus of the LAB-fed fish (Figures 5A,B and Figures 6A,B). The abundance of the phylum Tenericutes (content and mucus) was higher in RII-fed fish, than in the RIII-fed fish group (Figures 5A,B and Figures 6A,B). Proteobacteria (content and The beta diversity of the bacterial communities in the rearing tank water and biofilm samples were also analyzed. The bacterial communities in the water of the 3 study groups were not different (Supplementary Figure 2A, F-statistic = 0.753, R2 = 0.273, p = 0.684), as was the case with the bacteria in the biofilm (Supplementary Figure 3A, F statistic = 0.681, R2 = 0.185, p = 0.574). On the other hand, the bacterial communities in the water were significantly different from those of the fish (Supplementary Figures 2B–G). January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 5 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. FIGURE 2 | Diversity of the bacterial communities of the intestinal content. Boxplots show the species richness (A), Shannon index (B), Simpson index (C), and Faith’s phylogenetic diversity (D). The feed group codes are as follows: Control, CDC; RII, RIIDC; RIII, RIIIDC. FIGURE 2 | Diversity of the bacterial communities of the intestinal content. Boxplots show the species richness (A), Shannon index (B), Simpson index (C), and Faith’s phylogenetic diversity (D). The feed group codes are as follows: Control, CDC; RII, RIIDC; RIII, RIIIDC. mucus) decreased in the LAB-fed groups compared to the control group (Figures 5A,B; Figures 6A,B and Table 1). The abundance of Spirochaetes was higher in the DI mucus of RIII-fed fish and lower in the RII-fed fish (Figures 6A,B). The abundant phyla in water is shown in Supplementary Figure 5A. The dominant phyla in water were Bacteriodetes and Proteobacteria (Supplementary Figure 5B). The changes in the abundance of most bacterial taxa in both DI content and mucus of the LAB-fed groups compared to the control group is shown in Table 1. Brevinema, Methylobacterium (B. jicamae, P. phosphoreum, P. myrsinacearum, B. andersonii, M. Core Bacterial Communities of the Intestinal Microbiota Significantly Abundant and Relevant Bacterial Taxa of the Intestinal Microbiota ANCOM analysis detected the significantly abundant bacterial OTU in the DI content, which turned out to be L. fermentum in RIII-fed fish (Table 1). However, this bacterium was not detected as a significant feature in the DI mucus. We identified the core microbiota, i.e., the members of the bacterial communities that were commonly shared among 99% of the samples.The common core taxa–at prevalence (bacterial community population frequency) of 99% and abundance detection threshold of 20%–are shown in Figures 7A,B. In the DI content, the abundant genera in the LAB-fed fish, namely Lactobacillus, Ralstonia (L. paraplantarum, R. pickettii) and Mycoplasma were noted to be among the core bacterial members. Bradyrizhobium, Photobacterium, Phyllobacterium, Boruta analysis gave 9 and 8 relevant OTUs in the intestinal content and mucus, respectively. In the DI content, L. fermentum, L. paraplantarum, Streptococcus sobrinus, Corynebacterium simulans, Lactococcus plantarum, W. cibaria, C. amphilecti, and bacterial taxa belonging to Streptococcus and Xanthomonodales January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 6 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. FIGURE 3 | Diversity of the bacterial communities of the intestinal mucus. Boxplots show the species richness (A), Shannon index (B), Simpson index, (C) and Faith’s phylogenetic diversity (D). Different letters indicate statistically significant differences (P < 0.05) between the study groups. The feed group codes are as follows: Control, CDM; RII, RIIDM; RIII, RIIIDM. FIGURE 3 | Diversity of the bacterial communities of the intestinal mucus. Boxplots show the species richness (A), Shannon index (B), Simpson index, (C) and Faith’s phylogenetic diversity (D). Different letters indicate statistically significant differences (P < 0.05) between the study groups. The feed group codes are as follows: Control, CDM; RII, RIIDM; RIII, RIIIDM. assortativity (assortativity coefficient ca) of the phylum-level network associated with the three groups (control, RII- and RIII- fed fish) were 0.09, 0.19, and 0.10, respectively. The significantly abundant and relevant OTUs belonged to different phyla and modules (Figures 8A–C and Supplementary Figures 9 A–C). The degree distribution of the microbial network (for all OTUs) of the study groups (Supplementary Figure 11A) revealed that there are many highly connected hub nodes for the bacterial network of the RII-fed fish and the hubs of the control group have more node degrees. were the relevant bacteria. L. paraplantarum was found to be abundant in the RII-fed group, whereas L. Core Bacterial Communities of the Intestinal Microbiota fermentum and Xanthomonodales were found to be abundant in the RIII-fed group. S. sobrinus, C. simulans, L. plantarum, W. cibaria, C. amphilecti were reduced in abundance in the LAB-fed groups. In the mucus, Lewinella antarctica, L. paraplantarum, L. fermentum, Salinisphaera, Colwellia aestuarii and bacteria belonging to- Gammaproteobacteria, Rhodobacteraceae, and Clostridiales were found to be the relevant bacterial taxa (most of them were abundant in the mucus of the RIII-fed fish–Table 1). DISCUSSION FIGURE 4 | Beta diversity of the intestinal bacterial communities. Principal coordinate analysis plot (A) shows the differences in the composition of the bacterial communities in the intestinal content (Control, CDC; RII, RIIDC; RIII, RIIIDC). Principal coordinate analysis plot (B) shows the differences in the composition of the bacterial communities in the intestinal mucus (Control, CDM; RII, RIIDM; RIII, RIIIDM). Probiotics are live microbes that can impart health-benefiting effects on host organisms. For instance, feeding of some species belonging to genera Lactobacillus and Bifidobacterium can elicit positive effects on host health (Wang et al., 2015; Bagarolli et al., 2017). Probiotics alter the gut microbiota and interact with them to produce several types of metabolites, vitamins, and antimicrobial agents that affect the host physiology (Saulnier et al., 2011; O’Shea et al., 2012; LeBlanc et al., 2017). In the present study, we investigated the intestinal microbiota changes in Atlantic salmon after feeding them with dietary supplements of two Lactobacillus spp., named RII and RIII. To understand the differences in the microbial community associated with the content and mucus of the DI, the bacteria in the two samples were analyzed separately because the microbial niche in the DI mucus is distinct compared to the intestinal contents. Feeding LAB to the fish may facilitate their establishment in the intestine, although significant difference was noted for the abundance of only one of the two LAB species. The feed- delivered organisms also altered the diversity and composition of the DI bacteria differently. RIII supplementation caused a significant increase in the species richness and phylogenetic diversity of the bacterial community in DI mucus. Furthermore, both RII and RIII caused a shift in the community composition; bacteria belonging to different genera were altered in the two feed groups. The co-occurrence networks indicated differential bacterial associations in the control and LAB-fed groups. FIGURE 4 | Beta diversity of the intestinal bacterial communities. Principal coordinate analysis plot (A) shows the differences in the composition of the bacterial communities in the intestinal content (Control, CDC; RII, RIIDC; RIII, RIIIDC). Principal coordinate analysis plot (B) shows the differences in the composition of the bacterial communities in the intestinal mucus (Control, CDM; RII, RIIDM; RIII, RIIIDM). Water bacterial communities may have an effect on the microbiota of fish. To clarify this, we compared the microbial community composition in the intestinal and environmental samples. DISCUSSION Notwithstanding the fact that different extraction methods can cause small variations in the microbial profile (Wagner Mackenzie et al., 2015) studies have shown that rearing water has a minor effect on the GI microbiota in fish (Giatsis et al., 2015; Uren Webster et al., 2018). Betiku et al. (2018) have demonstrated that recirculating water systems have more diverse microbial composition compared to the flow- through system. However, similar to other reports (Yan et al., 2016, Lokesh et al., 2018, Gupta et al., under review) water bacterial communities might not have affected the intestinal bacterial profile in our study. Also, none of the dominant OTUs of water were detected in the DI of fish, suggesting that host-specific gut microbial species selection is modulated by the host gut habitat and host’s genotype (Giatsis et al., 2015). The connectivity pattern of the significantly abundant and relevant OTUs in the phylum-level co-occurrence network is shown in Supplementary Figures 10 A–C. The average node degrees were 4.12 (SD: 2.20), 2.29 (SD: 2.09), 2.74 (SD: 1.19) for the control, RII- and RIII-fed fish, respectively. The values for betweenness of the control, RII- and RIII-fed fish were 505 (SD: 664), 481 (SD: 596), 613 (SD: 766), respectively. Dunn’s test identified significant differences between the LAB- fed groups, and between control and RIII-fed fish; for node degree, but not for edge betweenness; p = 0.0002, p = 0.003 and p = 0.08, p = 0.07, respectively. The degree of assortativity (assortativity coefficient ca) of the phylum- level network for the three groups (control, RII- and RIII- fed fish) were −0.01, −0.07, and 0.13, respectively. The degree distribution of the microbial network (for all OTUs) of the three groups is shown in Supplementary Figure 11B. The node degree histogram showed that the hubs of the RII-fed groups have higher node degrees than the other groups. The DI Mucus Bacteria The single-domain bacterial (SDB) network derived from the DI content of the 3 groups comprised of one giant connected component (Supplementary Figure 7). The significantly abundant and relevant OTUs were labeled based on their membership in different modules (Figures 8A–C). The connectivity pattern of the significantly abundant and relevant OTUs in the phylum-level co-occurrence network is shown in Supplementary Figures 9 A–C. The average node degrees were 4.27 (SD: 3.44), 3.71 (SD: 1.52), 4.06 (SD: 2.48) for the control, RII- and RIII-fed fish, respectively. Similarly, the values for betweenness were 370 (SD: 369), 396 (SD: 351), 388 (SD- 391). The average node degrees and betweenness of the three groups were not significantly different. The degree of The SDB network derived from the DI mucus of the control, RII, and RIII groups comprised of one giant connected component (Supplementary Figure 8). In the bacterial network of RII-fed fish, we observed a singleton (C. aestuarii), a dyad (2 OTUs of Mycoplasma), and a triad (L. paraplantarum, W. cibaria, and P. piscicola) with no connection to the main network (Supplementary Figure 8). As for the RIII-fed group, there were 3 dyads (Sphingobacteriales + Myxococcales, 2 OTUs of Mycoplasma, and Xanthomonadales + Gammaproteobacteria) with no connection to the main network (Supplementary Figure 8). The significantly abundant and relevant OTUs were labeled based on their membership in different modules (Figures 9A–C). January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 7 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. Frontiers in Microbiology | www.frontiersin.org LAB Increases the Microbial Diversity in the Intestinal Mucus Color codes for the dominant genera: Proteobacteria—shades of green, Spirochaetes—dark blue, Firmicutes—shades of yellow, Actinobacteria—orchid, and Tenericutes—dark orange LAB Increases the Microbial Diversity in the Intestinal Mucus Corresponding to our observation on the content bacteria, a few previous studies have also shown that LAB supplementation does not alter the intestinal bacterial diversity (Chao1 and Shannon diversities); in humans (Van Zanten et al., 2014) and in mice with colon cancer (Mendes et al., 2018). On the other hand, species richness, Shannon and Simpson diversities, and PD of the bacteria in the DI mucus were higher in the RIII-fed g The main results of this study are summarized in Figure 10. January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 8 Gupta et al. Lactobacilli Modulate Salmon Intestinal Microbiota FIGURE 5 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B), in the intestinal content. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from greatest to smallest, and separated by a thin black border line. Color codes for the dominant genera: Proteobacteria—shades of green, Spirochaetes—dark blue, Firmicutes—shades of yellow, Actinobacteria—orchid, and Tenericutes—dark orange. sh. In the case of mucus bacteria of RII-fed fish, we noted li h d ( ) i l h di i d LAB Promotes the Abundance and D i f I t ti l L t b ill d FIGURE 5 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B), in the intestinal content. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from greatest to smallest, and separated by a thin black border line. Color codes for the dominant genera: Proteobacteria—shades of green, Spirochaetes—dark blue, Firmicutes—shades of yellow, Actinobacteria—orchid, and Tenericutes—dark orange. FIGURE 5 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B), in the intestinal content. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from greatest to smallest, and separated by a thin black border line. Color codes for the dominant genera: Proteobacteria—shades of green, Spirochaetes—dark blue, Firmicutes—shades of yellow, Actinobacteria—orchid, and Tenericutes—dark orange. FIGURE 5 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B), in the intestinal content. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from greatest to smallest, and separated by a thin black border line. LAB Promotes the Abundance and Dominance of Intestinal Lactobacillus and Other Firmicutes fish. In the case of mucus bacteria of RII-fed fish, we noted a slight decrease (p > 0.05) in alpha diversity compared to the control fish. Previous studies have shown that Lactobacillus can increase the bacterial PD in the gut of mice (Usui et al., 2018) and weaning piglets (Zhao et al., 2016). On the contrary, offering LAB in combination with Bifidobacterium breve and Bifidobacterium longum did not result in greater bacterial species diversity (Chao1, Shannon index and PD) in mice that received antibiotics (Grazul et al., 2016). fish. In the case of mucus bacteria of RII-fed fish, we noted a slight decrease (p > 0.05) in alpha diversity compared to the control fish. Previous studies have shown that Lactobacillus can increase the bacterial PD in the gut of mice (Usui et al., 2018) and weaning piglets (Zhao et al., 2016). On the contrary, offering LAB in combination with Bifidobacterium breve and Bifidobacterium longum did not result in greater bacterial species diversity (Chao1, Shannon index and PD) in mice that received antibiotics (Grazul et al., 2016). L. paraplantarum (LP) is related to L. plantarum (Curk et al., 1996). It was dominant in the RII-fed group and L. fermentum (LF) was found dominant in the RIII-fed group. Lactobacilli are a group of gram-positive ubiquitous LAB that produce organic acids as end products of their metabolic activity linked to carbohydrate fermentation (Bernardeau et al., January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org Frontiers in Microbiology | www.frontiersin.org 9 Gupta et al. Lactobacilli Modulate Salmon Intestinal Microbiota FIGURE 6 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B) in the intestinal mucus. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from highest to smallest, and separated by a thin black border line. Color codes for the dominant genera: Proteobacteria—shades of green, Spirochaetes—dark blue, Firmicutes—shades of yellow, Actinobacteria—shades of orchid, and Tenericutes—dark orange. FIGURE 6 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B) in the intestinal mucus. The height of each bar segment represents he abundance of individual operational taxonomic units (OTUs) stacked in order from highest to smallest, and separated by a thin black border line. Frontiers in Microbiology | www.frontiersin.org LAB Promotes the Abundance and Dominance of Intestinal Lactobacillus and Other Firmicutes Sample type Intestinal content Intestinal mucus XXXXXXXX Taxa Groups RII RIII RII RIII Acidobacteria ↑ ↑ ↑ ↑ Actinobacteria ↑ ↑ ↓ ↓ Fusobacteria ↓ ↑ ↓ ↓ Deinococcus-Thermus ↑ ↑ ↓ ↑ SR1 ↑ – – ↑ Chloroflexi ↑ ↑ – – Parcubacteria ↓ ↓ ↑ ↑ Planctomycetes ↓ ↓ ↑ – Lactobacillus fermentum ↑ ↑ ↓ ↑ Lactobacillus paraplantarum ↑ ↓ ↑ ↑ Colwellia aestuarii ↑ ↓ ↓ ↑ Streptococcus sobrinus ↓ ↓ ↓ ↑ Lewinella antarctica ↓ ↑ ↑ ↑ Lactobacillus plantarum ↓ ↓ ↓ ↓ Acinetobacter radioresistens ↓ ↑ ↓ ↓ Novosphingobium sediminicola ↓ ↑ ↓ ↓ Phyllobacterium myrsinacearum ↓ ↑ ↓ ↓ Ralstonia pickettii ↓ ↑ ↓ ↓ Stenotrophomonas maltophilia ↓ ↑ TND TND Undibacterium oligocarboniphilm ↓ ↑ TND TND Micrococcus luteus ↓ ↑ ↓ ↓ Enterococcus cecorum ↑ – TND TND Mycoplasma ↑ ↓ ↑ ↑ Aquabacterium ↓ ↓ ↓ ↓ Bradyrizhobium ↓ ↓ ↓ ↓ Brevinema ↓ ↓ ↓ ↑ Delftia ↓ ↓ ↓ ↑ Methylobacterium ↓ ↓ ↓ ↓ Aquabacterium parvum ↓ ↓ ↓ ↓ Pelomonas ↓ ↓ ↓ ↑ Photobacterium ↓ ↓ ↓ ↓ Sphingomonas ↓ ↓ ↓ ↓ Weissella ↓ ↓ TND TND Brevinema andersonii ↓ ↓ ↓ ↑ Pelomonas saccharophila ↓ ↓ ↓ – Bradyrizhobium jicamae ↓ ↓ ↓ ↓ Methylobacterium fujisawaense ↓ ↓ ↓ ↓ Photobacterium phosphoreum ↓ ↓ ↓ ↓ Aliivibrio logei TND TND ↓ ↓ Caulobacter segnis TND TND ↓ ↓ Cornybacterium ↓ ↓ ↓ ↓ Propionibacterium acnes TND TND ↓ ↓ Arrows indicate changes in abundance (blue arrow: increase, red arrow: decrease, bold black line: no change, TND: taxon not dominant). L. plantarum (LbOG1 strain) exhibit in vitro antibacterial activities against fish pathogens in Clarias gariepinus (Adenike and Olalekan, 2009). The higher abundance of intestinal Enterococcus cecorum, was also found to be dominant in the content of the RII-fed group compared to the control group (Table 1). Enterococcus spp. isolated from the intestine of rainbow trout (Oncorhynchus mykiss) are used as probiotics due to their antimicrobial activity against fish pathogens (Carlos et al., 2015). The functional potential of E. cecorum in Atlantic salmon has not yet come to light although one particular strain is known to cause infections in broilers (Herdt et al., 2009). Clostridiales (belonging to Firmicutes) were higher in the mucus of salmon offered diets with RIII. LAB Promotes the Abundance and Dominance of Intestinal Lactobacillus and Other Firmicutes Commensal Clostridiales are known to promote gut health by modulating gut homeostasis and taking part in immune activation (Lopetuso et al., 2013). LAB Favors Certain Members of Tenericutes, Spirochaetes, and Actinobacteria LAB significantly aided in altering the abundance of the genus Mycoplasma (Tenericutes) and B. andersonii (Spirochaetes) in the mucus, which are the common core members in the DI content of Atlantic salmon (Figure 7A). Mycoplasma has consistently been isolated from salmon intestine (Holben et al., 2002; Zarkasi et al., 2014) and its presence as a core microbiota suggests that it may be a commensal organism in the intestinal ecosystem. B. andersonii has been reported in the intestinal microbiota of flatfish, Solea senegalensis (Tapia-Paniagua et al., 2010). Although B. andersonii is known to digest lignocellulose and fix nitrogen in termite guts (Kudo, 2009), their functional importance needs to be elucidated. The abundance of the genus Micrococcus (M. luteus), a member of Actinobacteria, was higher in the DI content of the RIII-fed group (Table 1). Though M. luteus is known to be a pathogen for rainbow trout (Salmo trutta L.) and brown trout (Oncorhynchus mykiss) (Pkala et al., 2018) an in vivo feeding study has suggested that they can enhance the growth and health of Nile tilapia (Abd El-Rhman et al., 2009). LAB Promotes the Abundance and Dominance of Intestinal Lactobacillus and Other Firmicutes TABLE 1 | Changes in abundances of the bacterial taxa by LAB feeding. Sample type Intestinal content Intestinal mucus XXXXXXXX Taxa Groups RII RIII RII RIII Acidobacteria ↑ ↑ ↑ ↑ Actinobacteria ↑ ↑ ↓ ↓ Fusobacteria ↓ ↑ ↓ ↓ Deinococcus-Thermus ↑ ↑ ↓ ↑ SR1 ↑ – – ↑ Chloroflexi ↑ ↑ – – Parcubacteria ↓ ↓ ↑ ↑ Planctomycetes ↓ ↓ ↑ – Lactobacillus fermentum ↑ ↑ ↓ ↑ Lactobacillus paraplantarum ↑ ↓ ↑ ↑ Colwellia aestuarii ↑ ↓ ↓ ↑ Streptococcus sobrinus ↓ ↓ ↓ ↑ Lewinella antarctica ↓ ↑ ↑ ↑ Lactobacillus plantarum ↓ ↓ ↓ ↓ Acinetobacter radioresistens ↓ ↑ ↓ ↓ Novosphingobium sediminicola ↓ ↑ ↓ ↓ Phyllobacterium myrsinacearum ↓ ↑ ↓ ↓ Ralstonia pickettii ↓ ↑ ↓ ↓ Stenotrophomonas maltophilia ↓ ↑ TND TND Undibacterium oligocarboniphilm ↓ ↑ TND TND Micrococcus luteus ↓ ↑ ↓ ↓ Enterococcus cecorum ↑ – TND TND Mycoplasma ↑ ↓ ↑ ↑ Aquabacterium ↓ ↓ ↓ ↓ Bradyrizhobium ↓ ↓ ↓ ↓ Brevinema ↓ ↓ ↓ ↑ Delftia ↓ ↓ ↓ ↑ Methylobacterium ↓ ↓ ↓ ↓ Aquabacterium parvum ↓ ↓ ↓ ↓ Pelomonas ↓ ↓ ↓ ↑ Photobacterium ↓ ↓ ↓ ↓ Sphingomonas ↓ ↓ ↓ ↓ Weissella ↓ ↓ TND TND Brevinema andersonii ↓ ↓ ↓ ↑ Pelomonas saccharophila ↓ ↓ ↓ – Bradyrizhobium jicamae ↓ ↓ ↓ ↓ Methylobacterium fujisawaense ↓ ↓ ↓ ↓ Photobacterium phosphoreum ↓ ↓ ↓ ↓ Aliivibrio logei TND TND ↓ ↓ Caulobacter segnis TND TND ↓ ↓ Cornybacterium ↓ ↓ ↓ ↓ Propionibacterium acnes TND TND ↓ ↓ Arrows indicate changes in abundance (blue arrow: increase, red arrow: decrease, bold black line: no change, TND: taxon not dominant). TABLE 1 | Changes in abundances of the bacterial taxa by LAB feeding. LAB Promotes the Abundance and Dominance of Intestinal Lactobacillus and Other Firmicutes Color codes for he dominant genera: Proteobacteria shades of green Spirochaetes dark blue Firmicutes shades of yellow Actinobacteria shades of orchid and FIGURE 6 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B) in the intestinal mucus. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from highest to smallest, and separated by a thin black border line. Color codes for the dominant genera: Proteobacteria—shades of green, Spirochaetes—dark blue, Firmicutes—shades of yellow, Actinobacteria—shades of orchid, and Tenericutes—dark orange. FIGURE 6 | Barplots showing the abundance of all bacterial phyla (A), and dominant genera (B) in the intestinal mucus. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from highest to smallest, and separated by a thin black border line. Color codes for the dominant genera: Proteobacteria—shades of green, Spirochaetes—dark blue, Firmicutes—shades of yellow, Actinobacteria—shades of orchid, and Tenericutes—dark orange. acid-producing bacterial populations in mouse suffering from colitis (Rodriguez-Nogales et al., 2017). 2006). LP is known to produce bacteriocins, which are antimicrobial peptides produced as a defense response (Tulini et al., 2013). A Lactobacillus isolate (LP 11-1) stimulated the innate immune system and induced tolerance against the pathogenicity of Pseudomonas aeruginosa in silkworm (Nishida et al., 2017). LF has been found to restore the expression of markers associated with the maintenance of intestinal barrier function, and recover the SCFAs- and lactic acid-producing bacterial populations in mouse suffering from colitis (Rodriguez-Nogales et al., 2017). Lactobacillus is part of the normal intestinal flora of fish (Ringø et al., 1995; Spanggaard et al., 2000; Ringø and Olsen, 2003). In zebrafish, probiotic Lactobacillus helps to overcome infection (He et al., 2017). In Nile tilapia (Oreochromis niloticus), LF is known to improve fish immune response (Nwanna and Bamidele, 2014). LF (LbFF4 strain) along with Lactobacillus is part of the normal intestinal flora of fish (Ringø et al., 1995; Spanggaard et al., 2000; Ringø and Olsen, 2003). In zebrafish, probiotic Lactobacillus helps to overcome infection (He et al., 2017). In Nile tilapia (Oreochromis niloticus), LF is known to improve fish immune response (Nwanna and Bamidele, 2014). LF (LbFF4 strain) along with January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 10 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. LAB Largely Decreased the Abundance of Proteobacteria Proteobacteria is the most abundant phylum in many marine and freshwater fishes (Yan et al., 2016; Lokesh et al., 2018) and it is also known to dominate the gut microbiota of Atlantic salmon (Gajardo et al., 2016; Lokesh et al., 2018). Therefore, it was surprising to find this taxon in low abundance in the LAB- fed and the control fish. A general decrease in the abundance of intestinal Proteobacteria has also been reported in farmed Atlantic salmon that were transferred to seawater (Rudi et al., 2018). Taxa belonging to Proteobacteria are involved in metabolic pathways that participate in carbon and nitrogen fixation and in the stress response regulatory system (Vikram et al., 2016). They are also important in the digestive process in fish (Romero et al., 2014). P. phosphoreum, a known gut symbiont of marine fish, helps in chitin digestion and use luciferase- reoxidize reduced coenzymes and other molecules for metabolism (Nealson and Hastings, 1979). N. sediminicola and P. myrsinacearum are Arrows indicate changes in abundance (blue arrow: increase, red arrow: decrease, bold black line: no change, TND: taxon not dominant). L. plantarum (LbOG1 strain) exhibit in vitro antibacterial activities against fish pathogens in Clarias gariepinus (Adenike and Olalekan, 2009). The higher abundance of intestinal Lactobacillus members and the altered bacterial abundance in the LAB-fed fish confirms that LAB feeding can change the intestinal microbial composition. January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 11 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. Gupta et al. FIGURE 7 | Abundance of the core bacterial taxa in the intestinal content (A) and mucus (B). Color codes: Shades of green—Proteobacteria, yellow—Firmicutes, dark blue—Spirochaetes, and dark orange—Tenericutes. FIGURE 7 | Abundance of the core bacterial taxa in the intestinal content (A) and mucus (B). Color codes: Shades of green—Proteobacteria, yellow—Firmicutes, dark blue—Spirochaetes, and dark orange—Tenericutes. FIGURE 7 | Abundance of the core bacterial taxa in the intestinal content (A) and mucus (B). Color codes: Shades of green—Proteobacteria, yellow—Firmicutes, dark blue—Spirochaetes, and dark orange—Tenericutes. connectivity. The node degree histograms also communicate interesting information about the network; the mucus bacteria of RII-fed group had hubs with more node degree. However, the lower average node degree and lower selective linking of the RII-fed group indicate less interactions among the gut bacteria. Cooperative microbial communities are known to provide microbiome stability because of their functional dependence. LAB Largely Decreased the Abundance of Proteobacteria Studies have shown that the stability declines with an increase in microbial diversity and proportion of cooperative interactions (Coyte et al., 2015). However, higher cooperating microbial communities can cause a runaway effect that can collapse the competing microbial population due to over- representation of the most stable community (McNally and Brown, 2016). known as nitrogen-fixing bacteria (Gonzalez-Bashan et al., 2000; Muangthong et al., 2015). On the other hand, R. pickettii formerly known as Burkholderia pickettii has genes to biodegrade aromatic hydrocarbons (Ryan et al., 2007). In the current and in our recent (Gupta et al, under review) studies we found that P. myrsinacearum and R. pickettii are part of the core gut microbiota of Atlantic salmon; N. sediminicola was also significantly abundant in the intestinal mucus of the fish fed oligosaccharide. Functions of the aforementioned bacteria are not yet reported in fish. LAB Affects the Microbial Association We inferred single-domain networks using the SPEIC-EASI framework, and highlighted the significantly abundant and relevant OTUs in the intestinal microbiota. For DI mucus, the inferred SDB network for RII-fed fish showed lower overall The dyads in the mucus bacterial networks of LAB-fed fish were different, the exception being the one constructed January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 12 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. 8 | Network graphs showing the significantly abundant and relevant OTUs of the intestinal content in different modules of the network. Bacterial networks of (A), RII (B), and RIII (C) fish. Nodes represent OTUs and specific colors of the modules reveal the membership of the significantly abundant and relevant e left graph shows the location of the OTUs and in the right graph, the significantly abundant and relevant OTUs that belong to the same module are shown FIGURE 8 | Network graphs showing the significantly abundant and relevant OTUs of the intestinal content in different modules of the network. Bacterial networks of the control (A), RII (B), and RIII (C) fish. Nodes represent OTUs and specific colors of the modules reveal the membership of the significantly abundant and relevant OTUs. The left graph shows the location of the OTUs and in the right graph, the significantly abundant and relevant OTUs that belong to the same module are shown in callouts FIGURE 8 | Network graphs showing the significantly abundant and relevant OTUs of the intestinal content in different modules of the network. Bacterial networks of the control (A), RII (B), and RIII (C) fish. Nodes represent OTUs and specific colors of the modules reveal the membership of the significantly abundant and relevant OTUs. The left graph shows the location of the OTUs and in the right graph, the significantly abundant and relevant OTUs that belong to the same module are shown in callouts. January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 13 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. ork graphs showing the significantly abundant and relevant OTUs of DI mucus in different modules based on their membership for control fish (A), h (C). Nodes represent OTUs and specific colors of the modules reveal the membership of the significantly abundant and relevant OTUs. CONCLUSION with 2 OTUs of Mycoplasma which had higher abundance in the RII-fed fish and lower abundance in the RIII-fed fish. This result could be suggesting that intestinal Mycoplasma in the LAB-fed fish was not associated with other gut bacterial communities. In the content of LAB-fed fish, most of the labeled OTUs (except OTU 8) were existing in their respective modules (Figures 8B, C). In the mucus of RIII-fed fish OTUs belonging to C. aestuarii, L. paraplantarum and Clostridiales were found to exist in one module. Clostridiales and Rhodobacteraceae, which had same module membership in the network of the control fish were no longer closely associated after LAB feeding. So was the case with L. fermentum and C. aestuarii. Members affiliated to Rhodobacteraceae are known for their denitrification properties, and Kraft et al. (2014) have shown that Clostridiales indirectly participates in nitrate respiration by providing fermentation substrates (e.g., acetate, formate, or hydrogen) to Rhodobacteraceae-like denitrifiers. Our findings suggests that the taxa belonging to the same module can be functionally dependent but the alteration of their membership after LAB feeding has to be further investigated. In summary, LAB feeding promoted the dominance of intestinal Lactobacillus (Firmicutes) and certain members of the phyla Tenericutes, Spirochaetes, and Actinobacteria. Although the abundances of many members of Proteobacteria were decreased, the phylum remained dominant in the distal intestine of Atlantic salmon. Dietary supplementation with the two LAB strains shifted the intestinal bacterial community composition. Furthermore, the co-occurrence networks of the intestinal bacteria were also different for the LAB-fed fish. Taken together, our results show that the LAB influences the gut microbiota of Atlantic salmon. This information will help in future studies that explore the microbial interactions between LAB-modulated gut microbiota and the host. AUTHOR CONTRIBUTIONS MS and VK procured the funding for the study. VK, MS, JK, AF, and SG designed the study. JK provided the probiotics. AF and SG conducted the feeding experiment. SG performed the 16S rRNA sequencing studies. SG, VK, and JF analyzed the data. SG wrote the manuscript with the guidance of VK. All authors read, revised and approved the manuscript. The mucus bacteria of RIII-fed fish had higher species richness and PD, and the significantly abundant and relevant OTUs belonged to different modules. For the RIII-associated network, 2 OTUs each belonging to two modules (Rhodobacteraceae and L. fermentum; C. aestuarii, and Clostridiales) had higher abundances compared to the control group. In addition, significantly abundant and relevant bacteria had higher abundance in the RIII-fed fish compared to the control group. This abundance pattern does not indicate negative feedback loops (Coyte et al., 2015). These results of bacterial networks have to be validated through culture-based studies. LAB Affects the Microbial Association The left ocation of the OTUs and in the right graph the significantly abundant and relevant OTUs that belong to the same module are shown in callouts. twork graphs showing the significantly abundant and relevant OTUs of DI mucus in different modules based on their membership for control fish (A) FIGURE 9 | Network graphs showing the significantly abundant and relevant OTUs of DI mucus in different modules based on their membership for control fish (A), RII (B), and RIII fish (C). Nodes represent OTUs and specific colors of the modules reveal the membership of the significantly abundant and relevant OTUs. The left graph shows the location of the OTUs and in the right graph the significantly abundant and relevant OTUs that belong to the same module are shown in callouts. FIGURE 9 | Network graphs showing the significantly abundant and relevant OTUs of DI mucus in different modules based on their membership for control fish (A), RII (B), and RIII fish (C). Nodes represent OTUs and specific colors of the modules reveal the membership of the significantly abundant and relevant OTUs. The left graph shows the location of the OTUs and in the right graph the significantly abundant and relevant OTUs that belong to the same module are shown in callouts. January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 14 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. FIGURE 10 | Illustration summarizing the salient observations of the study. DI, distal intestine; C, control group; RII and RIII, LAB-fed groups. FIGURE 10 | Illustration summarizing the salient observations of the study. DI, distal intestine; C, control group; RII and RIII, LAB-fed groups. Frontiers in Microbiology | www.frontiersin.org ACKNOWLEDGMENTS = 16.291, R2 = 0.520, P = 0.003 (E), RII intestinal mucus and RII tank water: F-statistic = 2.934, R2 = 0.163, P = 0.051 (F), RIII intestinal mucus and RIII tank water: F-statistic = 3.910, R2 = 0.206, P = 0.03 (G). The Lactobacillus strains employed in this study are the property of The University of Veterinary Medicine and Pharmacy in Košice, Košice, The Slovak Republic. We thank Professors Peter Popelka (Department of Food Hygiene and Technology) and Dagmar Mudroová (Department of Microbiology and Immunology), The University of Veterinary Medicine Košice for providing the microorganisms for this study. We are thankful to Ghana Vasanth for her assistance in sample collection, Martina Kopp for her technical help in sequencing the libraries, and Nord University research station stafffor their help during the period of fish sampling. Special thanks to Bisa Saraswathy for her support in data analysis, scientific input, helpful discussions and preparation of the manuscript. The authors acknowledge the open access publication funding provided by Nord University. Supplementary Figure 3 | Double principal coordinate analysis plots showing the beta diversity of the bacterial communities. Tank biofilm bacteria (A), Control intestinal content and control tank biofilm: F-statistic = 2.061, R2 = 0.120, P = 0.082 (B), RII intestinal content and RII tank biofilm: F-statistic = 1.915, R2 = 0.113, P = 0.015 (C), RIII intestinal content and RIII tank biofilm: F-statistic = 4.171, R2 = 0.217, P = 0.043 (D), Control intestinal mucus and control tank biofilm: F-statistic = 5.807, R2 = 0.1279, P = 0.002 (E), RII intestinal mucus and RII tank biofilm: F-statistic = 1.476, R2 = 0.09, P = 0.146 (F), RIII intestinal mucus and RIII tank biofilm: F-statistic = 2.078, R2 = 0.121, P = 0.076 (G). Supplementary Figure 4 | Barplots showing the dominant bacterial phyla and species in the intestinal content (A,B) and mucus (C,D). Supplementary Figure 5 | Barplots showing the abundance of the bacterial phyla (A), dominant phyla (B) in the tank water. The height of each bar segment represents the abundance of individual operational taxonomic units (OTUs) stacked in order from largest to smallest, and separated by a thin black border line. Color codes: Proteobacteria—green, Bacteroidetes—light blue. Supplementary Figure 6 | DPCoA showing the differences in the composition of the core members of the intestinal content (A) and mucus (B) samples of the control and LAB-fed groups. FUNDING The study was undertaken as part of the project Bioteknologi– en framtidsrettet næring (FR-274/16), funded by the Nordland County Council, Norway. January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 15 Lactobacilli Modulate Salmon Intestinal Microbiota Gupta et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.03247/full#supplementary-material Supplementary Figure 7 | The single-domain network graph of the bacteria in the intestinal content. Nodes represent different phyla shown in different colors. The three panels represent the three feed groups: Control (A), RII (B), RIII (C). Supplementary Figure 8 | The single-domain network graph of the bacteria in the intestinal mucus. Nodes represent different phyla shown in different colors. The three panels represent the three feed groups: Control (A), RII (B), RIII (C). Supplementary Figure 1 | Sample-size-based rarefaction curves for the reads obtained from the intestinal content (A) and mucus (B). The shaded portion around each line represents the 95% confidence interval. Color code for the feed groups: green lines- control, orange lines- RII, pink lines- RIII. Codes for content samples: CDM-control, RIIDC-RII, RIIIDC-RIII. Codes for mucus samples: CDM-control, RIIDM-RII, RIIIDM-RIII. Supplementary Figure 9 | Network association graph showing the connectivity pattern of the significantly abundant and relevant OTUs in the intestinal content of the Control (A), RII (B), and RIII (C) groups. Supplementary Figure 2 | Double principal coordinate analysis plots showing the beta diversity of the bacterial communities. Tank and inlet water (A), control intestinal content and control tank water: F-statistic = 4.035, R2 = 0.211, P = 0.01 (B), RII intestinal content and RII tank water: F-statistic = 2.375, R2 = 0.136 P = 0.07 (C), RIII intestinal content and RIII tank water: F-statistic = 5.006, R2 = 0.250, P = 0.002 (D), Control intestinal mucus and control tank water: F-statistic Supplementary Figure 2 | Double principal coordinate analysis plots showing the beta diversity of the bacterial communities. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Frontiers in Microbiology | www.frontiersin.org January 2019 | Volume 9 | Article 3247 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Zarkasi, K. Z., Abell, G. C., Taylor, R. S., Neuman, C., Hatje, E., Tamplin, M. L., et al. (2014). Pyrosequencing-based characterization of gastrointestinal bacteria of Atlantic salmon (Salmo salar L.) within a commercial mariculture system. J. Appl. Microbiol. 117, 18–27. doi: 10.1111/jam.12514 Appl. Microbiol. 117, 18–27. doi: 10.1111/jam.12514 Zhao, X., Xian, Y., Li, C., Wang, C., Yu, D., Zhu, W., et al. (2016). Feeding Lactobacillus plantarum and Lactobacillus casei increased microbial diversity January 2019 | Volume 9 | Article 3247 Frontiers in Microbiology | www.frontiersin.org 19
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Valuation through Narrative Intelligibility
Valuation studies
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Valuation through Narrative Intelligibility Patrycja Kaszynska Valuation Studies 10(1) 2023: 148–166 Valuation Studies 10(1) 2023: 148–166 Abstract Abstract Narrative intelligibility is central to making sense of valuation. Narrative intelligibility is a framing device that combines empirical observation and situated interaction with teleological, purpose-oriented, normative inquiry. Thus understood, narrative intelligibility provides a useful analytical frame to explain how the phenomenon of valuation is practised. At the same time – and on the level of research – it bridges synthetically different traditions of thought, including actor–network theory’s descriptive accounts of valuation practices and humanities-grounded, normative theories of value. As such, narrative intelligibility offers a way of avoiding the alleged weakness of overstating the agency of devices and material actors in actor–network informed approaches, without however seeking to relocate analysis into the ‘ineffable’ realm of purely theoretical constructs, the way some humanities scholars are said to have done. The argument shows that the humanities- derived understanding of values, approached in terms of standards of justification and norms of criticism, can be combined with the vernacular concepts of valuation from actor–network theory in a way that promises a unified research agenda going forward. Keywords: valuation; Valuation Studies; narrative; normativity; humanities; actor–network theory Introduction If one of the ambitions of the Valuation Studies journal is to consolidate a field of inquiry under the same name – the question arises: what fragmented, pre-existing areas have enough proximity and relevance to enter the gravitational pull? Perhaps an obvious answer is: those concerned with value and with valuing. And yet, a little paradoxically, existing accounts of value and those concerned with valuing do not, at present, overlap in a productive way. The starting – contestable – point of this article is that the approach to valuation represented, or perhaps over-represented, in the present journal has not intersected in a sustained and systematic manner with the humanities’ ‘way’ of thinking about value. As explained in the next section, to talk about distinctive ‘schools’ is an oversimplification and possibly a reification. And yet, if one accepts that there is a notable distinction between descriptive and normative approaches – those concerned with describing how valuation practices unfold on the one hand, and those asking why they develop the way they do and how they should be conducted on the other – one can legitimately insist that there is a spectrum with two quite distinctive ends. Humanities literature, in particular, of a philosophical orientation, has traditionally aimed at articulating theories of value, and has often – but certainly not always (cf. Dewey 1939) – been conducted in a priori terms and based on speculative claims concerning which normative concepts should be applied. The descriptive ‘school’ can be characterised by empirical engagement with situated contexts and ‘devices’ used in these contexts. The latter, actor–network theory (ANT)-inspired tradition, downplays (Munk and Abrahamsson 2012) or eliminates outright (Roberts 2014; Heinich 2020) the normative concerns that form the core of the humanities approaches, its focus is on mapping ‘the systems, devices, instruments and infrastructures that underpin various kinds of valuation (rating, pricing, ranking, accounting, funding, and assessing)’ (Helgesson et al. 2017: 3). This article argues that the ANT-informed valuation studies and the humanities-grounded theories of value need one another: without any standards of justification or critique, ANT accounts are unable to characterise the kind of agency that is operative in valuation and thus, to genuinely act on the recognition that ‘to describe the real is always an ethically charged act’ (Law 2007: 17). Patrycja Kaszynska is Senior Research Fellow at University of the Arts London. © 2023 The authors This work is licensed under a Creative Commons Attribution 4.0 International License. https://doi.org/10.3384/VS.2001-5992.2023.10.1.148-166 Hosted by Linköping University Electronic press http://valuationstudies.liu.se Valuation Studies 149 Introduction On the other hand, humanities-grounded accounts of value need to appreciate the material and the empirical dimensions highlighted by ANT – this in order to curtail their propensity for abstraction and reification. The key contribution of this article is to present the notion of narrative intelligibility as a meeting point for the two ‘schools’ (ends of the spectrum) without compromising their central tenets. That is to say, narrative intelligibility allows making the most of the socio- material analysis of the ANT-informed approaches, while at the same Valuation through Narrative Intelligibility 150 time capitalising on the humanities to present valuation as an object susceptible to critique and thus something that can be analysed in normative terms. As explained in detail in what follows, narrative intelligibility is defined here in terms of teleological continuity and situated interaction and draws on the account of narrative developed by MacIntyre (2007), Haraway (2016) as well as, crucially, the Deweyan understanding of situational means-ends adjustment (Dewey 1939). The upshot of this article is a better framework for understanding valuation which does not rest on metaphysical speculation about value and values, nor does it reduce valuation to a multitude of patterns. This marks an approach to valuation that is both practical and normative (Horkheimer 1972; see also Lynch and Fuhrman 1991; Radder 1992, 2008). This is because the normativity in question is derived from the ‘know-how’ of everyday practice which is situationally embedded (Bohman 2004; see also Kaszynska 2021). In suggesting that the frame of narrative intelligibility should be added to the ‘toolbox’ of valuation studies, this article makes an important contribution to consolidating the field that cuts across the socio- material and humanistic analysis. The frame of narrative intelligibility is a means of bridging the ends of the spectrum which have remained too distant up until now. Between theories of value and valuation networks There is a long tradition of thinking about value in the humanities across axiology, ethics, theology and moral theory, aesthetics and theory of art, intellectual history and political philosophy and – of special interest in this article – the theory of value as related to action and pragmatics. Some canonical names in this lineage include: Aristotle([340BC] Crisp 2014); Thomas Aquinas ([1265–1274] 2012); Bentham ([1780] 1996); Kant ([1785] 2005); Marx ([1867–1883] 2010); Nietzsche ([1887] Nietzsche and Hollingdale 1989); Dewey (1939); Arendt [1958] 2013); Habermas (2015); Foucault (1980); Rorty (1989); Chang (1997); and Nussbaum (2009); more recently, Srinivasan (2015); McMullin (2018). The list is necessarily selective and partial (for one thing, it privileges philosophy over other humanities disciplines such as literature and history) but serves well the purpose of illustrating the historical expansiveness of thinking about value in the humanities. i In comparison, valuation studies as a field of inquiry consolidated around the journal of the same name established in 2013 – is in its infancy. Of course, if valuation is understood as ‘any social ... practice ... where ... the ... value ... or values of something are established, assessed, negotiated, provoked, maintained, constructed and/or contested’ (Doganova et al. 2014: 87) – an interest in Valuation Studies 151 understanding these practices predates 2013. The sociology of valuation can easily be traced to the pioneering work of Viviana A. Zelizer (1979) but has perhaps deeper roots in institutional economics (Veblen [1899] 1973) and Weberian sociology (Weber [1922] 2019). Interest in the social embedding and determination of value has since found articulation across the pragmatic valuation sociology (Espeland and Stevens 2008; Stark 2011; Lamont 2012), fed into the developments of economic sociology (Beckert and Aspers 2011; Muniesa 2011; McFall and Ossandón 2014) and the economics of convention (Thévenot 2001; Diaz-Bone 2011), got a stronghold in science and technology studies (Callon and Muniesa 2005; Haywood et al. 2014), and informed some strands of management and business (Ramirez 1999; Chandler and Vargo 2011). There are of course individual scholars working in the sociology of valuation who have been engaged with the journal and whose work has presented a sustained involvement with literature, history and literary criticism (e.g. Czarniawska 1997, 2004; Fourcade 2009, 2011). Valuation through Narrative Intelligibility 152 Valuation through Narrative Intelligibility 152 Valuation through Narrative Intelligibility 152 Guggenheim 2020). The sociology of valuation can be perhaps located somewhere in-between in that it shows a range of different points of emphasis, e.g. from Weber’s theoretical reconstructions to Stark’s empirically-underpinned conceptualisations (for the purposes of this article, the sociology of valuation will not be considered as a separate category but rather as redistributed on the spectrum). Also, it worth noting that there are internal differences within the ‘schools’ themselves, including differences between the ‘founding fathers’ of ANT: Latour and Law (see Munk and Abrahamsson 2012). Setting the question of internal unity aside, what is apparent is that the humanities and the ANT-inspired ‘schools’ do not meet in a productive way. This, as this article argues, results in partial accounts of valuation and puts constraints on what the ‘schools’ can individually accomplish. The humanities ‘school’ lacks empirical credibility; the ANT valuation studies ‘school’ is deprived of much ability to compare across different contexts and to critique individual accounts, indeed to issue value judgements in the traditional sense of the word (cf. Doganova et al. 2014). In other words, the ‘Normative– Descriptive’ (Radder 1998: 325) schism or, at very least, a spectrum arises – the theoretical accounts with standards of justification are set apart from the descriptive reports with empirical engagement. Between theories of value and valuation networks Indeed, it is worth noting that Latour himself presents an interesting case when it comes to recognising the cogency of a perspective spanning a plurality of different discourses in making sense of valuation. While, on the one hand, his commitment to deflating abstract constructs makes him suspicious of ‘values’ as construed by the modern western epistemology, ethics and metaphysics (Latour 2013), his central argument for why We Have Never Been Modern rests on observing the entanglement of different value registers in practical situations (Latour 2012) and the need for resolving those as quasi-ethical matters of concern (Latour 2004). It should also be acknowledged that the editors of Valuation Studies did openly recognise that there are different disciplinary ‘positions’ which raise different questions and give different answers about how the constructions of values and valuation are or should be studied (Doganova et al. 2014). In this sense, the seeds of cross-disciplinarity spanning the spectrum from ANT-informed descriptive accounts and humanities-grounded normative theories – have been planted. And yet, this cross-fertilisation has not been pervasive enough to benefit both ‘schools’: ANT approaches to valuation (which have been well represented in Valuation Studies) and humanities-grounded theories (which have not). Needless to say – as indeed observed by Helgesson and Muniesa (2013) in the inaugural editorial address to Valuation Studies – there is much to be gained from more collaborative approaches. This raises the question: where and how do these approaches meet? Oversimplifying, the humanities ‘school’ studies value and values starting with theoretical presuppositions and concepts; the valuation studies ‘school’ starts with material and semiotic networks and seeks to stay ‘local’, ‘situated’ and ‘empirically descriptive’ (Law 2004; Does ANT need normativity? First, a word of explanation about what is meant by ‘normativity’, since the word is used differently in ANT and in the humanities, including critical theory. To be normative in the sense of Annemarie Mol (Mol 1999) or John Law (Law 2004) is to make deliberate choices: to foreclose some alternatives. To be normative in the Critical Theory sense is to pay attention to how actors manifest the normative attitudes implicit in their practical knowledge (Geuss 1981; Horkheimer 1993; Habermas 2015) and in the everyday ‘know-how’ of human conduct (Bohman 2004). What is sometimes not fully understood is that normativity in the critical theory sense is not about proscribing or fixing courses of action, nor even about setting rules. Rather, it is based on capitalising on the fact that action coordination presupposes that some standards of criticism and justification are applied and can be collectively ascertained (Kaszynska 2021). pp y y ANT designates a method rather than a theory but it signals a predilection for a certain mode of inquiry. As John Law put it back in 1992, ‘actor–network theory almost always approaches its tasks empirically’ (Law 1992: 6). This often translates into the suspicion of ‘occult entities’ and the preference for the ontological ‘desert landscapes’, to borrow expressions from the philosopher Willard Van Orman Quine (1948). In other words, the default position of the ANT Valuation Studies 153 sympathisers is a commitment to flatter or flat ‘ontologies’ in the sense suggested by Quine and notably, in the opposite sense to that intended by Bhaskar (1978) who first coined the term. The emphasis in this tradition is very much on the mechanism – the nuts and bolts – of valuation, such as: ‘market technologies’ (Muniesa 2007), ‘political technology’ (Muniesa and Doganova 2020) and other kinds of ‘the systems, devices, instruments and infrastructures that underpin various kinds of valuation’ (Helgesson et al. 2017: 3: see also Orlikowski and Scott 2014). What this amounts to in practice is studies preoccupied with detailed description of the formation of socio-material-semantic networks, attentive to contexts and devices as a way of mapping out the trajectories along which valuation unfolds. Does ANT need normativity? Even though there is a growing interest in generalising from specific circumstances and in understanding the conditions of critique for the systems in question (see for instance Muniesa 2017, 2019; Muniesa and Doganova 2020; also Latour 2004, 2012), the main emphasis remains on mapping the actants and the circumstances in which they act. With reference to the Valuation Studies journal specifically, this can be exemplified by the influential study of the role of algorithmic valuation devices and verification mechanisms in the tourism and travel sector by Baka (2015). Forseth et al.’s account of ‘Reactivity and Resistance to Evaluation Devices’ (2019) in the banking sector is another example. These ANT-informed approaches are now well established in valuation studies – and indeed Valuation Studies – but with this success comes a growing recognition of potential limitations. One such an indication is the reaction against, or perhaps a growing self- awareness of, the excessive concentration on the ‘mechanics’ of valuation (Zuiderent-Jerak and van Egmond 2015). As Hauge points out, ‘while the many studies of valuation practices have drawn attention to the pervasive effects of valuation devices, only a few studies have taken into account the fact that many spaces, including organizations, are already flooded with practices and ideas that constitute what is valuable’ (Hauge 2016: 117). ( g ) Some commentators sympathetic to ANT were explicit about the no absence of normative considerations and standards of critique (Radder 1992; Marres 2009). An overt concern about ‘flat-land of relativism’ was noted in the editorial address in Valuation Studies back in 2014 (Doganova et al. 2014). The root can be traced back to the early 1990s. The ‘Epistemological Chicken’ controversy involved some of the prominent figures such as Latour and Callon (Fuller 1991; Lynch and Fuhrman 1991; Redder 1992) and centred on the claim made by Collins and Yearley (2010) that the ANT method leads to an infinite regress because it is unable to fix any parameters of the debate as standards of comparison and criticism. If there is nothing beyond the shifting networks of relationships and all actors and actants have the Valuation through Narrative Intelligibility 154 same status – questions such as: what motivates agency in valuation and what gives it direction? what binds the discrete moments of action into a valuation situation? and above all, which courses of action are to be preferred over others? – are difficult to settle. Does ANT need normativity? It should be noted that Latour’s talk of ‘ontopolitics’ (Latour 2012) and Mol’s ‘ontological politics’ (Mol 1999) reveal that these issues are cogent even within the ANT discourse. At some risk of overstating the case, the need to ponder politics in ANT is an admission that the ethical choices have not gone away. As Law puts it: ‘There is nowhere to hide beyond the performativity of the webs’: But since our own stories weave further webs, it is never the case that they simply describe. They too enact realities and versions of the better and the worse, the right and the wrong, the appealing and the unappealing. There is no innocence. The good is being done as well as the epistemological and the ontological. (Law 2007: 16). Think of the existence of racism and racial oppression in the USA. There are multiple accounts – many told by the oppressors, and few told by the oppressed. The current situation is precarious because of the polarisation fuelled by culture wars. As Charles Mills (2014) argues in The Racial Contract, the one constant is that racial contract is continually being reformatted. Once upon a time it was manifested as physical enslavement; later, more intangible forms of segregation and inequality were proposed; now, it is primarily based on immaterial forms of discrimination by institutional and cultural norms which maintain the same racialised hierarchy. In Mills’s (2014) opinion, not much has changed but the reality needs to change. How to achieve this? One way of handling this is to encourage some open-ended plurality of ‘webspinning’ without arbitration (cf. Stark 2011). But is this enough? Surely, justice cannot be achieved just by pitting the opposing factions against one another the way ANT might recommend. But how can this prevent the ‘might is right’ scenario? If, in the context of racial oppression, one finds oneself confronted with the question: What am I to do here and now? – it is far from obvious that the ‘laissez-faire’ attitude of ANT has a satisfactory answer. The point is that there is ‘no innocence’: there is the right and the wrong way of acting when it comes to racial oppression. Does ANT need normativity? pp Admittedly, as noted above, there have been some attempts in ANT to offer ‘embedded’ accounts of normativity and to answer the ‘where to’ question from within the contexts, networks and situations themselves: be they Situated Intervention and the Ethics of Specificity (Zuiderent-Jerak 2015), or Care in Practice (Mol et al. 2015), or Latour’s ‘ontopolitics’ (Latour 2012). These attempts however do not offer satisfactory answers on a scale capable of changing collective Valuation Studies 155 behaviours. As the next section discusses, it is by relocating the discussion from the realm of causes and patterns (the how) to the realm of purposes (the why), that the humanities opens up the possibility of normative analysis to ANT. This however does not need to mean betraying the instincts of ANT to stay situated and grounded. Narrative intelligibility is introduced as a meeting point for empirical social inquiry and normative philosophical argumentation. The notion of narrative intelligibility is presented as a frame which makes it possible to cross-fertilise the ANT-informed studies of valuing and the humanities tradition of thinking about value. Narrative and narrative intelligibility Some have taken MacIntyre to be making a metaphysical claim that individuals are narrative constructs; others understood him as saying that narratives are useful as reflective prisms for people to look at their life (Kupfer 2014; cf. Taylor 2016). Resolving this is not necessary for the purposes of this article. What matters is that MacIntyre defines narrative as a form that has a beginning, a middle and an ending, and crucially, an internal coherence that is instrumentally useful in making sense of valuation situations. Importantly, what is required for narrative coherence is more than just being a sequence. The chain of events has to unfold in a certain purpose- oriented way with parts interpreted in the light of the whole sequence and with the whole defined by its teleological orientation. To rephrase, what is at issue is not just a mechanical pattern where one thing causes the next (cf. Aristotle’s Poetics in Janko 1987) but that events unfold according to a unifying purpose. Some may at this point be alarmed by the prospect of resurrecting ‘occult entities’ such as the notion of ‘divine causes’, ‘absolute spirits’ and other ontological ‘pathologies’ that ANT has set to abolish. This alarm would be premature. This can be demonstrated by a quick look at how narrative – as a form that has a beginning, a middle and an ending, and crucially, some internal coherence – can be found in Donna Haraway’s work. Haraway’s account of the power of ‘fabulating’ in the influential Staying with the Trouble (2016) presents an account not dissimilar to MacIntyre’s. There Haraway sets out a programme which uses telling stories as a practice for generating imaginative patterns, something on which she elaborates with the example of string figures which can be transformed over and over again. Each moment of transformation is underpinned by its unique teleological drive. Each stringing moment has some – arguably purposive, even if implicit – unity, even though there is no overarching, universal purpose. To be clear, Haraway is concerned with the material semiotics (Haraway 2013). Even though she agrees that ‘for a material semiotics teleology may not reside in human intentions’ (Law 2007: 10) she retains the idea of teleology as the means-ends adjustment for every discrete moment of the string transformation. Narrative and narrative intelligibility Starting in the 1980s, there has been a growing interest in narratives across a wide range of disciplines. A number of publications set out to examine the use of narrative structures in different domains: history, literature, psychology and social science (Polkinghorne 1988) and literature, philosophy and science (Nash 2005 [1994]; see also Brosch and Sander 2015) to name but some. The interest in narratives as a subject matter has gone hand-in-hand with the development of narrative-based methodologies: narrative inquiry and narrative analysis (Mishler 1990; Plummer 2001; Riessman 2008). What has become known as the ‘narrative turn’ is usefully summarised by Hinchman and Hinchman (1997) as a rejection, among other things, of the atomistic understanding of social phenomena and the dis- embedding of individual acts from the ‘web of communication’ out of which they arise (Hinchman and Hinchman 1997: xiv). Narratives in this context are presented as a means of retrieving the plurality of stories that cultures and subcultures tell without submerging them into some uniform meta-narrative, while at the same time not giving up on the ambition of finding a common communicative ground. Not surprisingly, a number of interesting scholars have been developing different forms of engagement with narratives in relation to valuation studies (e.g. Czarniawska 1997, 2004; Smith 1988; Fourcade 2009, 2011). More recently, Beckert and Bronk (2018) revived the idea of narrative as a coordinating device that can be used to deal with the conditions of radical uncertainty when a stabilising of expectations is needed. Alasdair MacIntyre was one of the first to take an interest in the role of narrative in valuation. In broader terms, the key argument of After Virtue can be reconstructed as saying that without a minimal narrative structure, lives and actions of individuals will lack intelligibility. Actions and events need to be formulated as episodes in a narrative to have meaning. In other words, moral discourse has been cut adrift from the narrative understanding of purposes, and with this, it was rendered incoherent. This is a problem because: ‘I can only answer the question, Valuation through Narrative Intelligibility 156 “What am I to do?” if I can answer the prior question, “Of what story or stories do I find myself a part?’’’(MacIntyre 2007 [1981]: 216) – as MacIntyre put it. Narrative and narrative intelligibility To be fair, in a similar vein, MacIntyre recognises that narratives do not occur in a vacuum, rather they are historically and geographically situated. In this context, MacIntyre speaks evocatively that ‘we enter upon a stage which we did not design and we find ourselves part of an action that was not of our making. Each of us being the main character in his own drama plays subordinate parts in the dramas of others, and each drama constrains the others’ (2007 [1981]: 49). Even though the ontology of socio-material and technological networks does not figure Valuation Studies 157 in MacIntyre’s vocabulary, he admits that narratives are situated and contextually circumscribed. Taking MacIntyre’s and Haraway’s work together, it can be suggested that narrative intelligibility is where socio-material networks and teleology meet (cf. Bevir and Galisanka 2016). That is to say, to make a narrative intelligible both are needed: an understanding of a sequence as internally coherent and so directed towards a goal and also an understanding of how it is situated in a specific context. Accordingly, the notion of narrative intelligibility is defined in this article in terms of both: goal-oriented, teleological continuity and contextually circumscribed interaction. But, what kind of entity is narrative intelligibility? g y Narrative intelligibility could be thought a framing construct, to borrow a term from communication and media studies (cf. Clarke 2003). The framing at issue should not be understood as a phenomenon known as agenda-setting (Kuypers 2010) nor is it an attempt to forge uniformity through appealing to some set of universal values (Lakoff 2014). Rather, the frame is here understood in the sense presupposed by Kenneth Burke’s concept of terministic screens. Terministic screens are filters for photographs which accentuate different features of photographs. So, applying different screens to the same photograph may make different features apparent and prompt different interpretations. In the same way, Burke (1966) argued that individuals can represent issues based on the choice of some aspects fixing their attention – whether consciously or unconsciously – which they emphasise in language. Narrative intelligibility is a way of filtering valuation situations through the prism of the chosen goals as they become adjusted to the available means. This underscores that what is at issue with narrative intelligibility is not some universalistic grand narrative. Far from it as the next section demonstrates turning to a quintessentially Deweyan understanding of valuation. Situational interaction and teleological continuity in Dewey Dewey approaches valuation as a type of inquiry. Inquiry for Dewey is a social process that follows the following six steps: 1. Identify the problem 2. Plan possible solutions 3. Evaluate and test the various solutions 4. Decide on a mutually acceptable solution 5. Implement the solution 6. Evaluate the solution. Following this general pattern, valuation takes the form of a practical judgement, that is – as spelled out in Dewey’s The Logic of Judgments of Practise (1915) – the judgement characterised in terms of being geared towards future actions, based on an assessment of desired outcomes and using means- ends reasoning, and being grounded in some form of empirical inquiry. Valuation through Narrative Intelligibility 15 158 This, in a nutshell, is tantamount to meeting the conditions of situational interaction and teleological continuity. g y Thus, it could be suggested that narrative intelligibility is the grounding of Dewey’s valuation theory. According to Dewey, valuation is best thought of in terms of acts of interpretation and justification configured by specific socio-material contexts (Dewey 1939). Even though anchored in individual experiences, as these are shaped by moral norms and ethical values, value can be seen as constructed and reconstructed by groups and communities who themselves are shaped by their historical and geographical, social and cultural, institutional and technological contexts (cf. Foucault 1980; Latour 2005). This is consonant with ANT and, not surprisingly, Dewey has been embraced by key figures in valuation studies (e.g. Muniesa 2011). It is worth pointing out in this context that Dewey and his fellow George Herbert Mead were instrumental in the development of the Chicago School of Sociology. There they played a pivotal role in driving the work to understand the nature of relationships in regional geography (Singelmann 1972) later to be associated with the thinking about ‘social worlds’ (Strauss 1978) which, through science and technology studies, fed into ANT thinking (Clarke and Star 2008) and situational analysis (Clarke 2003). y Dewey’s thinking is thus informed by the considerations concerning ‘social worlds’ and ‘site ontologies’ that preoccupied the Chicago school. However, for Dewey the idea of situated ‘shared discursive spaces that are profoundly relational’ (Clarke and Star 2008: 120) is never separated from teleological analysis and never purely discursive. Simply put, according to Dewey, people value things because they are useful to them. And yet, not all desired outcomes and future actions are equal. Situational interaction and teleological continuity in Dewey On the one hand, Dewey admits that there are a number of normative standards that can in principle apply to any given situation. At the same time, the process of inquiry, as outlined in the six steps above, rests on the idea of justification. To value is to weigh the means and ends in specific situations and crucially, to demonstrate that the proposed course of action is justifiable in collective terms. This is the basis of normativity in Dewey. y y Thus, while faithful to some tenets of what this article dubbed the ANT-informed valuation, Dewey is also a member of the humanities tradition with his insistence on normativity. The problematic situations that trigger valuation are unmistakably a part of the socio-material networks. Yet, the teleology constitutive of valuation unfolds according to the means-ends reasoning and in line with normative constraints. His account brings together MacIntryre and Haraway. Situational interaction and teleological continuity underpinning narrative intelligibility are the ground and a way of bridging the descriptive and normative approaches to valuation in Dewey. In such a way, narrative intelligibility provides a solution to what Dewey dubs Valuation Studies 159 ‘the deepest problem of modern life’: ‘the problem of restoring integration and cooperation between man’s [sic] beliefs about the world in which he lives and his beliefs about values and purposes that should direct his conduct’ (1929: 204). Acknowledgments g I am grateful for the generous feedback from the two anonymous reviewers and for the thoughtful, careful, and supportive steer from the editors of this theme issue. I also wish to thank Christian Nold – Lecturer in Design at The Open University – for talking with me about ANT. Valuation through Narrative Intelligibility 160 Valuation through Narrative Intelligibility 160 Valuation through Narrative Intelligibility 160 Acknowledgments Concluding reflections on overcoming ‘the great divide’ This article argues that the ANT-informed descriptive approach to valuation and the humanities-grounded normative theories of value will not succeed in separation from one another. Moreover, they have much to gain from coming together, individually and collectively. For the humanities-grounded perspective, unless this tradition finds a way of keeping itself empirically embedded, the use of reasoning alone is likely to result in rarefied and reifying accounts of value. As for the ANT perspective, it too will fail in separation. By its own admission, ANT sees itself as ‘a toolkit for telling interesting stories about, and interfering in, those [world] relations’ (Law 2007: 2). But can ANT really tell stories rather than compose the proverbial shopping lists? Are the networks created just random collections or is there any orientation to them? Moreover, are some ways of composing these networks better than others – are some courses of affairs straightforwardly bad? The argument presented here is that, if ANT remains stranded with lists and not stories, it will fail in its attempts to account for valuation as a phenomenon in the world where some ways of acting are better than others, where norms of criticism and standards of justifications apply. This article argues that narrative intelligibility is a useful framing device that brings together the tradition concerned with value in the humanities and the ANT approach preoccupied with valuing. Narrative intelligibility binds the notions of teleological progression, orientation and purpose (MacIntyre 2007 [1987]) with those of the situated, material interconnectedness (Haraway 2013, 2016). Conceived in these terms, it allows valuation to be seen as normatively grounded and goal oriented, as well as empirically situated and contextually circumscribed (Dewey 1939). y y Narrative intelligibility provides thus a good frame to make sense of the phenomenon of valuation but also to set an agenda for research into valuation. Adding narrative intelligibility to the ‘toolbox’ of valuation studies opens up the possibility of integrating the normative and descriptive approaches and with this, the option of interrogating the assumptions and hegemonies present in the understandings around ‘good’ and ‘bad’ valuing (cf. Horkheimer 1972) without relying on some fixed metaphysical abstractions. This option is, arguably, needed in valuation studies and in Valuation Studies (Helgesson and Muniesa 2013; Helgesson 2021). Valuation through Narrative Intelligibility 160 References Aquinas, Thomas. 2012. Summa Theologica (Part 1). Milton Keynes: Authentic Media. Arendt, Hannah. 2013. 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Patrycja Kaszynska is Senior Research Fellow at University of the Arts London (UAL) and Research Associate at Culture, King’s College London. She is also Research Affiliate at Northeastern University London, where she was Head of the Art History Faculty before joining UAL. She has a track record of shaping the UK’s national discussion on how the value of arts, culture and heritage is articulated and measured in the context of decision making: first as Project Researcher Valuation through Narrative Intelligibility 166 for the Arts and Humanities Research Council (AHRC) Cultural Value Project, then Project Manager for the Cultural Value Scoping Project and most recently, Principal Investigator for the AHRC and Department of Digital Culture Media & Sport (DCMS)-funded Scoping Culture and Heritage Capital Project. Patrycja’s interests are at the cross section of critical theory, pragmatic philosophy, cultural studies and design with a key focus on the theory of value and valuation studies.
https://openalex.org/W1917219012
https://revistas.ucm.es/index.php/CUTS/article/download/CUTS0202110217A/7909
Spanish; Castilian
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Instinto de mejora.Diferencia entre dos movimientos migratorios: el éxodo rural de la década de 1960 y la inmigración actual.
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ISSN: 0214-0314 ISSN: 0214-0314 Cuadernos de Trabajo Social Vol 15 (2002): 217-234 Cuadernos de Trabajo Social Vol 15 (2002): 217-234 Abstract The present article tries to show the similitu- des and differences between two of the most im- portant and with more consequences migratory movements that have taken place in the last fifty years in Spain: the depopulation of the country- side that laid out and prepared for city develop- ment and industrialized the countre since 1950 and present immigration surrounded by discrimi- nation, racism and intolerance. El presente artículo intenta mostrar las simili- tudes y diferencias entre dos de los movimientos migratorios más importantes y con más conse- cuencias que se han producido en los últimos cin- cuenta años en España: el éxodo rural, que urba- nizó e industrializó el país a partir de 1950, y la inmigración actual rodeada de discriminación ra- cismo e intolerancia. Juan José MUÑOZ ORTEGA* Juan José MUÑOZ ORTEGA* * Sociólogo. Cuadernos de Trabajo Social Vol 15 (2002): 217-234 Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz Juan José Muñoz mentos más utilizados para entrar en el país, no es el elemento definitorio del fenómeno de la inmigración actual, sino más bien es el avión el medio más utilizado en conjunto para llegar a nuestro país. Concretamente, si en 1999 había un total de 801.329 inmi- grantes (legales) residiendo en Espa- ña, un 20% eran marroquíes mientras que un 32% eran europeos del este, sudamericanos y asiáticos1. Con ello quiero decir que, aunque los inmi- grantes marroquíes fueran los más nu- merosos, la patera no es el vehículo más utilizado. Son muchos los tipos de inmigración y, porque la televisión nos muestre imágenes lamentables de sucesos ocurridos en las costas an- daluzas, no vamos a generalizar y a to- mar por el todo lo que es una las par- tes de lo que es la inmigración. Sobre todo cuando los movimientos migra- torios nos han acompañado y ayuda- do en el propio devenir histórico de la sociedad humana. por sus posibles consecuencias po- tenciales en otros aspectos, como son las sociales (probables conflictos y di- ficultades de integración), las políticas (medidas y acuerdos políticos para la total integración de los inmigrantes) y las económicas (el paro estructural que sufre la sociedad española). La solución del actual problema in- migratorio viene de la mano de una de las variables que explican mejor el fe- nómeno: la legalidad o ilegalidad de los inmigrantes. Las pregunta es: ¿qué ha- cemos con los inmigrantes ilegales? Díez Nicolás advierte que España podría soportar la entrada de 100.000 inmi- grantes al año para mantener su creci- miento demográfico y económico. Para ello, se debería controlar más si cabe los flujos de entrada a nuestro país. Así pues, según los resultados de la encuesta del CIS, la inmigración ac- tual se presenta como un problema que se ha de resolver a los ojos de los ciudadanos españoles, aunque desde una esfera política se piense en la in- migración como una ventaja en su sen- tido demográfico, económico, político y cultural, sobre todo, cuando el nú- mero de inmigrantes que llegan a nues- tro país crece, haciendo de España un país de destino de inmigración. 1 Anuario Estadístico de la Comunidad de Madrid 2001. Instituto de Estadística. Consejería de Presiden- cia y Hacienda. 1. Introducción por la masiva entrada de inmigrantes en nuestro país; pero esta entrada es- tá condicionada por la imagen que la sociedad española tiene de la inmi- gración. Me refiero a las imágenes de la entrada ilegal de africanos, cruzan- do el Estrecho, alojados en miserables pateras que, un día sí y otro también, invaden las costas andaluzas y los no- ticiarios españoles. Como es sabido, ésta no es la única inmigración que se produce, ya que el fenómeno migra- torio ha existido siempre y no preci- samente gracias a pateras y norteafri- canos. Si nos atenemos a los datos, las pateras, siendo uno de los instru- S e ( S egún el barómetro del Centro de Investigaciones Sociológicas (CIS) de agosto de 2001, la in- migración se situaba como el segun- do problema nacional por detrás de ETA y por delante del paro aunque, analizando los datos por Comunida- des Autónomas, podíamos observar cómo en determinadas regiones (An- dalucía y Murcia), la inmigración se consideraba el principal problema que ha de ser resuelto. Como vemos, la opinión pública española está seriamente preocupada 217 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Dicho lo cual, aportando una nue- va visión sobre la inmigración, intenta- ré comparar ambos movimientos mi- gratorios partiendo de la base de que sus causas pueden ser parecidas: la búsqueda de un futuro mejor, el cam- bio de un ámbito rural, pobre, subde- sarrollado hacia otro urbano, desarro- llado, más atractivo que la sociedad de origen, bien sea los crecientes Madrid, Barcelona o Bilbao de los años 1960 o los ya consolidados Madrid, Barcelo- na o Bilbao del recién inaugurado siglo XXI; pero sin intentar llegar a conclu- siones explicativas generales que re- suelvan el problema, sino más bien aportando aproximaciones hipotéticas, ampliando el campo de estudio y mos- trando efectos descriptivos del fenó- meno de la inmigración. Para ello, he realizado dos entrevistas en profundi- dad interesándome por la historia de vida de dos sujetos que experimenta- ron la sensación de abandonar su ciu- dad natal, sus costumbres, y familias de origen para buscar destino en una ciudad completamente diferente, bien sea nacidos en un pueblo de Sala- manca o en el mismo Marruecos con el objeto de analizar las expectativas del viaje, las causas y los modos, así como las pautas de adaptación y la vi- da diaria en la sociedad de acogida a la que llegaron. Instinto de mejora. Diferencias entre dos movimientos migratorios... Tanto es así, que los expertos en de- mografía e incluso la ONU postulan la necesidad de acoger y facilitar la en- trada de inmigrantes para soportar el creciente ritmo demográfico actual de la sociedad, tanto española como eu- ropea (sobre todo en el caso de Espa- ña cuya tasa de natalidad es una de las más bajas de Europa). El profesor Díez Nicolás comentaba en el I Congreso Profesional de Sociólogos y Politólo- gos celebrado en Madrid en julio de 2001 que el fenómeno de la inmigración es interesante, no sólo por sus reper- cusiones demográficas sino también El proceso migratorio actual ad- quiere tintes semejantes a aquel otro que ocurrió en la España de la déca- da de 1950-1960, cuando centenares de personas abandonaron sus pueblos de origen y abastecieron de mano de obra las recién industrializadas ciuda- des importantes de España. Me refie- Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 218 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz uno quede sorprendido, pero exami- nando detenidamente ambos movi- mientos migratorios podemos observar que, en su génesis y en su desarrollo, son similares, salvando y procurando respetar las tres grandes diferencias que minimizan los efectos de la com- paración: el momento del viaje —el éxodo rural se da en torno a 1950- 1960 y la inmigración actual se pro- duce cuarenta años más tarde—; el te- rritorio —el éxodo rural se produce al interno de España, del campo a la ciu- dad, y la emigración es de un país a otro, de una cultura e idioma a otros—; y su consolidación en la sociedad de acogida —en 1960 el emigrante rural pudo mantener su nivel de vida, inclu- so mejorarlo, mientras que en la ac- tualidad el inmigrante encuentra serias dificultades para trabajar, formar una familia y educar a sus hijos en un cli- ma acorde para su desarrollo y forta- lecimiento—. ro al masivo éxodo rural que asoló los campos y urbanizó las ciudades. Po- demos decir que ambos procesos pre- sentan características comunes y no cabe duda de que son fuente de cam- bio social en el sentido que modifican las bases culturales de la sociedad. 2. Aproximaciones teóricas sobre las migraciones El fenómeno de la migración se puede definir como cualquier cambio permanente de residencia, que impli- que la interrupción de las actividades en un lugar y su reorganización en otro (Ravenstein, 1885). Si eso es así, po- demos ver que las migraciones han si- do un recurso muy importante para asegurar la subsistencia humana. A su vez, la movilidad social asociada a la geográfica ha sido durante siglos un ansia en el sentimiento de gran parte de los seres humanos, ya que el ins- A primera vista, puede parecer és- ta una comparación ficticia y más de Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 219 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz de las migraciones —a decir de R. E. Park— que centra la atención en quien emprende el viaje. “El protagonista de la migración es el migrante, la perso- na que experimenta y que sufre todo el proceso” (Park, 1950). Enfatiza las particularidades que conforman la per- sonalidad del migrante, como si tuvie- ra características especiales: una ini- ciativa superior, una capacidad para el trabajo y de adaptación mayor a los de su entorno; todo ello sumado a los agentes externos. tinto de mejora es innato en el ser hu- mano, es el motor de progreso y, cuan- do el hombre percibe la existencia de una posible mejora en sus condiciones de vida, a través de los desplazamien- tos geográficos se ponen en juego una serie de factores, cuya conjunción puede determinar que una persona emprenda la migración. Una vez producido el desplaza- miento, el individuo experimenta un proceso de adaptación que puede acarrear conflictos, en el sentido de confrontar dos concepciones de vida totalmente diferentes. Los valores aprendidos en la niñez y los nuevos valores asumidos en la nueva socie- dad deben confluir adecuadamente en el hábito de vida de la persona que mi- gra, aunque ello no impida que el sen- timiento de desarraigo y la pérdida de la antigua identidad traigan consigo particulares características que mar- quen al sujeto en cuestión. Otra concepción la ofrece Manga- lam, al entender la migración como un proceso adaptativo cuyo principal ob- jetivo es mantener el equilibrio diná- mico de la organización social con mo- vimientos de cambios destinados a proveer a sus miembros de recursos para superar sus privaciones. 2.1. Teorías sobre las migraciones Los primeros estudios sobre las mi- graciones datan de finales del siglo XIX, cuando las migraciones se veían como un proceso de expulsión-atracción (Ra- venstein, 1885, y Haddon, 1912), por el cual todo individuo se halla sometido a una serie de fuerzas que tienden a ex- pulsar a las personas de la comunidad en la que viven y, a su vez, en la mente de las mismas se desarrolla una idea acerca de la posible sociedad a la que dirige su migración. Esta idea es atra- yente y muestra las ventajas que se po- dían obtener con el cambio. 2. Aproximaciones teóricas sobre las migraciones De esta manera, presta más atención al pro- ceso de adaptación que a la persona y al proceso de expulsión o atracción de la gente (Mangalam, 1968). Por su parte, Kasdan presenta al migrante no como un sujeto pasivo de su historia personal, sino como un in- dividuo voluntarioso que comprende su entorno y toma sus propias deci- siones entre las alternativas que se le ofrecen. Esta cuarta forma de enten- der el proceso migratorio trata de va- lorar la propia iniciativa de la persona (Kasdan, 1970). Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 2.2. Causas de las migraciones Según uno de los expertos en eco- nomía y demografía española, Ramón Tamames, los movimientos migrato- rios se producen por diferentes ten- siones demográficas en los recursos Años más tarde, a mediados del si- glo XX, surge una nueva concepción Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 220 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz económicos disponibles. Tamames pone el dedo en la llaga y lleva la ex- plicación al terreno que le correspon- de, ya que, independientemente de los factores personales, es un hecho que los movimientos migratorios son pro- cesos de carácter esencialmente eco- nómico (Tamames, 1983). Todos los procesos migratorios que se produje- ron en España a partir de la década de los cincuenta y sesenta responden a la industrialización y a la demanda de mano de obra. gar de expulsión como en el lugar de recepción, ya que se crea un desequi- librio demográfico en ambos lugares, las diferencias culturales y sociales se acentúan y la diversidad de etnias, len- guas y religiones requieren una inte- gración para aliviar esas tensiones. Los expertos en migraciones han podido elaborar una serie de caracte- rísticas comunes de los procesos de migración: — Las migraciones son selectivas. Es un fenómeno discriminatorio, ya que no puede migrar todo el mundo, sino un tipo determinado de personas: los varones y las mujeres jóvenes prin- cipalmente. El gran conocedor de migraciones en España, el profesor Víctor Pérez Díaz, señala que cuando una crisis ame- naza con hacer estallar una sociedad tradicional, el éxodo rural contribuye a que desaparezca el trabajador de la tie- rra. La migración —dice— es una solu- ción particular a los problemas surgidos en una sociedad particularista y al em- pobrecimiento de zona rural. En defini- tiva, “la emigración es la manera más rotunda que tienen los campesinos de manifestar su crítica de las condiciones de vida y de trabajo que les ofrece el campo” (Pérez Díaz, 1974: 37). — Existen redes de migraciones. Tienden a migrar las personas más ca- pacitadas para luego comunicar la ex- periencia. — No son permanentes ni en el tiempo ni en el espacio. De un sitio se va a otro para luego buscar otro mejor. 2.2. Causas de las migraciones — Existe una teoría de red por la cual el que migra necesita tener una comunidad afín a su propia individuali- dad con el objeto de obtener un apoyo. — Las migraciones suponen un flujo de divisas de los emigrantes ha- cia su lugar natal con las que la fami- lia que se queda en el lugar de expul- sión continúa viviendo. — Las migraciones suponen un flujo de divisas de los emigrantes ha- cia su lugar natal con las que la fami- lia que se queda en el lugar de expul- sión continúa viviendo. 2.3. Características de las migraciones Como decíamos al principio, los movimientos migratorios son cualquier cambio permanente de residencia que implique la interrupción de las activi- dades en un lugar y su reorganización en otro. Su origen está en el desequi- librio entre la población y los recursos del lugar en los que se reside y del lu- gar al que se va. No cabe duda de que tal necesidad de recursos produce problemas y tensiones, tanto en el lu- 2.4. Marco teórico propio Parto de la idea de que las migra- ciones afectan a las personas que las experimentan y suponen un cambio radical en sus vidas. Lo más curioso es que siempre obedecen a una deci- sión enteramente personal, aunque el individuo desconozca las consecuen- Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 221 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz cisión de migrar: el factor económico. La miseria rural que produjo la Guerra Civil desoló los campos y proporcionó mano de obra a las fábricas de las ciu- dades más importantes de España. cias de su movimiento. El migrante percibe su entorno socioeconómico al igual que sus compatriotas, pero el emigrar responde a una necesidad personal y raramente percibe que esa decisión personal obedece a una cri- sis socioeconómica del entorno en el que vive y a un auge económico y de ampliación de posibilidades de elec- ción de la sociedad a la que se dirige. De esta manera, lo que he estudia- do es el fenómeno migratorio compa- rando las similitudes y diferencias entre dos de los movimientos más importan- tes ocurridos en España intentando mostrar las expectativas, los procesos, las causas y sus consecuencias. Pero, ¿en qué contextos se sitúan ambos fe- nómenos? Los dos siguientes epígrafes ofrecen un breve contexto histórico de ambas situaciones. De este modo, toda migración com- prende un marco socioeconómico de- terminado, un individuo, una sociedad de origen, una comunidad receptora, unas causas del movimiento, unas ex- pectativas, un intento de mejora y unos objetivos deseados. Como vemos, además de los factores personales que inciden en la toma de la decisión de mi- grar, encontramos factores externos que son los que acaban provocando los grandes movimientos de masa. Junto a todo ese proceso interno de la persona migrante no podemos olvi- darnos del componente clave de la de- 3. Contexto socio-histórico de la España de 1950 y 1960 La Guerra Civil supuso un cierto re- traso en las condiciones de vida de to- dos los españoles. No sólo por la des- Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 222 Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz tantes de España y la precaria situa- ción de vida y laboral de los campos provocaron un proceso migratorio de alto rango, en el que más de dos mi- llones de campesinos abandonaron los campos en poco tiempo. La vida rural se hacía cada vez más difícil y sobre todo los jóvenes ansiosos de mejoras vieron con buenos ojos la búsqueda de otras oportunidades, desembarazán- dose de las duras tareas agrícolas. aparición de hombres y mujeres que lu- charon sino por las míseras condicio- nes en las que se quedaron las familias que trabajaban en el campo. En este caso no hay que olvidar que la mayoría de los activos se dedicaban a la agri- cultura y las consecuencias de la Gue- rra Civil redujo su trabajo únicamente a la supervivencia física y a conseguir ali- mentos a través de un trabajo mal pa- gado en una nación bloqueada econó- micamente, en el interior y en el exterior, gracias al autárquico régimen político español que no gustó a las potencias aliadas de la Segunda Guerra Mundial. En paralelo al proceso de urbani- zación, se produjo el proceso de in- dustrialización. Los centros urbanos más importantes necesitaron mano de obra para las nuevas industrias recién creadas con las ayudas económicas exteriores. Ante ese efecto llamada, las personas que decidían emigrar a la ciudad veían compensado su esfuer- zo con un trabajo estable en la fábri- ca. En total, entre 1940 y 1960 dejaron su provincia de origen en torno a 2.743.000 personas (Del Campo y Na- varro, 1987). Con el objetivo de crear una Espa- ña autosuficiente se intentó industria- lizar el país y para ello se crearon mu- chas industrias nuevas con el apoyo del gobierno. Se intentó controlar has- ta el máximo el escaso comercio ex- terior y, por último, se trató de au- mentar la producción agraria. Pero el crecimiento industrial que se produjo no podía continuar dentro de la políti- ca restrictiva de autarquía. En años posteriores, el cambio de sistema llevó consigo una transforma- ción de la coyuntura económica. 3. Contexto socio-histórico de la España de 1950 y 1960 La ayuda económica de Estados Unidos en 1951 facilitó la adquisición de bie- nes y favoreció la expansión econó- mica que llegó hasta 1959, año en el que se publicó el Programa de Estabi- lización cuyo objetivo era alinear la po- lítica económica española a la inter- nacional. De esta manera, se ponía fin al aislamiento económico y el país se encaminaba a una economía libre de mercado según las pautas del capita- lismo occidental. 5. Del pueblo a Madrid Se llama María Inmaculada y nació en San Muñoz, como dice ella, un pue- blo pequeñito en la provincia de Sala- manca. Un día, con catorce años, des- pués de acabar el último año de escuela y de pasar todo un verano ayudando a su padre en las tareas del campo, sonó uno de los dos teléfonos de los que disponía el pueblo. Al otro lado estaba Isabel, su hermana mayor. Le dijo que la señora que vivía arriba buscaba una muchacha para las ta- reas del hogar y que la habló de ella. Isabel ya estaba en Madrid, trabajan- do para los maestros del pueblo. A partir de aquel día su futuro estaba es- crito: un día cualquiera se iría a traba- jar a Madrid al lado de su hermana. En nuestro país el hecho más sig- nificativo ha sido el aumento del tráfi- co de pateras entre las costas africa- nas y el territorio español. Durante el año 2000, alrededor de 15.000 perso- nas fueron detenidas y decenas de ellas perdieron la vida en el intento, mientras que durante el año 1999 fue- ron aproximadamente 3.500 los inter- ceptados. Estos inmigrantes llegaron en más de 780 pateras, sin contar los que lograron entrar ni los que entraron legalmente por la frontera con dinero suficiente y visado de turista para tres meses. Tampoco se contabilizaron los desaparecidos ni las decenas de inmi- grantes fallecidos en los naufragios. Al margen de los problemas de atención, los colapsos de los centro de acogida y de las comisarías de los puntos de llegada, la repatriación hacia los paí- ses de origen se presenta complicada ya que la expulsión no es posible le- galmente en menos de 72 horas. San Muñoz no era un pueblo muy rico y lo único que podían hacer los jó- venes era buscarse la vida en otro si- tio o quedarse trabajando en el cam- po ayudando a sus familias. Como su padre no disponía de tierras propias, Isabel primero y Mari después tuvie- ron que emigrar a Madrid para traba- jar y buscar un destino. Llegó el día pa- ra Mari, cogió un autobús en dirección a Salamanca y otro para Madrid y en Atocha estaba su hermana esperán- dola, la llevó a su nueva casa y así em- pezó la historia de Mari-emigrante en la capital. 4. Contexto de la inmigración actual El problema de la inmigración se centra sobre todo en el equilibrio en- tre la contención de flujos y la necesi- dad de trabajadores. En nuestro caso, España es uno de los países de la Unión Europea con más inmigrantes ilegales (cerca de 300.000) y, aunque el proceso de regulación concluido en el año 2000 permitió legalizar su si- tuación en torno a 140.000 inmigran- tes, otros 60.000 fueron rechazados sub conditione de revisión en algunos casos. El desarrollo industrial que se esta- ba dando en las ciudades más impor- Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 223 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz Juan José Muñoz Según los datos de la agencia es- tadística de la Unión Europea, Euros- tat, en España hay un inmigrante por cada 1.000 habitantes, un porcentaje bastante bajo comparado con el res- to de los países miembros de la UE, sólo por encima de Francia y de Fin- landia (por ejemplo, Holanda tienen 3 por cada 1.000 habitantes, Irlanda 6, Italia 3, Alemania 7, Bélgica 20 o Lu- xemburgo 114). Además, general- mente estos inmigrantes trabajan en economías sumergidas, se les paga menos, operan en peores condiciones y carecen en muchos casos de vivien- da digna. propio relato de dos mujeres (Mari y Babía) que experimentaron la sensa- ción de emigrar a otro lugar especial- mente diferente. Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 5. Del pueblo a Madrid Actualmente, tiene 53 años, vive en Leganés (Madrid), está sepa- rada, trabaja limpiando una oficina por Los siguientes epígrafes del artí- culo intentan poner de manifiesto to- do lo dicho anteriormente en base el Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 224 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz cha independiente, etc. hacían que la única salida fueran las redes sociales que se formaban alrededor del pueblo. De esta manera, a muchos de los jó- venes se les presentaba la oportuni- dad de emigrar amparados por otra persona que podía ayudarlos en su consolidación fuera de allí. La gente se iba a Logroño, San Sebastián, Bil- bao, Salamanca, Madrid, etc.; todos utilizaban a otra persona como puen- te, gracias a la buena amistad entre iguales y a la buena vecindad entre los pudientes del pueblo y las familias tra- bajadoras. las mañanas y tiene dos hijos: Antonio de 30 años, casado y con un niño, y Jesús de 28, que vive en un piso al- quilado con su novia. 5.1. Las raíces del pueblo La familia Ortega vivía en una casa al lado de la plaza del pueblo, no era la mejor casa de la zona, pero por lo menos les daba suficiente cobijo co- mo para soportar el helado frío inver- nal de la zona montañosa salmantina. La poca gente que quedaba en el pue- blo se dedicaba a la agricultura y a la ganadería. Jesús, el padre de Mari, trabajaba duro en una finca de los se- ñores más pudientes del pueblo y Amelia, su mujer, tenía bastante con las tareas propias del hogar. Por aquel entonces, Isabel y Mari eran muy jó- venes, pensaban en jugar con las ami- gas y en divertirse, aunque eso no qui- taba para que tuvieran que aportar algo a la familia sirviendo y limpiando en las casas del médico, del cura, del maestro o del alcalde. Como vemos, la familia se sustentaba gracias al es- fuerzo de todos los miembros del ho- gar, daba igual la edad que tuvieran las hijas: debían aportar de su parte para tener de comer al día siguiente. Como vemos, la vida de Mari se vio sometida a dos aspectos de vital im- portancia: la falta de recursos econó- micos y la ruralidad. El análisis que he hecho se ha basado en estos dos ejes intentando explicitar la visión de su vi- da. El siguiente gráfico muestra gros- so modo la historia de Mari desde su comienzo en San Muñoz —rodeada de dificultades económicas y del contex- to rural— hasta hoy en Leganés —sin dificultades económicas graves y en un contexto urbano radicalmente di- ferente al de sus comienzos—. 5.2. Una decisión tomada La vida en el pueblo transcurría monótonamente, la rutina y la reacción negativa a los cambios propiciaban una vida que giraba alrededor de la subsistencia económica y de las tare- as de la tierra y la cría de animales. En San Muñoz, cuando uno aca- baba la escuela con catorce años, se planteaba la primera elección impor- tante; aunque eran muy jóvenes, los/as chiquillos/as del pueblo hacían lo mismo tarde o temprano. La impo- sibilidad de continuar con las tareas del padre, la inviabilidad de aprender un oficio diferente al del campo, la in- alcanzabilidad de comprar tierras, las dificultades para emprender una mar- Poco a poco, las niñas iban cre- ciendo y empezaron a divertirse con las muchas muchachas del pueblo. Entre tanto un ambiente pesimista les rode- aba, los pocos mozos que había se Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 225 Juan José Muñoz Instinto de mejora. Diferencias entre dos movimientos migratorios. Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz una muchacha que se hiciera cargo de los críos, ya que ambos trabajaban, y pensaron en Isabel. Después de pedir permiso a sus padres y obtener su con- sentimiento, al acabar el curso, em- prendieron viaje hacia Madrid. marchaban a la capital a trabajar y las pocas muchachas mayores dejaban la casa de sus padres para buscar fortu- na más allá de las fronteras invisibles del pueblo. A los 14 años se acababa la escuela y, como pasa cuando se aca- ba una etapa, había que buscar nuevos horizontes. Las cohortes de edades cercanas a los 14 se fueron yendo a otros lugares con el consentimiento de sus padres ya que las posibilidades de hacer algo en el pueblo eran escasas. Unos se fueron primero y otros des- pués, pero no cabe duda de que la ló- gica de ser en el pueblo era abando- narlo para trabajar en otro sitio. Por otro lado, cuando acabó la es- cuela, Mari tenía unas ganas locas de salir de aquel ambiente rural, no era consciente del cambio que podía su- ponerle una modificación en el estilo de vida como aquél, pero ella quería irse. San Muñoz para Mari representa una buena parte de su personalidad. Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 5.2. Una decisión tomada Aquel ambiente tradicional, sin cam- bios, sin conflictos, donde la vecindad y la tranquilidad eran las señas de identidad de la gente que vivía allí con- figuraron desde el principio la infancia de Mari. De similar modo, la pobreza que debían soportar, no sólo su fami- lia sino todas aquellas que no dispo- nían de una fuente de ingresos cons- tante ni clara, estructuró su infancia en Isabel fue la primera de la familia que lo hizo. Era una chica muy cono- cida en el pueblo por lo vivaracha y no pasó desapercibida para los maestros, un matrimonio con tres hijos pequeños, que pidieron el traslado a Madrid para continuar su profesión y dar una mejor educación a sus hijos. Necesitaban Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 226 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz el sentido de luchar y trabajar por so- brevivir. ... pero como ya veníamos con los maestros... mi hermana fue la casuali- dad y gracias a eso me vine. Y de he- cho estabas contenta, estabas bien, añorando tus raíces y tus cosas... ... Entonces a los 14 años pasaba eso. O te ibas de allí o te quedabas, pues eso, a vestir santos o a nada por- que nosotros, en mi casa, no teníamos labor, no teníamos nada y como todas se iban pues tú igual te ibas... La forma de salir del pueblo era común: la utilización de otras gentes que hubieran pasado por ese proce- so. Bien sea por amigos de más edad, que buscaban acomodo en el lugar de atracción a los chicos más jóve- nes, o por recomendaciones de las gentes pudientes del pueblo. Todo ello, encaminado a ciudades en auge por los procesos de industrialización y de oferta de mano de obra de las ciudades. ... se veía normal, porque nosotros no teníamos tierras, no teníamos nada pero gente que sí las tenía y podía vi- vir de ello, también se venían... En el pueblo, el modelo estaba cla- ramente construido, la vida exigía bus- car una salida a la precariedad y en aquellos momentos, la única opción era irse y aumentar así las oportuni- dades de elección, la niñez en el pue- blo duraba poco, los 14 eran una edad en la que ya se podía salir adelante in- dependientemente de los padres. 5.2. Una decisión tomada Sa- lir del entorno rural se veía normal, co- mo un ciclo por el que había que pasar. Los padres querían a sus hijos como ahora, pero eran conscientes de que ellos tenían un trabajo, que eran po- bres, y que no podían supeditar la vi- da de un hijo a un destino tan poco va- lorado y con tanto sacrificio. Además, si hubiera tierras, ganado o alguna otra industria en donde poder trabajar, la gente se hubiera podido quedar, pero éste no era el caso de San Muñoz. Los padres de entonces sabían que la vi- da no estaba en el pueblo y las co- rrientes migratorias anteriores mos- traban el camino de los mozos siguientes. ... yo estaba loca por salir del pue- blo... ... yo estaba loca por salir del pue- blo... ... por cambiar, porque si nos hu- biéramos quedado allí no hubiera ha- bido nada que hacer... ... poco futuro y ningún aliciente, nada... Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz cién llegados. Según Mari, todo era grande, las dimensiones de la ciudad sobrepasaban lo imaginado por ella. Acostumbrada a cuatro calles, a la pla- za, la única carretera de acceso, los pisos bajos, la rutina, etc. salir del pue- blo para vivir en una ciudad de un mi- llón de habitantes, con edificios de diez plantas, el metro, los autobuses, las nuevas tecnologías, oportunida- des, consumo, etc. Se trataba de un cambio importante, sobre todo, cuan- do no había salido nunca de San Mu- ñoz, ni siquiera para ir a Salamanca que estaba a 42 kms. hacía compañía a cambio de propor- cionarle cama, comida y dinero para que ella pudiera aprovechar la opor- tunidad. Mari se encontró muy bien allí, no le faltó de nada, incluso pudo conocer cosas nuevas que en el pue- blo nunca podría haber encontrado: bicicletas, dulces, tebeos, etc. No era un trabajo tan duro como en el cam- po, pero exigía más tiempo del que Mari estaba acostumbrada. Al princi- pio le costó un poco la adaptación a la ciudad, ella sólo quería quedarse en casa, pasar el día conversando con Dª María o con alguna amiga, leer tebe- os, comer regaliz, etc. Los dos ámbitos en lo que Mari se encontraba tenían diferentes caracte- rísticas: por un lado estaba el pueblo donde se encontraba con su familia, con sus amigos de la niñez, con las ra- íces, con los juegos, con la compañía, etc.; mientras que en la ciudad dispo- nía de una segunda familia, la juven- tud, el desarraigo, la gente conocida, el trabajo, la soledad, etcétera. La relación con la gente de Madrid era excelente, contaba con su her- mana a un palmo de distancia y ella le ayudó a adaptarse más deprisa. El ca- riño mutuo que se tenían era y es constante, no sabrían vivir la una sin la otra. En los primeros meses, Isabel se preocupaba mucho por dar aco- modo a su hermana y, como ella ya llevaba mucho tiempo allí, le había da- do tiempo para hacer amistades, pre- ocupándose de presentárselas a su hermana. Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 5.3. Un nuevo hogar en Madrid La casa a la que llegó Mari estaba encima del hogar en que trabajaba su hermana. Se trataba de dos mujeres, Dª María, viuda desde hacía muchos años, y su hija que acababa de que- darse sola al fallecer a su marido en- fermo. Ambas habían estudiado en la Universidad y trabajaban fuera de ca- sa. Al estar solas necesitaban la ayu- da doméstica de una chica como Ma- ri. Al ser muy joven, las señoras la trataron como si fuera una hija más. Para Mari, aquellas personas signifi- caban todo. A pesar de ser sus amas, no miraron a Mari como a una niña que venía a trabajar para ellas, sino que les ... (hablando de los padres)... esta- ban conformes, lo primero porque cuando yo me vine, no me vine a la aventura, venías con una referencia, un poco para tranquilizarlos... Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 227 6. España, país de destino chaban para ir a ver a sus padres y a las amigas. Era un pretexto para vol- ver, pero también para mostrar lo bien que les iba en la ciudad, para afian- zarse moralmente en su elección de emigrar a Madrid y para mostrar el sta- tus adquirido a las demás gentes del pueblo. Por otra parte, Rabía nació en Lara- che, a pocos kilómetros al sur de Tán- ger (Marruecos) hace 31 años. Toda su vida la dedicó a ayudar a sus padres en el negocio familiar hasta que, en 1993, tuvo la posibilidad de cruzar el estrecho y buscarse una nueva vida en España. Por aquel entonces tenía 23 años y tar- dó poco tiempo en decidirse, puso en una balanza lo que tenía en Larache (la familia) y lo que podría tener en España (trabajo, dinero, su propia familia, etc.) y con el consentimiento de sus padres y ayudada por unos vecinos, que iban y venían de España frecuentemente, emigró hacia nuestro país. Hoy está ca- sada, tiene un niño y vive en un piso del barrio de La Latina en Madrid, en com- pañía de dos hermanos suyos que han venido también de Marruecos. ... locas por ir a las fiestas, por lle- var ropa bonita, por llevar cosas que a lo mejor en el pueblo no conocían... Entre tanto y tanto, un día cual- quiera, en el Retiro, intentando apren- der a montar en bicicleta conoció a un chico muy simpático llamado Antonio. Alternando el trabajo y el noviazgo buscaron un piso en las afueras de Madrid y encontraron uno en Leganés, una ciudad en auge poblacional. En poco tiempo se casaron y Mari dejó de vivir en la casa de Dª María. La vida transcurrió con normalidad, como ella dice “con etapas buenas y malas” y actualmente continúa vivien- do en Leganés, sus hijos son mayores y tienen la vida resuelta. Ella sigue tra- bajando y sintiendo algo más que nos- talgia por aquella infancia en el pue- blo. Instinto de mejora. Diferencias entre dos movimientos migratorios... El problema era que Mari te- nía un carácter distinto del de su her- mana, era más bien triste, su timidez, su tranquilidad y sencillez le hacía ser una chica muy formal y muy respeta- da en su círculo de amistades. Al pa- recer las características rurales toda- vía estaban presentes en Mari: el apego a la familia, la tradición, la reli- giosidad, etc. El tiempo pasaba con las tareas del hogar, pero también tenía momentos libres para divertirse. Era muy hogare- ña, se sentía bien en casa, con los su- yos, hablando y permitiéndose los ca- prichos que en el pueblo nunca tuvo. Pensando en el pueblo, tanto Mari co- mo Isabel, se compraban ropa, bolsos, zapatos, se cuidaban, iban a la pelu- quería, siempre pensando en volver en las fiestas del pueblo para ver a sus padres. La vuelta al pueblo era muy espe- rada por ambas. Significaba volver a las raíces, al lugar donde habían pa- sado su tierna infancia. Cada 24 de ju- nio se celebraban las fiestas de San Juan en el pueblo y ambas aprove- Por aquel entonces, Madrid estaba creciendo. La ciudad se llenaba de gente de todas partes y la zona del centro se convertía en escaparate de las vanguardias sociales para los re- Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 228 Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz Juan José Muñoz 6. España, país de destino El contraste se presenta cuando comparamos los espacios en los que vivió: de la subsistencia que ofrecía el pueblo, de la imposibilidad de movili- dad social que representaba un ámbi- to rural cerrado a la innovación a la po- sibilidad de ascensión en una ciudad grande, al trabajo remunerado base de todo el patrimonio que tiene hoy en día, al bienestar social y personal en un piso propio de Leganés represen- tante la lucha por sacar una familia adelante. Al igual que en el caso de Mari, la vida de Rabía se podría estudiar me- diante dos ejes: uno señalaría el paso de un ámbito subdesarrollado y de economía de subsistencia a otro más desarrollado y de economía de merca- do y hábitos de consumo; el otro eje señalaría el paso de una posición so- cial empobrecida y lejana del bienes- tar hacia una posición social más aco- modada y más libre de ataduras económicas. De este modo, podremos observar que la trayectoria vital de Ra- bía mejoró sustancialmente, no sólo por su coraje al intentar cruzar el Es- trecho, sino también por haber conse- guido un trabajo, mantenerlo, apren- der un idioma, obtener los papeles que legalizaban su estancia y lograr esta- bilidad emocional al lado de su familia: su marido, su hijo y sus dos hermanos. ... te metes en una rueda y la tienes ... te metes en una rueda y la tienes que seguir... que seguir... Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 229 Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz En relación al propio relato de Ra- bía, el texto se va estructurar en tres partes: una dedicada al viaje desde Larache a Madrid, haciendo referen- cia a las causas del movimiento y a las expectativas del viaje; otra dedicada a los primeros años de vida en la ca- pital y a su consolidación en una cul- tura diferente a la suya; y, por último, una tercera dedicada a la propia visión que tienen nuestra protagonista sobre el fenómeno de la inmigración. ta donde pasó por una segunda adua- na y la vigilancia de perros policía en busca de droga. Y, por último, ence- rrada durante dos horas en el trayecto en barco desde Ceuta hasta Algeciras. Allí, amigos marroquíes nacionalizados españoles del hombre que la trajo en el maletero la condujeron hacia la ca- pital. Una vez en Madrid, Rabía se di- rigió a la casa de sus vecinos de Ma- rruecos que la esperaban con un trabajo para empezar al día siguiente. Los primeros meses fueron muy di- fíciles para Rabía, se encontraba en un país diferente, no sabía nada de es- pañol y debía de ir todos los días a una casa a cuidar a un niño pequeño. Los problemas empezaron temprano, al primer mes de estancia en España, cuando cobró su primer sueldo, se compró ropa y sus vecinos, aquellos que la dieron una cama en su casa, la acusaron de robo en la casa donde trabajaba por envidia, según Rabía. El 6.1. La llegada Rabía llegó a España en octubre de 1993 con 23 años alojada en un male- tero de un coche. Lo pasó muy mal y fue una experiencia especialmente du- ra para ella que la marcó en toda su es- tancia en España. Allí encerrada estu- vo durante el viaje de Larache hasta Tetuán pasando por una primera adua- na. Después, desde Tetuán hasta Ceu- Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 230 Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz peles y en 1995 Rabía consiguió lega- lizar su estancia en España. Poco tiempo después, se casó con un chi- co marroquí que conoció en Madrid, trabajaba como portero en una casa y, al tener dinero ahorrado, ambos deci- dieron irse a vivir en un piso alquilado en el barrio madrileño de La Latina. Como ya no vivía en la casa de aque- lla señora, respetándola y queriéndo- la mucho, cambió de trabajo. Ahora debía cuidar a una niña desde las ocho de la mañana hasta las ocho de la tar- de. Las cosas se fueron arreglando ya que en esos dos años aprendió a uti- lizar con fluidez el castellano, pudo disfrutar de la oferta de ocio madrile- ña, incluso pudo viajar con su marido por España gracias a la compra de un coche de segunda mano. Por aquel entonces, con los papeles bajo el bra- zo hizo su primer viaje de regreso a Marruecos, porque a pesar de tener los papeles arreglados, el viaje fue una catástrofe; cuando llegó a la aduana en Marruecos la pidieron el visado de salida del país, pero como ella había entrado ilegalmente la primera vez tu- vo que pasar otra noche en la comi- saría de la aduana hasta que llegaron sus padres pagando su libertad. Para volver a España legalmente tuvo que ir al consulado español en Tánger y pedir visado para poder volver nor- malmente. incidente la llevó a comisaría. Allí pa- só toda la noche hasta que la señora del piso donde trabajaba desmintió to- do y pagó la fianza. La dejó en liber- tad bajo su tutela, la dio un contrato de trabajo y como Rabía no tenía pa- peles prometió a la policía que arre- glaría su situación. La señora contra- tó a un abogado y a los dos años ya tenía en regla sus papeles. 6.1. La llegada No volvió a casa de sus vecinos y se quedó du- rante esos dos años interna en la ca- sa de aquella señora. En aquellos primeros momentos, empezó a comprobar que lo que le con- taron de España no se asemejaba a la realidad. De las cosas que observó, lo más llamativo fue el choque cultural del idioma, la religión, el trato de los hom- bres hacia las mujeres y el trato de los hijos hacia los padres, muy diferentes a lo que conocía y practicaba en Ma- rruecos. Lo pasó muy mal, incluso has- ta tuvo miedo, ya que, aparte de no ha- blar castellano y no tener papeles, no tenía a sus padres ni a sus hermanos, no tenía a nadie, sólo a la señora y al niño que cuidaba. Aún pasándolo mal tenía la esperanza de tener una vida mejor que la que tenía en su país. ... porque en mi país no tenía ni tra- bajo ni nada, y por eso pensé venir aquí, para buscar una vida mejor que la mía, que la que tenía en mi casa y en mi país, pero siempre en esta vida te cuesta ... Pero lo que más echaba en falta en Madrid, además de su familia, era el tiempo. A veces no disponía de tiem- po para ella misma; se levantaba, iba a trabajar, volvía, preparaba la cena y a la cama para repetir el proceso al día siguiente. No podía aguantar así. Unos años más tarde, en 1998, llegó un in- 6.3. El fenómeno de la inmigración Este último aspecto que hemos presentado en la vida de Rabía, es de- cir, las ganas de regresar a Larache no sólo son porque allí están sus raíces, ni tampoco porque aquí lo haya pasado tan mal, sino por las diferencias cultu- rales y los hábitos de comportamien- tos tan diferentes entre lo conocido por Rabía en su estancia en Marrue- cos y en Madrid, además del senti- miento de rechazo y discriminación no manifiesto pero sí latente. Actualmente viven los cinco en un pisito pequeño intentando aportar los recursos suficientes para continuar vi- viendo en Madrid. Pero, hace poco, su marido se quedó sin trabajo. El edifi- cio en el que trabajaba fue comprado por otras personas y se quedó sin em- pleo. El niño tiene ya tres años y como su marido está buscando trabajo, Ra- bía se ha visto obligada a trabajar. Ahora se dedica a limpiar una casa en Fuencarral. Trabaja de ocho a seis y le gustaría pasar más tiempo con su ni- ño. Entre tanto, su marido y su her- mana, a la vez que buscan trabajo, se dedican a cuidar al pequeño mientras ella no está. ... siempre notas que eres dife- rente de la gente que te rodea, no es tu gente, no es tu religión, ni el idioma ni tu país..., con el tiempo noté que to- dos somos personas... Por ejemplo, al llegar a España le lla- mó especialmente la atención cómo se comportaban y cómo eran tratadas las mujeres aquí. Le parecía bien que des- de pequeñas, las niñas pudieran elegir qué hacer y cómo ser, lo que en Ma- rruecos no sucede. Por el contrario, un aspecto más negativo que llamó su atención fue la falta de respeto que tie- nen los jóvenes españoles con los pa- dres y mayores, lo que en Marruecos no sucede ya que un joven no hace na- da sin el consentimiento de los padres. Todo esto le da más razones a Ra- bía para pensar en su tierra y en su fa- milia. Piensa que cuando tenga dine- ro suficiente, cuando le toque la lotería volverá a Larache porque es su sitio, su lugar, sus raíces y porque no se vi- ve tan mal. 6.2. Su consolidación y la actualidad Rabía estuvo dos años en casa de aquella señora cuidando a su hijo. Con el tiempo, el abogado tramitó los pa- Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 231 Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz Juan José Muñoz de quiere vivir, si en España o en Ma- rruecos. ... siempre tengo pensado que al- gún día tengo que volver a mi país. ¡Al- gún día tengo que volver! No me voy a quedar toda mi vida aquí, aunque me va bien, pero siempre echas de menos tu tierra... vitado, en 1998 nació su primer hijo. Pero, otra vez lo pasó realmente mal, ya que tuvo que dejar de trabajar pa- ra cuidar a su pequeño y, a su vez, se sentía sola, a pesar de la compañía de su marido, echaba en falta a su pa- dres, sus hermanos y amigos. Para ayudarla y aprovechando la situación, llegaron dos hermanos suyos de La- rache. Uno tenía 29 años y encontró trabajo rápidamente en la construc- ción y otra de 20, la más pequeña de la familia, que se quedó en casa con ella, cuidando al niño. El tiempo solu- cionó los problemas. de quiere vivir, si en España o en Ma- rruecos. ... siempre tengo pensado que al- gún día tengo que volver a mi país. ¡Al- gún día tengo que volver! No me voy a quedar toda mi vida aquí, aunque me va bien, pero siempre echas de menos tu tierra... Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 6.3. El fenómeno de la inmigración Piensa tener su negocio propio, un trabajo y una casa, además quiere que su hijo conozca la cultura marroquí, después cuando sea más mayor le dará libertad para elegir don- A su vez, pudo comprobar el re- chazo y la discriminación, no sólo en Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 232 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz su incidente con los vecinos que le ha- bían acusado de robar en su trabajo, sino en otras muchas ocasiones cuan- do la gente se manifestaba en estos términos a la hora de coger el autobús o buscar trabajo. En las muchas oca- siones que iba en autobús, las seño- ras de alrededor agarraban el bolso y se apartaban de ella. A su vez, su ma- rido, cuando dejó de ser conserje y buscó trabajo, “con lo preparado que estaba, teniendo conocimientos y ex- periencia de mantenimiento, jardine- ría o electricidad, no ha conseguido en ninguna de las muchas entrevistas a las que ha ido ningún empleo”. sensación de novedad, de triunfo, de riqueza que llega a Marruecos se vuel- ve contra ellos aquí ya que son capa- ces de abandonar sus familias y su vi- da tranquila para venir a España, lugar en el cual no es oro todo lo que relu- ce, sobre todo si intentan llegar a Es- paña ilegalmente. Rabía nos contaba como muchos de sus amigos que han llegado ilegal- mente se arrepentían de su decisión, ya que en Marruecos tenían a su fa- milia y su trabajo digno y que lo deja- ron todo para mal vivir en España tra- bajando muchas horas al día. Incluso nos contaba que lo han intentado mu- chas veces por patera y que no lo han conseguido habiéndose gastado aho- rros de la familia para nada. Lo peor es que lo volverán a intentar. 6.3. El fenómeno de la inmigración vida que te pasa todo en ella, y pien- san, a lo mejor, que nada más llegar aquí van a trabajar, van a tenerlo todo, pues no es así, todo es difícil y todo te cuesta, todo... Aunque es verdad que ambos mo- vimientos están separados por cua- renta años y que uno sucede en Es- paña y en el otro media con fuerza un país diferente, no cabe duda de que el resultado lo es también, debido a que todavía la sociedad en la que vivimos tiene prejuicios acerca del origen y el modo de vestir de los que llegan a Es- paña. En definitiva, que el desarrollo de ambos procesos es idéntico, tiene el mismo esquema, pero el final reve- la cómo las barreras culturales entre unos y otros hacen que fracase la in- tegración actual de las personas que llegan a España procedentes de otros países. ... conozco a un profesor, que es de Marruecos, ha dejado a su mujer, sus dos hijos y vino hacia aquí, y nada, tra- baja en la construcción, trabaja en lo que sea, en coger butanos y cosas así, y se arrepintió muchísimo por dejar su trabajo allí... 6.3. El fenómeno de la inmigración Pero es al- go lógico, todos los seres humanos in- tentan mejorar su situación siempre que tengan expectativas de cumplir- las y la propia historia reciente dice que no es muy difícil llegar a España, otra cosa es que una vez en llegados salgan las cosas bien para seguir me- jorando, sobre todo cuando aquí, por ejemplo, el sueldo que cobraba Rabía cuidando al niño de la señora que la ayudó cuando la acusaron de robo, era el salario de un profesor de escuela de Larache. Ante estos datos, ¿qué ma- rroquí no quiere llegar a España? Otro de los aspectos que no en- tiende de la vida de un inmigrante en España tiene que ver con la propia re- lación entre estos colectivos. En este período de tiempo en que Rabía ha es- tado viviendo en España ha podido comprobar cómo la envidia y el rencor entre los propios inmigrantes crece a un ritmo arrollador. Como ya hemos comentado, Rabía contaba que fueron sus propios vecinos de Marruecos los que la acusaron de robar simplemen- te por haberse comprado ropa y exhi- birla ante ellos. También contaba có- mo sus cuñadas, también marroquíes, no le ayudaban cuando tuvo el niño y cómo llegaron a no hablar ni a preo- cuparse de su hermano cuando em- pezó a vivir con ella. Por lo tanto, nos encontramos an- te dos trayectorias: la del inmigrante que está en España y desea volver por diferentes motivos; y la del extranjero que quiere llegar a España invirtiendo todo su dinero y arriesgando su pro- pia vida. Todo esto al final se vuelve contra los propios marroquíes, ya que cuan- do Rabía y su marido volvían a Lara- che de vacaciones, la gente del lugar se alegraba de verlos tan bien, con dinero, con un coche, con piso, con ropas nuevas, con muchos objetos que en Marruecos nunca verían. Esta ... además ellos creen que aquí es una vida fácil, pero no es así, es una Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 233 Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Instinto de mejora. Diferencias entre dos movimientos migratorios... Juan José Muñoz Juan José Muñoz cruzaron el Estrecho para llegar a Es- paña tienen serias dificultades para en- contrar trabajo, ganar dinero, conocer la otra cultura, adaptarse o integrarse. 7. Puntos en común. Conclusiones La comparación entre ambos mo- vimientos se antoja sencilla y fácil de explicar. Se trata de dos procesos si- milares, con el mismo origen y similar desarrollo, pero desigual consolida- ción y resultado. Si ambos nacen ba- jo las dificultades de la precariedad económica y los hábitos de un espa- cio subdesarrollado, basado en la au- tosuficiencia, ambos también logran salir de este ámbito gracias a la emi- gración y a la búsqueda de mejores condiciones de vida en otro lugar. Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 Bibliografía Del Campo, S. y Navarro, M. (1987), Nuevo aná- lisis de la población española, Madrid, Ariel. Haddon, A. C. (1912), The wondering of peoples. Camdbridge. Kasdan, L. (1970), “Introduction” in Migration and Anthropology. Proceedings of the 1970 Anual Spring Meeting of the American Etnological Society, Washington. Ambos difieren en una cuestión muy importante: el resultado de la mi- gración. Si todas aquellas personas, como Mari, que dejaron su pueblo pa- ra llegar a una gran ciudad tuvieron fa- cilidades para trabajar, adaptarse, co- nocer gente y ganar dinero suficiente para tener una vida digna y autosufi- ciente, otras personas como Rabía que Mangalam, J. J. (1968), Human migration: a gui- de to migration literature in enghish (1955- 1962), Lexington. Park, R. E. (1950), “Human migrations an the mar- ginal man” in Race and Culture, Gleonce. Pérez Díaz, V. (1974), Pueblos y clases sociales en el campo español, Madrid, Alianza. Ravenstein, E. G. (1885), “The laws of migration” in Journal of Royal Stat. Society. Tamames, R. (1983), Estructura económica de Es- paña, vol. 1, Madrid, Alianza Universidad. Cuadernos de Trabajo Social Vol. 15 (2002): 217-234 234
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D5.5 CROSS-SECTOR POLICY LIFECYCLE MANAGEMENT: SOFTWARE PROTOTYPE 2
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CLOUD FOR DATA-DRIVEN POLICY MANAGEMENT Project Number: 870675 Start Date of Project: 01/01/2020 Duration: 36 months D5.5 CROSS-SECTOR POLICY LIFECYCLE MANAGEMENT: SOFTWARE PROTOTYPE 2 Dissemination Level PU Due Date of Deliverable 31/10/2021, Month 22 Actual Submission Date 02/11/2021 Work Package WP5 Cross-sector Policy Lifecycle Management Task T5.2, T5.3, T5.4, T5.5 & T5.6 Type Demonstrator Approval Status Version V0.7 Number of Pages p.1 – p.42 Abstract: This document provides a description of the software components of the Policy Management Framework Layer, and Policy Development Toolkit Layer. The information in this document reflects only the author’s views and the European Community is not liable for any use that may be made of the information contained therein. The information in this document is provided “as is” without guarantee or warranty of any kind, express or implied, including but not limited to the fitness of the information for a particular purpose. The user thereof uses the information at his/ her sole risk and liability. This deliverable is licensed under a Creative Commons Attribution 4.0 International License. CLOUD FOR DATA-DRIVEN POLICY MANAGEMENT Project Number: 870675 Start Date of Project: 01/01/2020 Duration: 36 months D5.5 CROSS-SECTOR POLICY LIFECYCLE MANAGEMENT: SOFTWARE PROTOTYPE 2 Dissemination Level PU Due Date of Deliverable 31/10/2021, Month 22 Actual Submission Date 02/11/2021 Work Package WP5 Cross-sector Policy Lifecycle Management Task T5.2, T5.3, T5.4, T5.5 & T5.6 Type Demonstrator Approval Status Version V0.7 Number of Pages p.1 – p.42 Abstract: This document provides a description of the software components of the Policy Management Framework Layer, and Policy Development Toolkit Layer. The information in this document reflects only the author’s views and the European Community is not liable for any use that may be made of the information contained therein. The information in this document is provided “as is” without guarantee or warranty of any kind, express or implied, including but not limited to the fitness of the information for a particular purpose. The user thereof uses the information at his/ her sole risk and liability. This deliverable is licensed under a Creative Commons Attribution 4.0 International License. MANAGEMENT: SOFTWARE PROTOTYPE 2 Dissemination Level PU Due Date of Deliverable 31/10/2021, Month 22 Actual Submission Date 02/11/2021 Work Package WP5 Cross-sector Policy Lifecycle Management Task T5.2, T5.3, T5.4, T5.5 & T5.6 Type Demonstrator Approval Status Version V0.7 Number of Pages p.1 – p.42 Abstract: This document provides a description of the software components of the Policy Management Framework Layer, and Policy Development Toolkit Layer. The information in this document reflects only the author’s views and the European Community is not liable for any use that may be made of the information contained therein. The information in this document is provided “as is” without guarantee or warranty of any kind, express or implied, including but not limited to the fitness of the information for a particular purpose. The user thereof uses the information at his/ her sole risk and liability. This deliverable is licensed under a Creative Commons Attribution 4.0 International License. Abstract: This document provides a description of the software components of the Policy Management Framework Layer, and Policy Development Toolkit Layer. The information in this document reflects only the author’s views and the European Community is not liable for any use that may be made of the information contained therein. The information in this document is provided “as is” without guarantee or warranty of any kind, express or implied, including but not limited to the fitness of the information for a particular purpose. The user thereof uses the information at his/ her sole risk and liability. This deliverable is licensed under a Creative Commons Attribution 4.0 International License. he information in this document reflects only the author’s views and the European Community is not liable for any use that may be f the information contained therein. The information in this document is provided “as is” without guarantee or warranty of an xpress or implied, including but not limited to the fitness of the information for a particular purpose. The user thereof uses the infor t his/ her sole risk and liability. This deliverable is licensed under a Creative Commons Attribution 4.0 International License. The information in this document reflects only the author’s views and the European Community is not liable for any use that may be made of the information contained therein. MANAGEMENT: SOFTWARE PROTOTYPE 2 The information in this document is provided “as is” without guarantee or warranty of any kind, express or implied, including but not limited to the fitness of the information for a particular purpose. The user thereof uses the information at his/ her sole risk and liability. This deliverable is licensed under a Creative Commons Attribution 4.0 International License. PolicyCloud has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 870675. D5.5 – v1.0 Versioning and Contribution History Version Date Reason Author 0.1 20/09/2021 Updated ToC and assignments Armend Duzha 0.2 27/09/2021 Contributions from ICCS Kostas Moutselos 0.3 04/10/2021 Contributions from LXS Luis Miguel Garcia Jesús Manuel Gallego 0.4 11/10/2021 Contributions from ATOS Ana Luiza Pontual Miquel Mila Prat 0.5 26/10/2021 Consolidated draft ready for internal review Armend Duzha 0.6 27/10/2021 Internal Review Giannis Ledakis (UBI) / Yosef Moatti (IBM) 0.7 28/10/2021 Addressing review comments Armend Duzha 0.8 02/11/2021 Quality Check Argyro Mavrogiorgou 1.0 02/11/2021 Final version ready for submission Submitted version Armend Duzha Author List Author List Author List Organisation Name MAG Armend Duzha UPRC Ilias Maglogiannis ICCS Kostas Moutselos LXS Luis Miguel Garcia Jesús Manuel Gallego OKS Chris Maragkos Stelios Mpetas ATOS Ana Luiza Pontual Miquel Mila Prat 2 www.policycloud.eu 2 D5.5 – v1.0 www.policycloud.eu Abbreviations and Acronyms Abbreviation/Acronym Definition API Application Programming Interface AT Analytical Tool DSS Decision Support System EC European Commission KPI Key Performance Indicator PDT Policy Development Toolkit PM Policy Model PME Policy Modelling Editor PP Public Policy SOA Service Oriented Architecture UI User Interface UX User eXperience UML Unified Modelling Language WP Work Package www.policycloud.eu www.policycloud.eu 3 D5.5 – v1.0 Contents www.policycloud.eu 4 Contents Versioning and Contribution History .................................................................................................................... 2 Author List ................................................................................................................................................................ 2 Abbreviations and Acronyms ................................................................................................................................ 3 Executive Summary ................................................................................................................................................ 7 1 Introduction ...................................................................................................................................................... 8 1.1 Purpose and Scope ................................................................................................................................ 8 1.2 Summary of changes ............................................................................................................................. 8 1.3 Structure of the Deliverable .................................................................................................................. 8 2 Prototype overview ......................................................................................................................................... 9 2.1 Main components of the prototype ................................................................................................... 10 2.1.1 Policy Modelling Editor .................................................................................................................... 10 2.1.2 Policy Development Toolkit ............................................................................................................ 10 2.1.3 Data Visualization ............................................................................................................................. 11 2.1.4 PDT Backend ..................................................................................................................................... 14 2.2 Interfaces ............................................................................................................................................... 16 2.2.1 Policy Modelling Editor .................................................................................................................... 16 2.2.2 Policy Development Toolkit ............................................................................................................ 20 2.2.3 Data Visualization ............................................................................................................................. 28 2.2.4 PDT Backend ..................................................................................................................................... 30 2.3 Baseline technologies and tools ......................................................................................................... 37 2.3.1 Policy Modelling Editor .................................................................................................................... 37 2.3.2 Policy Development Toolkit ............................................................................................................ 37 2.3.3 Data Visualization ............................................................................................................................. 37 2.3.4 PDT Backend ..................................................................................................................................... 37 3 Source Code ................................................................................................................................................... 38 3.1 Availability.............................................................................................................................................. 38 3.1.1 Policy Modelling Editor .................................................................................................................... 38 3.1.2 Policy Development Toolkit ............................................................................................................ 38 3.1.3 Data Visualization ............................................................................................................................. 38 3.1.4 PDT Backend ..................................................................................................................................... 38 D5.5 – v1.0 D5.5 – v1.0 3.2 Deployment ........................................................................................................................................... 39 3.2.1 Policy Modelling Editor .................................................................................................................... 39 3.2.2 Policy Development Toolkit ............................................................................................................ 39 3.2.3 Data Visualization ............................................................................................................................. 39 3.2.4 PDT Backend ..................................................................................................................................... 39 4 Conclusions .................................................................................................................................................... 41 References ............................................................................................................................................................. 42 3.2 Deployment ........................................................................................................................................... 39 3.2.1 Policy Modelling Editor .................................................................................................................... 39 3.2.2 Policy Development Toolkit ............................................................................................................ 39 3.2.3 Data Visualization ............................................................................................................................. 39 3.2.4 PDT Backend ..................................................................................................................................... 39 4 Conclusions .................................................................................................................................................... 41 References ............................................................................................................................................................. 42 List of Figures Figure 1 - PolicyCloud System Architecture ......................................................................................................... 9 Figure 2 - Example of the Heatmap for Maggioli use case (Event Locator) ................................................... 11 Figure 3 – Example of Heatmap for Maggioli use Case (“Hot” zones) ............................................................ 11 Figure 4 - Example of the line and gauge charts for the sarga use case ....................................................... 12 Figure 5 - Example of the Heatmap for The SOFIA use case ........................................................................... 12 Figure 6 - Example of the BAR CHART for The LONDON use case ................................................................. 13 Figure 7 - Example of BAR CHART for The SOFIA use case .............................................................................. 13 Figure 8 - Example of another BAR CHART for The SOFIA use case ............................................................... 14 Figure 9 - Polciy Model Editor Home Page ......................................................................................................... 16 Figure 10 - Existing KPIs selection ....................................................................................................................... 17 Figure 11 - Add New KPI ....................................................................................................................................... 17 Figure 12 - Add New Actor ................................................................................................................................... 18 Figure 13 - Analytical Tool selection ................................................................................................................... 18 Figure 14 - Set parameter value .......................................................................................................................... 19 Figure 15 - Review and Submit ............................................................................................................................ 19 Figure 16 - Policy Model Submitted .................................................................................................................... 20 Figure 17 – Policy selection-evaluation wizard .................................................................................................. 20 Figure 18 - Policy selection and policy properties ............................................................................................ 21 Figure 19 - KPI selecion......................................................................................................................................... 22 Figure 20 – KPI properties .................................................................................................................................... 22 Figure 21 – Evaluation – Analytics tool selection .............................................................................................. 23 Figure 22 – Analytics tool technical details ........................................................................................................ 23 Figure 23 – Analytics tool parameters & submission ...................................................................................... 24 Figure 24 – Analytics tool parameters details ................................................................................................... 25 Figure 25 – Policy overview / saving ................................................................................................................... 25 Figure 26 – PDT components menu ................................................................................................................... 26 Figure 27 - ONLINE help ....................................................................................................................................... 26 5 5 D5.5 – v1.0 Figure 28 - User Data protection information notice ....................................................................................... 27 Figure 29 - User data protection text ................................................................................................................. 27 Figure 30 - User profile properties / logout ....................................................................................................... 28 Figure 31 - Example for heat map chart data .................................................................................................... 29 Figure 32 – Example for Sarga Gauge Chart Data ............................................................................................ 29 Figure 33 – Example for London Bar Chart data............................................................................................... 29 Figure 34 – Example for One Bar Data for Sofia ............................................................................................... 30 Figure 35 - Web Methods related with Actors ................................................................................................... 32 Figure 36 - WEB METHODS RELATED WITH Analytical Tools ........................................................................... 33 Figure 37 - WEB METHODS RELATED WITH Data Models ................................................................................ 33 Figure 38 - WEB METHODS RELATED WITH Domains ...................................................................................... 33 Figure 39 - WEB METHODS RELATED WITH Goals ............................................................................................ www.policycloud.eu List of Figures 34 Figure 40 - WEB METHODS RELATED WITH Job Status..................................................................................... 34 Figure 41 - WEB METHODS RELATED WITH JOBs .............................................................................................. 35 Figure 42 - WEB METHODS RELATED WITH KPIS .............................................................................................. 35 Figure 43 - WEB METHODS RELATED WITH Policies ......................................................................................... 36 www.policycloud.eu www.policycloud.eu 6 6 D5.5 – v1.0 www.policycloud.eu 1.1 Purpose and Scope This document is the second iteration of “Cross-Sector Policy Lifecycle Management: Software prototype”, and covers tasks T5.2, T5.3, T5.4, T5.5 and T5.6. Its main purpose is to provide a description of the prototype implementation of the Policy Modelling Editor (PME) and the Policy Development Toolkit (PDT) to support policy makers in the modelling, creation, update, and evaluation of the policies. This document is consistent with deliverable D5.4 “Cross-Sector Policy Lifecycle Management: Design and Open Specification 2” [1], delivered in August 2021, which provided the overall architecture and design specifications of the PME and PDT. This report will be further updated during the last phase of the project duration (at month M34, October 2022) and will include further advancements and contributions from other tasks. Executive Summary This document is an incremental update of deliverable D5.3 Cross-sector Policy Lifecycle Management: Software Prototype 1, delivered in November 2020, and describes in detail the software components of the Policy Management Framework Layer and Policy Development Toolkit Layer, which are the frontend parts of the PolicyCLOUD platform and allow policy makers to create, update and evaluate policies. For each component, a description of its APIs, specification of the main functionalities, and description of the source code are provided. 7 www.policycloud.eu www.policycloud.eu 7 D5.5 – v1.0 1.2 Summary of changes The main changes in this deliverable regard the update of Section 2.1 and 2.2. In this second version of the demonstrator, all components have been integrated and are running on the testbed infrastructure. Furthermore, integration work has been performed for the Maggioli and Sarga use cases, which are now integrated with the Policy Development Toolkit layer. In addition, we have updated and revised the whole document (including figures) to reflect the current work done at M22 (October 2021). 1.3 Structure of the Deliverable 1.3 Structure of the Deliverable The rest of the document is structured as follows: The rest of the document is structured as follows: • Section 2 introduces the main components and sub-systems, their interfaces and technologies used for their development. • Section 2 introduces the main components and sub-systems, their interfaces and technologies used for their development. • Section 3 describes where the source code is made available. • Finally, Section 4 provides the conclusion of the document. 8 8 www.policycloud.eu D5.5 – v1.0 2 Prototype overview In this section we provide the description of the prototype implementation of the Policy Modelling Framework Layer and Policy Development Toolkit Layer, as illustrated in Figure 1. FIGURE 1 - POLICYCLOUD SYSTEM ARCHITECTURE FIGURE 1 - POLICYCLOUD SYSTEM ARCHITECTURE 9 www.policycloud.eu www.policycloud.eu 9 D5.5 – v1.0 2.1.1 Policy Modelling Editor The Policy Model Editor (PME) is the core component that supports and guides the end-user to effectively create a Policy Model safely. More specifically, the PME is a Single Page Application that relies on the PDT backend REST API to fetch or store related entities of the Policy Model. 2.1 Main components of the prototype 2.1.1 Policy Modelling Editor 2.1.2 Policy Development Toolkit As described in the deliverable D2.2 “Conceptual Model and Reference Architecture”[2], PDT interacts with components from Layer 3 - Policies Management Framework (T5.1, T5.2), Analytical Tools (WP4) and the PDT Backend. PDT provides the integrated prototype of the following components as described in the deliverable D5.4 “Cross-sector Policy Lifecycle Management: Design and Open Specification 2” [1]: • Policy Model Viewer • Policy Evaluations / Analytics Integration with the Backend has been implemented, utilising Docker images. This way, PDT can retrieve default policy models in the structured JSON format which represent the Policy Models (PMs). So, the functionalities included in this prototype are: Integration with the Backend has been implemented, utilising Docker images. This way, PDT can retrieve default policy models in the structured JSON format which represent the Policy Models (PMs). So, the functionalities included in this prototype are: • The policy maker can obtain a list of available policies and select one for further ex • Once a policy is selected, the properties of the policy are shown, along with the available the policy, • Once a KPI is selected, the relative KPI properties are also shown to the user, • The Analytical Tools (ATs) which can compute the selected KPI are presented. The policy maker can see the parameters related with each AT and invoke the respective AT. Finally, the policy maker can see a short overview of the current selected PMs. The aforementioned functionalities are implemented as a policy wizard component, comprised by three steps. The relative UI components are described in Section 2.2.2. The relative UI components are described in Section 2.2.2. www.policycloud.eu 10 D5.5 – v1.0 2.1.3 Data Visualization The Data Visualization component has been successfully integrated with the PDT. This second prototype includes all graphs defined in the first prototype and now can be seen through the PDT. They are: • A Heatmap as requested by the “Participatory Policies Against Radicalization” Use Case of Maggioli. This chart uses data extracted from the Data Analytics of WP4. • A Heatmap as requested by the “Participatory Policies Against Radicalization” Use Case of l h h d d f h l f FIGURE 2 - EXAMPLE OF THE HEATMAP FOR MAGGIOLI USE CASE (EVENT LOCATOR) FIGURE 2 - EXAMPLE OF THE HEATMAP FOR MAGGIOLI USE CASE (EVENT LOCATOR) This Heatmap is intended to show, at a glance, all incidents that occurred in a chosen region. Currently, there are two possible regions: “North America” and “Eastern Europe”; but the list will be enlarged in final version. Whenever a region is selected, the heatmap shows all incidents that took place in it. It can be filtered by location and by year, and it is possible to see the incident details, when clicking on it. Users can change regions as desired. The heat map can also show “hot” zones (zones with a bigger number of incidents) using colours, instead of numbers: FIGURE 3 – EXAMPLE OF HEATMAP FOR MAGGIOLI USE CASE (“HOT” ZONES) FIGURE 3 – EXAMPLE OF HEATMAP FOR MAGGIOLI USE CASE (“HOT” ZONES) www.policycloud.eu 11 D5.5 – v1.0 • A Line and Gauge charts as requested by the “Intelligent Policies for Denomination of Origin” Use Case of Sarga, that plots the evaluation of the Sentimental Analysis considering some Keywords. • A Line and Gauge charts as requested by the “Intelligent Policies for Denomination of Origin” Use Case of Sarga, that plots the evaluation of the Sentimental Analysis considering some Keywords. • A Line and Gauge charts as requested by the Intelligent Policies for Denomination of Origin Use Case of Sarga, that plots the evaluation of the Sentimental Analysis considering some Keywords. FIGURE 4 - EXAMPLE OF THE LINE AND GAUGE CHARTS FOR THE SARGA USE CASE FIGURE 4 - EXAMPLE OF THE LINE AND GAUGE CHARTS FOR THE SARGA USE CASE Considering a set of keywords, the Sentiment Analysis Line chart shows the sentiments for these keywords during a given period. The Accumulated Sentiment Gauge chart shows the average of these sentiments, during the same period. 2.1.3 Data Visualization In order to achieve the internal milestone of having at least one chart by Use Case scenario, the visualization component has been incremented and has integrated other charts. They are: In order to achieve the internal milestone of having at least one chart by Use Case scenario, the visualization component has been incremented and has integrated other charts. They are: • A heatmap, requested by the “Facilitating urban policy making and monitoring through crowdsourcing data analysis” Use Case of Sofia. In this case, an adaptation of previous Heatmap has been done, following Sofia Use Case’s requirements. In this way, the adapted Heatmap is focused on Sofia city and can filter by Sofia’s districts, by year, and, also, differently from Maggioli Use Case, by months and days. FIGURE 5 - EXAMPLE OF THE HEATMAP FOR THE SOFIA USE CASE FIGURE 5 - EXAMPLE OF THE HEATMAP FOR THE SOFIA USE CASE www.policycloud.eu 12 D5.5 – v1.0 • A Bar Chart, required both for Sofia and London Use Cases. In the case of London, it is intended to show unemployment data by gender: • A Bar Chart, required both for Sofia and London Use Cases. In the case of London, it is intended to show unemployment data by gender: • A Bar Chart, required both for Sofia and London Use Cases. In the case of London, it is intended to show unemployment data by gender: FIGURE 6 - EXAMPLE OF THE BAR CHART FOR THE LONDON USE CASE FIGURE 6 - EXAMPLE OF THE BAR CHART FOR THE LONDON USE CASE FIGURE 6 - EXAMPLE OF THE BAR CHART FOR THE LONDON USE CASE And for Sofia, the chart represents the number of incidents per month and per district (the 24 districts of Sofia): FIGURE 7 - EXAMPLE OF BAR CHART FOR THE SOFIA USE CASE FIGURE 7 - EXAMPLE OF BAR CHART FOR THE SOFIA USE CASE FIGURE 7 - EXAMPLE OF BAR CHART FOR THE SOFIA USE CASE Both are the same Bar charts, with different data, but that can be seen in two different formats: cluster and stacked. Both are the same Bar charts, with different data, but that can be seen in two different formats: cluster and stacked. 2.1.3 Data Visualization 13 www.policycloud.eu D5.5 – v1.0 Also, for Sofia Use Case, there’s another Bar Chart, where the data that can be visualized is the number of incidents by subcategories per districts: D5.5 – v1.0 Also, for Sofia Use Case, there’s another Bar Chart, where the data that can be visualized is the number of incidents by subcategories per districts: Also, for Sofia Use Case, there’s another Bar Chart, where the data that can be visualized is the number of incidents by subcategories per districts: FIGURE 8 - EXAMPLE OF ANOTHER BAR CHART FOR THE SOFIA USE CASE FIGURE 8 - EXAMPLE OF ANOTHER BAR CHART FOR THE SOFIA USE CASE 2.1.4 PDT Backend Those tools can be retrieved either by name, by the domain they are related to or according to the datasets that they are compatible with www.policycloud.eu 14 D5.5 – v1.0 • Retrieve information about the available datasets, stored in the central data repository of PolicyCLOUD and managed by the Data Acquisition and Analytic layer • Add, remove, modify and retrieve domains that can be relevant to the entities of the policies • Add, remove, modify and retrieve goals of a KPI, and associate those tools with stakeholders • Check the status of a job, which has been submitted as the result of the invocation of an analytical tool • Get information about a job, submitted by the invocation of an analytical tool • Get the result of an analysis, related to its job • Add, remove, modify and retrieve KPIs, based on their name, their corresponding domain or their relevant actors • Add or remove actors, goals, domains and data models from a KPI • Add, remove, modify and retrieve policies, according to their names, the user that stored the policy or their corresponding domain they belong to. • Add or remove KPIs from policies • Store combined types of results of analytical tools, thus giving the opportunity to the end-user to switch between different views of the same results • Correlate policies with the end-users that have defined them • Retrieve policies depending on the user that has issued the request • Correlate jobs with the users that initially submitted a request to execute an analysis • Retrieve the results of the jobs owned by a given end-users • Cancel the execution of an analysis if it exceeds the defined maximum response time allowed • Provides a warning when the result of an analysis exceeds the maximum number of characters that can be efficiently processed by the visualization tools at a later phase Regarding the interaction with the Data Acquisition and Analytic layer, the corresponding APIs have been defined in deliverable D4.4. The Backend of the PDT is integrated with the latter, in order to implement the higher-level functionality, as described above. The backend of the PDT consists of a single process that runs on a servlet container. It relies on a relational datastore to store the relational model of the policy, its attributes, related entities, and relations between them. 2.1.4 PDT Backend The PDT backend provides support to the whole PDT, and can be considered as the layer between the user interfaces that the policy maker can use to have access the tool, the policy modelling editor, and the Data Acquisition and Analytics layer of the PolicyCLOUD platform, provided by the components of WP4 and WP3. It exposes its functionality via REST APIs and web sockets, to allow for the integration with the aforementioned components, while it also makes use of the REST API provided by the Data Acquisition and Analytics layer. As a result, it serves four main purposes: • Allows storing, modifying and retrieving policies, along with their relevant corresponding entities (i.e., KPIs, stakeholders, etc.) (i.e., KPIs, stakeholders, etc.) • Hold policy meta-information that can be used by the policy modelling editor, in order for the latter to propose new policies to the domain experts • Invoke an analytical tool to perform an analysis through the Data Acquisition and Analytics layer, given the input parameters defined by the KPIs of the involved policy • Allow for the retrieval of the results of the analytical tools, which can be later fed to the visualization component of the tool. This involves again the integration of the backend with the Data Acquisition and Analytics layer More precisely, regarding the functionality implemented at this phase of the project for the PDT and the Policy Model Editor, the backend provides the ability to: More precisely, regarding the functionality implemented at this phase of the project for the PDT and the Policy Model Editor, the backend provides the ability to: • Store, and retrieve actors, based on their name or the domain they are related to • Retrieve information about the Analytical Tools that are available in the Data Acquisition and Analytic layer. 2.1.4 PDT Backend It should be highlighted that the datastore which is being used by this component is not the central data repository of PolicyCLOUD, as it is used for storing the raw data coming from the data providers, along with the results of the analytical tools. The purpose of the data storage element of this component is to store the policies and the metadata information that are related with those, along with the results of the analytical tools, while at the same time it aims at providing a standard way for communicating with the backend. www.policycloud.eu 15 D5.5 – v1.0 2.2.1 Policy Modelling Editor Upon the end-user’s entrance into the PME, the end-user has to both indicate the domain of its Policy Model and to provide a short description. Furthermore, the end-user has to provide to the PME the existing KPIs that might fit in the Policy Model under consideration. Figure 9 illustrates the Home Page interface of the PME. It fetches from the PDT backend KPIs that belong in the same domain with the Policy Model; and then prompted by the component to continue in the next page to select existing KPIs. FIGURE 9 - POLCIY MODEL EDITOR HOME PAGE Figure 10 shows the Existing KPIs selection interface, in which the end-user has the capability to add or 2.2 Interfaces 2.2.1 Policy Modelling Editor 2.2.1 Policy Modelling Editor FIGURE 9 - POLCIY MODEL EDITOR HOME PAGE Figure 10 shows the Existing KPIs selection interface, in which the end-user has the capability to add or to select existing Actors that are included in the Policy Model domain. www.policycloud.eu 16 www.policycloud.eu D5.5 – v1.0 D5.5 – v1.0 D5.5 – v1.0 FIGURE 10 - EXISTING KPIS SELECTION FIGURE 10 - EXISTING KPIS SELECTION In the Add new KPI interface (see Figure 11) the end-user has the capability to create a new KPI; while he/she can enter the relevant info of the KPI, describe its Goals, and integrate the additional Stakeholders. FIGURE 11 - ADD NEW KPI FIGURE 11 - ADD NEW KPI It should be noted that the user has the following capabilities: (i) to provide description of the Data Model and add or select Actors, (ii) to select the relevant Analytical Tools from a specific list (drop-down menu) (Figure 12), (iii) to provide the parameter values that comes for the Analytical Tools (Figure 13), (iv) to review and submit the Policy Model (Figure 14). FIGURE 11 - ADD NEW KPI It should be noted that the user has the following capabilities: (i) to provide description of the Data Model and add or select Actors, (ii) to select the relevant Analytical Tools from a specific list (drop-down menu) (Figure 12), (iii) to provide the parameter values that comes for the Analytical Tools (Figure 13), (iv) to review and submit the Policy Model (Figure 14). It should be noted that the user has the following capabilities: (i) to provide description of the Data Model and add or select Actors, (ii) to select the relevant Analytical Tools from a specific list (drop-down menu) (Figure 12), (iii) to provide the parameter values that comes for the Analytical Tools (Figure 13), (iv) to review and submit the Policy Model (Figure 14). 17 www.policycloud.eu www.policycloud.eu D5.5 – v1.0 FIGURE 12 - ADD NEW ACTOR FIGURE 13 - ANALYTICAL TOOL SELECTION FIGURE 12 - ADD NEW ACTOR FIGURE 13 - ANALYTICAL TOOL SELECTION FIGURE 13 - ANALYTICAL TOOL SELECTION www.policycloud.eu 18 D5.5 – v1.0 FIGURE 14 - SET PARAMETER VALUE Finally, if the process was successful the user is redirected to the last page that promotes the user to visit the PDT and evaluate the submitted Policy Model or compose a new one. FIGURE 15 - REVIEW AND SUBMIT FIGURE 14 - SET PARAMETER VALUE FIGURE 14 - SET PARAMETER VALUE FIGURE 14 - SET PARAMETER VALUE Finally, if the process was successful the user is redirected to the last page that promotes the user to visit the PDT and evaluate the submitted Policy Model or compose a new one. www policycloud eu 19 FIGURE 15 - REVIEW AND SUBMIT www.policycloud.eu 19 D5.5 – v1.0 2.2.2 Policy Development Toolkit 2.2.2 Policy Development Toolkit The following figures present the UI implementations of the PDT functionalities, as listed in Section 2.1.2. The web pages are served locally communicating with the dockerised backend API. Figure 17 shows the landing web page of PDT (after the pending Authentication procedure). The page shows the three-step policy wizard, with the first step as the default location. Here the user selects a policy from a list of available policy models. FIGURE 17 – POLICY SELECTION-EVALUATION WIZARD www.policycloud.eu 20 D5.5 – v1.0 Figure 18 shows the properties of the policy model selected by the user. After the selection of a policy, the related KPIs are displayed, and the PM can select one KPI for evaluation. Figure 18 shows the properties of the policy model selected by the user. After the selection of a policy, the related KPIs are displayed, and the PM can select one KPI for evaluation. FIGURE 18 - POLICY SELECTION AND POLICY PROPERTIES FIGURE 18 - POLICY SELECTION AND POLICY PROPERTIES Figure 18 displays the step for the KPI selection and Figure 19 shows the properties of the selected KPI, e.g., formula, data, domain, goal, actors and stakeholders. 21 www.policycloud.eu www.policycloud.eu D5.5 – v1.0 FIGURE 19 - KPI SELECION FIGURE 20 – KPI PROPERTIES D5.5 – v1.0 FIGURE 19 - KPI SELECION D5.5 – v1.0 D5.5 – v1.0 FIGURE 19 - KPI SELECION 22 www.policycloud.eu www.policycloud.eu D5.5 – v1.0 Figure 21 shows the third step of the wizard, which is the evaluation of the selected KPI, by using the proper Analytics Tool. Figure 21 shows the third step of the wizard, which is the evaluation of the selected KPI, by using the proper Analytics Tool. FIGURE 21 – EVALUATION – ANALYTICS TOOL SELECTION FIGURE 21 – EVALUATION – ANALYTICS TOOL SELECTION FIGURE 21 – EVALUATION – ANALYTICS TOOL SELECTION Figure 22 shows technical details of the Analytics Tool in an expandable card, as to not distract the PM. Figure 22 shows technical details of the Analytics Tool in an expandable card, as to not dis FIGURE 22 – ANALYTICS TOOL TECHNICAL DETAILS FIGURE 22 – ANALYTICS TOOL TECHNICAL DETAILS 23 www.policycloud.eu D5.5 – v1.0 Figure 23 shows the parameter list that the selected Analytics Tool is expecting for invocation. A parameter’s values overview is also presented, which includes the default values for the calculation of the KPI. . FIGURE 23 – ANALYTICS TOOL PARAMETERS & SUBMISSION FIGURE 23 – ANALYTICS TOOL PARAMETERS & SUBMISSION 24 www.policycloud.eu www.policycloud.eu D5.5 – v1.0 Figure 24 shows the details for each parameter of the Analytical tool, also in the form of an expandable card. FIGURE 24 – ANALYTICS TOOL PARAMETERS DETAILS e 25 shows the fourth step of the wizard, presenting a short overview of the selected policy FIGURE 25 – POLICY OVERVIEW / SAVING FIGURE 25 – POLICY OVERVIEW / SAVING 25 www.policycloud.eu D5.5 – v1.0 Figure 26 shows the UI Menu for selecting different PDT components. Figure 26 shows the UI Menu for selecting different PDT components. Figure 26 shows the UI Menu for selecting different PDT components. FIGURE 26 – PDT COMPONENTS MENU For each step of the wizard help menu is available. Figure 27 shows the online help for the 1st step of the wizard. Figure 26 shows the UI Menu for selecting different PDT components. FIGURE 26 – PDT COMPONENTS MENU For each step of the wizard help menu is available. Figure 27 shows the online help for the 1st step of the wizard FIGURE 26 – PDT COMPONENTS MENU For each step of the wizard help menu is available. Figure 27 shows the online help for the 1st step of the wizard. For each step of the wizard help menu is available. Figure 27 shows the online help for the 1st step of the wizard. FIGURE 27 - ONLINE HELP FIGURE 27 - ONLINE HELP www.policycloud.eu 26 D5.5 – v1.0 At the footnote of each PDT page, the link for the End Users Data Protection Information has been added, as shown in Figure 28 and Figure 29. FIGURE 28 - USER DATA PROTECTION INFORMATION NOTICE At the footnote of each PDT page, the link for the End Users Data Protection Information has been added, as shown in Figure 28 and Figure 29. FIGURE 28 USER DATA PROTECTION INFORMATION NOTICE FIGURE 28 - USER DATA PROTECTION INFORMATION NOTICE FIGURE 28 USER DATA PROTECTION INFORMATION NOTICE FIGURE 29 - USER DATA PROTECTION TEXT FIGURE 29 - USER DATA PROTECTION TEXT 27 www.policycloud.eu D5.5 – v1.0 A user-friendly guide / FAQ on how PolicyCLOUD users should address each parameter required from them for the registration (in particular, those specified in order to ensure legal/ethical compliance) is being developed as part of the implementation of the WP4 Legal/Ethical Checklist. After the authentication of the User by providing his/her credentials to the Authentication server, the User Profile is available to the PDT page. Figure 30 shows the User Profile, as well as the Logout action button. FIGURE 30 - USER PROFILE PROPERTIES / LOGOUT FIGURE 30 - USER PROFILE PROPERTIES / LOGOUT www.policycloud.eu 2.2.3 Data Visualization From the initial page, users will select the different options offered by the tool, and among them, the graphics to visualize the results of the selected analytics. When a HeatMap is selected to visualize the analytics results, depending on the Use Case, it will receive the data in one or another format. These data format has been changed to respect the initial prototype of the Visualization Tool, but has been also agreed with WP4, and contain data for both, Maggioli and Sofia Use Cases. The json is like: www.policycloud.eu 28 www.policycloud.eu D5.5 – v1.0 FIGURE 31 - EXAMPLE FOR HEAT MAP CHART DATA FIGURE 31 - EXAMPLE FOR HEAT MAP CHART DATA FIGURE 31 - EXAMPLE FOR HEAT MAP CHART DATA Where data field is the data returned by the Analytics Tool, and the other fields are added by the PDT Backend, which is the one that does the communication between Analytics and the Visualization. The input data used for Sarga Use Case has been provided by the Sentimental Analysis component from WP4, and the agreed format is: Backend, which is the one that does the communication between Analytics and the Visualization. The input data used for Sarga Use Case has been provided by the Sentimental Analysis component from WP4, and the agreed format is: FIGURE 32 – EXAMPLE FOR SARGA GAUGE CHART DATA In the case of the Bar chart for London Use Case, the data received has the format: FIGURE 33 – EXAMPLE FOR LONDON BAR CHART DATA 29 www.policycloud.eu D5.5 – v1.0 In the case of Sofia’s Bar charts, the data received has the format: In the case of Sofia’s Bar charts, the data received has the format: @Path("/starts") @ApiOperation(value="Returns the list of domains that starts with the given input", notes="Returns the list of domains that starts with the given input", responseContainer = "List", response = DomainDTOImpl.class) @ApiResponses(value= { @ApiResponse(code=500, message="Exception's message") }) @Produces(MediaType.APPLICATION JSON) @Path("/starts") 2.2.4 PDT Backend As described in the previous subsection, the backend of the PDT exposes its interfaces via a REST API. In order to provide an always up-to-date documentation of the definition of the interface to the developers of other components, we automated this process by making use of the swagger [3] open source documentation UI. Swagger allows the developer to annotate her source code and provides tools and plugins that auto-generate the documentation during compilation time. It generates JSON files with all the relevant information. It also provides a web application that can be used as the UI tool for the consumer of the REST services to see their documentation. This web application makes use of those auto-generated JSON files to build the user interface. Moreover, it provides to the REST service consumer the ability to test the interface online, by invoking them in real-time and check the results. The following code snippet indicates how the developer of a REST service can annotate the latter via the swagger framework. @Path("domains") @Api(basePath="http://localhost:54735/pdt", value = "/domains", description = "REST Service that provides functionalities for domains") public class DomainResource { @Path("domains") @Api(basePath="http://localhost:54735/pdt", value = "/domains", description = "REST Service that provides functionalities for domains") public class DomainResource { The class DomainResource implements the web methods related with the functionalities for the domains, and it has been annotated to use the REST path “/domains”. As it can be noted, the developer can additionally annotate the class with the @Api that informs the swagger that this REST resource should be documented. Moreover, at the level of web methods, the following code snippet shows how the developer can document the details of each invocation 30 www.policycloud.eu D5.5 – v1.0 @SuppressWarnings("UseSpecificCatch") public Response getDomainsStartingWith( @ApiParam(value = "the start with parameter", required = true) @QueryParam("name") String name) { @SuppressWarnings("UseSpecificCatch") public Response getDomainsStartingWith( @ApiParam(value = "the sta @QueryParam("name") String This web method that returns all domains whose name starts with a given value. The swagger annotation indicates to the framework that this should be an operation that will return a List of serialized objects of the DomainDTOImpl class. Besides the default HTTP code 200 that indicates a successful invocation, this web method can also return an HTTP error code 500 and provides the parameter that is required. 2.2.4 PDT Backend Swagger, uses Java Reflection to retrieve additional information like the type of the invocation (GET, POST, PUT etc.) the name and type of the input parameters, the media type used in the body of the request or/and the response of the HTTP call etc. Finally, the DTOs that will be transferred via the REST calls, can also be annotated, as the following code snippet indicates. Finally, the DTOs that will be transferred via the REST calls, can also be annotated, as the following code snippet indicates. @JsonIgnoreProperties(ignoreUnknown = true) @JsonIgnoreProperties(ignoreUnknown = true) public class DomainDTOImpl implements DomainDTO, Serializable { private static final long serialVersionUID = 1L; @ApiModelProperty(value="domain id", required=true) private Long id; @ApiModelProperty(value="domain name", required=true) private String name; @JsonCreator public DomainDTOImpl( @JsonProperty("id") Long id, @JsonProperty("name") String name) { this.id = id; this.name = name; } public class DomainDTOImpl implements DomainDTO, Serializable { private static final long serialVersionUID = 1L; private static final long serialVersionUID = 1L; The aforementioned DomainDTOImpl used by the web method explained before, has two attributes, the id and the name of the domain. The ApiModelProperty can be used by swagger to provide this kind of information to the documentation. After having annotated all our web services, web methods and DTOs, the swagger plugin for maven was used to auto-generate the stub information in JSON files. The following code snippet was used to do this work. <plugin> <groupId>com.github.kongchen</groupId> <artifactId>swagger-maven-plugin</artifactId> <version>2.3.4</version> <configuration> <apiSources> <apiSource> <locations>eu.policycloud.rest.resources</locations> <apiVersion>${project.version}</apiVersion> <basePath>/resources</basePath> <groupId>com.github.kongchen</groupId> www.policycloud.eu 31 D5.5 – v1.0 <swaggerDirectory>${project.basedir}/src/main/webapp/</swaggerDirectory> <outputTemplate>${project.basedir}/src/main/resources/markdown.mustache</outputTemplate> <mustacheFileRoot>${project.basedir}/src/main/resources/</mustacheFileRoot> <outputPath>${project.basedir}/src/main/webapp/document.html</outputPath> </apiSource> </apiSources> </configuration> <executions> <execution> <phase>compile</phase> <goals> <goal>generate</goal> </goals> </execution> </executions> </plugin> <swaggerDirectory>${project.basedir}/src/main/webapp/</swaggerDirectory> <outputTemplate>${project.basedir}/src/main/resources/markdown.mustache</outputTemplate> <mustacheFileRoot>${project.basedir}/src/main/resources/</mustacheFileRoot> <outputPath>${project.basedir}/src/main/webapp/document.html</outputPath> </apiSource> </apiSources> </configuration> <executions> <execution> <phase>compile</phase> <goals> <goal>generate</goal> </goals> </execution> </executions> </plugin> It creates the JSON files during compile phase, and adds them to corresponding directory where the swagger app is expecting to retrieve this information, while it also modifies the markdown mustache template used by the web app. After setting up everything, the online documentation is now available. The following screenshots show this documentation per REST Web Service implemented. FIGURE 35 - WEB METHODS RELATED WITH ACTORS FIGURE 35 - WEB METHODS RELATED WITH ACTORS 32 www.policycloud.eu D5.5 – v1.0 FIGURE 36 - WEB METHODS RELATED WITH ANALYTICAL TOOLS FIGURE 36 - WEB METHODS RELATED WITH ANALYTICAL TOOLS FIGURE 36 - WEB METHODS RELATED WITH ANALYTICAL TOOLS FIGURE 37 - WEB METHODS RELATED WITH DATA MODELS FIGURE 36 - WEB METHODS RELATED WITH ANALYTICAL TOOLS FIGURE 37 - WEB METHODS RELATED WITH DATA MODELS FIGURE 37 - WEB METHODS RELATED WITH DATA MODELS FIGURE 38 - WEB METHODS RELATED WITH DOMAINS FIGURE 38 - WEB METHODS RELATED WITH DOMAINS FIGURE 38 - WEB METHODS RELATED WITH DOMAINS 33 www.policycloud.eu D5.5 – v1.0 FIGURE 39 - WEB METHODS RELATED WITH GOALS FIGURE 40 - WEB METHODS RELATED WITH JOB STATUS D5.5 – v1.0 www.policycloud.eu FIGURE 39 - WEB METHODS RELATED WITH GOALS FIGURE 39 - WEB METHODS RELATED WITH GOALS FIGURE 39 - WEB METHODS RELATED WITH GOALS FIGURE 40 - WEB METHODS RELATED WITH JOB STATUS FIGURE 39 - WEB METHODS RELATED WITH GOALS FIGURE 40 - WEB METHODS RELATED WITH JOB STATUS FIGURE 40 - WEB METHODS RELATED WITH JOB STATUS 34 www.policycloud.eu www.policycloud.eu D5.5 – v1.0 FIGURE 41 - WEB METHODS RELATED WITH JOBS FIGURE 42 - WEB METHODS RELATED WITH KPIS D5.5 – v1.0 FIGURE 41 - WEB METHODS RELATED WITH JOBS FIGURE 41 - WEB METHODS RELATED WITH JOBS FIGURE 41 - WEB METHODS RELATED WITH JOBS FIGURE 41 - WEB METHODS RELATED WITH JOBS FIGURE 42 - WEB METHODS RELATED WITH KPIS FIGURE 41 - WEB METHODS RELATED WITH JOBS FIGURE 42 - WEB METHODS RELATED WITH KPIS FIGURE 42 - WEB METHODS RELATED WITH KPIS 35 www.policycloud.eu D5.5 – v1.0 www.policycloud.eu FIGURE 43 - WEB METHODS RELATED WITH POLICIES FIGURE 43 - WEB METHODS RELATED WITH POLICIES FIGURE 43 - WEB METHODS RELATED WITH POLICIES Finally, the Backend allows for asynchronous communication with the PDT via web sockets. This is crucial when other components perform updates that the Backend should be aware of, so that the PDT can modify the UI in order to reflect those updates, without having to continuously invoke the Backend to ask for potential pending updates. This helps to improve the overall UX of the end-user. For instance, when an analytical tool finishes its executions and produces some results, the Data Acquisition and Analytic API notifies the Backend, which will be triggered in order to send a message via this web socket to the PDT. By doing this, the PDT can be in position to update the status of a pending job, without periodically ask this status from the backend. The DTOs that are exchanged have the following format: { "id": 3242, "notification": "UPDATED", "job": { "id": 3242, "name": "job name", "description": "the description of the job", "status": "FINISHED", "dateCreated": "2021-10-01T09:23:43" { "id": 3242, "notification": "UPDATED", "job": { "id": 3242, "name": "job name", "description": "the description of the job", "status": "FINISHED", "dateCreated": "2021-10-01T09:23:43" . . . . } } In this DTO, the id is the unique identifier of the job its status has been changed and the notification can accept one of the following values: ADDED, UPDATED or DELETED. Finally, the job contains all information about the corresponding job, like its id, name, description, status, etc. "description": "the description of the job" } In this DTO, the id is the unique identifier of the job its status has been changed and the notification can accept one of the following values: ADDED, UPDATED or DELETED. Finally, the job contains all information about the corresponding job, like its id, name, description, status, etc. 36 D5.5 – v1.0 2.3.3 Data Visualization As the Data Visualization is part of the PDT, it has been developed using the same base technology, AngularJS v10 [4]. The main JavaScript libraries used for this first prototype are: As the Data Visualization is part of the PDT, it has been developed using the same base technology, AngularJS v10 [4]. The main JavaScript libraries used for this first prototype are: • Leaflet [7] is used to plot the heatmap • Amcharts [8] is used to plot the line and the gauge charts Although the component is integrated into the PDT, a standalone version can also be used, and the detailed invocation instructions are detailed into the readme file that accompanies the source code of the component. Still, the service can be started either with a NodeJS server or using a Docker container (see Section 3.1.3). Although the component is integrated into the PDT, a standalone version can also be used, and the detailed invocation instructions are detailed into the readme file that accompanies the source code of the component. Still, the service can be started either with a NodeJS server or using a Docker container (see Section 3.1.3). To visualize it as part of the PDT, this component has to be started and all previous steps must be taken. 2.3.2 Policy Development Toolkit As described in the deliverable D5.2 “Cross-sector Policy Lifecycle Management: Design and Open Specification 1” Section 4.2, PDT is built as a Single Page Application developed using the open-source Angular framework [4][3]. For UI components and controls we use the Angular Material library [5]. The PDT is using Docker to encapsulate the web serving functionality along with NGINX [6] web server. The PDT is using Docker to encapsulate the web serving functionality along with NGINX [6] web server. 2.3.1 Policy Modelling Editor As described in the deliverable D5.4 “Cross-sector Policy Lifecycle Management: Design and Open Specification 2”[1], the PME is developed using the open-source Angular framework [4]. For UI components and controls we use the Angular Material library [5]. 2.3 Baseline technologies and tools 2.3.1 Policy Modelling Editor 3.1.1 Policy Modelling Editor 3.1.1 Policy Modelling Editor The source code of the prototype of the Policy Modelling Editor component is available at: 3.1.2 Policy Development Toolkit The source code of the prototype of the Policy Development Toolkit component is available at: 3.1.3 Data Visualization The source code of the Data Visualization component is available separately at: • Project GitLab repository: https://registry.grid.ece.ntua.gr/ana.pontual/visualization Or through the PDT at: • Project GitLab repository: https://registry.grid.ece.ntua.gr/kostas/pdt • Project GitLab repository: https://registry.grid.ece.ntua.gr/kostas/pdt The Backend of the PDT is currently released under open source license, and its source code has been uploaded and is publicly available at: • Project GitLab repository: https://registry.grid.ece.ntua.gr/pavlos_LXS/pdt-backend It also provides the tools to compile the source code and also build a docker image that can deploy and run this component. As already mentioned, the PDT Backend requires the use of a relational data store to persistently store its policies, policy related meta-information and results of the analytical tools. We relied on the datastore provided by LXS, which can be found at: As already mentioned, the PDT Backend requires the use of a relational data store to persistently store its policies, policy related meta-information and results of the analytical tools. We relied on the datastore provided by LXS, which can be found at: • Project GitLab repository: https://registry.grid.ece.ntua.gr/pavlos_LXS/policy-store 2.3.4 PDT Backend The backend of PDT has been implemented using Java SDK 8. It does not make use of any framework like Spring, but it relies on the standard JDK. For the implementation of the REST services, it makes use of the javax servlet API 3.1.0 while it also makes use of an embedded tomcat, as the servlet container that the web services are deployed. We rely on the Apache Embedded Tomcat 8 [9]. By doing this, there is no need to maintain an additional standalone container like Tomcat, Glassfish or similar, rather than the java virtual machine starts the embedded one inside its process. Moreover, Maven 3 was used as the tool for the automation of the build process. For the persistent storage of this component, we relied on the LXS relational datastore. However, this is not mandatory and therefore, the Backend component of the PDT can switch to other relational datastore, with minimal changes in the source code. www.policycloud.eu 37 D5.5 – v1.0 www.policycloud.eu • Project GitLab repository: https://registry.grid.ece.ntua.gr/pavlos_LXS/policy-store As the datastore is published with LXS IPR licence, its source code cannot be available. However, the GitLab repositories for the datastore contains all binaries and scripting files necessary for packaging the distribution and creating a docker container that the datastore can start within. As the datastore is published with LXS IPR licence, its source code cannot be available. However, the GitLab repositories for the datastore contains all binaries and scripting files necessary for packaging the distribution and creating a docker container that the datastore can start within. 38 D5.5 – v1.0 3.2 Deployment 3.2.1 Policy Modelling Editor The Policy Modelling Editor will follow the Cloud provider setups and Kubernetes staging. 3.2.1 Policy Modelling Editor The Policy Modelling Editor will follow the Cloud provider setups and Kubernetes staging. 3.2.2 Policy Development Toolkit The Policy Development Toolkit will follow the Cloud provider setups and Kubernetes staging. 3.2.4 PDT Backend git clone http://snf-877903.vm.okeanos.grnet.gr/pavlos/pdt-backend.git And later compile the source code. Assuming maven 3 and Java 8 are pre-installed, it needs to execute the following: And later compile the source code. Assuming maven 3 and Java 8 are pre-installed, it needs to execute the following: mvn clean install The component can be executed via a java JRE tools, but we also provide a Dockerfile to fully containerize the deployment and integrate it with the LXS datastore. Firstly, they would need to locally build a docker image. Assuming that docker has been already installed in the host machine, they should execute the following: docker build –t pdt-backend . This will create a docker image that can be found in the host machine’s catalogue. To execute the component, a docker-compose file has also been provided in the GitLab, which makes use of the LXS datastore. The latter must have been already available in the host machine’s docker catalogue. To start everything, the administrator should execute the following: docker-compose up docker-compose up The docker-compose file exposes the 54735 port to the host machine, where the servlet container listens to. Therefore, the administrators can now open their favorite browser and put the following URL, which will open the swagger documentation: www.policycloud.eu www.policycloud.eu 39 D5.5 – v1.0 An alternative option for starting the PDT backend is to manually start firstly the LXS relational datastore, and then the PDT backend. With the assumption that the docker images for both the relational datastore and the PDT backend has been already created and available, then the user needs to first start the datastore by executing the following: docker run -d -p 2181:2181 -p 1529:1529 --name datastore --env KVPEXTERNALIP='datastore!9800' policy-store www.policycloud.eu Once the datastore is running, then the user can start the PDT backend with: docker run -d -p 54735:54735 --name pdt_backend --env DATASTORE_HOST='172.17.0.2' --env swagger_path=/tmp/pdt-backend Once the backend has been started, then the administration can still open a browser and check that everything is working by invoking the swagger documentation, as shown before at: http://localhost:54735 http://localhost:54735 www.policycloud.eu www.policycloud.eu 40 D5.5 – v1.0 www.policycloud.eu 4 Conclusions This deliverable provided a description of the software components of the Policy Management Framework Layer and Policy Development Toolkit Layer, which consists of the frontend part of the PolicyCLOUD platform and allow policy makers to create, update and evaluate policies. For each component, a description of its APIs, specification of the main functionalities, and description of the source code is provided. www.policycloud.eu 41 www.policycloud.eu 41 D5.5 – v1.0 www.policycloud.eu References www.policycloud.eu 42 [1] PolicyCLOUD. D5.4 “Cross-sector Policy Lifecycle Management Design and Open Specification 2”. Armend Duzha et al 2021. [2] PolicyCLOUD. D2.2 “Conceptual Model and Reference Architecture”, Panayiotis Michael et al 2021. [3] Swagger [Online], Available at: https://swagger.io/tools/swagger-ui/ [4] Angular [Online], Available at: https://angular.io [5] Angular Material [Online], Available at: https://material.angular.io/ [6] NGINX [Online], Available at: https://www.nginx.com/ [7] Leaflet [Online], Available at: https://leafletjs.com/ [8] Amcharts [Online], Available at: https://www.amcharts.com/ [9] Apache Tomcat [Online], Available at: http://tomcat.apache.org/ [1] PolicyCLOUD. D5.4 “Cross-sector Policy Lifecycle Management Design and Open Specification 2”. Armend Duzha et al 2021. [2] PolicyCLOUD. D2.2 “Conceptual Model and Reference Architecture”, Panayiotis Michael et al 2021. [3] Swagger [Online], Available at: https://swagger.io/tools/swagger-ui/ [4] Angular [Online], Available at: https://angular.io [5] Angular Material [Online], Available at: https://material.angular.io/ [6] NGINX [Online], Available at: https://www.nginx.com/ [7] Leaflet [Online], Available at: https://leafletjs.com/ [8] Amcharts [Online], Available at: https://www.amcharts.com/ [9] Apache Tomcat [Online], Available at: http://tomcat.apache.org/ [2] PolicyCLOUD. D2.2 “Conceptual Model and Reference Architecture”, Panayiotis Michael et al 2021. [3] Swagger [Online], Available at: https://swagger.io/tools/swagger-ui/ [4] Angular [Online], Available at: https://angular.io [5] Angular Material [Online], Available at: https://material.angular.io/ [6] NGINX [Online], Available at: https://www.nginx.com/ [7] Leaflet [Online], Available at: https://leafletjs.com/ [8] Amcharts [Online], Available at: https://www.amcharts.com/ [9] Apache Tomcat [Online], Available at: http://tomcat.apache.org/ www.policycloud.eu www.policycloud.eu 42
https://openalex.org/W4386125940
https://www.frontiersin.org/articles/10.3389/fpls.2023.1235686/pdf
English
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Copper boosts the biostimulant activity of a vegetal-derived protein hydrolysate in basil: morpho-physiological and metabolomics insights
Frontiers in plant science
2,023
cc-by
11,898
TYPE Original Research PUBLISHED 24 August 2023 DOI 10.3389/fpls.2023.1235686 COPYRIGHT © 2023 Rouphael, Carillo, Ciriello, Formisano, El-Nakhel, Ganugi, Fiorini, Miras Moreno, Zhang, Cardarelli, Lucini and Colla. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. OPEN ACCESS OPEN ACCESS EDITED BY Pushp Sheel Shukla, Dalhousie University, Canada REVIEWED BY Sławomir Kocira, University of Life Sciences of Lublin, Poland Arup Ghosh, Council of Scientific and Industrial Research (CSIR), India Sumera Javad, Lahore College for Women University, Pakistan *CORRESPONDENCE Giuseppe Colla giucolla@unitus.it Petronia Carillo petronia.carillo@unicampania.it RECEIVED 06 June 2023 ACCEPTED 31 July 2023 PUBLISHED 24 August 2023 CITATION Rouphael Y, Carillo P, Ciriello M, Formisano L, El-Nakhel C, Ganugi P, Fiorini A, Miras Moreno B, Zhang L, Cardarelli M, Lucini L and Colla G (2023) Copper boosts the biostimulant activity of a vegetal-derived protein hydrolysate in basil: morpho-physiological and metabolomics insights. Front. Plant Sci. 14:1235686. doi: 10.3389/fpls.2023.1235686 EDITED BY Pushp Sheel Shukla, Dalhousie University, Canada REVIEWED BY Sławomir Kocira, University of Life Sciences of Lublin, Poland Arup Ghosh, Council of Scientific and Industrial Research (CSIR), India Sumera Javad, Lahore College for Women University, Pakistan Youssef Rouphael 1, Petronia Carillo 2*, Michele Ciriello 1, Luigi Formisano 1, Christophe El-Nakhel 1, Paola Ganugi 3,4, Andrea Fiorini 4, Begoña Miras Moreno 3, Leilei Zhang 3, Mariateresa Cardarelli 5, Luigi Lucini 4,6 and Giuseppe Colla 5* 1Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy, 2Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Caserta, Italy, 3Department for Sustainable Food Process, Università Cattolica del Sacro Cuore, Piacenza, Italy, 4Department of Sustainable Crop Production, Università Cattolica del Sacro Cuore, Piacenza, Italy, 5Department of Agriculture and Forest Sciences, University of Tuscia, Viterbo, Italy, 6CRAST Research Centre, Università Cattolica del Sacro Cuore, Piacenza, Italy In addition to be used as a plant protection agent, copper (Cu) is also an essential micronutrient for plant growth and development. The bioavailability of Cu in agricultural systems can be limited due to its specific physical–chemical characteristics, leading to imbalances in plant production. To address this issue, an experimental trial was conducted on Genovese basil (Ocimum basilicum L.) in protected conditions to comparatively evaluate the effects of a vegetable protein hydrolysate (VPH), free Cu and Cu complexed with peptides and amino acids of vegetal origin (Cu and Cu-VPH, respectively), and a combination of VPH and Cu-VPH (VPH+Cu-VPH). The study showed that the combined application of VPH+Cu-VPH led to a significant average increase of 16.3% in fresh yield compared to the untreated Control and Cu treatment. This finding was supported by an improved photosynthetic performance in ACO2 (+29%) and Fv/Fm (+7%). frontiersin.org Frontiers in Plant Science 1 Introduction Hatamian, 2019). However, in recent years, the unsustainability of the massive use of synthetic chelating agents in agriculture has emerged because the main artificial chelating agents used are persistent in natural systems because of their low biodegradability (Wu et al., 2004). In addition to this disadvantage, these compounds can alter the natural evolution of metals in groundwater and soil, making toxic metals bioavailable and compromising the quality of agricultural products and food safety (Zuluaga et al., 2023). A balance of nutrients, especially between macro- and micronutrients, is essential to ensure plant health and the sustainability of agricultural productions (Souri et al., 2017). Despite being required in smaller amounts than primary nutrients, micronutrients play a crucial role in plant metabolism and physiology. Accordingly, plants must efficiently control their mobilization, absorption, distribution, and storage (Hänsch and Mendel, 2009). Among micronutrients, Cu, a redox-active transition metal, plays a fundamental role in various physiological and biochemical processes (Kaewchangwat et al., 2017). At optimal levels (between 5 and 30 mg kg–1), this microelement participates in primary metabolic processes (mitochondrial respiration, electron transport chain, and photosynthesis), lignin biosynthesis, hormone signaling, and response to oxidative stress (Mir et al., 2021). However, since the 1880s, the role of Cu has been relegated to an essential active ingredient in fungicidal and bacteriostatic formulations (Lopez-Lima et al., 2021). Its broad-spectrum fungicide action has made Cu essential, especially for organic agriculture where the management of fungal disease depends almost exclusively on its use (La Torre et al., 2018). The necessary shift towards sustainable agricultural systems has highlighted the potential of innovative technical solutions, such as plant-based biostimulants. These latter can increase nutrient use efficiency and improve crop productivity and quality (Rouphael and Colla, 2020). The ability to produce biostimulants from plant waste has made vegetal protein hydrolysate (VPH) one of the most promising products (Xu and Geelen, 2018). Their biostimulant activity is attributable to a mixture of bioactive compounds such as amino acids, peptides, carbohydrates, minerals, phytohormones, phenols, and other organic compounds (Ciriello et al., 2022a). As Colla et al. (2015) stated, protein hydrolysates (PHs) can improve plant nutrition through three modalities: (i) promoting the growth of the root system, (ii) stimulating absorption processes, and (iii) increasing the availability of nutrients in the soil by increasing soil cation exchange and thus inorganic minerals’ solubility in water (Zuluaga et al., 2023). KEYWORDS vegetal protein hydrolysates, sustainable agriculture, primary metabolism, oxidative stress, metabolomics, photosynthetic performance, ionomics Frontiers in Plant Science OPEN ACCESS Furthermore, mineral analysis using ICP OES demonstrated that Cu and Cu-VPH treatments determined, on average, a 15.1-, 16.9-, and 1.9-fold increase in Cu in plant tissues compared to control, VPH, and VPH+Cu-VPH treatments, respectively. However, the VPH+Cu-VPH treatment induced the highest contents of the other analyzed ions, except for P. In particular, Mg, Mn, Ca, and Fe, which take part in the constitution of chlorophylls, water splitting system, and photosynthetic electron transport chain, increased by 23%, 21%, 25%, and 32% compared to respective controls. Indeed, this improved the photosynthetic efficiency and the carboxylation capacity of the plants, and consequently, the physiological and productive performance of Genovese basil, compared to all other treatments and control. Consistently, the untargeted metabolomics also pointed out a distinctive modulation of phytochemical signatures as a function of the treatment. An accumulation of alkaloids, terpenoids, and phenylpropanoids was observed following Cu treatment, suggesting an oxidative imbalance upon metal COPYRIGHT © 2023 Rouphael, Carillo, Ciriello, Formisano, El-Nakhel, Ganugi, Fiorini, Miras Moreno, Zhang, Cardarelli, Lucini and Colla. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science 01 frontiersin.org Rouphael et al. 10.3389/fpls.2023.1235686 exposure. In contrast, a mitigation of oxidative stress was highlighted in Cu-VPH and VPH+Cu-VPH, where the treatments reduced stress-related metabolites. Overall, these results highlight an interaction between Cu and VPH, hence paving the way towards the combined use of Cu and biostimulants to optimize agronomic interventions. Rouphael et al. 2.2 Determination of CIELab colorimetric and SPAD indexes Color coordinates (L, a*, and b*) were recorded using a Minolta Chroma Meter CR-300 handheld colorimeter (Minolta Camera Co. Ltd., Tokyo, Japan). Ten measurements were taken on the surface of fully expanded young leaves of eight plants per experimental unit on the day before harvest (36 DAT). The same leaves were used to determine the SPAD index using the SPAD-502 optical device instrument (Minolta Corp. Ltd., Osaka, Japan). The experimental trial was conducted on the experimental farm “Torre Lama” (lat. 43°31’N, long. 14°58’E; alt. 60 m above sea level) of the University of Naples “Federico II” - Department of Agriculture (DIA). The test was conducted in an unheated greenhouse with a galvanized iron structure and a glass roof. The structure has an aisle width of 10 m and a length of 30 m; the ridge height is 4.5 m, and the height at the eaves is 3.0 m. The plant material used for the trial was the Ocimum basilicum L. Genovese cultivar Mammolo (L’Ortolano, Cesena, Italy), grown in 1.2-L plots filled with agricultural soil at a density of 20 pt m−2, with 25 and 20 cm inter- and intra-rows, respectively. The main physical and chemical soil characteristics at the experimental site were clay loam texture (46% sand, 24.2% silt, and 29.8% clay), electrical conductivity (EC): 0.16 dS m−1, pH: 7.7, total nitrogen (N): 0.11%, and organic matter: 1.21% (w/w). The Olsen phosphorus and exchangeable potassium were 88 and 980 mg kg−1, respectively. The seeds were transplanted on 12 June 2019, in the two- to the three-leaf stage and irrigated using a micro-irrigation system with 16-mm inner diameter drip wings and a 10-cm pitch with self- compensating drippers of 2 L h–1 flow rate. The experimental trial involved randomized blocks with five treatments replicated three times. The experimental unit consisted of 15 plants. Seven days after transplanting (DAT), the crop was subjected to the following treatments: copper sulfate pentahydrate CuSO4-5H2O (0.23 g L–1; henceforth Cu), vegetal-protein hydrolysate at the manufacturer’s recommended dose (4 ml L–1 of Trainer®; henceforth VPH; Hello Nature, Rivoli Veronese, Italy), Cu complexed with peptides and amino acids of vegetal origin (Scudo® - Hello Nature, Rivoli Veronese, Italy—at the dose of 0.5 ml L–1; henceforth Cu-VPH), Cu complexed with peptides and amino acids of vegetal origin (Scudo®) enriched with vegetal-protein hydrolysate Trainer® (0.5 ml L–1 and 3.7 ml L–1, respectively; henceforth VPH+Cu- VPH), and untreated control. 1 Introduction Combining biostimulants and innovative fertilizers such as chelates may represent a step forward in solving nutritional problems in agricultural systems. With their ability to act as ligand via non-covalent bonds, peptides may function as biochelators for specific microelements (Zuluaga et al., 2023). The use of biochelates makes micronutrients more available than standard fertilization practices (Souri et al., 2017). Moreover, the enhanced biodegradability of metal biochelates, compared to synthetic counterparts, provides a more favorable environmental impact. In the specific case of Cu, PHs might also provide additional benefits in terms of mitigation of metal-related stress in plants. Despite their potential, no in-depth studies have been conducted on the single and combined effects of biostimulants and the corresponding biochelates. The definition of synergism between Cu and PHs may offer novel solutions in the agronomic management of both plant nutrition and disease management. For this reason, our research aimed to compare the applications of copper sulfate pentahydrate, a VPH, Cu complexed with a On the other hand, excessive use of Cu in agriculture and industry has significantly increased its concentration in cultivable soils. High levels of Cu inhibit plant development and growth due to irreversible morphological, physiological, and biochemical alterations (Mir et al., 2021). In fact, a complex and articulated system of utility, toxicity, absorption, and transport in plants has led to a dual outlook for Cu, as a harmful or an essential element (Shabbir et al., 2020). Predominantly present as a cupric ion (Cu2+), its bioavailability depends not only on the absolute concentration but also on the physicochemical characteristics of the soil. Organic and inorganic colloids immobilize Cu, making it unavailable to plants, especially in alkaline soils that represent approximately 30% of cultivable soils worldwide (Kumar et al., 2021; Wairich et al., 2022). In these cases, the foliar application of Cu is preferable to the soil application as it improves its own uptake and use efficiency, thus promoting a more sustainable agriculture. Although introduced to improve the nutritional status of animal organisms, synthetic chelating agents (EDTA and EDDHA) have become essential for the balanced nutrition of crop systems (Souri and 02 frontiersin.org Rouphael et al. 10.3389/fpls.2023.1235686 10.3389/fpls.2023.1235686 At 37 DAT, eight plants per replicate were sampled to determine biometric parameters. Specifically, leaves and stems were separated to fresh weight (fw; g plant–1). The fresh yield was expressed as kg m–2. 2.2 Determination of CIELab colorimetric and SPAD indexes VPH was produced through enzymatic hydrolysis of legume biomass as reported by Colantoni et al. (2017) while the Cu complexed with peptides and amino acids (Cu-VPH) was obtained by chemical reaction of copper sulfate with peptides and amino acids contained in the VPH. Copper concentration in all Cu treatments (Cu, Cu-VPH, and VPH-Cu- VPH) was 58.5 mg L−1. In both VPH and VPH-Cu-VPH treatments, nitrogen as free amino acids and soluble peptides was 0.24 g L−1 while Cu-VPH treatment provided 0.02 g L−1 of N as amino acids and soluble peptides. Treatments were made by foliar application, always in the early morning, using separators to prevent drift contamination. By weekly applications, four treatments were made throughout the crop cycle. 2.3 Determination of leaf gas exchange and chlorophyll fluorescence At 37 DAT, between 10:30 a.m. and 1:30 p.m., in order to check the physiological status of plants at harvest, gas exchange and chlorophyll fluorescence measurements were made. Measurements were performed on fully expanded young basil leaves, avoiding the midrib. The maximum quantum efficiency of photosystem II (PSII) Fv/Fm was assessed on the leaves of nine plants per experimental unit after their adaptation to darkness (for at least 10 min) using leaf clips specific for the portable fluorometer Fv/Fm Meter (Opti-Sciences Inc., Hudson, USA). On the same leaves, the assimilation rate (ACO2) and transpiration rate (E) were determined using a portable gas exchange analyzer (LCA 4; ADC BioScientific Ltd., Hoddesdon, United Kingdom). The ratio between ACO2 and E was used to determine the instantaneous water use efficiency (WUEi). For leaf gas exchange measurements, all parameters [CO2 concentration, photosynthetically active radiation (PAR), and relative humidity (RH)] were set to ambient values while airflow was set to 400 ml s–1. Frontiers in Plant Science 1 Introduction The sampled plant material was then placed in a ventilated oven (for 72 h at 65°C) to assess the total dry weight (dw) of leaves and stems and thus the dry matter (%). vegetable protein hydrolysate (Cu-VPH), and a mixture of VPH and Cu-VPH on the yield and biochemical traits of Genovese basil grown in a glass greenhouse. frontiersin.org 2.6 Statistics All data were analyzed with IBM SPSS Statistics software version 26.0 (SPSS Inc., Chicago, IL, USA) and are presented as mean ± standard error, n = 3. All mean effects were subjected to one-way ANOVA analysis. Statistical significance was determined with Tukey’s HSD test at the p = 0.05 level. Macro- and trace element analysis of the digested samples was performed by ICP OES. The plasma source was provided by 99.999% purity argon (SOL, Marcianise, Italy). The instrument was optimized daily for the maximum signal and tuned with a specific solution for the wavelength range studied. For non-alkali elements (Fe, Mn, Cu, Zn, and P), the calibration curve was constructed in the 1.0–100 mg L–1 range. For alkaline elements (K, Ca, and Mg), the calibration curve was constructed from 2 mg L–1 to 1,000 mg L–1. Macro (K, Ca, Mg, and P)- and trace elements (Cu, Fe, Mn, and Zn) were expressed as mg g–1 fw and µg g–1 fw, respectively, based on the original dw of each basil sample. Mass Profiler Professional 15.1 software (Agilent Technologies) was used to elaborate metabolomic data. Compound abundance was Log2 transformed and normalized at the 75th percentile, then baselined against the median. Thereafter, the unsupervised hierarchical cluster analysis (Ward linkage method and Euclidean distance) was carried out to get an overview of phytochemical patterns and investigate relatedness among treatments. Then, the raw dataset was imported into SIMCA 13 (Umetrics, Malmo, Sweden), pareto scaled, and subjected to the supervised orthogonal projection to latent structures discriminant analysis (OPLS-DA). The OPLS-DA model was cross- validated (CV- ANOVA) and R2Y and Q2Y parameters (degree of correlation and prediction ability, respectively) were recorded. Moreover, overfitting was excluded through a permutation test (n = 100), outliers were inspected through Hotelling’s T2, and discriminant compounds selected by variable importance in projection (VIP) in the predictive model were noted. Finally, Volcano plot analysis (p- value < 0.05, Benjamini correction; fold change ≥2) was used to identify differential compounds, and the PlantCyc pathway tools software [http://www.plantcyc.org/; Caspi et al. (2014)] was used for biochemical interpretations. 3 Results Untargeted metabolomic analysis was performed using a 1290 ultrahigh-pressure liquid chromatographic system coupled to a G6550 electrospray quadrupole-time-of-flight mass spectrometry (UHPLC-ESI/QTOF-MS) from Agilent Technologies (Santa Clara, CA, USA). Reverse-phase chromatographic separation used an Agilent 120 PFP column (100 mm × 2.1 i.d., 1.9 mm particle size) and a mobile phase linear gradient of water and acetonitrile (6-94% acetonitrile in 33 min). Both mobile phases were acidified with 0.1% (v/v) formic acid. The mass spectrometer operated in full scan mode (range 100–1200 m/z), positive polarity (ESI+), and source parameters were previously optimized (Mastinu et al., 2021). 2.4 Determination of macro- and trace elements The determination of macro- and trace elements was carried out as detailed by Ciriello et al. (2023). All standards and reagents used to determine macro- and trace elements by inductively coupled plasma optical emission spectrometric analysis (ICP OES) were purchased from Sigma-Aldrich (Milan, Italy). Calibration standards were prepared from the multi-element standard solution ICP Calibration mix EH61 PrimAg (Romil, Cambridge, UK). For accuracy determination, Standard Reference Material (SRM) 1570a (NIST) was prepared for trace elements in spinach (Spinacia oleracea L.) leaves, which was analyzed in triplicate in the same manner as the samples. All glassware and 03 frontiersin.org Rouphael et al. 10.3389/fpls.2023.1235686 10.3389/fpls.2023.1235686 instruments were decontaminated by soaking them in a sulfuric acid solution overnight and then rinsed several times, first with deionized water and then with ultrapure water (Merck Millipore, Darmstadt, Germany). Compound annotation was achieved with MussHunter Profinder 10.0 software (Agilent Technologies) by applying the “find-by-formula” algorithm, based on the combination of monoisotopic accurate mass (tolerance of 5 ppm) and isotopic spacing and ratio, following mass and retention time alignment. The PlantCyc 9.6 [http://www.plantcyc.org/; Hawkins et al. (2021)] database was used for annotation, and post-annotation filtering retained the compounds present in three replications within at least one treatment. According to COSMOS Metabolomics Standard Initiative, Level 2 of annotation, corresponding to putatively annotated compounds, was adopted (Salek et al., 2015). The basil leaves were washed with ultrapure water and placed in a forced-ventilated oven at 65°C until they reached a constant weight and were finely ground. Before the analysis of the ICP OES (Spectroblue, Spectro Ametek, Berwyn, PA, USA), 0.5 g of the dried plant sample was weighed and subjected to laboratory analysis. Five grams of the dried plant sample, weighed in PTFE (polytetrafluoroethylene) vessels, was treated by microwave-assisted digestion (MLS-1200, Microwave Laboratory Systems, Milestone, Shelton, CT, USA) in a mixture of 9 ml of nitric acid (70%, for trace analysis), 3 ml of hydrochloric acid (30%, for trace analysis), and 0.5 ml of H2O2. The heating program consisted of switching from 20°C to 180°C in 15 min and then staying at 180°C for an additional 10 min, with a power setting of 800 W. The fully digested samples, after cooling, were transferred to a volumetric flask and the volume was increased to 50 ml with ultrapure water (Volpe et al., 2015). 2.5 UHPLC-ESI/QTOF-MS untargeted metabolomics Basil leaves were collected, ground with liquid nitrogen using pestle and mortar, and successively extracted and analyzed according to Rouphael et al. (2020). Briefly, a 1-g aliquot per sample was extracted in 15 ml of an aqueous solution with 80% (v/v) methanol acidified with 0.1% formic acid using an Ultra- Turrax (Polytron PT, City, Switzerland). Thereafter, the extracts were centrifugated at 8,000 × g for 10 min and 1 ml of each resulting supernatant was transferred into a glass vial by filtering through a 0.22-mm cellulose syringe filter for analysis. Frontiers in Plant Science 3.1 Yield and yield parameters With the exception of dry matter, the yield and yield components presented in Table 1 showed significant differences among the treatments (Control, Cu, Cu-VPH, VPH+Cu-VPH, and VPH). Specifically, when compared to the Control group, the VPH +Cu-VPH treatment exhibited notable increases in leaf fresh weight, total fresh weight, stem dry weight, total dry weight, and fresh yield, with improvements of 16.80%, 18.62%, 29.50%, 22.70%, and 18.48%, respectively. 04 frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. TABLE 1 Leaf, stem, and total fresh weight; leaf, stem, and total dry weight; fresh yield; and dry matter of basil under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu- VPH) treatments. Treatments Leaf fresh weight Stem fresh weight Total fresh weight Leaf dry weight Stem dry weight Total dry weight Fresh yield Dry matter g plant–1 kg m–2 % Control 62.56 ± 0.23 c 29.39 ± 0.42 b 91.95 ± 0.53 c 6.97 ± 0.07 b 3.39 ± 0.04 d 10.35 ± 0.08 c 1.84 ± 0.01 c 11.26 ± 0.03 Cu 64.22 ± 0.80 c 31.25 ± 0.93 b 95.47 ± 1.67 c 6.94 ± 0.06 b 3.87 ± 0.07 bc 10.80 ± 0.12 bc 1.91 ± 0.03 c 11.32 ± 0.12 Cu-VPH 66.38 ± 0.52 b 34.47 ± 0.73 a 100.85 ± 1.25 b 7.42 ± 0.14 b 3.71 ± 0.08 c 11.14 ± 0.19 bc 2.02 ± 0.03 b 11.04 ± 0.06 VPH+Cu- VPH 73.07 ± 0.14 a 36.00 ± 0.43 a 109.07 ± 0.57 a 8.32 ± 0.26 a 4.39 ± 0.07 a 12.70 ± 0.32 a 2.18 ± 0.01 a 11.64 ± 0.24 VPH 67.52 ± 0.27 b 34.69 ± 0.28 a 102.21 ± 0.37 b 7.67 ± 0.19 ab 4.00 ± 0.02 b 11.68 ± 0.19 b 2.04 ± 0.01 b 11.42 ± 0.15 Significance *** *** *** *** *** *** *** ns ns and *** denote nonsignificant or significant at p ≤0.001, respectively. Different letters within each column indicate significant differences according to Tukey HSD test (p = 0.05). All data are expressed as mean ± standard error, n = 3. 3.3 Protein, carbohydrate, and polyphenols concentration Relative to stem fresh weight, the highest values were obtained from the Cu-VPH, VPH+Cu-VPH, and VPH treatments, with no significant differences. Compared with the Cu-VPH treatment, the combined VPH+Cu-VPH treatment significantly increased leaf dry weight (+12.13%). Cu-VPH and VPH biostimulant application significantly affected soluble protein content. The protein content was significantly decreased by applying Cu-VPH and VPH, which was 25.6% and 31.6% lower than in untreated basil plants, respectively (Table 3). None of the treatments significantly affected starch (avg. 144.8 µmol g–1 dw) and sucrose (2.3 µmol g–1 dw) concentrations. (Table 3). However, the application of VPH+Cu-VPH averaged over other treatments positively affected the glucose concentrations in basil leaves (an increase of 76.2% compared to control), even if also Cu and Cu- VPH determined an increase of this soluble sugar of approximately 40% (Table 3). Fructose increased 24.4% and 30.8% compared to control when treated with Cu-VPH and VPH+Cu-VPH, respectively (Table 4). Finally, Cu-VPH and VPH significantly decreased the polyphenols content, with no significant differences among the three treatments (Table 3). Frontiers in Plant Science *** significant at p ≤0.001. Different letters within each column indicate significant differences according to Tukey HSD test (p = 0.05). All data are expressed as mean ± standard error, n = 3. Frontiers in Plant Science frontiersin.org 05 3.2 Physiological parameters All physiological parameters, shown in Table 2, were significantly affected by the treatments under investigation. The highest values of net CO2 assimilation rate (ACO2; 14.38 mmol CO2 m–2 s–1) and fluorescence (0.81) were obtained in plants of the VPH+Cu-VPH treatment. In comparison with the Control, the Cu-VPH and VPH+Cu-VPH treatments increased transpiration (E) by 49.10% (on average). In contrast, the Cu- VPH treatment reduced WUEi (–29.91%) compared with the Control. TABLE 2 Net CO2 assimilation rate (ACO2), transpiration (E), water use efficiency (WUE), and chlorophyll fluorescence (Fv/Fm) of basil under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments. Treatments ACO2 E WUEi Fluorescence Fv/Fm mmol CO2 m−2 s−1 mol H2O m−2 s−1 mmol CO2 mol−1 H2O Control 11.15 ± 0.11 b 1.66 ± 0.04 c 6.72 ± 0.12 a 0.76 ± 0.00 d Cu 11.96 ± 0.13 b 2.18 ± 0.18 b 5.57 ± 0.45 bc 0.77 ± 0.00 c Cu-VPH 12.19 ± 0.13 b 2.59 ± 0.04 a 4.71 ± 0.10 c 0.78 ± 0.00 b VPH+Cu-VPH 14.38 ± 0.45 a 2.36 ± 0.03 ab 6.09 ± 0.12 ab 0.81 ± 0.00 a VPH 12.14 ± 0.19 b 2.00 ± 0.02 bc 6.07 ± 0.15 ab 0.77 ± 0.00 c Significance *** *** *** *** 05 frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. TABLE 3 Soluble proteins, glucose, fructose, sucrose, starch, and polyphenols concentration of basil under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments. 3.2 Physiological parameters Treatments Soluble proteins Glucose Fructose Sucrose Starch Polyphenols mg g–1 fw µmol g–1 dw mg GAE g–1 dw Control 9.11 ± 0.59 a 36.96 ± 1.41 c 22.90 ± 2.87 b 2.65 ± 0.50 146.27 ± 10.43 20.21 ± 0.22 a Cu 7.85 ± 0.34 ab 51.65 ± 2.33 b 23.17 ± 1.12 b 2.76 ± 0.30 152.57 ± 24.54 18.92 ± 0.91 a Cu-VPH 6.78 ± 0.47 b 51.23 ± 0.27 b 28.50 ± 0.89 a 2.10 ± 0.31 148.91 ± 25.37 16.40 ± 0.48 b VPH+Cu-VPH 7.26 ± 0.33 ab 65.12 ± 3.75 a 29.96 ± 0.50 a 2.37 ± 0.28 160.45 ± 3.04 15.79 ± 0.42 b VPH 6.23 ± 0.14 b 44.88 ± 0.89 bc 27.40 ± 1.58 ab 1.68 ± 0.20 115.66 ± 19.12 15.38 ± 0.36 b Significance ** *** * ns ns *** ns, *, **, and *** denote nonsignificant or significant at p ≤0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Tukey HSD test (p = 0.05). All data are expressed as mean ± standard error, n = 3. ns, *, **, and *** denote nonsignificant or significant at p ≤0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Tukey HSD test (p = 0.05). All data are expressed as mean ± standard error, n = 3. 3.6 Principal component analysis To obtain an in-depth overview of growth parameters, mineral profile, and nutritional and functional quality traits of the studied basil leaves in response to the diverse treatments, a principal component analysis (PCA) was carried out for all the analyzed parameters discussed above. The first three principal components (PCs) were related with eigenvalues higher than 1 and explained 92.6% of the total variance, with PC1, PC2, and PC3 accounting for 54.2%, 25.1%, and 13.3%, respectively (data not shown). The increase in metabolism reshaping (activation of more metabolic pathways) contributed to the clear separation of PC1 with VPH+Cu-VPH in the positive side of PC1. In contrast, the distribution on PC2 was more linked to the efficient use of water with the control and Cu-VPH in the positive and negative side of PC2, respectively (Figure 1). PC1 was positively correlated to amino acids [in particular phenylalanine, asparagine, valine, tryptophan, branched-chain amino acids (BCAAs), tyrosine, threonine, and serine], dry matter, leaf fresh weight, stem dry weight, leaf dry weight, total dry weight, and ACO2 (Figure 1). PC1 negatively correlated with P, polyphenols, and L* and a* parameters. PC2 was positively correlated with lysine and WUEi, and negatively correlated with E (Figure 1). In addition, basil plants treated with VPH+Cu-VPH positioned on the positive side of PC1 close to the x- axis between the lower and upper right quadrant produced leaves of premium quality with higher fresh yield and concentrations of free amino acids and, in particular, essential amino acids. Control and Cu-treated plants in the upper left quadrant were instead characterized by the lowest fresh yield but the highest polyphenols content. Finally, Cu-VPH showed the lowest amino acid content, lower soluble proteins, and polyphenols than the control, but a high mineral concentration (K, Mg, Cu, and Fe) (Figure 1). 3.4 Free amino acid concentration reduced Zn by 21.93% compared to the VPH+Cu-VPH treatment (Table 5). The free amino acid profile of basil plants as a function of the different treatments is reported in Table 4. Irrespective of the treatments, glutamate and alanine were quantitatively the major amino acids, representing, on average, approximately 31.0% and 9.9% of total free amino acids in basil plants, respectively. Essential amino acids (the sum of isoleucine, histidine, leucine, lysine, methionine, threonine, and valine) accounted, on average, for 15.5% of total amino acids. Interestingly, the VPH and VPH+Cu- VPH determined a significant increase of all amino acids, particularly glutamate, glutamine, aspartate, and asparagine, which are involved in nitrogen assimilation and transport (Table 4). In contrast, the application of Cu-VPH alone determined the opposite effect, with a strong decrease of total amino acid content and of GABA (–61.6%), MEA (–67.5%), phenylalanine (–35.3%), glutamate (–42.9%), and alanine (– 22.8%); even the latter two were not significant (Table 4). In this condition, the only amino acids that did not decrease compared to the respective controls were the amides asparagine and glutamine, proline, and essential amino acids (Table 4). Frontiers in Plant Science 3.5 Leaf mineral composition Among the analyzed minerals, Mn and K were by far the most abundant micro- and macroelements. Regardless of the treatments, K values ranged from 52.1 to 59.7 mg g–1 dw, while Mn values ranged from 56.5 to 74.2 µg g–1 dw (Table 5). None of the treatments affected the concentration of P (averaging at 2.4 mg g– 1 dw). However, the Cu, Cu-VPH, and VPH+Cu-VPH treatments significantly increased the Cu concentration by 15.3-, 15.0-, and 7.8- fold, respectively, compared to the control (Table 5). The Cu-VPH treatment also significantly increased K (+14.3%), Mg (+18.0%), and Fe (+29.2%), while the VPH+Cu-VPH treatment significantly increased Ca (+24.7%) and Mn (+20.8%) compared to their respective control treatments. The VPH treatment significantly Frontiers in Plant Science 06 frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. TABLE 4 Aminogram, essential amino acids, branched-chain amino acid (BCAAs), and total amino acids of basil under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu- VPH) treatments. 3.5 Leaf mineral composition Treatments Control Cu Cu-VPH VPH+ Cu-VPH VPH Significance Ala 3.64 ± 0.34 b 5.41 ± 0.10 b 2.81 ± 0.39 b 9.68 ± 1.63 a 5.93 ± 0.73 ab *** Arg 0.908 ± 0.13 b 1.771 ± 0.08 a 1.436 ± 0.11 ab 0.959 ± 0.13 b 1.757 ± 0.22 a ** Asn 1.142 ± 0.226 b 0.970 ± 0.087 b 1.061 ± 0.087 b 2.634 ± 0.486 a 1.627 ± 0.204 ab ** Asp 0.513 ± 0.023 b 0.374 ± 0.041 b 0.410 ± 0.047 b 0.957 ± 0.137 a 1.018 ± 0.106 a *** GABA 1.825 ± 0.367 ab 2.666 ± 0.339 a 0.700 ± 0.126 b 2.816 ± 0.439 a 2.206 ± 0.374 ab ** Gln 2.028 ± 0.273 ab 1.356 ± 0.099 b 1.209 ± 0.234 b 3.619 ± 0.594 a 3.805 ± 0.532 a ** Glu 18.49 ± 1.17 ab 16.43 ± 1.85 ab 10.55 ± 0.68 b 20.15 ± 2.78 a 20.49 ± 2.06 a * Gly 0.511 ± 0.068 abc 0.443 ± 0.024 bc 0.275 ± 0.044 c 0.714 ± 0.055 a 0.685 ± 0.067 ab *** His 0.040 ± 0.006 b 0.165 ± 0.018 b 0.136 ± 0.014 b 0.369 ± 0.052 a 0.357 ± 0.040 a *** Ile 0.781 ± 0.099 ab 0.896 ± 0.048 ab 0.543 ± 0.036 c 1.155 ± 0.090 a 1.006 ± 0.125 a ** Leu 1.454 ± 0.130 ab 1.465 ± 0.082 ab 1.088 ± 0.096 b 1.850 ± 0.094 a 1.641 ± 0.186 ab * Lys 2.454 ± 0.068 2.411 ± 0.097 2.176 ± 0.221 2.318 ± 0.146 2.464 ± 0.057 ns MEA 0.919 ± 0.156 a 0.773 ± 0.052 ab 0.299 ± 0.034 b 1.207 ± 0.183 a 1.149 ± 0.163 a ** Met 0.028 ± 0.003 ab 0.033 ± 0.006 a 0.015 ± 0.002 b 0.031 ± 0.003 ab 0.031 ± 0.004 ab * Orn 1.108 ± 0.098 1.217 ± 0.125 0.904 ± 0.070 1.313 ± 0.099 1.214 ± 0.127 ns Phe 0.201 ± 0.028 bc 0.238 ± 0.004 bc 0.130 ± 0.013 c 0.442 ± 0.044 a 0.322 ± 0.066 ab ** Pro 2.584 ± 0.158 2.051 ± 0.055 2.261 ± 0.215 2.332 ± 0.107 2.067 ± 0.304 ns Ser 0.611 ± 0.048 bc 0.517 ± 0.049 bc 0.394 ± 0.048 c 0.897 ± 0.037 a 0.634 ± 0.055 b *** Thr 0.382 ± 0.054 ab 0.250 ± 0.021 b 0.166 ± 0.028 b 0.632 ± 0.095 a 0.465 ± 0.092 ab ** Trp 0.403 ± 0.083 ab 0.495 ± 0.002 ab 0.251 ± 0.026 b 0.752 ± 0.116 a 0.530 ± 0.101 ab * Tyr 0.354 ± 0.063 ab 0.338 ± 0.009 ab 0.218 ± 0.018 b 0.518 ± 0.057 a 0.407 ± 0.076 ab * Val 0.850 ± 0.174 ab 1.044 ± 0.057 ab 0.743 ± 0.038 b 1.471 ± 0.128 a 1.231 ± 0.204 ab * Essential AA 7.50 ± 0.74 bc 8.77 ± 0.28 abc 6.68 ± 0.54 c 9.98 ± 0.65 ab 10.14 ± 0.44 a ** BCAAs 3.08 ± 0.40 ab 3.40 ± 0.19 ab 2.37 ± 0.17 b 4.48 ± 0.29 a 3.88 ± 0.51 ab * Total AA 51.81 ± 4.34 bc 53.49 ± 2.12 ab 36.83 ± 2.72 c 67.94 ± 4.29 a 68.05 ± 3.02 a *** ns, *, **, and *** denote nonsignificant or significant at p ≤0.05, 0.01, and 0.001, respectively. Frontiers in Plant Science 3.5 Leaf mineral composition Different letters within each row indicate significant differences according to Tukey HSD test (p = 0 05) All d t d ± t d d 3 All d t d l –1 d ns, *, **, and *** denote nonsignificant or significant at p ≤0.05, 0.01, and 0.001, respectively. Different letters within each row indicate significant differences according to Tukey HSD test (p = 0.05). All data are expressed as mean ± standard error, n = 3. All data were expressed as µmol g–1 dw. 3.7 UHPLC-ESI/QTOF-MS metabolomic profile of basil 3.7 UHPLC-ESI/QTOF-MS metabolomic profile of basil frontiersin.org 3.7 UHPLC-ESI/QTOF-MS metabolomic profile of basil Cu-VPH- and VPH-treated samples—was created, indicating a more comparable metabolic profile in the basil plant after the two treatments. UHPLC-ESI/QTOF-MS untargeted metabolomics was carried out to understand the effect of the different treatments on the basil metabolome. Overall, more than 3,200 leaf compounds were putatively annotated and provided as Supplementary Material, together with individual abundances and composite mass spectra (Supplementary File 1). Successively, the supervised OPLS-DA multivariate analysis allowed us to better outline the differences between treatments and to highlight discriminant (VIP) compounds underlying these differences. The OPLS-DA score plot, provided in Figure 3, revealed distinct phytochemical profiles in basil leaves after treatments. In particular, the first latent vector discriminated VPH+Cu-VPH from the control, with the treated replicates being considerably separated from the untreated ones. This result indicated the more pronounced effect of VPH+Cu-VPH on metabolic reprogramming of basil leaves, compared to the other treatments. Differently, the second latent vector pointed out a differential profile of VPH from Cu-VPH and control. The OPLS model was validated through CV-ANOVA Based on these metabolomic signatures, unsupervised hierarchical clustering was first performed to describe similarities/ dissimilarities among the treatments. As shown in Figure 2, two main clusters were generated by the fold-change-based heatmap, one represented by Cu and VPH+Cu-VPH, and another by control, Cu-VPH, and VPH. Within this last one, a subcluster—containing 07 frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. 3.7 UHPLC-ESI/QTOF-MS metabolomic profile of basil Treatments P K Ca Mg Cu Fe Mn Zn mg g–1 dw mg g–1 dw Control 2.61 ± 0.11 52.10 ± 1.05 b 16.54 ± 0.62 b 3.20 ± 0.06 c 7.78 ± 0.65 c 34.06 ± 1.18 b 61.37 ± 0.54 bc 19.19 ± 0.59 ab Cu 2.41 ± 0.12 53.02 ± 1.29 b 16.76 ± 0.48 b 3.30 ± 0.12 c 118.66 ± 6.81 a 33.41 ± 1.58 b 66.10 ± 2.23 b 18.40 ± 0.55 b Cu-VPH 2.42 ± 0.14 59.59 ± 1.56 a 18.65 ± 0.58 ab 3.78 ± 0.05 ab 116.87 ± 8.78 a 44.01 ± 1.98 a 65.80 ± 1.99 b 18.76 ± 0.74 ab VPH+Cu-VPH 2.36 ± 0.01 57.73 ± 1.39 ab 20.63 ± 0.98 a 3.94 ± 0.13 a 60.66 ± 2.53 b 44.83 ± 2.76 a 74.17 ± 0.87 a 21.56 ± 0.77 a VPH 2.32 ± 0.07 59.72 ± 0.93 a 17.17 ± 0.16 b 3.50 ± 0.08 bc 6.98 ± 0.64 c 40.32 ± 1.20 ab 56.52 ± 0.66 c 16.83 ± 0.50 b Significance ns ** ** *** *** ** *** ** ns, **, and *** denote nonsignificant or significant at p ≤0.01 and 0.001, respectively. Different letters within each column indicate significant differences according to Tukey HSD test (p = 0.05). All data are expressed as mean ± standard error, n = 3. (p-value = 1.87E-15), and adequate accuracy parameters (R2Y = 0.99 and Q2Y = 0.88) were obtained. secondary metabolites, terpene and nitrogen-containing compounds were markedly modulated, followed by phenylpropanoid derivates. Notably, Cu elicited the highest accumulation of metabolites of each of these classes, reporting above all higher concentration of alkaloids [(S)-laudanosine, (S)- N-methylcoclaurine, 16-hydroxytabersonine, and galanthamine] and terpenoids, including carotenoids (lactucaxanthin, b- citraurin, and 9′-cis-neoxanthin), diterpenoids, and sesquiterpenoids (desoxyhemigossypol and 2-dehydrolubimin). Interestingly, increased levels of terpenoid phytoalexins (e.g., momilactone A and 2-dehydrolubimin) were noticed in Cu- treated samples. Nevertheless, an up-accumulation for both these two classes was observed with the other treatments, except for VPH, which exhibited a slight decrease of alkaloid and glucosinolate compounds (2-benzoyloxy-3-butenylglucosinolate and 2- sinapoyloxy-3-butenylglucosinolate). The list of VIP compounds, which mostly discriminated the treatments (VIP > 1.5), is provided in Supplementary File 1. Overall, isoprenoids were the most represented class of metabolites, mainly including carotenoids, sesquiterpenes, and gibberellins. Frontiers in Plant Science 3.7 UHPLC-ESI/QTOF-MS metabolomic profile of basil Finally, Volcano plot analysis (p-value < 0.05; fold change > 2) was carried out to select those compounds involved in basil plants’ response to the treatments. A total of 251 differential metabolites were identified and provided in Table S3 together with their function class. Therein, compounds related to secondary metabolite biosynthesis were the most represented in all treatments, followed by fatty acid and lipid biosynthesis-related metabolites. The graphical interpretation obtained with the PlantCyc pathway tool analysis allowed us to get insight into the metabolic pathways mostly affected in basil leaves (Figure 4). Notably, secondary metabolism was strongly modulated, showing an overall upregulation after each treatment. Among Differently, improved contents of phenylpropanoids were highlighted only after VPH and Cu treatments. However, the FIGURE 1 Principal component loading plot and scores of principal component analysis (PCA) of growth parameters, mineral elements, SPAD index, starch, soluble carbohydrates, polyphenols, and total amino acids in basil leaves untreated (Control) or foliarly treated with copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH). FIGURE 1 Principal component loading plot and scores of principal component analysis (PCA) of growth parameters, mineral elements, SPAD index, starch, soluble carbohydrates, polyphenols, and total amino acids in basil leaves untreated (Control) or foliarly treated with copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH). 08 Frontiers in Plant Science frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. FIGURE 2 Fold-change heatmap obtained from the unsupervised hierarchical cluster analysis (Euclidean distance; linkage rule: Ward) on UHPLC-ESI/QTOF-MS data of basil leaf chemical profiles under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments. FIGURE 2 Fold-change heatmap obtained from the unsupervised hierarchical cluster analysis (Euclidean distance; linkage rule: Ward) on UHPLC-ESI/QTOF-MS data of basil leaf chemical profiles under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments. total fresh and dry weight that increased the basil fresh yield. Probably, it was due to its higher carboxylation activity (ACO2) and maximum potential quantum efficiency of Photosystem II (Fv/ Fm) compared to all other treatments. 3.7 UHPLC-ESI/QTOF-MS metabolomic profile of basil It may be supposed that the VPH+Cu-VPH treatment improved the Cu use efficiency, as well as the Ca and Mn ones. In particular, it enhanced Cu and Fe integration as cofactors in thylakoid electron transporters like plastocyanin and Fe-proteins (e.g., cytochromes, Fe-S clusters, and ferredoxin), and that of Ca and Mn in the water splitting system of the photosynthetic electron transport chain, which became capable of accommodating faster and higher rates of electron flow under high light (Printz et al., 2016). Also, photochemical quenching saturated at higher irradiance in these Cu-sufficient plants, thus increasing the effective quantum yield of photosynthesis compared to other treatments, particularly to strongest modulation was shown with Cu, highlighting particularly enhanced concentrations of (2E,4Z,6E)-5-formyl-2-hydroxy-8- oxoocta-2,4,6-trienoate, (+)-sesaminol, apigeninidin, and (+)-6a- hydroxymaackiain. In contrast, Cu-VPH and VPH showed a differential pattern, showing a general downregulation of phenylpropanoid compounds. Following both treatments, decreased levels of 8-C-glucosyl chrysin, rutin, (+)-dihydrokaempferol, and diphyllin were observed. 4 Discussion Accordingly, in control, the highest levels of polyphenols were found, since they are a powerful antioxidant particularly efficient in scavenging ROS and counteracting oxidative stress damage (Caldwell et al., 1983). Nevertheless, considering that treatments with higher levels of phenols are also less productive, it is probable that both the control plants and the Cu-treated plants have bolstered secondary metabolic pathways at the expense of primary ones (Prinsloo and Nogemane, 2018; Ciriello et al., 2022b). Phenolic compounds have an antioxidant activity higher than vitamins since they can prevent ROS formation and improve antioxidant cell response due to their ability to chelate metal ions (Faraloni et al., 2021). The problem of excess light correlated to high polyphenols content was not present in the other plant treatments, which showed higher transpiration and carboxylation activity, but significantly lower polyphenols, except for Cu treatment, even if at a lower extent, but without negative consequences on plant fresh weight. Interestingly, VPH +Cu-VPH-, VPH-, and Cu-VPH-treated plants also showed a high SPAD index accompanied by the deepest coloration resulting from more intense yellowness (b* values; Supplementary Table S1) and the highest content of glucose and free amino acids. In particular, in VPH+Cu-VPH plants, the increase in the amounts of leaf glutamate, aspartate, and amides (glutamine and asparagine) evidenced not only a higher N use efficiency but also (i) a higher energy availability, probably deriving by the more efficient photosynthesis, and by the capacity to store nitrogen in glutamine and asparagine instead of soluble proteins, with a much lower energy expense (Diab and Limami, 2016), and (ii) the possibility to use these two amides as long-distance nitrogen transport molecules, given their high N/C ratio, to supply organic nitrogen to roots to be used as amine donor for the synthesis of all protein amino acids boosting root growth (Van Oosten et al., 2019). In contrast, the effect of Cu-VPH alone deserves particular attention. It determined the highest transpiration and the lowest WUEi and free amino acid levels, maintaining a high SPAD index, ACO2, Fv/Fm, mineral nutrients, and fresh yield. Noteworthy, among amino acids, GABA and MEA strongly decreased. It seems that GABA, which has a well-known antioxidant activity against ROS and is useful to stabilize and protect membranes and macromolecules, was not needed under Cu-VPH treatment (Carillo, 2018). 4 Discussion Remarkable changes in morpho-physiological and biochemical traits were observed under VPH+Cu-VPH treatments compared to other ones. VPH+Cu-VPH treatment showed the highest leaf and FIGURE 3 Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) score plot for basil leaf under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments. FIGURE 3 Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) score plot for basil leaf under untreated control, copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments. 09 frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. B A FIGURE 4 Leaf basil metabolic processes (A) and the relative details of secondary metabolism (B) as affected by copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments compared to untreated control. The compounds resulting from Volcano plot analysis (p value < 0.05; fold change > 0.01) were loaded into the PlantCyc Pathway Tool (https://www.plantcyc.org/). The x-axis represents each set of metabolic subcategories, while the y-axis corresponds to the cumulative log fold change (FC). The large dots represent the average (mean) of all FCs for the different metabolites in the class, while the small dots represent the individual log FC. A A B FIGURE 4 Leaf basil metabolic processes (A) and the relative details of secondary metabolism (B) as affected by copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH) treatments compared to untreated control. The compounds resulting from Volcano plot analysis (p value < 0.05; fold change > 0.01) were loaded into the PlantCyc Pathway Tool (https://www.plantcyc.org/). The x-axis represents each set of metabolic subcategories, while the y-axis corresponds to the cumulative log fold change (FC). The large dots represent the average (mean) of all FCs for the different metabolites in the class, while the small dots represent the individual log FC. control (Kim and Price, 2017). Instead, in the other treatments, particularly in control, the absorbed light energy might exceed the photosynthetic capacity, and over-reduce the photosynthetic electron transport chain, thus leading to the generation of reactive oxygen species (ROS) (Sperdouli et al., 2021). Frontiers in Plant Science frontiersin.org 4 Discussion VPH+Cu-VPH treatment increased, even if not significantly compared to the VPH one, the tryptophan content of plants, and determined an inhibition of stem elongation and an increase of compactness of plants, with a more equilibrated leaf enlargement (especially that of older leaves) and consequent increase of yield compared to all other differently treated plants (Figure 5). This is puzzling since Colla et al. (2014) demonstrated that VPH (Trainer®) was able to induce coleoptile elongation in detached corn (Zea mays L.) seedlings due to the high tryptophan content equal to 3 g kg–1 that exerted an auxin-like activity (Rouphael et al., 2018). However, tryptophan, which is the main precursor of indole- 3-acetic acid (IAA), not only can modulate shoot length, but also, in synergy with the peptides contained in VPH, can certainly contribute via auxin- and/or gibberellin-like activity, as shown in in vitro bioassays by Ertani et al. (2009) and Colla et al. (2014), to modulate/improve root architecture (Fukaki and Tasaka, 2009). This reshaping of root architecture has been correlated with an enhancement of N uptake and translocation known as “nutrient acquisition response” and can also be associated with the higher root assimilation of Ca, Mg, Mn, and Fe, and certainly also Cu, and their translocation to shoots of basil plants, as previously seen in Rouphael et al. (2017) and Carillo et al. (2019). This higher availability of N and other minerals can be responsible for the better leaf nutritional status and yield of VPH+Cu-VPH treatment. In particular, the higher availability of amino acids and Mg can contribute to the higher photochemical efficiency and activity of photosystem II (PSII), possibly due to the increase of photosynthetic proteins, particularly Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and their Mg-dependent activation, thus increasing CO2 assimilation (ACO2) and translocation of recent photosynthates via the phloem to sink tissues (Carillo et al., 2019). Therefore, the interaction between Consistently, untargeted metabolomics unraveled a distinctive reprogramming of basil leaf metabolome in response to the different treatments, generally displaying a remarkable modulation of secondary metabolite concentrations. The exposure of basil plant to Cu treatment induced significant changes in its alkaloid, terpenoid, and phenylpropanoid profiles, showing the most marked accumulation compared to the other treatments. This result can be interpreted as plant protective effect against physiological processes’ damage and oxidative stress, due to the excess presence of Cu. 4 Discussion This is also confirmed by the low levels of monoethanolamine (MEA), a serine derivative important for synthesizing or regenerating phospholipids (Carillo et al., 2020). This suggests that under Cu-VPH treatment, the higher Cu concentration, probably not yet toxic as in Cu treatment, can modulate plant metabolism in a way that photo-assimilates are promptly diverted to roots. In roots, N is mainly used to boost root growth and soil exploration, instead of remaining in leaves and being invested in proteins, particularly RUBISCO proteins that make up a large fraction of N in leaves. The higher amounts of water 10 frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. VPH and Cu-VPH exerted a positive effect on the electron transport chain, CO2 carboxylation, and translocation of newly synthetized soluble sugars to young leaves, decreasing the photooxidative damage, the consequent ROS production, and the need to synthesize polyphenols, while increasing N use efficiency, amino acid synthesis, and growth. This phenomenon was phenotypically exteriorized by the change in plant shape and higher crop yield. Moreover, when Cu is supplied by foliar spraying, high contents of free cytosolic Cu+ in leaves may cause a massive generation of ROS and oxidative stress or activate the programmed cell death (PCD). To prevent this copper-dependent oxidative burst, Cu+ must be chelated by intracellular cysteine-rich metallothioneins (MTs) or specific chaperones whose synthesis has a high energy cost, thus jeopardizing plant growth (Printz et al., 2016). In contrast, VPH peptides, particularly amino acids and small peptides that can be absorbed by plants (Colla et al., 2015), can complex not only Cu+, but also Cu2+, maintaining its redox capability, and facilitate Cu transport and allocation to Cu proteins, as also recently seen in medical studies on Cu-based chemotherapeutics (Peña et al., 2022). and mineral nutrients acquired exert a positive effect on plant metabolism, positively affecting processes such as chloroplast and mitochondrial electron transports, ROS detoxification, and redox status. However, since the high availability of water, Cu-VPH plants less strictly control transpiration, but without being able to further increase carboxylation activity; consequently, the lower ACO2-to- transpiration (E) ratio determines lower WUEi values compared to that of VPH+Cu-VPH (Liu et al., 2018). 5 Conclusions The widespread awareness of the impact of anthropogenic activities on the environment calls for careful and critical reflection on the agricultural sector’s future. The unconditional use of chemical fertilizers and pesticides is no longer sustainable, requiring growers to use innovative techniques that are increasingly eco-friendly. Cu, despite being a broad-spectrum fungicide and nutritionally essential element for plants, is a heavy metal that may accumulate and enter trophic chains. For this reason, its use with new formulations may be a necessary sustainable means of reducing dosages and increasing crop uptake and use efficiency. Regarding the mineral profile, the plants treated with Cu-VPH had the same copper concentration as those treated with inorganic copper. Still, at the same time, the use of Cu-VPH significantly improved the concentrations of K, Mg, and Fe, whereas VPH+Cu-VPH treatments, which showed half of leaf Cu concentration compared to Cu and Cu-VPH treatments, underwent a better Ca and Mn uptake capacity, and Cu and N use efficiency. It is likely that Cu integration as a cofactor in transporters of the photosynthetic electron transport chain, and that of Mn in the water splitting system, improved the maximum potential quantum efficiency of Photosystem II (Fv/Fm) and the electron transport flow under high light. At the same time, the carboxylation capacity of the plants and the synthesis and translocation of amides increased, thus enhancing the physiological and productive performance of Genovese basil and its fresh production compared to the other treatments and the untreated control. Finally, untargeted metabolomics pointed out increased concentrations of stress-related compounds (mainly alkaloids, terpenoids, and phenylpropanoids) following Cu supply, differently from VPH-Cu, VPH, and VPH+Cu-VPH treatments, which showed low levels of these compounds. This suggested the VPH ability to alleviate the negative effect of oxidative stress due to plant exposure to substantial amount of Cu. Therefore, in light of the promising results obtained in this study and with a view to enhancing agricultural sustainability, producers should promote the use of complex micronutrients combined with biostimulants. Likewise, plant production of terpenoids with antioxidant activities to cope and overcome abiotic stress is increasingly described in the scientific literature (Toffolatti et al., 2021). When exposed to the above threshold concentration of trace metals, plants can modify metabolic processes by synthesizing them (Anjitha et al., 2021). Kunwar et al. 5 Conclusions (2015) accounted significant changes in basil terpenoid composition by the addition of Cu, showing improved concentrations of linalool, chavicol, and methyl cinnamate when compared to the control (Kunwar et al., 2015). Interestingly, Hojati et al. (2017) investigated the response of Tanacetum parthenium L. to copper chloride dihydrate (CuCl2·2H2O) by associating stress-related responses with changes in terpenoids: low Cu (5 mM) supply increased the proportion of sesquiterpenes, both hydrocarbons and oxygenated, while elevated Cu levels reduced the total content of isoprenoids, by depressing the uptake of divalent cations, which are essential cofactors of enzymes involved in the biosynthesis of these metabolites. In contrast, VPH-, Cu-VPH-, and VPH+Cu-VPH-treated plants displayed lower contents of alkaloids and terpenoids. Overall, we infer that the lower presence of these compounds following VPH supply is related to healthier plants with reduced levels of ROS. These results are in accordance with the recent findings of Sorrentino et al. (2021) where Trainer® VPH treatment on Arabidopsis thaliana showed a clear downregulation of terpenoid compounds (Sorrentino et al., 2021). Likewise, Lucini et al. (2020) pointed out a general decrease of defense compounds, including alkaloids and terpenoids following the application of the same VPH on Solanum lycopersicum L. (Lucini et al., 2020). Moreover, it is interesting to note that phenylpropanoid metabolites were markedly accumulated only in Cu-treated samples, since VPH+Cu-VHP induced just a weak increase and noteworthy downregulations were observed with VPH and Cu- VPH treatments. It is proven that the phenylpropanoid biosynthetic pathway is triggered under heavy metal stress, leading to the accumulation of phenolic compounds with ROS scavenging capacity (Park et al., 2022). Mira et al. (2002) extensively described flavonoid ability to exert pro-oxidant effects under Cu metal stress through the metal chelating process. This clearly explains our finding that the levels of phenylpropanoid and flavonoid compounds were found to be increased by Cu excess. 4 Discussion Several studies on many other plant species (Brassica napus L., Brassica juncea L., Cucumis sativus L., Oryza sativa L., and Coriandrum sativum L.) showed that plant exposure to substantial amount of Cu may result in increased ROS production—hydrogen peroxide (H2O2), superoxide anion radicals (O2−), and hydroxyl radicals (OH−)—causing damage to cellular constituents (Shaw and Hossain, 2013; Sunita and Shekhawat, 2016; Nair and Chung, 2017; Mosa et al., 2018; Alquraidi et al., 2019). ROS produced may cause lipid peroxidation, stimulating the production of highly active signaling compounds competent of triggering the production of secondary metabolites in the plant (Anjitha et al., 2021). FIGURE 5 Illustrative picture of basil plants untreated (Control) or foliarly treated with copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH). FIGURE 5 Illustrative picture of basil plants untreated (Control) or foliarly treated with copper sulfate (Cu), copper complexed with amino acids and peptides (Cu-VPH), vegetal protein hydrolysate (VPH), and Cu-VPH enriched with VPH (VPH+Cu-VPH). 11 11 Frontiers in Plant Science Frontiers in Plant Science frontiersin.org Rouphael et al. 10.3389/fpls.2023.1235686 Rouphael et al. In this regard, it is well known that alkaloids represent key components of the plant defense system, being released by the plant to face stress conditions. In particular, the antioxidant activity of alkaloids includes the upregulation of ROS scavenging, the inhibition of ROS-producing enzymes, and the chelation of metals (Yoon et al., 2006). Lala (2020) reported a significant increase in the total content of alkaloid metabolites following Cu- based treatment in Bacopa monnieri L., accomplished by an increase in ROS markers. Moreover, enhanced production of alkaloids was found in O. basilicum L. and Catharanthus roseus L., respectively, following copper sulfate (CuSO4) and copper oxide (CuO) treatments (Pan et al., 2015; Nazir et al., 2021). Our results are in accordance with previous reports in which CuO nanoparticles enhanced total flavonoid content in Stevia rebaudiana, Brassica nigra L., and O. basilicum L. (Zafar et al., 2017; Javed et al., 2018; Nazir et al., 2021). Moreover, our study is coherent with the previous work of Sorrentino et al. (2021), which ascribed to the same VPH a significant downregulation of flavonoid compounds. 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This manuscript reflects only the authors’ views and opinions; neither the European Union nor the European Commission can be considered responsible for them. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1235686/ full#supplementary-material Funding All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. This study was carried out within the Agritech National Research Center and received funding from the European Union Next-Generation EU (PIANO NAZIONALE DIRIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022). This research work was partially funded by Università degli Studi della Campania Luigi Vanvitelli in the frame of project 'VAnviteLli pEr la RicErca (VALERE)'. This research work was partially funded by MUR in the frame of the project ‘Digitali, Intelligenti, Verdi e Sostenibili (D.I.Ver.So)’ managed by DAFNE of Tuscia University. Data availability statement The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. Frontiers in Plant Science 12 frontiersin.org 10.3389/fpls.2023.1235686 Rouphael et al. 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PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.
Journal of the American Medical Association
1,904
public-domain
8,170
PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. The group of twenty-five cases reported in this paper represents about one-half of the private and hospital cases which have come under my observation during the past ten years. Unfortunately, the records of a number of cases are so faulty as to render them unavailable for careful analysis. In presenting these reports the aim is made to consider the disease from each of three stand¬ points : First, the general condition of the patient ; sec¬ ond, the gastric digestion ; third, the blood. y j Third, in a few cases where the anemia was marked, there was not found a complete achylia gastrica, but merely hypochlorhydria. Since Austin Flint's original observation that there is an absence of gastric secretion in pernicious anemia, this relation has been constantly recognized; but it is apparent that the failure of gastric juice is the result of depression in the blood. It is not a cause of pernicious anemia, but, on the other hand, in these cases pernicious anemia is the cause of achylia gastrica. In most cases of achylia gastrica the motor activity of the stomach is exaggerated, as a result of which the stomach usually empties itself more quickly than normal. This rule holds true in those cases which result from pernicious anemia, but, according to my ob¬ servation, not so often as in other cases of achylia gas¬ trica. This is probably true for the reason that with the failure in the blood there comes about a failure in the strength of the muscular coat of the stomach, and in some cases there is the development of a real gastric atony and sometimes gastritis. Perhaps for this reason some patients complain of food stagnation and anorexia —conditions which are rarely observed in achylia gas¬ trica from other causes. In some of the cases reported in this paper diarrhea was an occasional and troublesome symptom. It appears to me that it occurs less often in this group than in the same number of cases of achylia gastrica occurring without pernicious anemia. PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. When present it is usually associated with excessive gastric motility and the disturbance of intestinal peristalsis by the untimely emptying of the stomach onward ; also, the exaggerated motor impulse which empties the stomach is apparently carried over to the intestine, which influ¬ ences result in the appearance of frequent liquid stools showing undigested food substances. , gast c digestion ; , The stomach examinations, so far as relates to the chemistry, were made mostly by Dr. Allen Jones; the blood examinations by Dr. A. E. Woehnert. Nearly all of these cases were referred to me for diagnosis, and in· many it became impossible to follow systematically the subsequent state of the digestion. As will be seen in the history of individual cases, the patient almost uniformly complained of dyspnea, weakness and digestive dis¬ turbance, sometimes relating to the stomach, at other times to the intestines. All of them presented the pic¬ ture of marked pallor, associated with a more or less marked lemon-colored tint of the skin, differing from jaundice, and yet in some instances strongly suggesting it. In most the loss of weight was not great. In almost all cases the tongue was strikingly pale, usually showing a loss of epithelium, and therefore not coated, or but slightly so. A proportion of the cases suffered from marked gastric distress; others complained of no stom¬ ach trouble. In some there was anorexia, but in most there was a fairly good appetite, and in a few instances the appetite was increased. In all the gastric digestion was greatly depressed, and in the majority there was complete achylia gastrica, no appreciable digestion tak¬ ing place in the stomach. A number of times I have seen moderate improvement in digestion at intervals corresponding with improvement in the state of the blood, but this does not apply to the secretion of gastric juice. In those cases of pernicious anemia in which the secretion of hydrochloric acid has disappeared, I have not seen it return, even when gastric motility has be¬ come apparently normal. The cases in which complete achylia gastrica was present did not differ, so far as the stomach examinations are concerned, from the ordinary type of achylia gastrica. From a careful history of these cases I am convinced that the achylia gastrica is not an etiologic factor in the development of pernicious anemia. *Read at the Fifty-fifth Annual Session of the American Med- ical Association, in the Section on Practice of Medicine, and ap- proved for publication by the Executive Committee : Drs. J. M. Anders, Frank Jones and W. S. Thayer. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User on 05/25/2015 I have observed, not in the gastric digestion. Possibly, if all cases had been studied with this in view for a pro¬ longed period, some instances of improved gastric secre¬ tion, corresponding with betterment in the blood, might have been noted. However, in a few cases in which the condition of the stomach has been ascertained at regular intervals during several years, no change for the better was found, although at times the blood has made re¬ markable improvement. Dr. Frank . Wynn, Indianapolis—Answering Dr. Bergey's question, so far as organic changes are concerned, there were none observable. I mentioned in the paper symptomatic evi¬ dences of failure in nutrition in the animals, loss of appetite and of playfulness. The two groups were near at hand and could be compared with each other. The .experimental rabbits were not frolicking about as the other animals were, and most of all, perhaps, the physical evidence of failure of nutri¬ tion was most clearly manifest in the loss of luster of the hair. p Second, in a large number of cases of achylia gastrica which I have studied, many of which have been under observation' during periods of several years, there has not been found in a single instance a change in the blood that was suggestive of pernicious anemia. In several cases of achylia gastrica, associated with diarrhea, there sometimes develops a severe secondary anemia, but the characteristics of pernicious anemia have been found wanting. In this respect my experience agrees with that of Riegel, Einhorn, and most others who have care¬ fully studied the two subjects. PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. While I hoped for a return of the combined chlorids during these periods of im¬ provement in the patient, I have been so far disappointed. Starch digestion is moderately well performed when the pa¬ tient is doing well, but even this is greatly diminished when the blood count is very low. y Following this is a brief statement of each of the re¬ ported cases. A report of the study of the stomach ex¬ aminations is inserted, and, as will be seen, they repre¬ sent practically what is found in ordinary cases of gas¬ tric anacidity. I very much regret that the records of some of the stomach examinations have been lost, and the paper is more incomplete in this connection than I anticipated ; nevertheless, the uniformity of the results is so evident that I feel the same conclusion may be drawn from these reports that would follow a much longer series. o ge Most of the patients had an incorrect idea as to the nature of their disease, some complaining of the liver, others of cancer of the stomach, heart disease, locomotor ataxia, and so on. g Most of the patients had an incorrect idea as to the nature of their disease, some complaining of the liver, others of cancer of the stomach, heart disease, locomotor ataxia, and so on. Case 1.—G. K., German, aged 47, carpenter, married, fam¬ ily history negative; personal history negative until the begin¬ ning of the present illness, two years ago. Patient is weak, short of breath, has tinnitus aurium, numbness of the ex¬ tremities, slow reflexes, gaseous pulse. First sound of the heart at apex is weak, with slight systolic bruit; aortic sound exaggerated; arteries sclerosed moderately; capillary circula¬ tion poor; skin pale, lemon tint; appetite fair, afraid to eat because of subsequent distress; tongue pale, shiny and tender; spleen not palpable. Treatment, Donovan's solution, etc. Im¬ provement wonderful on two occasions. Died after an illness of four years. y Blood.—Red cells, 1,021,000; large number of megalocytes, few megaloblasts and normoblasts; hemoglobin, 26 per cent.; leucocytes, 3,200; polymorphonuclear neutrophiles, 60 percent. y p y p p Case 6.—A. M., aged 38, laborer. Mother died at 45 of un¬ known trouble, one brother from hemoptysis, one sister from hemoptysis, one of Bright's disease, and the third from sudden failure of the heart. The patient's previous history has been good. PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. Complained that for some months he had been weak, and of late had shortness of breath; distress after eating, although he had a good appetite; lost somewhat in flesh, exact amount unknown. His pulse was weak and gaseous; arteries very com¬ pressible; heart rather enlarged; apex diffuse; hemic bruit heard over preeordium; also bruits de Diable; râles at bases of both lungs; tongue pale, epithelium largely absent; bowels constipated; liver large, one inch below ribs; spleen and lymph glands normal. Patient's skin was dry, scaly, with bran-like exfoliations and characteristic lemon-colored tint. A letter from the patient March 28, 1903, reports that he had been at work since May last without the loss of a day and appears to be in fair health. y Blood.—Red cells, 980,000 ; hemoglobin, 25 per cent. ; megalo- blasts, megalocytes, microcytes, poikilocyt.es marked; hemo¬ globin index high; leucocytes, 3,300. Case 2.—S. J. M., American, aged 45, physician. Family history: Mother probably died of tuberculosis; in other re¬ spects negative. Patient always well until present illness. Had great mental strain for a year, followed by weakness, dyspnea, and later by digestive disturbances. Pulse frequent, gaseous; heart normal in size, systolic bruit at apex, bruits de Diable. Appetite fair, tongue pale, bowels loose, often diar¬ rhea ; skin lemon tint and pale ; occasionally slight cough ; lungs normal ; spleen not palpable. Three periods of marked improvement. Died two years and a half after beginning of illness. Blood.—Red cells, 1,199,000; hemoglobin, 23 per cent.; many microcytes, poikilocytes and megalocytes; normoblasts and megaloblasts. Leucocytes, 8,000; 50 per cent, polymorphonu¬ clear neutrophiles. Blood.—Red cells, 1,130,600; hemoglobin, 25 per cent.; ma- crocytes, microcytes and poikilocytes were numerous; megalo- blasts and normoblasts; leucocytes, 2,933; polymorphonuclear cells, 84 per cent. p Case 7.—S. F., patient of Dr. Roos, Wellsville, N. Y., aged 60, merchant; negative family history; suffered from typhoid fever 25 years ago, subsequently well until 12 years ago, when he had a decline and gave up business. He improved again and was fairly well. Five years ago had what was apparently an epileptic seizure; since then has had several such attacks. Two months ago began to suffer greatly with his stomach, nausea, distress especially after eating, occasionally followed by vomiting. PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. There have been four distinct relapses, followed by striking improvement in general health, still at no time has there been any evidence of gastric juice. The gastric motility has varied markedly with the condition of the blood. When the anemia is at its worst the stomach is found to empty itself almost immediately after eating, but when the blood is better the stomach retains the food for a longer period, and with this return of a more natural gastric motility the general nutrition improves. What rela¬ tions exist between these facts I am unable to say, but it seems probable that the intestine is better prepared to carry on the digestion when the stomach discharges itself more slowly, and, therefore, when the intestinal peristalsis, especially in the upper part of the tract, is less disturbed. While I hoped for a return of the combined chlorids during these periods of im¬ provement in the patient, I have been so far disappointed. Starch digestion is moderately well performed when the pa¬ tient is doing well, but even this is greatly diminished when the blood count is very low. p p Case 5.—-Mrs. W. D. R., American, aged 45, patient of Dr. Benninghoff of Bradford, Pa., suffers from weakness, anorexia, dyspnea. A remarkable case, which I have studied for five years, and which is still under observation ; has shown great oscillations in the blood counts—from a few over one million at the lowest to over four millions at the best, the hemoglobin ranging from 26 per cent, to 92 per cent. There have been four distinct relapses, followed by striking improvement in general health, still at no time has there been any evidence of gastric juice. The gastric motility has varied markedly with the condition of the blood. When the anemia is at its worst the stomach is found to empty itself almost immediately after eating, but when the blood is better the stomach retains the food for a longer period, and with this return of a more natural gastric motility the general nutrition improves. What rela¬ tions exist between these facts I am unable to say, but it seems probable that the intestine is better prepared to carry on the digestion when the stomach discharges itself more slowly, and, therefore, when the intestinal peristalsis, especially in the upper part of the tract, is less disturbed. PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. My reasons for reaching this view are : First when in g g I am somewhat surprised, in reviewing these cases, to find so little evidence of marked cardiac disease. In those engaged in heavy manual work the heart was usually slightly dilated, and sometimes there was a rela¬ tive mitral insufficiency, hemic bruits were often heard, and sometimes bruits de Diable, but the latter I have not found so commonly present as in chlorosis. There has been found no constant change in the liver, spleen or lymph glands. Often the liver has been found slightly enlarged, sometimes markedly so ; the spleen sometimes increased in bulk, but rarely very large. No other strik¬ ing features have been found present with sufficient fre- quencv to warrant notice, with the exception of symp¬ toms of spinal cord disease. Since Billings called atten- et o og c p p My reasons for reaching this view are : First, when in a given case the blood shows marked improvement for several weeks together, there usually appears evidences of improvement in the general nutrition, but, so far as number of microcytes, poikilocytes and megalocytes, no nor- moblasts; an occasional megaloblast; leucocytes, 3,800; poly¬ morphonuclear cells, 68 per cent. number of microcytes, poikilocytes and megalocytes, no nor- moblasts; an occasional megaloblast; leucocytes, 3,800; poly¬ morphonuclear cells, 68 per cent. tion to its frequency I have found it in the majority of cases. The personal history of the patient is remarkable in the fact of the absence of previous severe illness. Most of the patients insist that they had never before been seriously ill. The family histories were negative, so far as relate to pernicious anemia, and I do not know of an instance in which it has occurred in the second generation. The results of study of the urine have not been very notable in these cases. p p Case 5.—-Mrs. W. D. R., American, aged 45, patient of Dr. Benninghoff of Bradford, Pa., suffers from weakness, anorexia, dyspnea. A remarkable case, which I have studied for five years, and which is still under observation ; has shown great oscillations in the blood counts—from a few over one million at the lowest to over four millions at the best, the hemoglobin ranging from 26 per cent, to 92 per cent. PERNICIOUS ANEMIA AND ITS RELATION TO GASTRIC DIGESTION, BASED ON TWENTY-FIVE CASES.* CHARLES G. STOCKTON, M.D. BUFFALO, N. Y. He now complains of great weakness; has a lemon tinted skin, suffers from nausea ; the pulse is very weak ; ar¬ teries not deeply sclerosed; heart dilated, with musical sys¬ tolic bruit at apex; tongue pale; bowels constipated; liver large palpable below ribs; spleen and lymph glands negative. Former weight 150, now 138 pounds. A letter, March, 1903, reports that the patient was still living and in fair health. p Case 3.—A. S., German, aged 40, engineer. Good family history. The present is the patient's first illness ; began a year ago, gradually losing strength ; is now very weak, short of breath; digestion disturbed; pulse gaseous, weak; heart nega¬ tive ; anorexia ; tongue pale ; bowels constipated ; skin very pale, lemon tint; liver and spleen negative; one period of marked improvement. Died after two years' illness. p y Blood.—Red cells, 1,366,000; hemoglobin, 35 per cent.; macrocytes in great number ; microcytes, poikilocytes marked ; leucocytes, 2,400 ; polymorphonuclear cells, 54 per cent. y p y p p Case 4.—J. M.. American, farmer, aged 70. Family history good. Present illness began eight months ago. He was pale, weak, had dyspnea; pulse weak, somewhat gaseous; heart sounds weak, arteries sclerosed; appetite good; tongue pink; digestion bad; bowels loose: skin pale, sallow, sodden; liver and spleen negative, reflexes active. Made no improvement. Died a year after the beginning of the illness. p p g Blood.—Red cells, 1,058,000 ; hemoglobin, 45 per cent. ; megalocytes, microcytes, normoblasts; leucocytes, 8,400; poly¬ morphonuclear neutrophiles, 66 per cent. p p , Case 8.—Captain S., patient of Dr. C. W. Howe, lumber dealer, aged 70; served three years in Civil War; has suffered from rheumatic arthritis ; for five years has had much disturb¬ ance with stomach, much worse for past two years; for a y g g Blood.—Red cells, 1,248,000; hemoglobin, 30 per cent.; large y g g Blood.—Red cells, 1,248,000; hemoglobin, 30 per cent.; large aded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User on 05/25/2015 Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o The pulse was weak and frequent; heart not indicative; blood vessels normal; liver and spleen negative; tongue almost col¬ orless; appetite poor; bowels irregular. She died in Febru¬ ary, five months after being discharged from the hospital after an illness of about three years. g Blood.—Red cells, 1,250,000"; hemoglobin, 40 per cent.; gigantoblasts, normoblasts, microcytes; poikilocytes marked; leucocytes, 4,000; polymorphonuclear neutrophiles, 70 per cent. Case 11.—B. P. B., aged 42, caterer, married; claims to have been well until four weeks ago, then began to suffer from progressive weakness and shortness of breath on exertion. No emaciation; marked lemon-tinted skin; tongue pale; appetite poor; bowels loose; pulse gaseous, frequent; apex of heart at the nipple line; systolic bruit at apex and heard in axilla; liver and spleen normal. Case 11.—B. P. B., aged 42, caterer, married; claims to have been well until four weeks ago, then began to suffer from progressive weakness and shortness of breath on exertion. No emaciation; marked lemon-tinted skin; tongue pale; appetite poor; bowels loose; pulse gaseous, frequent; apex of heart at the nipple line; systolic bruit at apex and heard in axilla; liver and spleen normal. Case 11.—B. P. B., aged 42, caterer, married; claims to have been well until four weeks ago, then began to suffer from progressive weakness and shortness of breath on exertion. No emaciation; marked lemon-tinted skin; tongue pale; appetite poor; bowels loose; pulse gaseous, frequent; apex of heart at the nipple line; systolic bruit at apex and heard in axilla; liver and spleen normal. Blood.—Red cells, 949,333 ; hemoglobin, 35 per cent. ; mega¬ locytes, poikilocytes marked, normoblasts and megaloblasts; leucocytes, 3,240; polymorphonuclear neutrophiles, 63 per cent. Blood.—Red cells, 1,118,000; hemoglobin, 25 per ent. ; mi¬ crocytes, macrocytes, poikilocytes present; leucocytes, 6,000; polymorphonuclear neutrophiles, 75 per cent. p Blood.—Red cells, 949,333 ; hemoglobin, 35 per cent. ; mega¬ locytes, poikilocytes marked, normoblasts and megaloblasts; leucocytes, 3,240; polymorphonuclear neutrophiles, 63 per cent. p Blood.—Red cells, 949,333 ; hemoglobin, 35 per cent. ; mega¬ locytes, poikilocytes marked, normoblasts and megaloblasts; leucocytes, 3,240; polymorphonuclear neutrophiles, 63 per cent. p y p p Case 16.—B. W., German, aged 42, indoors, machinist; fam¬ ily history negative; personal history good; worked in an ice¬ house for 15 years; lost slightly in weight; present illness began two months ago with weakness, dyspnea on exercise, biliousness and slight edema of the ankles. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o He died in 1902, two and a half years after beginning of illness. y g g Blood.—Red cells, 1,100,000; hemoglobin, 40 per cent.; megalocytes, numerous; megaloblasts, 1; leucocytes, 4,463; polymorphonuclear neutrophiles, 46 per cent. y g g Blood.—Red cells, 1,100,000; hemoglobin, 40 per cent.; megalocytes, numerous; megaloblasts, 1; leucocytes, 4,463; polymorphonuclear neutrophiles, 46 per cent. p y p p , p Case 9.—C. J. M., aged 33, farmer; family history negative; patient had no previous illness; complained of weakness, dysp¬ nea, faintness; pulse weak, gaseous; has hemic bruit over the heart; arteries very compressible; tongue is denuded of epi¬ thelium, pale; appetite capricious; bowels irregular with oc¬ casional diarrhea; skin lemon tint; liver and spleen negative. Dr. Shugert, of Tidioute, Pa., his physician, writes me that three months after the patient's visit to me he had so far improved as tó resume his work, and that he has continued, with slight interruptions, to work ever since that time, and seems to be in very good condition. His work is out-of-doors, quite laborious, and he walks several miles a day. His color, June, 1904, is good, and the man is at work. g , Blood.—Red cells, 2,118,400; hemoglobin, 51 per cent. Large number of microcytes, poikilocytes and megalocytes ; one mega- loblast; leucocytes, 12,334; polymorphonuclear neutrophiles, 55 per cent. p Case 10.—H. B., English, aged 46, plumber, married; family history good; never sick until present trouble; complained of his stomach first one year ago; had chills with fever several days in succession eight months later. Present condition, weak, cachectic, losing flesi,, dyspnea; pulse gaseous; heart of normal size; pulmonary second sound exaggerated; appe¬ tite poor; tongue pale; bowels irregular (constipation or diar¬ rhea) ; skin pale, sallow, with lemon tint; spleen just palp¬ able; mentality good; sleep undisturbed. Had several peri¬ ods of improvement, some of them quite remarkable. Treat¬ ment, Donovan's solution, etc.; died three years after begin¬ ning of illness. y p y Blood.—Red cells, 1,700,000; hemoglobin, 33 1/3 per cent.; megaloblasts, normoblasts, macrocytes, microcytes, poikilo¬ cytes; some leucocytosis ; polymorphonuclear neutrophiles, 38 per cent. Case 15.—Mrs. I. J. S., patient of Dr. C. A. Ellis, Sherman, N. Y., aged 34. Patient had been ill for two and a half years, sometimes showing betterment, followed by periods of depres¬ sion. Her phief symptoms were weakness and dyspnea. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o The-e was a marked lemon-colored tint to the skin; tongue very smooth, with absence of papillae; muscles flabby; pulse weak, small; lungs normal ; heart of normal size, systolic bruit at apex ; right heart slightly dilated, slight bruit de Diable; liver con¬ siderably enlarged; spleen palpable. Case 12.—J. K., telegraph operator, aged 33, single; present illness began within six months with weakness and dyspnea; marked pallor, with lemon-colored tint to skin; tongue pale; appetite fair; bowels very loose, with yeasty, dark colored stools; patient appears well nourished; pulse soft, about 100; temperature, 101 degrees; apex of heart diffused; systolic bruit heard over the base of the heart, with a loud venous hum of the vessels of the neck; liver a trifle full; spleen normal; reflexes somewhat exaggerated. y g ; p p p Blood.—Red cells, 1,560,000; hemoglobin, 30 per cent.; a few normoblasts; leucocytes, 2,600. y , Case 17.—J. L., aged 49, laborer; family history negative; patient has handled nitroglycerin for 25 years; no illness save that two years ago he froze his hands and feet while shoveling snow; has not been well since; complains of dyspnea on exercise; great weakness; numbness of extremities; lost 30 pounds in weight; has poor appetite; no cough or hemorrhage; heart of normal size, apical systolic bruit, no bruit de Diable; liver slightly enlarged, two inches below ribs; spleen not pal¬ pable; skin sallow and pigmented; tongue smooth and pale; bowels constipated. y Case 17.—J. L., aged 49, laborer; family history negative; patient has handled nitroglycerin for 25 years; no illness save that two years ago he froze his hands and feet while shoveling snow; has not been well since; complains of dyspnea on exercise; great weakness; numbness of extremities; lost 30 pounds in weight; has poor appetite; no cough or hemorrhage; heart of normal size, apical systolic bruit, no bruit de Diable; liver slightly enlarged, two inches below ribs; spleen not pal¬ pable; skin sallow and pigmented; tongue smooth and pale; bowels constipated. gg Blood.—Red cells, 1,127,000; hemoglobin, 31 per cent; ma- crocytes, microcytes and poikilocytes marked; normoblasts present; leucocytes, 3,900; polymorphonuclear neutrophiles, 75 per cent. Case 13.—-J. D., aged 40, laborer; duration of illness not known; complained of extreme weakness and digestive dis¬ turbance; some dyspnea; has marked lemon tint to skin. Case 13.—-J. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o Case 14.—S. 0., Freedom, . ., aged 46, excellent family history; patient had pneumonia when he was 21 years old, otherwise perfectly well until a year ago, when his health gradually declined; he complained of weakness, shortness of breath, disturbance of the stomach; pain above the pubes, re¬ lieved by the action of the bowels. His pulse was extremely weak, arteries thickened, the heart slightly enlarged, systolic bruit at the apex, the pulmonary second sound exaggerated. His appetite was good, at times craving; tongue smooth, de¬ nuded of epithelium, pale; bowels very constipated; skin pale, almost lemon tint; liver large, extending 1% inches below the ribs; spleen and lymph glands negative; patient weighed 155 pounds, now 137. Patient was given the bichlorid of mercury for two months and made marked improvement; he had com¬ plete achylia gastrica. Two months later he was given potas¬ sium iodid; he suffered greatly from proctitis. After six months patient was not so well. During the following year he again made improvement under the influence of bichlorid of mercury, nux vomica and iron, and on the 9th of January, 1895, he was discharged as cured. During the following sum¬ mer, 1895, he returned with all his former symptoms. This time the blood showed the characteristics of pernicious anemia. An incomplete examination made two years before showed 35 per cent, hemoglobin, the red corpuscles very large and de¬ formed. This case possesses special interest for the reason that after passing through my hands he was treated by an¬ other physician, who claimed to have found the ova of anky- lostoma, and furthermore he reported that the patient made great improvement under treatment by thymol. The stools were examined by Dr. Woehnert, who was unable to find the ova of ankylostoma, and the blood showed no eosinophilia. The case may be put down as not being one of ankylostomiasis. The patient died in 1900, eight years after the beginning of the attack, after at least three periods of distinct improve¬ ment. Not under my observation for the past three years. year has vomited at times excessively; none for past three months; suffered from exaggerated salivation; suffers most from eating solid food; has weakness and dyspnea; is pale; has arteriosclerosis ; pulse of fair tension ; heart slightly large ; aortic second sound exaggerated; tongue coated and flabby; bowels constipated; liver and spleen negative. p Blood.—Red cells, 1,150,000; hemoglobin, 50 per cent. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o He was very weak, but finally made great improvement and went to work. A year ago he had a second severe illness, with great weakness, lemon-colored tint and bleeding from hemor¬ rhoids; marked anasarca lasting two or three months. This again disappeared and the man was able to return to work. During each of these attacks the liver was large. The present attack occurred in September last; became yellow, had chills and fever, great weakness; became nervous with dyspnea on exercise; bleeding from the hemorrhoids and moderate ana¬ sarca. Pulse was weak and gaseous; temperature from 98 to 102; respirations, 24 to 26. Heart was moderately dilated, systolic bruit at the apex and over pulmonary areas. Lungs were normal ; arteries slightly sclerosed ; had paresthesia in both legs, some weakness in gait; appetite was poor, with dis¬ tress after eating; tongue was smooth and shiny; liver was very large, as also was the spleen; bowels were loose; absence of ova of parasites. Case 22.—W. O'D., aged 48, lumberman ; family history neg ative; personal history, had been well previous until two years ago, when he began to suffer from weakness, with vomiting, dizziness, dyspnea on exercise, edema of the extremities and numbness of the legs and feet. Had a marked lemon-colored tint; pale tongue; slightly enlarged liver; bowels loose. His heart was slightly dilated, pulse weak, but not gaseous. He had had one period of improvement. The urine showed a few hyaline casts, a trace of albumin; otherwise negative; stomach contents showed complete achylia gastrica. p y g Blood.—Red cells, 755,300; hemoglobin, 25 per cent; there were many macrocytes, microcytes, normoblasts and megalo¬ blasts; leucocytes, 5,200; the white cells showed 73 per cent, of the polymorphonuclear form. p y p Case 23.—Mrs. T. H. R., aged 41, farmer's wife; patient of Dr. Rich, Kennedy, N. Y. Family history good ; personal his¬ tory good; never been sick until present illness. Trouble be¬ gan one year ago, suffering from weakness, dyspnea on exer¬ cise, some dizziness, but especially from paresthesia and some anesthesia of the extremities ; some difficulty in walking. Ap¬ petite was good; pulse very weak, not gaseous. SUMMARY. Of the 24 cases here reported, the youngest was 32. the oldest 70 ; average, 45 years + ; there were 20 males and 4 females; 13 had outdoor occupation and 11 in¬ door ; the longest duration of illness was in one 5 years, the shortest 6 months, and fatal; 12 are known to be dead, 5 living, the others unknown; all had dyspnea on exercise; all suffered from faintness and weakness; 16 had gaseous pulse, 8 did not; 16 had heart slightly en¬ larged, 8 did not; 16 had systolic apical bruit, in 8 it was absent; 6 had bruit de Diable, in 18 it was absent; 8 had other bruit, 16 had no other; 22 had a distinct lemon-colored tint of the skin, in 2 it was absent ; 15 had the tongue strikingly denuded of epithelium, 9 did not ; 10 had good appetite, 7 capricious, and 7 anorexia; 18 complained of symptoms of stomach trouble, 6 did not ; 10 had constipation, 8 diarrhea, and 6 irregularity of the bowels; in 11 the liver was enlarged, in 13 it was not enlarged, small in none ; 1 had four periods of improve¬ ment, 3 two periods, 8 one period, and 8 no period of improvement. After my attention was called to the sub¬ ject by Billings' paper, I found signs of spinal cord in¬ volvement in 6 out of 9 cases. Nine cases showed slight dilatation of the stomach; in 13 the gastric digestion was absent, in 8 it was very low, in the 3 others it was fairly good. Fnye showed evidences of gastric catarrh ; p Blood.—Red cells, 1,363,000; hemoglobin, 38 per cent.; ma¬ crocytes, microcytes, poikilocytes numerous; no nucleated cells; there were 8,000 leucocytes, of which 62 per cent, were of the polymorphous form. After the first month considerable improvement was shown. The patient is now undergoing a period of improvement. p p Case 21.—P. J. K., aged 35, cabinet-maker; family history negative; personal history, had been well previous to present illness, which began about 18 months ago with the usual symptoms of weakness, dyspnea on exercise, indigestion, occa¬ sional vomiting with constipation. The heart was of normal size; weak systolic apical bruit, but no bruit de Diable. The stomach contents showed complete achylia gastrica. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o McN., aged 46, liquor dealer; family his¬ tory negative; personal history good until the beginning of the present illness last summer, at which time he began to lo>e strength; became sallow; suffered from shortness of breath on exercise; had vertigo and diarrhea. His pulse was very soft; heart of normal size, with apical systolic bruit and systolic bruit at the aortic orifice and bruit de Diable; blood vessels were in good condition; lungs sound; appetite fair; moderate complaint of stomach; skin has the characteristic lemon tint. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o Heart was moderately dilated, no bruit; had had nose bleed once; little complaint of the stomach; had complete achylia gastrica; food present unchanged three hours after meal; tongue pale, but not denuded; liver large, two inches below ribs; bowels fre¬ quently loose; skin pale, but absence of lemon tint; urine scant, urea low. No period of improvement. p Blood.—Red cells, 640,000; hemoglobin, 30 per cent.; a few megaloblasts and normoblasts, with many large and small cells; leucocytes, 8,200. Patient made no improvement and died April 1. Blood.—Red cells, 1,964,000; hemoglobin, 40 per cent.; ma¬ crocytes, microcytes, poikilocytes abundant, with a few megalo¬ blasts; lymphocytes showed 57 per cent, of the polymorphous form. p Case 19.—Mrs. C. H., aged 39; father died of cancer of the stomach; in other respects family history good; personal his¬ tory good save that three months ago suddenly lost strength and color ; had dyspnea on exercise, dizziness, bleeding from the nose; tinnitus; moderate anasarca, and some tremor and weakness in her extremities; pulse gaseous; temperature slightly raised; heart slightly enlarged; systolic apical bruit; bruit de Diable; blood vessels good; râles at base of both lungs; slight cough; mucous expectoration; stomach irritable; vomiting in the morning, and distress after eating; tongue was pale; had had diarrhea for two months; skin distinct lemon tint; some edema; regular but pale menstruation. Pa¬ tient had steadily failed since first attack, with no improve¬ ment; now in critical condition. Case 24.—B. J. H., aged 39, male, railroad engineer; family history negative; personal history, health uniformly good until a year ago, when he began to grow weak and suffer from his stomach. These symptoms have been progressive until the present time. Former weight, 225 pounds, now 184. He suf¬ fers from dyspnea on exercise, weakness, dizziness; the tongue is denuded and dry; appetite poor, gastric distress, occasional vomiting and constipation. He came supposing that he suf¬ fered from some disease of the stomach. His heart is dilated, relative mitral insufficiency and moderate bruit de Diable. Stomach shows achylia gastrica. Urine is negative. History of past luetic infection 20 years before. On Donovan's solu¬ tion he made improvement during the first six months, but his trouble became worse and he died April 1, 1904. Blood.—Red cells, 1,735,000; hemoglobin not taken; many normoblasts, megaloblasts. There were 5,000 leucocytes and 70 per cent, of polymorphonuclear cells. p y p Case 20.—G J. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User o D., aged 40, laborer; duration of illness not known; complained of extreme weakness and digestive dis¬ turbance; some dyspnea; has marked lemon tint to skin. y p Blood.—Red cells, 1,352,000; hemoglobin, 25 per cent; poi¬ kilocytes, macrocytes, megaloblasts, numerous; leucocytes, 4,000; polymorphonuclear neutrophiles, not given. y p Blood.—Red cells, 1,352,000; hemoglobin, 25 per cent; poi¬ kilocytes, macrocytes, megaloblasts, numerous; leucocytes, 4,000; polymorphonuclear neutrophiles, not given. p Blood.—Red cells, 1,150,000; hemoglobin, 50 per cent. p Blood.—Red cells, 1,150,000; hemoglobin, 50 per cent. ded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User on 05/25/2015 Case 18.—F. D., aged 47, farmer; patient of Dr. Tompkins of Randolph, N. Y. Father died of diabetes; in other respects family history is good. Patient had for years experienced severe bilious attacks with vomiting and sallowness, accom¬ panied by chills and fever. Four years ago, after severe influ¬ enza, he had one of these attacks with jaundice, accompanied by general anasarca, at which time the scrotum was drained. He was very weak, but finally made great improvement and went to work. A year ago he had a second severe illness, with great weakness, lemon-colored tint and bleeding from hemor¬ rhoids; marked anasarca lasting two or three months. This again disappeared and the man was able to return to work. During each of these attacks the liver was large. The present attack occurred in September last; became yellow, had chills and fever, great weakness; became nervous with dyspnea on exercise; bleeding from the hemorrhoids and moderate ana¬ sarca. Pulse was weak and gaseous; temperature from 98 to 102; respirations, 24 to 26. Heart was moderately dilated, systolic bruit at the apex and over pulmonary areas. Lungs were normal ; arteries slightly sclerosed ; had paresthesia in both legs, some weakness in gait; appetite was poor, with dis¬ tress after eating; tongue was smooth and shiny; liver was very large, as also was the spleen; bowels were loose; absence of ova of parasites. Case 18.—F. D., aged 47, farmer; patient of Dr. Tompkins of Randolph, N. Y. Father died of diabetes; in other respects family history is good. Patient had for years experienced severe bilious attacks with vomiting and sallowness, accom¬ panied by chills and fever. Four years ago, after severe influ¬ enza, he had one of these attacks with jaundice, accompanied by general anasarca, at which time the scrotum was drained. DISCUSSION. Dr. George Dock, Ann Arbor, Mich.—I agree with the re¬ marks made concerning the condition of the stomach and its relation or lack of relation to the disease. I have had a great deal of experience with hydrochloric acid. I have treated these cases with hydrochloric acid, fresh air and diet. Many of them have done as well as those with Fowler's solution and as others treated without any medicine at all. I have seen many patients with advanced pernicious anemia get well tem¬ porarily under fresh air and the same sort of diet as given to tuberculous patients. These patients may take 40, 50 or 60 minims of hydrochloric acid, diluted, after meals. It has been my experience that if they could not take the acid well they did not do well. The effect of the hydrochloric acid does not seem to have to do so much with stomach as with intes¬ tinal digestion. The condition of the intestine predominates in nutrition. In all cases the condition of the intestinal diges¬ tion should be determined by an examination of the stools. Dr. George Dock, Ann Arbor, Mich.—I agree with the re¬ marks made concerning the condition of the stomach and its relation or lack of relation to the disease. I have had a great deal of experience with hydrochloric acid. I have treated these cases with hydrochloric acid, fresh air and diet. Many of them have done as well as those with Fowler's solution and as others treated without any medicine at all. I have seen many patients with advanced pernicious anemia get well tem¬ porarily under fresh air and the same sort of diet as given to tuberculous patients. These patients may take 40, 50 or 60 minims of hydrochloric acid, diluted, after meals. It has been my experience that if they could not take the acid well they did not do well. The effect of the hydrochloric acid does not seem to have to do so much with stomach as with intes¬ tinal digestion. The condition of the intestine predominates in nutrition. In all cases the condition of the intestinal diges¬ tion should be determined by an examination of the stools. Dr. R. C. Cabot, Boston—Of 150 eases of pernicious anemia seen by me, five-sixths showed the absence of hydrochloric acid and yet digestion went on perfectly well. DISCUSSION. One is almost in¬ clined to say that hydrochloric acid is a luxury and not a ne¬ cessity. In cases with poor digestion the trouble seems due rather to motor insufficiency of the stomach, and not to the absence of hydrochloric acid. It has been my experience that there has often been an absence of hydrochloric acid even dur¬ ing periods of improvement. There is also a curious lack of cardiac symptoms in some cases, even when we know that advanced fatty degeneration is present. y g p Dr. Max Einhorn, New York—While some time ago the opinion prevailed that pernicious anemia was due to atrophy of the stomach, recently this view had to be changed. This point has been brought up by Dr. Stockton and myself. -There have been met many cases of pernicious anemia in which the gastric digestion is not impaired. Again, most cases of achylia gastrica do not show a condition of pernicious anemia. Per¬ nicious anemia may be complicated with achylia gastrica and atrophy of the stomach. But the latter condition is not the cause, but rather the result of pernicious anemia. y Dr. Charles G. Stockton—I can not wonder, considering the strange capriciousness shown in this disease, at the con¬ clusion reached by Dr. Cabot that hydrochloric acid is a lux¬ ury and not a necessity. However, I believe, perhaps for the first time, that I must take different ground from Dr. Cabot, as I regard hydrochloric acid as more than a luxury, though perhaps not a necessity. The fact brought out by Dr. Einhorn that in pernicious anemia we so often find serious atrophy of. the gastric mucosa seems to me to answer the point raised by Dr. Osier, who suggests that in some cases the gastric atony may bear a causal relation to the pernicious anemia. I would like to ask Dr. Osier if he does not think that in view of the serious degenerations of other organs in pernicious anemia, it might easily account for the gastric atrophy, such, for in¬ stance, as he described some years ago in a case reported in conjunction with Dr. Henry; that this atrophy is more likely to be the result of the pernicious anemia than the cause of that disease. I am sorry to hear Dr. DISCUSSION. Osier make the state¬ ment, for it leads me to feel that I must be wrong in my view; nevertheless, my belief is strongly to the contrary. It seems to me that we should draw a distinct line between ordinary achylia gastrica with atrophy of the gastric mucosa and those cases of gastric atrophy which appear in the course of per¬ nicious anemia. Attention has been called by Dr. Dock to the fact that these cases improve under hydrochloric acid, and I wish to say that I have universally given that remedy where patients could take it. In some instances improvement has fol¬ lowed, but it is difficult to say what actually does good in this disease. Formerly patients apparently did well under arsenic, and now we wonder why they sometimes do well without it. One patient whom I have had under observation for three years wrote me of his condition a month ago. He is still at work and under no treatment. However, there are cases that improve under hydrochloric acid, and this I can not deny. In some of my cases examinations of the stools have been made with nega¬ tive results as regard evidences of parasites. I am sorry that more new knowledge has not been added by our consideration of this disease. , p De. Allen Jones, Buffalo—I studied the gastric contents in these cases that Dr. Stockton has reported and I feel like confirming the view that pernicious anemia is not caused by achylia gastrica so far as we can tell. For a number of years after the idea was exploited that pernicious anemia was due to failure of the secretory functions of the stomach, it was found that achylia gastrica appeared without pernicious anemia more often than with it, and yet I felt like hesitating before stating positively that this condition of the stomach was not the cause of pernicious anemia. It is very gratifying to hear Dr. Stockton and others with larger experience than mine make this statement as a clinical fact. The point I wish particularly to emphasize is that in gastric work achylia gas¬ trica without pernicious anemia is more often encountered than with it. Db. William Osler, Baltimore—My experience agrees largely with Dr. Stockton's and Dr. Cabot's. It is wonderful to see, in the great majority of cases, when a diagnosis has been made, how rapidly these cases improve. SUMMARY. He died in the spring of 1903 after an illness of little over two years, with one period of improvement. p p Blood.—Red cells, 950,000; hemoglobin, 35 per cent.; many megalocytes and poikilocytes; there were four normoblasts and 10 macroblasts in one field. There were 2,500 leucocytes, of which only 15 per cent, belonged to the polymorphonuclear type. Downloaded From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User on 05/25/201 nicious anemia and not with a secondary anemia due to un- einariasis or other intestinal infection. Little has been said regarding treatment. It has long been thought that arsenic did good because of its antiseptic effect, and those who have laid especial stress on the frequent presence of pyorrhea alveo- laris have thought that here the antiseptic effect was especially prominent, yet I have recently seen one patient improve won¬ derfully, so that he has resumed his practice, under the hypo¬ dermic use of one-half grain of cacodylate of sodium daily. He had utterly failed to improve under the long-continued use of Fowler's solution. If the arsenic did any good here it cer¬ tainly was not through its antiseptic effect in the intestinal canal as when given by mouth. Pyorrhea was present in this case. in the others it was absent. Six showed no acidity of the gastrici contents, 1 (the highest) showed a total acidity of 46, one 26, and the average about 10. Four showed the presence of combined chlorids; in the others it was absent. One showed free hydrochloric acid, .05 per cent., one .06 per cent., and one .022 per cent. ; in all the others it was absent. Six showed lactic acid present; in the others it was absent. Four showed other organic acids; the others, none. The acid salts were very low, 18 being the highest, 5 having none whatever; 7 showed biuret reaction ; six showed rennet present, in the others it was absent. The digestion of starch was poor in all save two cases. DISCUSSION. There are cer¬ tain cases in which from the outset the stomach symptoms so dominate the whole scene as to suggest that the profound and increasing anemia is due to the gastric condition. Such cases, though, are extremely rare. In a case reported some years ago the entire mucous membrane of the stomach was cuticular, similar to that found in the esophageal end of the stomach in a horse. I should like to ask Dr. Stockton if he has made any observations on the condition of the mouth, if oral sepsis had anything to do with it. I do not think it has in the majority of cases. We have had a large number of cases this spring, almost an epidemic of pernicious anemia, but in not one was the oral condition extreme. We are still "at sea" as to the exact pathology of pernicious anemia. p gy p Dr. J. N. Hall, Denver—Much has been said regarding the absence of hydrochloric acid. I have, in such cases, seen fail¬ ure to improve when not administered and yet improvement was striking when hydrochloric acid was given in 25-minim doses. The absence of any excess of eosinophiles should be noted, as it generally indicates that we are dealing with per- What to eat and what to drink will always be decided by national custom and individual preference, so far as the public is concerned, but both may be influenced in the right direction by the guidance of skilled medical opinion.-—Brit. Med. Jour. d From: http://jama.jamanetwork.com/ by a Penn State Milton S Hershey Med Ctr User on 05/25/2015
https://openalex.org/W1978643214
https://zenodo.org/records/2410530/files/article.pdf
German
null
Ueber den inneren Widerstand des Westonelementes
Annalen der Physik
1,900
public-domain
1,928
848 848 11. Ueber dem i m e r e m Wiclerstamd des Weston- e Iernerbtes; vom Ipn ax K I e m eln d i E. 1) I. KlemenEi6, Wied. Ann. 66. p.917. 1898. 2) E. H o s e , Zeitschr. f. Elektrochemie Nr. 35. 1900. 2) E. H o s e , Zeitschr. f. Elektrochemie Nr. 35. 1900. 1) I. KlemenEi6, Wied. Ann. 66. p.917. 1898. 2) I. KlemenbiZ, Ber. d. naturw.-med. Yereinee in Innsbruck, 23. Jahrg. 1896-1897. 11. Ueber dem i m e r e m Wiclerstamd des Weston- e Iernerbtes; vom Ipn ax K I e m eln d i E. Vor einiger Zeit habe ich in einer Notiz') auf die prak- tische Bedeutnng des inneren Widerstandes der Normalelemente hingewiesen. Im allgenieinen wird durch einen kleineii inneren Widerstand die praktische Brauchbarkeit des Elementes wesent- lich erhiiht. Es sind schon von verschiedenen Seiten Versuche gemacht worden, den Elementen eine Form zu geben, bei welcher dieser Widerstand thatskchlich sehr klein ausfallt. Insbesondere ware in dieser Richtung die Form zu erwahnen, welche vor nicht langer Zeit Hr. Emil Bose2) beschrieben hat. g In vielen Fallen muss man jedoch bei der Construction auf andere Factoren , z. B. die Versandfahigkeit Riicksicht nehmen und kann daher nicht zugleich auch einen kleinen inneren Widerstand erzielen. Ein grosser Teil der Experimentatoren wird die An- schaffung fertiger Elemente vorziehen. Die Fabriken , welche solche Elemente lieferii, werden dabei natiirlich ' immer die Versandfahigkeit im Auge behalten miissen, es wird daher auch der innere Widerstand ziemlich betrachtlich ausfallen. Dieser Uebelstand ist jedoch nicht so gross, wenn nur der Wider- stand wenigstens niit der Zeit constant bleibt, ein Umstand, der bei den bisher meistenteils gebrauchten Clarkelementen nicht zutrifft. In neuerer Zeit liefert die Firma ,,The European Weston Electrical Instrument Co." in Berlin ein Normalelement, welches von der Firma kurz als Westonelement bezeichnet wird. Es ist gebaut wie das Clarkelement, nur hat es Cadmium statt des Zinkes und eine bei 4 concentrirte Cadmiumsulfatlosung ohne iiberschiissige Sulfatkrystalle. 8 49 Annalen cler Physlk. IV. Polge. 2. 1) W. J iiger, Centralblatt fur Accumulatoren uud Elementenkunde 1900. . .. ._ . . lnnerer Widerstand des Westoneleme7ites. lnnerer Widerstand des Westoneleme7ites. Dieses Element zeichnet sich durch viele wertvolle Eigen- schaften dern Clarkelemente und auch dem Cadmiumelemente Init stets conce~~t~-irter Sulfatlosung gegenuber aus. Ks ver- dient riach Hrn. W. Jager’) vor allen ancleren Elementen den Vorzug Einige Angaben fiber dieses Element, insbe- sondere ub er seinen inneren Widerstand durften daher allgemein interessiren. Jn erster Linie waren meine Messungen auf die Unter- suchung cles inlieren W-iderstandes gerichtet. Der innere Widerstand wurde nach einer Methode be- stimmt, welche ich vor einiger Zeit beschrieben hsbe.2) Die Methode ist besoriders gut durchfiihrbar, wenri man zwei gleiche Normalelemente zur Verfiigung hat. Sie besteht in Folgendem: Kin Galvanometer init feindrahtiger Rolle vom Wider- stande Q sei durch ein Element voii der elektromotorischen Kraft 4 und durch den Widerstaaid W geschlossen. Vor dem Galvanometer befinde sich ein Nebenschluss vom Widerstande w, wobei w gegen und W sehr klein ist. Dann ist bekanntlich die Stromstirke im Galvanometerzweige j gegeben durch . Ew j = ?-w- = & a , wo R der Reductionsfactor und cc der Ausschlag des Galvano- meters ist. wo R der Reductionsfactor und cc der Ausschlag des Galvano- meters ist. Ich lege nun statt des Widerstandes 10 ein Normalelement mit dem Widerstande T und der elektromotorischen Kraft 71 in den Nebenschluss. Um die Wirkung dieser elektromotori- schen Kraft auf das Galvanometer teilweise zu compensiren, lege ich ein zweites Normalelement mit der elektromotorischen Kraft e und passender Richtung derselben in den Galvano- meterzweig. g Die Stromstirke im Qalvanometerkreise ist danii wobei der Widerstand des compeilsirenden Elementes gegen 0 vernachlassigt ist. Durch Commutiren von B bekommt man wobei der Widerstand des compeilsirenden Elementes gegen 0 vernachlassigt ist. Durch Commutiren von B bekommt man 1) W. J iiger, Centralblatt fur Accumulatoren uud Elementenkunde 1900. . .. - ._ -. - . 54 54 850 I. KlemenEi8. leicht den der elektromotorischen Kraft B und dem Wider- stande T entsprechenden Ausschlag. Bas /?, a und w h ~ sich der Widerstand T berechnen. Bei den vorliegenden Mes- sungen bewegte sich der Widerstand W zwischen 40000 und 100000 Q, p zwischen 30000 und 70000 9, und T zwischen 100 und 300 Q. Nur im Falle eines Clark'schen Nementes war T an 9OO.Q. Es wurden untersucht: Zwei Westonelemente, geliefert von ,,The European Weston Electrical Instrument Co." in Berlin. Sie werden hier be- zeichnet rnit /Pi6 und W,6 ist am 17. October 18A8 und IV,, am 29. lnnerer Widerstand des Westoneleme7ites. October 1898 von Berlin angekommen. ,, g Zwei Westonelemente, H-Form, rnit uberschussigen Cad- mium - Sulfatkrystallen , verfertigt im hiesigen physikalischen Institute am 8. ,Juli 1898 nach den Vorschriften der Physi- kalisch-Technisclien Reichsanstalt und bezeiclinet mit W, und WI,. Zwei Clarkelemente. Das eine, H-Form, bezeichnet mit 11, ebenfalls verfertigt am 25. Mai 1898 im hiesigen physikalischcn Institute nach den Normen der genaniiten Anstalt; das zweite, bezeiclinet mit Nr. 160, coristruirt von Fiiss, spater (Sommer 1898) reconstruirt von H a r t m a n n und Braun. Da der innere Widerstand der Elemente von der Tem- peratur rtbhiingt, musste bei jeder Restimmung auch die Tem- peratur gemessen werden. Sie wurde im allgemeinen bei den Westonelementen an einem neben den Ellementen gelegenen Thermometer abgelesen, was natiirlicli lteine sehr genaue An- gabe gestattet,; diese diirften daher wohl nur eine Genauig- keit auf etwa 0,5-1,0" haben. In Tab. I sind die zu verschiedenen Zeiten geniachten Messungsresultate verzeichnet. Unter 1' sind die Temperaturen 1. XI. 1898 16. XII. 1895 22. 11. 1899 13. XI. 1899 7. VI. 1900 162 __ 161 ~ 159 158 153 150 147 177 I 169 159 I , 152 7' - .. .. -_ - - - 16,5 17,O 19'0 19,5 16,4 85 1 Innerer Widerstand des Westonelementes. Fur 77, und W,, giebt Tab. II: die Resultate. Fur 77, und W,, giebt Tab. II: die Resultate. Fur 77, und W,, giebt Tab. II: die Resultate. Zum Vergleiche folgeri in Tab. 111 noch die Widerstands- bestimmungen mit den Clarltelementen ,,H" und Nr. 160. 11 befand sich in Paraffin61 und die Temperatur wurde an einem im Bade befindlichen Thermometer abgelesen. Nr. 160 ist von der Fabrik aus mit einem eingesenkten Thermometer versehen. m 1 1 1 1 T T m 1 1 1 1 T T . ' Anschliessend moclite ich einige Beobachtungen mitteilen, welche niit gcniltclit wurden, um die Aenderung des innereii Widerstandes mit der Temperntur zu bestimmen. Zu diesem Zwecke wurdc IV,, in ein Bad von Paraffinol getaucht, welches auf verschiedene Temveraturen gebracht werden ltonnte. g l!!s wurde die Temperatur jedcsmal nur langsam erhijht oder erniedrigt und erst dann beobachtet, wenn die Temperatur 2-3 Stunden constant auf gleicher Hohe war. * 54 * 54 * 852 I. Klem en EiE. Aus den angefuhrten Uaten ergiebt sich ein Temperatur- coefficient e fur den iiineren Widerstand gleich 0,025. Der Widerstand bei einer gewissen niedrigen Temperatur wird durch die Temperaturerhohung nicht geandert, im Gegen- sake Zuni Clark'schen Elemente und vermutlich auch zum Westonelemente lriit uberschussigen Cadmium-Sulf~tltrystalleii. DAY Clnrltelement 11 hatte z. B. eineri Widerstand von 281 9 bei 18,7 O, nnch einer TemperaturerhGhung auf ca. '28" liatte es 261 S! bei 1(3,5(' und nach ciner abermaligen ErhGhung auf 29O tien Widerstand von 243 fi, bei 16,9O. Der Wider- stand fallt riucli der Erwkmung und es ist ja bekannt, dass in der Physikalisch- Technischen Reiclisanstnlt der mit der Zeit zunehmende innere Widerstand der Clarkelemente anf diese Weise wieder auf einen niederen Wert gebrxcht wird. Der Widerstand bei einer gewissen niedrigen Temperatur wird durch die Temperaturerhohung nicht geandert, im Gegen- sake Zuni Clark'schen Elemente und vermutlich auch zum Westonelemente lriit uberschussigen Cadmium-Sulf~tltrystalleii. DAY Clnrltelement 11 hatte z. B. eineri Widerstand von 281 9 bei 18,7 O, nnch einer TemperaturerhGhung auf ca. '28" liatte es 261 S! bei 1(3,5(' und nach ciner abermaligen ErhGhung auf 29O tien Widerstand von 243 fi, bei 16,9O. Der Wider- stand fallt riucli der Erwkmung und es ist ja bekannt, dass in der Physikalisch- Technischen Reiclisanstnlt der mit der Zeit zunehmende innere Widerstand der Clarkelemente anf diese Weise wieder auf einen niederen Wert gebrxcht wird. Die Teniperaturerhiihung wurde mit dem Clarkelemente 11 zwischen dem 22. Pebruar und 14. November vorgenommen. Ohne diese waren wahrscheinlich die Werte fur H vom 14. Kovember 1899 und 28. Xai 1900 in der Tab. I11 noch hoher ausgefalleii. Aus den angefuhrten Tabellen ergiebt sich also, dass der Widerstarid der Westonelemente, wie sie von der Firrna ,,The Kuropean Weston Electrical Instrument Co." geliefert werden, zwar im Verlaufe von 1,s ,Jahren ein wenig gestiegen ist, dass aber dieser Anstieg weitaus nicht jene Hohe erreicht, die bei den Elementen mit uberschussigen Cadmium- Sulfatkryatallen vorkommt. Auch die Clarkelemente zeigeii rnit der Zeit eine sehr starke Zunahme des inneren Wider- standes; iiberdies ist der absolute Wert desselben bei den in den Handel gebrachten Clurk'schen Elementeii vie1 grosser als bei den aus der gemarinten E'abrik stnmmenden Weston- elenienten. Die Teniperaturerhiihung wurde mit dem Clarkelemente 11 zwischen dem 22. Pebruar und 14. November vorgenommen. Ohne diese waren wahrscheinlich die Werte fur H vom 14. Kovember 1899 und 28. Xai 1900 in der Tab. I11 noch hoher ausgefalleii. Aus den angefuhrten Tabellen ergiebt sich also, dass der Widerstarid der Westonelemente, wie sie von der Firrna ,,The Kuropean Weston Electrical Instrument Co." geliefert werden, zwar im Verlaufe von 1,s ,Jahren ein wenig gestiegen ist, dass aber dieser Anstieg weitaus nicht jene Hohe erreicht, die bei den Elementen mit uberschussigen Cadmium- Sulfatkryatallen vorkommt. Auch die Clarkelemente zeigeii rnit der Zeit eine sehr starke Zunahme des inneren Wider- standes; iiberdies ist der absolute Wert desselben bei den in den Handel gebrachten Clurk'schen Elementeii vie1 grosser als bei den aus der gemarinten E'abrik stnmmenden Weston- elenienten. Die Westonelernerite der ,,The Europeaii Weston Electrical Instrument Co." siiid also nicht nur n i t Rucksicht auf die Unabhangigkeit der elektromotorischen Kraft von der Tem- peratur, sondern auch vori dem fur die praktische Bedeutung allerdings nur secundar wichtigen Staridpunlrte der Constanx und des niederen Wcrtes des inneren Widerstandes alleii anderen Normalelementen iiberlegen. Schliesslich mochte ich noch einige Daten uber die elektro- motorischen Kriifte der uritersuchten Elemente mitteilen. Hier Innerer Nrider.stand des Westonelementes. 853 kommen nur die Verldtnisse und nicht die absoluten Werte in Betracht. Das Verliiiltnis der elektromotorischen Krafte von iV, und 7YIl wurde am 17. (Eingegangen 9. Juli 1900.) October 1898 auf ca. 0,002 Proc. gleich gefunden. An diesem Tage war hei der Zimmertemperatur. Ilics stimmt mit. den Angnben der Physilralisch- Technischen Reichsaiistalt, wonach die Differenz zwischen der elelrtro- motorischen Kraft fur die beiden Sorten der Elemente etwa 0,04 Proc. bei 20" betragen soll. Bei 4') solleii die elelrtro- motorischen Krafte gleich sein. g Am 7 . Juni 1900 wurde gefunden 1 = 1,00001, 1 'VII WI = 1,00001, 1 'VII WI Die Xlemente behielten dalier durch mehr als 1,5 Jalire ein constantes Vcrh~ltnis der elektromotorischen Kriifte. Die Xlemente behielten dalier durch mehr als 1,5 Jalire ein constantes Vcrh~ltnis der elektromotorischen Kriifte. AUS den vorliegenden Daten kann man aber auch er- sehen, dass der Verschluss hei den Westonelementen ein guter ist, sodass Iteine Verduiistung der Llisung eintritt, was auf die elektromotorische Krzlft, nicht ohne EinHuss ware. Zwar sind die Verhaltnisse dcr elel~tromotorischen Kriifte gegen CY,, irn Verlaufe von 1,5 Jahren etwas gefallen, doch diirfte dies in dcr Verschiedenheit der Beobachtungsteinperaturen liegen.') I n n s bruck, Physikal. Inst. d. Univ., Juli 1900. 1) Wiihrcmd sich bci den Westonelementen mit eincr bci 4 O ge- sattigten Lijsung die elrlrtromotorischc Kraft mit der Temperatur gar nicht Bndert, ist bei den Elemcnten mit iiberschhsigen Krystallen ein lrleinrr Temperaturcoefficient vorhanden. (Eingegangen 9. Juli 1900.)
https://openalex.org/W4312061311
https://zenodo.org/records/7465069/files/KlGrBr_XXIII-Plenary-7-13.pdf
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Стратегия и структура инклюзивного управления предприятием
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Г.Б. Клейнер Г.Б. Клейнер Пленарное заседание Пленарное заседание ПЛЕНАРНОЕ ЗАСЕДАНИЕ ПЛЕНАРНОЕ ЗАСЕДАНИЕ DOI: 10.34706/978-5-8211-0809-8-7-13 СТРАТЕГИЯ И СТРУКТУРА ИНКЛЮЗИВНОГО УПРАВЛЕНИЯ ПРЕДПРИЯТИЕМ Клейнер Георгий Борисович, член-корреспондент РАН, руководитель научного направле- ния «Мезоэкономика, микроэкономика, корпоративная экономика» ЦЭМИ РАН, зав. кафед- рой «Системный анализ в экономике» Финансового университета при Правительстве РФ, зав. Кафедрой институциональной экономики Государственного университета управления, george.kleiner@inbox.ru Ключевые слова: инклюзивное управление, расщепление власти, социальное лидерство, экономика замкнутого цикла. Отечественная экономика вступает в сложный и ответственный период сво- его развития. Политика санкций, ограничений и давления, которую применяют мно- гие страны – традиционные партнеры России в области экономики – существенно затрудняет функционирование и развитие экономики России. Проблемы продолже- ния деятельности предприятий в сферах производства, науки, спорта и других сфе- рах заставляют искать новые способы управления экономическими агентами, позво- ляющие не только выдержать условия стресса, в которых оказались российские предприятия, но и создать предпосылки для дальнейшего развития в средне- и дол- госрочной перспективе. 7 По данным Национального агентства финансовых исследований (НАФИ) на март 2022 г., доля предприятий, считающих свое положение неудовлетворительным, выросла с 22% в январе – начале февраля до 40% в марте. Доля предприятий, рас- сматривающих свое состояние как устойчивое, снизилась с 36 до 25%. При этом 51% предприятий дают негативные прогнозы на ближайшие три месяца, 22% считают, что ситуация останется без изменений, 27% ожидают улучшений (Оперативный со- циально-экономический мониторинг…, 2022). По прогнозам Центра стратегических разработок, экономике предстоит лишиться около 2 млн рабочих мест (Мануйлова, 2022). Меняется, таким образом, и состав самостоятельных экономических агентов (предприятий) и обстановка внутри предприятия. Это приводит к необходимости ак- тивизировать выявление новых резервов производства, недостаточно эффективно использовавшихся ранее. Основным направлением такого поиска является опреде- ление возможностей перехода к инклюзивному управлению предприятием, т.е. управлению, ориентированному на вовлечение в хозяйственную деятельность всех 7 XXIII Всероссийский симпозиум «Стратегическое планирование и развитие предприятий» материальных и нематериальных ресурсов, работников и др. участников деятельно- сти предприятия. При этом в хозяйственную деятельность включается и координа- ционно-управленческая деятельность, так что тенденция демократизации управле- ния рассматривается как одна из составляющих инклюзивного управления. В настоящей статье излагаются результаты поиска новой структуры управ- ления предприятием, которая в максимальной степени способствовала бы активиза- ции деятельности работников предприятий по преодолению последствий изоляции экономики России в пространстве международного разделения труда. Задача со- стоит в том, чтобы максимально реализовать потенциал человеческих ресурсов на предприятиях, приблизиться к инклюзивному управлению, интегрирующему все по- зитивные факторы деятельности предприятия. Пути решения этой задачи видятся в оптимизации сочетания формальной структуры управления и неформальных инсти- тутов социального лидерства на предприятии. СТРАТЕГИЯ И СТРУКТУРА ИНКЛЮЗИВНОГО УПРАВЛЕНИЯ ПРЕДПРИЯТИЕМ Отличительной особенностью функционирования экономики России в крат- косрочной перспективе является ее частичная изоляция, работа в режиме «эконо- мики замкнутого цикла» (Валько, 2020). В таком режиме реализация продукции и пополнение израсходованных ресурсов осуществляются главным образом на терри- тории самого государства. Понятия «страна-производитель» и «страна-потреби- тель» данного вида продукции практически смыкаются. 8 Здесь возникает вопрос общего характера: может ли какая-либо страна или группа стран существовать в условиях автаркии? Общий положительный ответ дает сама жизнь: такая экономическая система, как мировая экономика, с самого начала и до сегодняшнего дня функционирует в условиях автаркии, поскольку не ведет обмена товарами и услугами ни с одной инопланетой цивилизацией. Если мировая экономика в течение тысяч лет находится в изоляции, то и Россия как самая большая страна в мире, видимо, тоже может существовать в режиме частичной изоляции достаточно длительное время. Главным фактором, обеспечивающим возможность функциониро- вания в таком режиме, является разнообразие условий экономической деятельности на территории страны (что аналогично разнообразию экономик стран, входящих в ми- ровое экономическое сообщество). Такое разнообразие должно учитывать админи- стративно-территориальное деление страны и распространяться на институциональ- ные, отраслевые природно-географические и ментальные особенности регионального развития. Инклюзивное управление страновой экономикой в значительной мере должно концентрироваться на управлении разнообразием, т.е. поиске и применении управленческих средств, обеспечивающих оптимальный (не слишком высокий, но и не слишком низкий) уровень разнообразия. Представляется, что определение опти- мальных границ (в более общем случае – оптимальное распределение фрагментов уплотнения и разрежения) условий экономической деятельности является одной из 8 Пленарное заседание актуальных задач современной экономической науки. Проблема разнообразия отно- сится и к усилению экономической специализации регионов, и к определению раз- мерностной структуры предприятий и организаций, и в целом к организации разме- щения и развития производительных сил в страновом пространстве и времени. Здесь должны взаимодействовать такие отрасли экономической науки, как пространствен- ная экономика, экономическая динамика и экономика разнообразия. В настоящее время страна должна готовиться к режиму частичной изоляции, т.е. к экономике замкнутого цикла. Следует подчеркнуть, что Россия обладает огромными запасами природных ресурсов, включая уникальные природные ре- сурсы; гигантским запасом человеческого ресурса, включая интеллектуальный, ко- гнитивный и абсорбционный потенциал; значительным опытом развития за «желез- ным занавесом». Основными ресурсами производства являются, как известно, труд (включая предпринимательский и организаторский потенциалы) и капитал (включая физиче- ский, финансовый, социальный и др. виды). В условиях продолжения изоляционист- ких тенденций следует ожидать сокращения доступа к физическому капиталу, к со- временным технологиям и техническим устройствам. СТРАТЕГИЯ И СТРУКТУРА ИНКЛЮЗИВНОГО УПРАВЛЕНИЯ ПРЕДПРИЯТИЕМ Единственным способом про- должения производственной деятельности для большинства российских предприя- тий становится расширение и активизация процессов замещения: во-первых, им- портозамещения; во-вторых, замещения трудом капитала. При этом процесс соб- ственного производства импортируемых комплектующих и устройств требует не- мало времени, в то время как процесс замещения недостающих элементов капитала за счет активизации трудовой деятельности и модернизации процессов управления может быть выполнен в достаточно сжатые сроки. Инклюзивное управление пред- полагает, в том числе, и решение последней задачи. По сути дела, речь идет об инвестициях в эластичность замещения между физическим капиталом и человеческим трудом. Задача управления эластичностью замещения не является новой для экономической науки. В 1970–1980 гг., в период расцвета теории производственных функций, были заложены основы построения производственных функций с переменной эластичностью замещения факторов (Клейнер, 1986; Revankar, 1971), см. также (Хацкевич, Проневич, 2017) В настоящее время внимание к моделям производства с управляемой эластичностью замещения должно быть усилено; эластичность замещения должна войти в состав предметов инклюзивного управления. 9 Более конкретно речь идет о замещении физического капитала интеллекту- альным трудом. Необходимо усилить внимание к уникальным специалистам («умельцам»), способным развивать производство в условиях сокращения доступа к импорту сырья, материалов, оборудования; западное управление талантами XXIII Всероссийский симпозиум «Стратегическое планирование и развитие предприятий» превратить в российское управление «умельцами». Управление «умельцами» при- звано организовать внутри предприятия непрерывные цепочки добавленной ценно- сти, в настоящее время терпящие разрывы из-за слома традиционных, внешних для предприятия, цепочек поставок. В определенном смысле проблемы логистического обеспечения предприятий должны быть сняты или, по крайней мере, ослаблены за счет интериоризации – «точечного» управления внутрифирменной логистикой. Та- ких «умельцев» немного, но в сегодняшних условиях к ним надо относиться более внимательно. Следует более бережно относиться к таким работникам, обеспечивать для них определенные стимулы привилегии и в целом более бережно относиться к человеческим ресурсам предприятий и организаций. Если мы сможем активизиро- вать креативный потенциал работников, мы сможем преодолеть ограничения, свя- занные с физическим недостатком капитала, технологий, сырья и т.д. Как же должно быть организовано инклюзивное управление предприятием в этих условиях? Чтобы обеспечить охват всех аспектов деятельности предприятия, обратимся к универсальной структуре предприятия, представленной четырьмя базо- выми секторами (подсистемами) предприятия: объектным, процессным, проектным и средовым (Клейнер, 2011). Объектный сектор составляют организационные под- разделения; процессный – совокупность бизнес-процессов, протекающих на пред- приятии; проектный – краткосрочные локализованные, значимые для предприятия, мероприятия различного характера; средовой – различные виды таких внутрифир- менных сред, как институциональная, информационная, социальная и т.п. Эти че- тыре сектора предприятия нуждаются во внутрисекторном управлении и межсектор- ной координации. СТРАТЕГИЯ И СТРУКТУРА ИНКЛЮЗИВНОГО УПРАВЛЕНИЯ ПРЕДПРИЯТИЕМ Для целенаправленного эффективного управления предприятием необходимо, чтобы указанные секторы были представлены в административной структуре управления и имели бы административных руководителей. Этого, однако, недостаточно для инклюзивного управления. Необходимо привлечение сил социаль- ного регулирования внутрифирменной среды. Предлагается двухкомпонентная структура инклюзивного управления, основанная на сочетании структур и процес- сов административного управления, с одной стороны, и социально-лидерского управления, с другой. В формальной структуре управления выделяются администра- тивные лидеры каждого из 4-х секторов: объектный лидер, процессный лидер, про- ектный лидер и средовой лидер. В обязанности объектного лидера входит обеспече- ние развития предприятия в пространственно-временном аспекте и обеспечение без- опасности функционирования предприятия. Процессный лидер осуществляет кон- троль за протеканием внутрифирменных процессов обмена товарно-материаль- ными, информационными и трудовыми ресурсами между различными подразделе- ниями предприятия. Сфера действия проектного лидера – технологические и иные 10 Пленарное заседание нововведения и мероприятия. Должностные обязанности средового лидера связаны с созданием и поддержанием в работоспособном состоянии внутренней инфраструк- туры предприятия, обеспечением благоприятного социального климата и повыше- нием привлекательности предприятия для новых работников и контрагентов. нововведения и мероприятия. Должностные обязанности средового лидера связаны с созданием и поддержанием в работоспособном состоянии внутренней инфраструк- туры предприятия, обеспечением благоприятного социального климата и повыше- нием привлекательности предприятия для новых работников и контрагентов. Параллельно со структурой административного управления на предприятии, переходящем к инклюзивному управлению, создается структура неформального ли- дерства, также включающая четырех лидеров, осуществляющих свою деятельность в рамках социальной среды предприятия. В структуру социального лидерства вхо- дят: инспиративный лидер, обладающий способностями влиять на поведение работ- ников с помощью личного примера или убеждения; культурный лидер, обладающий знаниями в сфере технологии и организации производства; интеллектуальный ли- дер, владеющий информацией о состоянии и динамике рынка в зоне хозяйствования предприятия и определяющий состав товаров и услуг, предлагаемый предприятием; духовный лидер, способный определить положение предприятия в перспективной внешней среде (Клейнер, 2022). Данная структура социального лидерства коррелирует со структурой функ- ционалистского лидерства, предложенной И. Адизесом (Адизес, 2007) (табл. 1) Системное лидерство, социальное лидерство и целевые функции лидеров Системные лидеры Социальные лидеры Функции по И. Адизесу Характеристика деятельности организа- ции, обеспечиваемая данной функцией Объектный Инспиративный Е-функция Результативность в долгосрочной перспек- тиве Процессный Культурный А-функция Эффективность в краткосрочной перспек- тиве Проектный Интеллектуаль- ный Р-функция Результативность в краткосрочной перспек- тиве Средовой Духовный I-функция Эффективность в долгосрочной перспек- тиве Системное лидерство, социальное лидерство и целевые функции ли В целом социальная сфера предприятия может рассматриваться как своего рода полевая структура, индуцированная влиянием соответствующих социальных лидеров: сфера инспиративного капитала; сфера культурного капитала; сфера интел- лектуального капитала; сфера духовного капитала. СТРАТЕГИЯ И СТРУКТУРА ИНКЛЮЗИВНОГО УПРАВЛЕНИЯ ПРЕДПРИЯТИЕМ При этом система управления утра- чивает свойство иерархической расслоенности и консолидируется. 2. Системное инклюзивное управление предприятием предполагает опору на сочетание формальной (административной) структуры управления и неформальной структуры социального лидерства. Такой подход обеспечит максимальную эффек- тивность использования уникальных специалистов, чей интеллект способен решить возникающие в условиях изоляции задачи. 3. Принципы бережливого производства должны быть распространены на бережное отношение к работникам предприятия. В особенности это должно отно- ситься к тем специалистам, чья квалификация позволит преодолеть разрывы во внут- рифирменных технологических и внешнефирменных рыночных цепочках поставок полупродуктов, сырья и комплектующих. 4. Органической частью инклюзивного управления на предприятиях должны стать процессы демократизации управления, создания на каждом предприятии атмо- сферы солидарности и взаимного уважения. Особое внимание требуется уделить процессам расширения в стране популяции бирюзовых, а впоследствии и перламут- ровых предприятий (Клейнер, 2020). СТРАТЕГИЯ И СТРУКТУРА ИНКЛЮЗИВНОГО УПРАВЛЕНИЯ ПРЕДПРИЯТИЕМ Соответственно, полевая струк- тура предприятия может быть представлена как совокупность четырех полей: поля влияния силы власти; поля влияния культуры; поля влияния интеллекта; поля влия- ния силы духа. Инклюзивное управление интегрирует интересы, возможности и им- перативы участников хозяйственной деятельности, обусловленные их принадлежно- стью к соответствующим внутрифирменным силовым полям. 11 XXIII Всероссийский симпозиум «Стратегическое планирование и развитие предприятий» Социальные лидеры принимают на себя неформальную ответственность за содержание ответов на следующие вопросы: «что производить и кому продавать?» (стратегия) – интеллектуальный лидер; «кто должен производить?» (организация производства) – инспиративный лидер; «как производить?» (технология производ- ства) – культурный лидер; «зачем производить?» (высшие цели и смыслы производ- ства) – духовный лидер. Мы видим, что номенклатура производства, состав участ- ников производства, технология и высшие цели деятельности находятся под идей- ным руководством неформальных социальных лидеров. Описанное выше распределение функций управления между социальными лидерами можно назвать «расщеплением власти». Такое расщепление, однако, не ослабляет власть, а, наоборот, способствует ее укреплению, поскольку опирается на структуру социальных сил предприятия. В том или ином виде подобное расщепле- ние распространяется не только на микроуровень (предприятие и его подразделе- ния), но и на мезоуровень (региональное и отраслевое управление), и на макроуро- вень (управление социально-экономическим развитием страны). Можно найти эле- менты подобного расщепления, скажем, в структуре управления судном и в других управленческих структурах армии. Учитывая, что данная статья отражает содержание пленарного доклада, сде- ланного 12 апреля 2022 г. в День космонавтики на XXIII Всероссийском симпозиуме «Стратегическое планирование и развитие предприятий», в качестве примера при- ведем распределение социальных лидерских ролей в команде С.П. Королева по со- зданию первого искусственного спутника Земли (реконструкция выполнена на ос- нове книги воспоминаний С. Хабарова (2021), работавшего вместе с С.П. Короле- вым, а также ряда других источников): инспиративный лидер – В.П. Мишин, первый заместитель и впоследствии наследник дела С.П. Королева; духовный лидер – Л.А. Вознесенский, ответственный за испытания техники; культурный лидер – С.О. Охапкин, заместитель по конструированию; интеллектуальный лидер – К.Д. Бушуев, начальник проектно-исследовательского отдела. Формальные институты управления (Chief Executive Officer (CEO), Chief Technology Officer (CTO), Chief Operating Officer (COO), Chief Financial Officer (CFO)) должны быть дополнены неформальными институтами лидерства (Leading Executive Officer (LEO), Leading Spiritual Officer (LSO), Leading Cultural Officer (LCO), Leading Intelligence Officer (LIO)). Изложенное выше позволяет сделать следующие выводы. Изложенное выше позволяет сделать следующие выводы. 1. Переориентация управления предприятиями на принципы инклюзивного управления, согласно которым внутрифирменное социальное пространство стано- вится более однородным, является тем более необходимой, чем более 12 Пленарное заседание изолированной становится экономика страны. СПИСОК ИСПОЛЬЗОВАННЫХ ИСТОЧНИКОВ Адизес И. (2007). Идеальный руководитель: почему им нельзя стать и что из этого следует. М.: Альпина Бизнес Букс. 262 с. Адизес И. (2007). Идеальный руководитель: почему им нельзя стать и что из этого следует. М.: Альпина Бизнес Букс. 262 с. Валько Д.В. (2020). Циркулярная экономика: основные бизнес-модели и экономические возможности // Журнал экономической теории. Т. 17. № 1. С. 156–163. DOI: 10.31063/2073-6517/2020.17-1.12 Валько Д.В. (2020). Циркулярная экономика: основные бизнес-модели и экономические возможности // Журнал экономической теории. Т. 17. № 1. С. 156–163. DOI: 10.31063/2073-6517/2020.17-1.12 Клейнер Г.Б. (1986). Производственные функции: теория, методы, применение. М.: Финансы и стати- стика. 238 с. Клейнер Г.Б. (2011). Новая теория экономических систем и ее приложения // Вестник Российской акаде- мии наук. Т. 81. № 9. С. 794–808. Клейнер Г.Б. (2020). Спиральная динамика, системные циклы и новые организационные модели: перла- мутровые предприятия // Российский журнал менеджмента. Т. 18. № 4. С. 471–496. DOI: 10.21638/spbu18.2020.401 Клейнер Г.Б. (2022). Социальное лидерство, расщепление власти и инклюзивное управление организа- цией // Вопросы экономики. № 4. С. 26–44. DOI: 10.32609/0042-8736-2022-4-26-44. Клейнер Г.Б. (2022). Социальное лидерство, расщепление власти и инклюзивное управление организа- цией // Вопросы экономики. № 4. С. 26–44. DOI: 10.32609/0042-8736-2022-4-26-44. Мануйлова А. (2022). Безработица примеряется к росту // Коммерсант. 2022. 29 марта. URL: р ( ) р р у р р цией // Вопросы экономики. № 4. С. 26–44. DOI: 10.32609/0042-8736-2022-4-26-44. Мануйлова А. (2022). Безработица примеряется к росту // Коммерсант. 2022. 29 марта. URL: https://www.kommersant.ru/doc/5281886 (дата обращения: 11.07.2022). Мануйлова А. (2022). Безработица примеряется к росту // Коммерсант. 2022. 29 марта. URL: https://www.kommersant.ru/doc/5281886 (дата обращения: 11.07.2022). Оперативный социально-экономический мониторинг. Результаты первой волны мониторинга среди предпринимателей. (2022). Март 2022 г. НАФИ // Сайт НАФИ. URL: https://nafi.ru/projects/predprinimatelstvo/pervaya-volna-operativnogo-sotsialno-ekonomicheskogo- monitoringa-sredi-predprinimateley/ (дата обращения: 11.07.2022). Хабаров С. (2021). Сюжет в центре. М.: Litres. Хацкевич Г.А., Проневич А.Ф. (2017). Квазиоднородные производственные функции единичной эла- стичности замещения факторов по Хиксу // Экономика. Моделирование. Прогнозирование. № 11. С. 135–140. Revankar N.S. (1971). A class of variable elasticity of substitution production functions. Econometrica, Vol. 39, No. 1, pp. 1–71. Revankar N.S. (1971). A class of variable elasticity of substitution production functions. Econometrica, Vol. 39, No. 1, pp. 1–71. 13
https://openalex.org/W1496605296
https://www.biodiversitylibrary.org/itempdf/130314
English
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The bird, its form and function,
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public-domain
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O; ii m ir CD 1 =O r=\ a m a THE BIRD THE BIRD THE BIRD esperornis, — a wingless, toothed, diving bird, about five feet in length, which inhabited the great seas during the Cretaceous period, some four millions of years ago. Hesperornis, — a wingless, toothed, diving bird, about five feet in length, which inhabited the great seas during the Cretaceous period, some four millions of years ago. ROBERT DRDMMOND, PRINTER, NEW YORK American j]5ature Series Group II. The Functions of Nature THE BIRD ITS FORM AND FUNCTION BY C. WILLIAM BEEBE Curator of Ornithology of the New York Zoological Park and Life Member of the New York Zoological Society ; Member of the American Ornithologists' Union and Fellow of the New York Academy of Sciences Author of " Two Bird- Lovers in Mexico" WITH OVER THREE HUNDRED AND SEVENTY ILLUSTRATIONS CHIEFLY PHOTOGRAPHED FROM LIFE BY THE AUTHOR NEW YORK HENRY HOLT AND COMPANY 1906 Copyright, 1906 BT HENRY HOLT AND COMPANY Published September, 1906 DEDICATED IN GRATITUDE AND ESTEEM TO professor "fccnrg ffairfielo ©sborn BY HIS FORMER PUPIL THE AUTHOR PKEFACE WE find to-day some thirteen or fourteen thousand different forms, or species, of birds upon the earth. For many years ornithologists have laboured to name, and to arrange in some rational order, these multitudinous forms of bird life. Some such arrangement is, of course, a neces- sity— without a handle we should indeed be handicapped in studying a bird; but let us not forget that classification is but a means to an end. Far too many students of birds follow some such mode of procedure as this: When a new bird is found, it is shot, labelled, preserved in a collection and forgotten; or, if studying the bird with a glass, all effort is centred in finding some characteristic by which it can be named, and, succeeding in this, search is at once made for still another species, whose name can in turn be added to a list. Observing the habits, the courtship and nest-building, and memorizing the song, is a third phase of bird-study — the best of all three methods; but few indeed have ever given a moment's thought to the bird itself. given a moment's thought to the bird itself. I have lectured to an audience of teachers, every one of whom was able to identify fifty birds or more, but not one among them knew the significance of the scales on Preface Vlll a bird's foot. It is to bridge this gap that this book is intended — an untechnical study of the bird in the abstract. This, it seems to me, is the logical phase of bird life, which, with an earnest nature-lover, should follow the handbook of identification — the study of the physical life of the bird itself preceding the consequent phase of the mental life, with its ever-varying outward ex- pression. pression. Far from considering this treatment exhaustive, one must remember that any chapter subject could easily be elaborated into one or more volumes. I have intended the book more as an invitation than aught else: for each to observe for himself the marvellously fascinating drama of evolution; to pass on from the nature stories of ideal- ized composite animals and birds to the consideration of the evolution of all life; to the tales of time and truth which have been patiently gleaned by the life-long labours of thousands of students. PKEFACE Whenever possible I have illustrated a fact with a photograph from a preparation or from a living bird, believing that, where verbal exposition fails, pictorial interest will often fix a fact in the memory. First of all we must consider a few of the more important and sig- nificant of the bird-forms of past ages; because no one who is interested in living birds from any standpoint should be entirely ignorant of a few facts concerning the ancestors of these creatures: Otherwise it is as if one, entirely ignoring the rest of the plant, studied certain leaves and flowers, knowing not whether they came from tree or vine. Pre NEW YORK ZOOLOGICAL PARK, May, 1906. Pre ix In my treatment of the various phases of the bird's physical life I have been considerably influenced by the many questions which I have heard asked by visitors to the New York Zoological Park. The short list of books in the Appendix will indicate the sources whence much more detailed information may be obtained by those who desire it. Some two dozen of the illustrations are from outside sources, and for permission to use these I am indebted to Dr. William T. Hornaday, the American Museum of Natural History, Prof. A. Smith-Woodward, Prof. R. S. Lull, A. E. Brown, Esq., Mr. R. H. Beebe, Mr. T. H. Jackson, Mr. Harold Whealton, and Mr. E. H. Baynes; and for the use of specimens to Dr. F. A. Lucas, Dr. Robert Ridgway, and Dr. Jonathan Dwight, Jr. Unless otherwise indicated, the illustrations were taken by the author. The work of Mr. Walter King Stone in the paint- ing for the frontispiece and a number of text cuts is gratefully acknowledged; and for the skilful printing of many of the photographs my thanks are due to Mr. E. R. Sanborn. To my wife, for constant and valuable help, criticism, and suggestion in all departments of the book, I render my sincere appreciation. To take a few dead facts and clothe them with the living interest which will make them memorable and full of meaning to any lover of birds, and at the same time to keep them acceptable in tenor and truth to the most critical scientist — this has been my aim. Preface Preface x A few chapters of this volume have already appeared in print in "Outing," "Bird-Lore," and the "New York Evening Post." C. W. B. CONTENTS CHAPTER PAOB 1. ANCESTORS 1 II. FEATHERS 19 III. THE FRAMEWORK OF THE BIRD 62 IV. THE SKULL 103 V. ORGANS OF NUTRITION 116 VI. THE FOOD OF BIRDS 142 VII. THE BREATH OF A BIRD 1 65 VIII. MUSCLES AND NERVES 188 IX. THE SENSES 203 X. BEAKS AND BILLS 223 XI. HEADS AND NECKS 252 XII. THE BODY OF A BIRD 285 XIII. WINGS 319 XIV. FEET AND LEGS 353 XV. TAILS 398 XVI. THE EGGS OF BIRDS 427 XVII. THE BIRD IN THE EGG 462 APPENDIX — Brief List of Useful Books 483 INDEX 485 xi 39966 39966 ANCESTORS I ITH the exception of Astronomy, the science which most powerfully dominates our imagina- tion is Palaeontology, or the study of the life of bygone ages. Of all things in Nature, the stars symbolize absolute immensity, their distances stretching out beyond our utmost calculation. So the revelations of Palaeon- tology take us far beyond the sciences of life on the earth to-day, and open vistas of time reaching back more than five-hundred-fold the duration of the sway of mankind. Fossil bones — philosophically more precious than any jewels which Mother Earth has yielded — are the only certain clews to the restoration of the life of past ages, millions of years before the first being awakened into human consciousness from the sleep of the animal mind. Until recently, Palaeontology has been popularly con- sidered one of the dryest and most uninteresting of the 'ologies, but now that the fossil collections in our museums are being arranged so logically and so interestingly, the most casual lover of Nature can read as he runs some of The Bird 2 the "poems hidden in the bones." As Professor Huxley once said, "Palaeontology is simply the biology of the past, and a fossil animal differs only in this regard from a stuffed one, that the one has been dead longer than the other, for ages instead of for days." the other, for ages instead of for days." A great many more fossil mammals and reptiles have been discovered than birds, and the reason may perhaps be conjectured. The bones and bodies of birds were in former times as now very light, and if death occurred on the water, the body would float and probably be de- voured by some aquatic reptile. Then, again, when some cataclysm of nature or change of climate obliterated whole herds and even races of terrestrial creatures, the birds would escape by flight, and when death eventually came, they would be stricken, not in flocks, but singly and in widely scattered places as to-day. and in widely scattered places as to-day. For perhaps a million years in the past, birds have changed scarcely at all, — the bones of this period belong- ing to the species or at least genera of living birds. ANCESTORS But in the period known as the Cretaceous, when the gigantic Dinosaurs flourished and those flying reptile-dragons — the Pterodactyls— flapped through the air, a few remains of birds have been found. Some of these are so com- plete that almost perfect skeletons have been set up, enabling us vividly to imagine how the bird looked when swimming through the waters of our globe, or flying through the air, perhaps four millions of years ago. through the air, perhaps four millions of years ago. The most remarkable peculiarity of these birds was the possession of teeth. Two of the most well-known examples are called Ichthyornis and Hesperornis. The Ancestors 3 bones of these birds were discovered by Professor Marsh imbedded in the rocks of western Kansas, and they are now preserved in the museum of Yale University. Pro- fessor Marsh tells us that Hesperornis, the Bird of the IG. 1. — Restored skeleton of Ichthyornis (after Marsh). 1/2 natural size. FIG. 1. — Restored skeleton of Ichthyornis (after Marsh). 1/2 natural size. West, "was a typical aquatic bird, and in habit was doubtless very similar to the loon, although, flight being impossible, its life was probably passed entirely upon the water, except when visiting the shore for the purpose of breeding. The nearest land at that time was the sue- The Bird 4 cession of low islands which marked the position of the present Rocky Mountains. In the shallow tropical sea, extending from this land five hundred miles or more to the eastward, and to unknown limits north and south, there was the greatest abundance and variety of fishes, and these doubtless constituted the main food of the present species. Hesperornis, as we have seen, was an admirable diver; while the long neck, with its capabilities of rapid flexure, and the long slender jaws armed with sharp recurved teeth, formed together a perfect instru- ment for the capture and retention of the most agile fish. The lower jaws were united in front only by cartilage, as in serpents, and had on each side a joint which admitted of some motion, so the power of swallowing was doubt- less equal to almost any emergency." less equal to almost any emergency." Hesperornis had numerous teeth set in grooves like those of serpents and crocodiles, but in Ichthyornis ('Fish- bird/ so called because its vertebra? ANCESTORS are biconcave like those of a fish) the teeth were in separate sockets as in alligators. The latter bird was not large, being about the size of a pigeon, and it had well-developed wings. the size of a pigeon, and it had well-developed wings. It is interesting to compare Hesperornis with the group of penguins, both being highly specialized, although in ways so different, for an almost wholly aquatic life. Hesperornis swam by strong strokes of its great webbed, or lobed, toes, its wings dangling uselessly for genera- tion after generation, until all trace, save a vestigial humerus, of their bony support disappeared. Penguins, however, make but little use of their feet in swimming, only occasionally aiding the tail in steering; but they Ancestors Ancestors literally fly through the water by means of their flipper- like wings. The large size of the leg and toe bones of Hesperornis shows that great speed was attainable in the water, FIG. 2. — Lower jaw of Ichthyornis (after Marsh). 4/5 natural sizf. FIG. 2. — Lower jaw of Ichthyornis (after Marsh). 4/5 natural sizf. FIG. 3.— Lower jaw of Alligator. 1/6 natural size. The teeth are set in distinct sockets both in the extinct bird and in the living reptile. FIG. 3.— Lower jaw of Alligator. 1/6 natural size. The teeth are set in distinct sockets both in the extinct bird and in the living reptile. while only a single bone remained to show where the wings of its ancestors were situated. It is doubtful if it could stand erect upon land, being in this respect more helpless even than a grebe. Its nest, if it made one, must have been at the very edge of the shore, from which The Bird 6 it could wriggle or push itself with its powerful toes into the water. The thought of the untold generations of birds which must have preceded this toothed, wingless, feathered being, makes the mind falter at the vast stretches of time during which evolution has been unceasingly at work. When we examine the skull of Hesperornis we get a clew to the reason why this great creature, nearly as large as a man, succumbed when some slight change in its environment called for new adjustments in its habits of life. ANCESTORS Its brain was comparatively smaller than that of any existing bird; and this absence of brain power im- plied a total lack of that ingenuity, so prominent in the crow, which, when man alters the face of the land, changes its habits, and with increasing wit holds its own against guns and traps. guns and traps. When Hesperornis passed, it was succeeded by birds much smaller in size but of greater wit — loons and grebes —which hold their own even to the present day. —which hold their own even to the present day. When in the depth of the winter, a full hundred miles from the nearest land, one sees a loon in the path of the steamer, listens to its weird, maniacal laughter, and sees it slowly sink downward through the green waters, it truly seems a hint of the bird-life of long-past ages. seems a hint of the bird-life of long-past ages. We must now pass back, as nearly as can be estimated, over two millions of years, through the ages when the Iguanodonts and Megalosaurs lived, long before the first serpents had evolved and about the time when the first timid forerunners of the mammals made their appear- ance,— tiny insect-eating creatures which were fated to Ance 7 remain so long subordinate to the masterful giant reptiles. This was about the middle of the Jurassic period, and in deposits of this epoch have been found remains of the very first birds of which we know anything. Two specimens have been discovered and named Archce- opteryx (ancient-winged-creature). From these two little stone slabs, one in the British Museum and the other at Berlin, we know that these birds were about the size of a crow. Instead of the broad, fan-shaped tail of modern birds, the tail of the ArcJiceopteryx was a long, jointed affair like that of a lizard, and was fringed with large feathers — a pair growing from each of the twenty joints. The wings were not large, and instead of the fingers being concealed by feathers, there were three entirely free digits, each armed with a. claw, in front of each wing. The skin-covered jaws were furnished with teeth, but the feet and legs were much like those of an ordinary crow. ANCESTORS Taken all in all, this was a most wonderful discovery, linking birds and reptiles together, and proving beyond all dispute the fact of their common origin. Perhaps the most surprising fact was the remarkable development of the plumage of the wings and tail, showing that perfect feathers were in existence at least six millions of years ago. ago. In the rocks deposited in very ancient epochs are found many footprints which were supposed to be those of huge birds, but it is more probable that they were made by certain three-toed reptiles which, like birds, walked or hopped on two feet. Indeed Nature seems to have made several abortive attempts to produce bird- The Bird 8 like creatures before she struck the right adjustments. Pterodactyls failed to become birds because they depended on a broad web of skin, like the wing of a bat, thus miss- ing the all-necessary feather-ideal; Dinosaurs began at the wrong end, learning to stand on their hind feet and to hop, but never the delights of flight. These offshoots sooner or later were forced to the wall, but Archceop- teryx seems to have been very near the true line of descent. But after all, what a meagre record we have of the un- told myriads of generations of birds which have succeeded each other through ages past! It is to be hoped that many more fossils may be discovered, for the hints given us in the anatomy of birds, and the glimpses of past his- tory which flash out from the development of the chick within the egg, — all this evidence is becoming ever more and more clouded and illegible. Having learned that birds are descended from a rep- tile-like ancestor, it is interesting to search among living reptiles for the one which most resembles birds, and we have no choice but to select the alligator — cold-blooded, scaly, bound to the earth though he is. A second near relation is to be found in the group of long-extinct Dino- saurs. A complete record of past ages would show the ancestral stems of alligators, Dinosaurs, and birds grad- ually approaching each other until somewhere, at some time, they were united in a common stock. * There are one or two reasons for regarding Archceopteryx as merely the tip of a parallel branch, but one sprouting close to the base of the avian tree. ANCESTORS But we must guard against the notion that birds are descended from any group of living reptiles; which is as fallacious an idea as that we Americans trace our direct descent from FIG. 4. — Anhceopteryx preserved in the British Museum. The pelvic girdle, le and tail show best in this specimen. 1/5 natural size. FIG. 4. — Anhceopteryx preserved in the British Museum. The pelvic girdle, leg and tail show best in this specimen. 1/5 natural size. The Bird i the Chinese, or that mankind is descended from the chim- panzee or gorilla. For the purpose of making more clear and interesting the ways in which birds have become especially adapted to their surroundings and needs, we may consider Archce- opteryx as resembling closely the typical original bird- type from which all others have at least indirectly evolved ; and thus having obtained a definitely fixed starting-point, we may consider how some of the more representative birds of the present day came to acquire their widely differing structure and characteristics.* differing structure and characteristics.* The tree of evolution of reptiles may be compared to a growth where several great trunks spring from the ground close together, towering up separately but equally high; the topmost twigs of which are represented by the living species of serpents, turtles, lizards, and crocodiles respectively. A very different arboreal structure is pre- sented in the genealogical tree of the Class of birds. Here, from a short trunk, we have many radiating branches, widely spreading and with thickly massed twigs, confu- sedly intermingled ; so slight are the divergences between adjoining groups and so equally do almost all share be- tween them various reptilian characteristics. It is not necessary to concern ourselves now with the processes of evolution, especially as scientists are still in doubt as to the exact methods. Let us read our Darwin, and hope for another, philosophically as great, to com- FIG. 5. — Archoeopteryx preserved in the Berlin Museum. The skull, vertebrae, forelimbs and flight-feathers are remarkably distinct. 1/3 natural size. FIG. 5. — Archoeopteryx preserved in the Berlin Museum. The skull, vertebrae, forelimbs and flight-feathers are remarkably distinct. 1/3 natural size. ize. The Bird 1 plete the work, meanwhile adding our own mite of truth- ful observation to swell the whole, and help prepare the way for this other. ANCESTORS For even Darwin's theory of evolu- tion was but the consummation of theories of former years and centuries, — beginning with Thales and Anaxi- mander, in the days of early Grecian civilization: in- deed Aristotle, coming but two hundred years later, is the only name in the history of zoology worthy of a place with that of Darwin. From the fragmentary evidence afforded by Archceop- teryx we may conclude that this Bird of Old had a short, blunt, skinny bill of moderate size, furnished with teeth which would enable the owner to feed upon Jurassic berries and fruit, or more probably a carnivorous diet of lizards and insects. Its wings were weak, hinting that it was a flutterer rather than a true flyer, perhaps only scaling like a flying squirrel from the summit of one tree to the base of the next. Even this would give it an immense advantage over its terrestrial and arboreal non-flying enemies. The three free fingers on each wing would allow it to climb easily, to pry into crevices for insects, or to draw a berry-laden branch close to its bill. Doubtless it frequently walked or ran on all fours, the more probably from its weak-loined condition, — the bones of the thigh-girdle not being fused together as in modern birds. Its tail has already been mentioned — a long double-feathered appendage, composed of a score of little vertebrae jointed together, — as we will later see the true forerunner of the modern fan-like tails. Its FIG. 6. — Restoration of Archceopteryx (adapted from Smit). Notice the teeth, three fingers, and lizard-like tail. 13 FIG. 6. — Restoration of Archceopteryx (adapted from Smit). Notice the teeth, three fingers, and lizard-like tail. The Bird H feet and legs were little different from those of perching birds of to-day, with strong toes well adapted to cling to a branch. Finally, from a cast of the brain, which fortunately was found with one of the fossils, we know that, although small, it was that of a true quick-witted bird. As yet science has no more to tell us. bird. As yet science has no more to tell us. ANCESTORS Our fancy may add an archaic attempt at song — a lizard's croak touched with the first harmony, which was to echo through all the ages to follow; we may also imagine, if we will, leathery eggs deposited in a rotten knot-hole of a Jurassic conifer. In both islands of New Zealand well-preserved remains of giant birds have been discovered, to which has been given the name of moas. One species must have reached a height of ten or eleven feet, which would make it tower above the largest living ostrich. They were, in fact, not unrelated to these latter birds and, like them, were flightless (in some cases absolutely wingless), and they had great massive feet and legs. Native legends among the Maoris hint that these birds were in existence during the last few centuries before the coming of the white men. In South America also, giant birds lived in ages past. One, the Phororhacos, stood seven to twelve feet in height, with a head and beak like that of a gigantic eagle. Unlike all eagles, however, this bird could not fly and doubtless ran down its prey, as a chicken runs down a grasshopper. grasshopper. It is an interesting fact that in South America there lives to-day a bird known as the Seriema, which is prob- Ance 1 5 ably at least an indirect descendant of the Phororhacos. The Seriema defies exact classification, sharing characters of cranes, bustards, and eagles. Its beak and inner claw are like those of a bird of prey, while in form of body, and in the other claws of the toes, and in the legs it is crane-like. One of these birds which I have ob- served for years in captivity is as gentle and as fearless as a bird can be. It will chase insects and field-mice outdoors in the Zoological Park, and will occasionally stalk solemnly into my office and, coming close to my desk, watch me closely. It has most beautiful gray- blue eyes, with long eyelashes (Fig. 199), and if the birds of past ages were as comely and as lovable as this interesting species, I regret that only their fossil bones are left to us. As the Seriema runs down and kills a mouse, so the giant Phororhacos, doubtless, overtook and slew creatures as large as a deer. Its skull (Fig. ANCESTORS 7) is drawn to the same scale as that of the living Seriema (Fig. 8). The evolution which has gone on since these epochs of old, bringing into being the wonderfully varied forms of penguin, ostrich, albatross, peacock, and humming- bird, may be summed up in two words which it is well to know and remember, — Adaptive Radiation. This is the spreading out or radiating of bird-forms descended from the ancient stem, into all parts of the earth, each coming into contact with a particular environment, to adjust itself to which, its various organs and parts exer- cise different functions, until the friction of the "struggle for existence" has moulded each to its particular niche. The Bird i6 If its lines lie in happy places, its race is established, and it pursues and flees, it fights and plays, it sings with joy or pants with fear, and Evolution marks another success in its inexorable movement onward and upward, — a new species is born! — a new species is born! Earth has few secrets from the birds. With wings and legs there is hardly a spot to which they cannot and indeed have not penetrated. Some find food and con- tentment in the desolate wastes of the far North; others spend almost all of their life on or above the sea far from FIG. 7. — Skull of Phororhacos, drawn to scale with Fig. 8. 1/6 natural size. IG. 7. — Skull of Phororhacos, drawn to scale with Fig. 8. 1/6 natural size. land; thousands revel in the luxuriance of reeking trop- ical jungles; a lesser number are as perfectly suited to the blazing dust of the desert; and there are birds which burrow deep into the very earth itself. Day and night; heat and cold; water, earth, and air, have all been con- quered by the thirteen or fourteen thousand species of birds which share the earth with us at the present day. These brethren of ours, whose clans have so bravely conquered the dangers of millions of years, and at last have gained a foremost rank in the scale of living crea- Ance 1 7 tures, now find themselves face to face with the culmi- nating effort of Nature, — Mankind. They cannot escape 8. — Seriema, a living descendant of Phororachos , with characters Bustards, and Eagles. 1/6 natural size. FEATHERS ANY definitions of the Class of birds have been given, but all fall short in some particular, or are weak in having exceptions. FEATHERED is the one word which always holds true. All birds have feathers, and nowhere else in the world are similar struc- tures found. A feather, like an egg, is perfect in its adaptation to the bird's requirements, and also, like the egg, its structure is rather complicated. A ANCESTORS m us, though the least among them laughs to FIG. 8. — Seriema, a living descendant of Phororachos , with characters of Cranes, Bustards, and Eagles. 1/6 natural size. from us, though the least among them laughs to scorn our efforts at following through the air. Yet all must from us, though the least among them laughs to scorn our efforts at following through the air. Yet all must The Bird 1 8 return sooner or later to earth for rest and food, and thus all are at our mercy. Let us beware of needlessly destroying even one of the lives — so sublimely crowning the ages upon ages of evolving; and let us put forth all our efforts to save a threatened species from extinction; to give hearty aid to the last few individuals pitifully struggling to avoid absolute annihilation. The beauty and genius of a work of art may be recon- ceived, though its first material expression be destroyed; a vanished harmony may yet again inspire the composer; but when the last individual of a race of living beings breathes no more, another heaven and another earth must pass before such a one can be again. Structure and Development First let us look at the skin itself in which the feathers grow. To skin a bird is an easy matter, for the skin, or integument as it is called, is very slightly attached to the muscles underneath. The skin of a dove is almost like tissue-paper, and tears so easily that it is a marvel how the hundreds of feathers find a sufficiently strong attachment. Thin as is this skin, it is made up of three separate layers, but in order to make our feather-study enjoyable by not overburdening it with too many details, we will consider only the two more important layers of the skin — a deeper one, the dermis, and an outer, more horny covering, the epidermis. The Bird 20 A list of all the structures of animals which are prod- ucts of the outer layer alone would be a long and sur- prising one, and we would be very ready to grant the importance of skin. Such an enumeration would include all claws and talons, nails and teeth, the rattles of a snake, spurs, hairs, the scales of fishes and reptiles, \\ FIG. 9. — Tarpon-scale, shark-tooth, and peacock-feather; showing diversity of structure derived from the skin alone. 1/2 natural size. FIG. 9. — Tarpon-scale, shark-tooth, and peacock-feather; showing diversity of structure derived from the skin alone. 1/2 natural size. spines, whalebone, beaks and feathers. Even the horn of a rhinocerus is only a solid mass of agglutinated hairs, while as the antithesis to this may be mentioned all down and feathers : the tiniest fluff from a humming- bird to the great pinion of a condor. spines, whalebone, beaks and feathers. Even the horn of a rhinocerus is only a solid mass of agglutinated hairs, while as the antithesis to this may be mentioned all down and feathers : the tiniest fluff from a humming- bird to the great pinion of a condor. If we examine a newly hatched dove or sparrow, the little, ugly, sprawling creature, at first glance, seems Feathers 21 to be entirely naked; but a closer inspection shows scanty tufts of down scattered irregularly over the body. This, like the set of milk-teeth in mammals, is useful only for a time, and is later pushed out by the second or true plumage. Even more numerous than the down- FIG. 10. — Brown Pelican nestlings, showing feather papillae on body and wings. About 1/4 natural size. FIG. 10. — Brown Pelican nestlings, showing feather papillae on body and wings. About 1/4 natural size. tufts are little pimples or dots, many hundreds of which cover certain parts of the skin. Each of these will event- ually give rise to a perfect feather — quill, vane, barbs, and all. tufts are little pimples or dots, many hundreds of which cover certain parts of the skin. Each of these will event- ually give rise to a perfect feather — quill, vane, barbs, and all. The under layer of skin, or dermis, is very thin in birds, much more so than in reptiles and other animals. The Bird The Bird 22 The first intimation of the appearance of a feather, or of down, is shown by a thickened group, or pimple, of cells in this under layer of skin, which grows and presses upward toward the outer layer — the epidermis. This is exactly the way in which the scales of fishes and reptiles begin to form; and if, at this stage, the tiny projection should flatten out, the shining scale of a carp, the armor FIG. 11. — Sprouting feathers of a 12-day embryo chick. Magnified 25 diameters. FIG. 11. — Sprouting feathers of a 12-day embryo chick. Magnified 25 diameters. of an alligator, or the cobble-scale of an iguana lizard might result. Indeed, in the feathers of a penguin we find transition stages of flat, almost unsplit feather- scales; while on the legs and feet of birds are reptile-like scales. The evolution of scales, hair, and feathers is a most interesting problem, most of the details of which are beyond the scope of this work. Suffice it to say that Feat 23 in sharks, which are among the most primitive forms of fishes, the skin is covered with tiny denticles or spines, which consist of enamel and dentine, and which rest on small bony plates. This form of scale is the most ancient known, and the hint of teeth which the description con- veys is not misleading; for we find that in some of these voracious fishes the spines in the skin become enlarged near the edge of the mouth, merging imperceptibly into the rows of cruel teeth which, to a certain extent, are homologous with the teeth of all higher animals. In other fishes the denticles become flattened scales, and many of these fish have teeth of corresponding plate- like form. So it is interesting to know that the scales of fishes and reptiles, the feathers of birds, and the teeth of animals have all evolved from skin structures which at an early stage of growth bear considerable resemblance to each other. But, in our young bird, the slender finger of cells which reaches upward, and whose base at the same time sinks deeply into the dermis, does not broaden out, but splits longitudinally into a number of folds, which grad- ually dry apart and harden into the slender, silky fila- ments which we know collectively as down. The Bird At the base of, and in fact attached to, the little pro- jection which gives rise to the nestling down is a small circular body of cells, which grows but little while the down plumage is serving its use; but when the bird is ready for a coat of true feathers this lower cellular mass begins to grow upward into a second finger, or column, of cells, pushing the base of the down feather out of its The Bird 2 socket. This growth continuing, the down is lifted clear of the skin, being supported on the new structure, and FIG. 12. — Early stages in the development of a down feather, showing close resemblance to scale of fish or reptile. FIG. 12. — Early stages in the development of a down feather, showing close resemblance to scale of fish or reptile. FIG. 12a. — Later stages of Fig. 12, showing the first splitting up of the feather pulp. FIG. 12a. — Later stages of Fig. 12, showing the first splitting up of the feather pulp. soon brushed off and lost. Thus, little by little, in shreds and tatters, the baby plumage is shed and replaced soon brushed off and lost. Thus, little by little, in shreds and tatters, the baby plumage is shed and replaced Feat 25 by true feathers, which overlap, protecting the body from heat and cold, dust and rain. heat and cold, dust and rain. FIG. 126. — Last stages in the formation of a down feather, showing the plumes well above the surface of the skin, as in a newly hatched chick. All greatly enlarged. — Last stages in the formation of a down feather, showing the above the surface of the skin, as in a newly hatched chick. All ged. the time of the first moult, this successi s can be observed in almost any young bird, FIG. 126. — Last stages in the formation of a down feather, showing the plumes well above the surface of the skin, as in a newly hatched chick. All greatly enlarged. At the time of the first moult, this succession of feathers can be observed in almost any young bird, being more noticeable in large species, which have very thick The Bird 2 or lengthened down, as gulls and ducks. The Bird A Red-winged Blackbird, or for that matter almost any passerine nest- ling, looks very odd when it rises up in the nest, gaping for food; the long gray streamers of down waving like an aureole around its head. In some water-birds this nest- ling down retains its usefulness for nearly two months. FIG. 13. — Feather from the head of a young Bobolink, with down still attached to its tip. Twice natural size. FIG. 13. — Feather from the head of a young Bobolink, with down still attached to its tip. Twice natural size. The feathers which replace the down are, when they first appear above the skin, rolled tightly and bound up in the thin tissue of the horny sheaths, so that they resemble a bundle of withes wrapped together in a cloth. In many young birds the feathers remain in this condi- tion until they are nearly full grown, and a young cuckoo Feat 27 or kingfisher is a curious-looking object, most of the bird's body seeming to be tiled with small, bluish sticks. FIG. 14. — Tip of feather from the crown of a young Song Sparrow, showing connection with down. Magnified 25 diameters. FIG. 14. — Tip of feather from the crown of a young Song Sparrow, showing connection with down. Magnified 25 diameters. FIG. 15. — Duck Hawk moulting into juvenal plumage, with the natal down coming away in shreds and tatters. 1/4 natural size. FIG. 15. — Duck Hawk moulting into juvenal plumage, with the natal down coming away in shreds and tatters. 1/4 natural size. When the folds of the developing feathers are sufficiently dry, they burst their sheaths and rapidly spread out. The Bird 2 The appearance of a young kingfisher or heron may be completely changed within a few hours time, so quickly and simultaneously does the first suit of feathers unroll. The condition of young birds when hatched varies greatly in birds of different groups. Nestlings are, in many ways, like human babies, and there are as many differences in the one class as there are in the other, FIG. 16. — Growth of an Ostrich feather from sheath to plume. 1/5 natural size. FIG. 16. — Growth of an Ostrich feather from sheath to plume. 1/5 natural size. between those from different countries, only Nature does for the little birds what parents do for the babies. The Bird We see American babies wrapped in furs and blankets, wheeled in carriages, and rocked to sleep; while a tiny savage is strapped tightly to its mother's back, and as soon as possible allowed to run where it pleases, find its own toys and -develop its little muscles, gaining a degree of health and strength which many a civilized child would envy. So with birds, the highest — such as crows and thrushes — are hatched almost naked and must be warmed Feat 29 and cuddled and fed for many weeks, before they learn to take care of themselves; while birds lower in the scale — as our quail — are born covered thickly with down and with wings nearly feathered, and in a few days can fly and find their own food. So a bird naked at birth is very helpless, one covered with down is more capable of taking care of itself, while FIG. 17. — Nestling Kingfisher with feathers still in their sheaths. 2/3 natural size. FIG. 17. — Nestling Kingfisher with feathers still in their sheaths. 2/3 natural size. the few which are completely feathered when hatched may be said to have no chickhood except in the egg. In the Crested Screamer (Fig. 264) the down-like character of the body-feathers of the adult birds may be a hint of the plumage of very ancient types of birds such as Archceopteryx. Now we are ready to begin our study of the perfect feather itself, and we will, for once, have to disregard our rule of starting with the simpler form — the scale of The Bird 3° a reptile — and working up to a feather; for, if we except the down, there seems to be no connecting link left. FIG. 18. — Young Brown Pelicans; hatched naked and helpless (altricial). 1/4 natural size. FIG. 18. — Young Brown Pelicans; hatched naked and helpless (altricial). 1/4 natural size. FIG. 19. — Young Red Jungle Fowl one day old; hatched covered with down and able within a few hours to help itself (precocial). Almost natural size. FIG. 19. — Young Red Jungle Fowl one day old; hatched covered with down and able within a few hours to help itself (precocial). Almost natural size. The Bird Although that old, old fossil bird Archceopteryx still retained reptile-like teeth, fingers and tail, it had Feathers feathers which were apparently as perfect as any we may examine to-day. When some form of scale had once changed so that it was of use in flight, the hollow elastic vane took first place at once, and all intermediate stages, which perhaps had been acquired merely for warmth, went to the wall. A creature could have flight if pro- vided with perfect feathers, or it could retain its scales and find existence possible along the old reptilian planes of life, but no awkward scale-flutterer could long be tolerated. All through the evidences of evolution we find instances like this, — a change for the better beginning slowly, through many channels, then the one best suited forging ahead with inconceivable swiftness, and crushing out all other less adapted structures. Hence the rarity of "missing links." of "missing links." Feathers are certainly among the most beautiful objects in Nature; and when we learn a little about their structure, they will be still more interesting. No matter how closely we may examine them, with hand-lens or microscope, their beauty and perfection of structure only increase. If we study a feather, say from the wing of a pigeon, we see that its whole structure is subservient to two characteristics — lightness and strength. What won- derful elasticity it has! We can bend the tip so that it touches the base and it will spring back into shape with- out breaking. If we look closely, we will see that each feather is composite — feathers within feathers. The quill gives off two rows of what are called barbs which together form the vane of the feather; each of these barbs has two The Bird 3 rows of barbules, and these give rise to a series of curved hooks, known as barbicels, which work into opposite series of grooves, so tightly that air cannot force its way through the feather. When the wings are pressed down- ward, the phenomenon flight is made possible by the accumulated resistance which the flight-feathers offer to the air. At the lower end of our pigeon's feather, bar- bicels are present only near the quill. Therefore the FIG. 20. — Two interlocked barbs from the vane of a Condor's wing-feather, show- ing barbules and barbicels. Magnified 25 diameters. FIG. 20. The Bird — Two interlocked barbs from the vane of a Condor's wing-feather, show- ing barbules and barbicels. Magnified 25 diameters. tips of the barbs are loose and fluffy, unconnected and useless for flight. This is the condition in all down and in the feathers of the ostrich and cassowary. We might naturally think that feathers stiffened by so many close rows of interlocking barbicels would be useful in many ways beside flight. But fluffy feathers are evidently just as efficient in keeping warmth in and rain out as the other kind; so Nature, economical to the most micro- scopic degree, has lessened the number of, or has never provided, barbules and barbicels wherever a feather is not needed for flight or steering. tips of the barbs are loose and fluffy, unconnected and useless for flight. This is the condition in all down and in the feathers of the ostrich and cassowary. We might naturally think that feathers stiffened by so many close rows of interlocking barbicels would be useful in many ways beside flight. But fluffy feathers are evidently just as efficient in keeping warmth in and rain out as the other kind; so Nature, economical to the most micro- scopic degree, has lessened the number of, or has never provided, barbules and barbicels wherever a feather is not needed for flight or steering. Feathers Feathers 33 The two lines of barbs which grow out on each side of the quill are very elastic and so intimately hooked to each other that they will bend some distance before sepa- rating. If we ever tried to force our way through a FIG. 21. — Model showing interlocking barbules and barbicels of feather, greatly enlarged. FIG. 21. — Model showing interlocking barbules and barbicels of feather, greatly enlarged. bramble of sweet-brier or blackberry- vines, we can more readily appreciate how these barbs and the interlocking barbules clutch each other. The thorns in the bramble catch our clothes and, when we move, the elasticity of the long stems tends to make them hold the tighter. We notice that one line of barbs — that along the inner curve of the quill — is much longer than that on the outer curve and we might think the air would force this The Bird 3 upward and escape beyond the edge. So it would, if it were not for the arrangement of the feathers on the wing, which overlap like the tiles on a roof, each vane over- lying and holding down the long barbs of the feather in front, while, above and below, other shorter feathers help to bind the whole tightly, thus enabling the bird at every stroke to whip a wingful of air downward and backward. A feather and its parts, like all the rest of the bird, is composed of cells — empty and hollow ones in this in- stance, as we can easily see for ourselves by placing a barb from a pigeon's feather in a drop of water and looking at it under a low-power magnify ing-lens. The network of horny cells is very plain. It is a simple matter to say that a feather consists of quill, barb, barbules, etc., but to appreciate the wonder- ful complexity of this structure let us make a little cal- culation. Suppose we have a wing-feather from a com- mon pigeon with a vane about six inches long. If we have patience enough to count the barbs on one side of the quill, we will find there are about six hundred of them. So the vane of the entire feather has twelve hundred of these little side featherlets. FIG. 22. — Feathers illustrating conditions where barbicels are unnecessary and are hence reduced or entirely lost, causing downiness. 3/5 natural size. (a) Primary of Pigeon — an important flight-feather; hence possessing a stiff vane. (6) Under wing-covert of a Great Blue Heron; downy portion was over- lapped by the adjoining feather, (c) Wing-covert of Owl; the downy edge makes possible the all-important noiseless flight of this bird. (d) Feather of Ostrich; the power of flight being lost, the feathers are downy throughout the entire vane. 35 Feathers One of these, from a narrow part of the vane, will show under the micro- scope about two hundred and seventy-five pairs of bar- bules, which multiplied by the number of barbs on that side amounts to three hundred and thirty thousand. Making a very low estimate of the whole vane, we have nine hundred and ninety thousand separate barbules on this one feather, and when we think of the innumerable . I l*L' FIG. 22. — Feathers illustrating conditions where barbicels are unnecessary and are hence reduced or entirely lost, causing downiness. 3/5 natural size. (a) Primary of Pigeon — an important flight-feather; hence possessing a stiff vane. (6) Under wing-covert of a Great Blue Heron; downy portion was over- lapped by the adjoining feather, (c) Wing-covert of Owl; the downy edge makes tire 35 tire 35 The Bird finer booklets, and then the number of feathers on the pigeon's body, we can echo the exclamation of Solomon: "The way of an eagle in the air" is "too wonderful for me!" Another beautiful adaptation to flight is seen in our FIG. 23. — Feathers of Condor and Emeu. The aftershaft in the former is reduced to a downy filament at the base of the vane; in the latter it equals the feather itself in size. FIG. 23. — Feathers of Condor and Emeu. The aftershaft in the former is reduced to a downy filament at the base of the vane; in the latter it equals the feather itself in size. feather. The upper part of the wing must of course be perfectly level, with no projections to catch the air and retard motion. So, on the upper side of the feather, we notice that the lines of barbs spring out flush with the flattened quill-top, while below, the shaft projects promi- nently from the vane. The obliquely forward direction in which the barbs grow, the change in shape of the feather. The upper part of the wing must of course be perfectly level, with no projections to catch the air and retard motion. So, on the upper side of the feather, we notice that the lines of barbs spring out flush with the flattened quill-top, while below, the shaft projects promi- nently from the vane. Feathers The obliquely forward direction in which the barbs grow, the change in shape of the Feat 37 quill — round where the body or body-feathers conceal it, square where it supports the vane, — and many other niceties which we can each detect for ourselves, show how exquisitely exact is the adaptation of a feather to its uses. FIG. 24. — Powder-down patch on the breast of a live Great White Heron. 2/3 natural size. FIG. 24. — Powder-down patch on the breast of a live Great White Heron. 2/3 natural size. Growing from the under side of the quill, at the be- ginning of the vane, is a tiny feather known as the after- shaft. In an ordinary down-feather of a young bird this is of considerable size, but it is either small or entirely absent in an ordinary feather. It reaches its greatest development in the emeu and the cassowary, where it is The Bird 3 8 as long and as perfect as the main feather. The origin and use of this feather-double is not known. and use of this feather-double is not known. Parrots, herons, and some other birds have a most convenient arrangement — a kind of automatic clothes- cleaner and valet combined. Concealed by the long body- plumage are several dense patches of down-feathers which grow quite rapildy, but instead of constantly increasing in length, the tips break up into a fine, white, greasy powder. This works its way through the entire plumage, and is doubtless of use in keeping the feathers in good condition and the body dry. Most of the birds possess- ing this convenience are comparatively free from lice, so this natural dressing may be as unpleasant to these ver- min as camphor-balls are to clothes-moths. min as camphor-balls are to clothes-moths. The forms and textures of feathers are innumerable, and the uses to which they are put, more than we would ever imagine, but these will be spoken of under the chap- ters treating of the different parts of the body where they are found. Arrangement In examining a nestling we will notice that the feather- dots are not scattered at random over the surface of the skin, but grow in lines and tracts, whose limits are very sharply defined. In an adult bird, say an English Spar- row, this is even more noticeable. If we part the feathers on the centre of the breast, a broad, bare area is seen, with only a thin scattering of soft downy feathers. Under the wings are other naked spaces, and several more are on other parts of the body. The most ancient birds were Feat 39 probably covered uniformly with scale-feathers, but as these increased in length there was less need for an un- broken covering, the feathers of one portion overlapping and protecting the surrounding parts, and besides, for ease in active motions, bare patches of skin were required. It has been found that the arrangement of the feathers on a bird's body varies in different groups, and, such variation being rather characteristic of these larger divi- FIG. 25. — Nestling Crow, showing feathered and infeathered portions of the body (pterylae and apteria). 1/2 natural size. FIG. 25. — Nestling Crow, showing feathered and infeathered portions of the body (pterylae and apteria). 1/2 natural size. sions, pterylosis — as it is called — is of some importance in classification. Penguins only, of existing birds, have feathers growing uniformly on all parts of the body. In the ostrich, which has given up flight and taken to run- ning, the body feathers have grown over almost all the bare spaces which existed in its flying ancestors. There are two marked exceptions due to the present habits of these birds. Like the camel, when resting, these giant birds lean upon their breasts. This portion of the body sions, pterylosis — as it is called — is of some importance in classification. Penguins only, of existing birds, have feathers growing uniformly on all parts of the body. In the ostrich, which has given up flight and taken to run- ning, the body feathers have grown over almost all the bare spaces which existed in its flying ancestors. There are two marked exceptions due to the present habits of these birds. Like the camel, when resting, these giant birds lean upon their breasts. Arrangement This portion of the body The Bird 4 is provided with a thick, callous pad, which, by constant use, is thus kept bare of feathers. In addition, the under sides of the degenerate wings are also free of plumage, owing no doubt to the continual close application of these organs to the sides of the body. The other bare areas are almost obliterated, but the legs are bare, thus allowing perfect freedom in action. Some birds, such as vultures and cassowaries, have lost all feathers on the head and neck, or other portions of the body, from various causes, as for cleanliness, or, in some cases, probably for ornament. This will be spoken of more in detail in a later chapter. Moult The waste of internal tissues and organs in animals is repaired by means of the blood which brings them fresh material and carries away worn-out cells, as it traverses arteries and veins. Entire parts, as the tails of tadpoles, may even be absorbed; but, in general, skin structures when old and worn out are cast off and renewed from the lower, or derm, layer. This takes place in various ways. The skin, even to the covering of the eyeballs, may come off entire, as is the case among snakes, or por- tions peel off and tear away, as in lizards. Warm-blooded animals also shed, or cast, their outside covering; mam- mals shedding their coats of hair, and birds their feathers. In the latter class this process is called moulting. The nestling down and the feather which replaces it can hardly be considered as separate structures, as the Feat 41 same channel perforates both and the nutriment pith which supplies the down traverses the hollow quill of the succeeding feather. A bird's swaddling-clothes and his first full dress are cut from the same piece. But when these perfect feathers reach full size, the aperture at the base closes, all blood-supply is cut off, and the feather at the commencement of its usefulness becomes a dead thing. There is no vital connection between the feathers of all the following moults. Each is separate, the papilla or feather-cells reawakening to new activity every time the process occurs. So when a bird's wing is clipped, no pain is felt, any more than when a person's hair is cut. Such feathers are of course not renewed until the succeed- ing moult. If a feather in a living bird be pulled out, it will be replaced immediately by another, and this will be repeated as often as the feather is removed. In cassowaries, each moult is advertised by dangling streamers of the old plumage still attached to the tips of the incoming feathers, but this connection is not a living one, the adult feathers being as lifeless as those of other birds. As powerful savages often exhibit very childlike traits, so these great birds are absurdly marked with what, in other species, are sure signs of recent chick- hood. The changing of plumage of the Brown Pelican is well shown by the illustrations. The naked young (Fig. 18) become covered with papillae (Fig. Moult 10) which soon burst into a coating of the softest white down (Fig. 36) ; this in turn gives place to the juvenile plumage of gray, the features of the wings and shoulders appearing first (Fig. The Bird 4 37). This is also the winter plumage of the adult birds, both sexes moulting alike into the rich-hued breeding plumage (Fig. 38) of yellow, chocolate, and silver-gray. plumage (Fig. 38) of yellow, chocolate, and silver-gray. The feathers of the entire bird are moulted or fall out naturally at least once a year, and in some cases twice or even three times. If we were asked at what season the FIG. 26. — Flight-feathers of Chimney Swift clogged with soot, showing necessity for moulting. FIG. 26. — Flight-feathers of Chimney Swift clogged with soot, showing necessity for moulting. principal annual moult would be most likely to occur, the fall of the year would suggest itself, and such is the case, for a number of good reasons. First, the hardest work which birds have to do, hatch- ing and caring for their young, has, at this season of the year, just been accomplished, and has doubtless told heavily on their plumage. Breast-feathers are worn thin, tails are badly frayed, and wing-pinions are broken and ragged. Two alternatives confront birds at this period. Those species which are to take their migratory flight Feat 43 over hundreds of miles of land and water must have perfect wings and rudders to carry them safely, against contrary winds and sudden accidents. Others which are contented with the food found near their homes, and elect (by the laws of their kind) to remain, must be pre- pared to withstand the blasts of winter. Their plumage must be abundant and thick to keep out the cold and snow, and to enable them to bury their tender eyes and feet in its warm mass. Otherwise the tiny round fluffs huddled close to the trunks in the evergreens would drop stiffened to the ground during some long winter night. So a renewal of plumage in the fall is most necessary to the life of birds. A baby robin, secure from most enemies in his nest, with parents to supply his every want, acquires his wing- quills only when his nestling down is shed. Moult He is care- fully watched and tended during his first flights, and takes such good care of these flight-feathers that they serve to carry him to his winter home far to the south- ward. But a brood of a dozen or more little Bob-whites whose wing-feathers sprout with the most marvellous rapidity, from the moment the birds tumble out of their white shells, would fare ill indeed if they had to trust to these nursery quills all the first winter, with hungry foxes sniffing for their scent, and more-to-be-dreaded owls shadowing their trembling covey. Nature has come to their aid, and when they have fairly worn out their wings in the first awkward attempts at flight, new feathers come in, and this succession of quills keeps them in fine flying condition until full grown. Indeed so solicitous is The Bird 4 Mother Nature about the ground-nesters that she puts strength and vigor into the coverts, or upper feathers on the little wings; so that these shoot forth with an energy far beyond what is usual, for a time lending their aid in flight, although they are not true primaries. Later they FIG, 27. — Iridescent feather from the breast of a Rufous Humming-bird, showing wearing off of the tips of the barbs, caused perhaps by rubbing against the petals of flowers. Magnified 25 diameters. FIG, 27. — Iridescent feather from the breast of a Rufous Humming-bird, showing wearing off of the tips of the barbs, caused perhaps by rubbing against the petals of flowers. Magnified 25 diameters. are far outgrown by the flight primaries, and then func- tion only as protectors of these more important feathers. The extreme in this precocious development of chicks is found in those strange Australian birds, the mound- builders, which are left from the first to shift for them- selves; even the duties of incubation being shirked by the parents. This necessitates a perfect ability on the Feat 45 part of the young birds to take care of themselves as soon as hatched. They pass the entire first moult within the egg itself, and are covered with perfect feathers and fully developed flight-quills when they emerge from the shell. Moult A wild duckling, although provided with a thick waterproof coat of down, has, like the robin, to wait a long time for his flight-feathers; but his aquatic habits and powers of diving make the dangers to which he is exposed far less than is the case with the young Bob- white. The causes of wear and disablement to feathers would make a long list if we but knew them all. As one instance take the wings of a Chimney Swift after she has reared her brood in the depths of some blackened chimney, or even a lightning-struck hollow tree. Her primaries are so matted and clogged with balls of soot that she would often find the migratory flight difficult indeed, were the feathers not replaced by new ones. When birds return from the South, and when a hint of spring warns winter residents to cease their roving, they prepare to develop all the advantages which may in any way aid them in securing a mate. Some indus- triously practise dance-steps, others flight-evolutions, a larger number rehearse their songs under their breath, while still others passively await the development of plumes, gorgets, spots and splashes of colour which, if the feathers come out large and brilliant, may stand them in as good stead in their wooing as any song or antic. Thus we find a class of birds which have a partial or com- plete moult in the spring. These feathers may last all The Bird 4 summer, or may drop out as soon as begins the hard work of building the nest or feeding the young, with which labor they might interfere. To return for a moment to the fall moult. If a spar- row or lark should shed all of its large wing-feathers simultaneously, it would have slight hope of ever living long enough for new ones to grow out again. If such defenceless birds were compelled to hop helplessly along FIG. 28. — Wings of English Sparrow, showing two feathers of each wing being moulted simultaneously. FIG. 28. — Wings of English Sparrow, showing two feathers of each wing being moulted simultaneously. the ground, weasels and cats would be able to catch hundreds of them without effort. This is avoided in all land birds by the moulting of only a pair of primaries, as the large flight-feathers are called, at a time, one from each wing. Moult This process usually starts with the pair farthest from the front of the wing, and the second pair does not fall out until the first pair of new feathers is nearly of full size. Thus all danger of a crippled flight is avoided. the ground, weasels and cats would be able to catch hundreds of them without effort. This is avoided in all land birds by the moulting of only a pair of primaries, as the large flight-feathers are called, at a time, one from each wing. This process usually starts with the pair farthest from the front of the wing, and the second pair does not fall out until the first pair of new feathers is nearly of full size. Thus all danger of a crippled flight is avoided. One of the most interesting phases of Nature is the way she provides for exceptions to what we are pleased to call her laws. Some birds, unlike those mentioned Feathers above, shed every primary in their wings at once, so that their angular stump-feathered wings are perfectly useless for flight. In this class are many water birds — ducks, geese, flamingoes, snake-birds and others. Just before this wholesale moulting occurs, a flock of wild ducks will FIG. 29. — Wing of adult Mallard Duck, with the new set of flight-feathers just appearing. FIG. 29. — Wing of adult Mallard Duck, with the new set of flight-feathers just appearing. make their way, by an unfailing instinct, to some large body of water where they can swim and dive in safety and, if need be, never come within reach of enemies on the shore until the new feathers are strong enough to bear them up. Associated with this temporary disablement is another provision for the safety of certain birds of this class. Our common Mallard Duck, for example, is sometimes com- The Bird 4 pelled to undergo the fall moult in a rather small body of water, where danger menaces on all sides. Although when flightless he swims low among the thick water-reeds, yet his brilliant colours — iridescent green and white — would too frequently mark him out. Moult So the invisible cloak of his brooding mate is dropped over him for a while — his colours vanish, and by a partial moult thus sandwiched in, the hues of his plumage change to an inconspicuous mottling of brown, hardly distinguishable from the female. Then when the splitting of his quill- sheaths hints of coming power to take care of himself again, the dusky mantle is lifted, and, triumphantly treading water, he stands upright and shakes his glisten- ing wings, daring his enemies to catch him if they can. This has been happily termed the " eclipse" plumage. In certain portions of the Old World where foxes are scarce and the ducks have been persistently pursued by men in boats, the knowing birds have changed their habits and, when their wing-quills fall, they make their home in deep woods, finding greater safety there than on ponds or lakes. A somewhat similar condition occurs in the Black Grouse of Europe, which loses the conspicuous black feathers of the head and neck during the helpless period caused by the moult of its tail-feathers. caused by the moult of its tail-feathers. This additional moult brings us to the consideration of the birds which have no less than three changes of plumage, and here we find the cause intimately connected with the colour of the birds' surroundings. Ptarmigans, which are species of grouse living in the far North, moult Feathers 49 FIG. 30. — Eclipse plumage of Mallard Duck. Male in full breeding p (the brilliant green of the head and neck is lost in the photograp FIG. 30. — Eclipse plumage of Mallard Duck. Male in full breeding plumage (the brilliant green of the head and neck is lost in the photograph). FIG. 31. — Male in eclipse plumage during moult of wing-feathers. FIG. 32.— Female Mallard. FIG. 32.— Female Mallard. The Bird 5° after the breeding season into a special gray or dark plumage, harmonizing well with the autumnal shades of the grass and lichened rocks. In the late fall a second plumage of immaculate white is assumed, affording these birds great protection on the snowy wastes where they FIG. 33. — Willow Ptarmigan in early spring, with brown feathers beginning to replace the white. 1/4 natural size. FIG. 33. — Willow Ptarmigan in early spring, with brown feathers beginning to replace the white. 1/4 natural size. live. Moult In spring a third suit is donned — brown and parti- coloured like the environment, which late in the year is still covered with patches of snow here and there. This too is the nuptial plumage, and lasts until the gray garb completes the cycle of the year's changes. The wing- feathers are white all the year, but when the wings are live. In spring a third suit is donned — brown and parti- coloured like the environment, which late in the year is still covered with patches of snow here and there. This too is the nuptial plumage, and lasts until the gray garb completes the cycle of the year's changes. The wing- feathers are white all the year, but when the wings are Feat 5 1 closed they telescope so neatly beneath the feathers of the shoulder that they are not noticeable while the bird is in either the autumnal or vernal plumage. As the feathers on the flipper-like wings of a penguin resemble the scales of reptiles in appearance, so this FIG. 33a. — Ptarmigan in the fall, showing the gray autumnal plumage (which has replaced the brown of summer on the upper parts of the body and wings), gradually giving place to the white of the corning winter feathers. Wild birds in Alaska. (Harold Whealton, photographer.) FIG. 33a. — Ptarmigan in the fall, showing the gray autumnal plumage (which has replaced the brown of summer on the upper parts of the body and wings), gradually giving place to the white of the corning winter feathers. Wild birds in Alaska. (Harold Whealton, photographer.) homology is carried out in the method of shedding them. Unlike the dropping out of feathers one by one, as in other birds, these come off in flakes, like the skin of a lizard. The feathers of the back loosen, shrivel up, and fade to a brownish hue before they peel away. homology is carried out in the method of shedding them. Unlike the dropping out of feathers one by one, as in other birds, these come off in flakes, like the skin of a lizard. The feathers of the back loosen, shrivel up, and fade to a brownish hue before they peel away. The Bird The Bird 5 We have seen how birds, by moulting their feathers, change the colour of their plumage; in some cases several times each year. There is, however, still another way in which the appearance of new colour is brought about. Not by increase of pigment, for the feather when once full grown is dead; but by the mere breaking or fraying FIG. 34. — The three moults of the Ptarmigan, shown in three individuals. (Courtesy of American Museum.) FIG. 34. — The three moults of the Ptarmigan, shown in three individuals. (Courtesy of American Museum.) of the edges of each feather. It is thus that the Snow- flake brushes off the rusty trimmings of his winter's suit and returns to his home in the far North, dressed in spick- and-span black and white. A much more familiar exam- ple is to be seen at our very doorstep. The cock English Sparrow in midwinter is even more sombrely clad than usual; but as spring approaches, although he can attain to no elaborate song or flowing plume, yet even this of the edges of each feather. It is thus that the Snow- flake brushes off the rusty trimmings of his winter's suit and returns to his home in the far North, dressed in spick- and-span black and white. A much more familiar exam- ple is to be seen at our very doorstep. The cock English Sparrow in midwinter is even more sombrely clad than usual; but as spring approaches, although he can attain to no elaborate song or flowing plume, yet even this Feathers 53 commoner feels the call of love for beauty, and day by day the dusty brown tips of his throat-feathers wear away one by one, and leave exposed the clear black centres; and behold, the vulgar frequenter of our streets FIG. 35. — Two male English Sparrows, showing the difference in colour caused by wear of the feather-tips between October and April. FIG. 35. — Two male English Sparrows, showing the difference in colour caused by wear of the feather-tips between October and April. and alleys, flaunts a jet cravat before the eyes of his lady- love! and alleys, flaunts a jet cravat before the eyes of his lady- love! Colour. The very interesting uses which the colours of birds serve, the part they take in courtship, in evading danger, or in enabling birds to find each other, are many. These uses have been much written about, but of the nature and formation of colour less is known. Few of us have The Bird 5 probably ever given a thought to the colours themselves Why is that feather blue? Why — because it is blue! Why is that feather blue? Why — because it is blue! There are two principal ways in which colours are produced in feathers: first, when a real colour-pigment is present, and again when the structure of the feather is more or less like miniature prisms in shape, breaking up FIG. 36. — Young Brown Pelicans in the downy plumage. 1/6 natural size. FIG. 36. — Young Brown Pelicans in the downy plumage. 1/6 natural size. the rays— rainbow-like — into the iridescence of the spec- trum. In the case of almost all the beauties of Nature, the more closely we examine them, the more beautiful they become. But this is not true of the iridescent colours of birds such as hummingbirds, unless we con- sider the structure. The colour itself disappears under the microscope, and only gray or black tints are seen. the rays— rainbow-like — into the iridescence of the spec- trum. In the case of almost all the beauties of Nature, the more closely we examine them, the more beautiful they become. But this is not true of the iridescent colours of birds such as hummingbirds, unless we con- sider the structure. The colour itself disappears under the microscope, and only gray or black tints are seen. The black, red, brown, and yellow colours of feathers Feat 55 are almost always due to pigment or colouring-matter in the shaft or vane. If we take a black feather and hold it to the light, it will still look black; if we pound it with a hammer, it will not change. a hammer, it will not change. Green is never found as a pigment except in the feathers of a small family of birds called plantain-eaters or turacous, which inhabit West Africa. Colour. For some time it was thought that the natives dyed the birds artificially, as when these birds were kept captive, the magnificent scarlet patch on the wing would gradually fade and become a dull gray. It is a fact that this colouring- matter washes out when the feather is washed in alkaline water. Even ordinary water will be slightly tinged if the feather is soaked in it. The pigment contains about ten per cent of copper, and this can be extracted chemically in the form of a metallic powder. The plumage of almost all brightly coloured birds will fade in the course of years, if the feathers are left exposed to direct sunlight; but, like photographic plates, the hues of some birds are more sen- sitive than others to the light. The delicate reds and yellows on the lower parts of Mexican Trogons are par- ticularly evanescent, and the rose-pink of the African Fairy Warbler disappears a short time after death. We might speak of a third class of colours, which are due to both pigment and structure. For instance, no blue pigment is known to exist in the feathers of birds, but blue feathers contain a brown or yellowish pigment which is encased in the horny coating of the feather. Between this outer sheath and the underlying pigment is a layer of many-sided cones or small projections which The Bird 5 have numerous little ridges extending down the sides, and in some way, by reflection, these change the yellow or black to blue. If we take a parrot's feather and pound the blue portion, that colour will disappear and the vane will become black. It is surprising to see how the colours of many beau- tiful feathers will vanish when we hold them between our eye and the light. When we look at feathers under the microscope, and see their horny rays, we forget, for a time, the delicacy and fluffiness which the bird's plumage as a whole exhibits, and we are constantly reminded of the scales of reptiles. And in colour we have another similarity between the two: lizards have both pigment and prisms, and the scales of large snakes glow like opals when the sunlight falls on them. Colour. White never exists as a pigment in the feathers of birds, but is always due to innumerable air-spaces in the substance of the feather, by which the rays of light are reflected and deflected until, as in snow or foam, all colour is lost and white results. In any one Order of birds there may often be found a series of species with colour patterns grading into each other and connecting two extremes, perhaps very diverse in appearance. But it is seldom that we can examine such a series at once, and, except in a large collection of birds' skins in a museum, these wonderful life-chains, or twig-tips of the tree of evolution seldom appeal to us very forcibly. But in a feather it is different. We may find on one bird a most delicately graduated series, show- ing every step in the process by which simple unicoloured i 05 m n-i 83 a> *» 8PS SoO i 05 m n-i 83 a> *» 8PS SoO i *• 05 m n-i 83 a> *» 8PS SoO m m The Bird 5 or spotted feathers assume most intricate and complex colour masses and patterns. Darwin illustrates this very plainly in the case of the Argus Pheasant, and pays a fitting tribute to the evolu- tion of the marvellous colour patterns among birds. "The ocelli on the wing-feathers of the Argus Pheasant are shaded in so wonderful a manner as to resemble balls lying loose within sockets. That these ornaments should have been formed through the selection of many succes- sive variations, not one of which was originally intended to produce the ball-and-socket effect, seems as incredible as that one of Raphael's Madonnas should have been formed by the selection of chance daubs of paint made by a long succession of young artists, not one of whom in- tended at first to draw the human figure. In order to discover how the ocelli have been developed we cannot look to a long line of progenitors, nor to many closely allied forms, for such do not now exist. Colour. But fortunately the several feathers on the wing suffice to give us a clue to the problem, and they prove to demonstration that a graduation is at least possible from a mere spot to a finished ball-and-socket ocellus." finished ball-and-socket ocellus." Two feathers from the wing of a Vulturine Guinea- fowl have been chosen to illustrate a more simple but no less beautiful colour evolution. On the less exposed side of one of the feathers are three or four series of irregu- lar white spots which tend in places to form transverse bands. On the opposite side of the shaft near the tip these spots are still distinct, but as our glance passes gradually toward the base of the feather, the spots con- FIG. 38. — Adult Brown Pelicans in full breeding plumage. 1/8 natural size. FIG. 38. — Adult Brown Pelicans in full breeding plumage. 1/8 natural size. ral The Bird 6o verge more and more, until two distinct longitudinal lines are formed, with traces of a third near the quill. A smaller feather from the same wing is marked with spots which are nearly circular and which show faint traces of encircling bands of white pointing toward a still more elaborate system of decoration. FIG. 39. — Evolution of a colour pattern upon two feathers of a ulturme Gun.ea- fowl; a stripe breaking up into dots, these forming cross-vars, and on the second feather a regular series of dots encircled with white. FIG. 39. — Evolution of a colour pattern upon two feathers of a ulturme Gun.ea- fowl; a stripe breaking up into dots, these forming cross-vars, and on the second feather a regular series of dots encircled with white. FIG. 39. — Evolution of a colour pattern upon two feathers of a ulturme Gun.ea- fowl; a stripe breaking up into dots, these forming cross-vars, and on the second feather a regular series of dots encircled with white. It is interesting to conjecture in which direction the decoration of feathers is proceeding. In the case of the guinea-fowl, are the spots converging into lines or are the lines the more ancient, and for some reason grad- ually splitting up into smaller divisions? This is hard to decipher, and if we look at the rest of the guinea-fowl's body, the matter becomes only the more complicated. Colour. Feat 6 1 For, higher up on the wings, and on the shoulders, we find that the fine specks which were barely noticeable on the tips of some of the wing-feathers, are in the ascend- ant, and absorb or replace the white spots over the whole feather. The faint trace of the third line near the shaft of which I spoke, has suddenly assumed an unexpected importance and has spread out into a broad central band. The young or the female might give us a clew; for in rnany birds the coloration of these shows a more ancient arrangement of colour pattern than the feathers of the male. The Indian Wood Ibis — what an imbecile it looks to our eyes when we observe it in a zoological garden; what a fishy smell it generally diffuses, how unpleasant are its feeding habits, and what a dull black and white colora- tion it has! Surely here is a bird with nothing which could possibly appeal to our aesthetic sense. But we are mistaken. Some of the innermost feathers of its wings, seldom visible, except when the bird partly spreads them, are of the most beautiful rose hue, shading at the tip into a deeper pink. Seldom, even in Nature, will we find tints comparable to the delicacy and bloom of these hidden feathers. We have gone into these details only to show the possibilities of a little feather-study. Even our common Plymouth Rock chickens and hundreds of other birds will show us unthought-of beauties, and in the fields or in a zoological park we have only to use our eyes more carefully to realize how ^auch we usually pass by un- noticed. FIG. 40. — X-ray photograph of the front view of a homing Pigeon, showing the bones clearly through the surrounding flesh. Observe the light, spongy character of the skull and the bones of the limbs, the latter appearing almost hollow. The crop filled with corn is visible spread out at the base of the neck, and low down in the body, near the right thigh, the grit and pebbles within the gizzard are very distinct. About the tarsus of the right leg is seen the metal tag which was used for the identification of the living bird. (Photographed by Dr. Henry G. Piffard.) THE FRAMEWORK OF THE BIRD H HEN we look at a living bird, we see only feathers, horn, and skin, and we sometimes forget that hidden beneath all these are many bones, — the framework of the body. If we wish to alter the style of architecture of a house, we need only to change the ex- terior, columns, arches and windows, while the stone foundation and brick walls may remain as they are. So in fashioning new forms of life, Nature has often altered the covering, and even the muscles and organs, of ani- mals to such an extent that we would have little clew as to the relations of these creatures, were it not for the underlying bones, which are so deeply seated that they react less slowly to changes in the outside life. If a fish, a lizard, a bird, a whale, and a man should be presented to us for classification, we might well hesitate until we had seen their bones, when there would flash upon us the same moulded type running through all. The study of the skeleton, or Osteology, is like all other 'ologies; it can be made as dry as the bones them- selves; or the very opposite, by leaving the minor details and less important particulars to text-books, choosing only the most significant facts. One may smile at the at FIG. 40. — X-ray photograph of the front view of a homing Pigeon, showing the bones clearly through the surrounding flesh. Observe the light, spongy The Bird 6 thought of bestowing an encomium on a jaw-bone, and yet the history of the lower part of a sparrow's beak opens a vista so far-reaching that the mind of man falters at the thought; it shows the last roll of an evolving which, could we follow it back, would merge the man, the whale, the bird, the lizard, the fish, into one. Let us look at some of the bones of a sparrow or dove or chicken. One way to do this is to place a dead bird in a box pierced with numerous holes, leave it near an ant-hill, and wait for the industrious insects to do their work. Another way is to clean as much flesh as possible from the skeleton and deposit the bones in a pail of water. In a few days they can be washed white and clean. THE FRAMEWORK OF THE BIRD Perhaps the easiest way of all is to save what bones you can of a boiled chicken. These are of large size and will show us all we wish to know. The framework of a bird consists of a long jointed string of bones called vertebrae, with the brain-box or skull at one end and a blunt tail at the other. Near the middle, the outcurving ribs extend around the organs of the body, and, with the breast-bone, form an encircling protective sheath. Two short series of bones project in front of the ribs — the bones of the wings, — and two more behind the ribs — those of the legs and feet; while at the point of attachment of each of these four limbs there radiates a trio of bones. The back-bone is the fundamental and oldest part of the skeleton, and though we cannot follow its evolution directly backward through the long ages, yet there is sufficient gradation among living creatures to give us FIG. 41. — Common Fowl, showing relation of the bony framework or skeleton to the contour of the body. Notice large eye, long and mobile neck, the knee wholly within the body plumage, and the well-developed keel hinting of ancestors with strong powers of flight. 1/4 natural size. 6? FIG. 41. — Common Fowl, showing relation of the bony framework or skeleton to the contour of the body. Notice large eye, long and mobile neck, the knee wholly within the body plumage, and the well-developed keel hinting of ancestors with strong powers of flight. 1/4 natural size. The Bird 6 hints of the way it originated. In the lowest of fish-like creatures — the Amphioxus, a tiny animal, an inch or two in length, living in the sand along our shores — there is a thread-like cord of a gelatinous substance (not carti- lage, however) extending down the back, known as the notochord. He looks like some kind of worm, but this little gristle is his badge of nobility and lilts him clear FIG. 42. — Amphioxus, one of the lowest vertebrates, with a mere thread of gristle foreshadowing the back-bone of higher animals. This creature bur- rows in the sand along the Atlantic coast. FIG. 42. — Amphioxus, one of the lowest vertebrates, with a mere thread of gristle foreshadowing the back-bone of higher animals. THE FRAMEWORK OF THE BIRD This creature bur- rows in the sand along the Atlantic coast. of corals, snails, insects, and worms, into the realm of back-boned animals. This notochord lies underneath a thin white line which is all the spinal chord he has, and, at the front end of this, a tiny dot of pigment stands for brain, eye, and ear. Indeed Amphioxus has neither skull, brain, nor limbs. The history of the back-bone, like human history, is not altogether a majestic upward evolution; it has its tragedies and set-backs, its hopes and failures. In the waters along our Northern seashores are creatures, some sponge- or lichen-like, others with strange bulb-like bodies * The actual evolution of birds was of course not through fish, tadpoles, and reptiles as we know them, but by some line of creatures unknown to us forever, and resembling some of these other living Classes at least in the pos- session of gills, scales, etc. The Framework of the Bird growing on the end of long stalks. We call them almost plants. But they hold a secret from the crabs and snails which crawl about, and when the fishes brush against IG. 43. — A colony of living Boltenia, photographed by the author in the Bay of Fundy. The Boltenia is one of Nature's failures to make a vertebrate. The larva is active and has a notochord; the adult is degenerate and fixe on a stem. Found in five fathoms and deeper off rocky coasts north of Cap Cod. FIG. 43. — A colony of living Boltenia, photographed by the author in the Bay of Fundy. The Boltenia is one of Nature's failures to make a vertebrate. The larva is active and has a notochord; the adult is degenerate and fixed on a stem. Found in five fathoms and deeper off rocky coasts north of Cape Cod. them — if their poor dull senses only knew it — they might claim a blood-brotherhood. When they were young, for a little while, a gelatinous notochord was theirs also, but this, with all the hopes that such a be- 6 The Bird ginning brings, of fish, of bird, of man even, soon melted away and there they nod and sway in the watery cur- rents, never to know of the opportunity Nature has snatched from them — why, who can tell? snatched from them — why, who can tell? In adult sharks, the back-bone has become jointed and flexible, and a crude kind of skull is present, but still more important is the presence of four fins which correspond to the four legs of lizards and to the wings and legs of birds. A curious basket-like skeleton pro- tects the delicate gills, and it is probable that this existed FIG. 44. — Back-bone of Dogfish, with simple cartilaginous vertebrae. FIG. 44. — Back-bone of Dogfish, with simple cartilaginous vertebrae. long before the limbs appeared. All of this is composed of gristly cartilage. In the higher fishes, bone replaces the cartilage, and when the lowly tadpole — fish-like at first, swimming about by means of the fin around his tail — pushes forth his legs and climbs upon the land, our skeleton is well on its way birdwards.* Reptiles of old took to trees; their back-bones grew less flexible, so that they might safely sail through the air; feathers replaced The Framework of the Bird The Framework of the Bird scales, two ringers of each hand were lost, and one from each foot; teeth disappeared; a beak of horn proved best; intelligence increased and the forehead rose high, and behold, — a bird! Can we then despise even an Eng- lish Sparrow? All these things we have learned from a comparison with creatures other than birds, and we may, without trouble, take one more glimpse into the dim past. Let us go to the hencoop, where for three days the patient biddy has been sitting on her precious eggs. We will FIG. 45. — Neck vertebrae of an Ostrich, highly complex and bony in structure. FIG. 45. — Neck vertebrae of an Ostrich, highly complex and bony in structure. rob her of one — she will not miss it — while from it we may learn many wonderful things. Rest the warm egg in a dish of sand, carefully picking away the shell from the upper part. A glance at the tiny embryo lying on the yolk within will show a double series of tiny squares extending down the long diameter of the body. These are the first hints of the spinal column, and if we could follow its further development we would see something of great interest. The squares are now divided up like beads, just as are the bones of our bird's vertebrae; but in reality this first segmentation is a false one. It is sim- The Bird 7° ply a copy of the primitive flakes or joints of the tiny muscle-beginnings, and is comparable to the joints or rings in the body of a beetle, butterfly, or earthworm. In a short time all the squares will fuse together, and not until later will they separate again into divisions which will ultimately form the real bones of the spinal column. Every little chick, before it hatches, goes through the same strange changes, — living reminders of the evolution which has gone on in past ages of the earth. It is inter- FIG. 46. — Muscle-plates, or false vertebrae, of third-day embryo chick. Magnified 25 diameters. FIG. 46. — Muscle-plates, or false vertebrae, of third-day embryo chick. Magnified 25 diameters. esting to note that the vertebrae of the embryo chick pass through a stage when they are biconcave, — a condi- tion found both in Amphioxus and Archseopteryx. The Framework of the Bird This digression upon the back-bone history may seem out of place, but in reality such a bird's-eye survey of the past, imperfect as it is, will add a new interest to our handful of chicken-bones. The Framework of the Bi * This bone is formed chiefly of two intercentra, which are small bones, very characteristic of reptiles (chevron-bones of the tail) and are not uncom- mon among the lower Orders of birds. t In Hornbills the atlas and axis are fused together. The Framework of the Bi 71 Let us suppose that we have strung a wire through the hollow centre of the back-bone of our chicken, to which the ribs are still attached, and that we have be- sides the skull and the bones of one wing and one leg. Compare them with those in the illustrations and we will see if they can tell us aught of interest. The bones of the neck are all separate, and slide back and forth on the wire, like beads on a string. How unlike FIG. 47. — Front and rear views of seventeenth and eighteenth cervical vertebrae of Ostrich, showing complicated saddles and sliding surfaces, giving great freedom of motion. FIG. 47. — Front and rear views of seventeenth and eighteenth cervical vertebrae of Ostrich, showing complicated saddles and sliding surfaces, giving great freedom of motion. the long smooth ribs are these vertebrae, bristling with spines and projections! How is it that a bird can be comfortable with a string of such irregular-looking ob- jects run through its body? But fit two of these bones together and see how beautifully they saddle end to end, every convexity or projecting knob exactly adjusted to a corresponding concave portion of the neighbouring bone. These saddles are characteristic of birds alone. Every one of the sixteen bones of the neck is different from its the long smooth ribs are these vertebrae, bristling with spines and projections! How is it that a bird can be comfortable with a string of such irregular-looking ob- jects run through its body? But fit two of these bones together and see how beautifully they saddle end to end, every convexity or projecting knob exactly adjusted to a corresponding concave portion of the neighbouring bone. These saddles are characteristic of birds alone. Every one of the sixteen bones of the neck is different from its The Bird 7 fellows and exactly suited to the requirements of its position, but the first two following just behind the skull are so radically unlike the others that we know at once that they must serve some particular purpose. The first is little more than a simple ring * of bone, and is called the atlas, after the mythological giant who held up the heavens upon his shoulders; named very aptly too, for FIG. 48. — Atlas and axis of Jabiru, separated FIG. 49. — Atlas and axis of Jabiru, joined. The Framework of the Bi FIG. 48. — Atlas and axis of Jabiru, separated FIG. 49. — Atlas and axis of Jabiru, joined. this tiny collar of bone supports the skull itself. The next vertebra is ring-like too, but has a curious knob in front, which projects forward through the atlas and forms a pivot on which the head turns, hence its name, — the axis.f Let us compare the neck-bones with those of a reptile and a man. Although, as a whole, the bones of the The Framework of the Bi 73 skeleton of a bird are more or less soldered together, yet the neck is far more flexible than in either of the other examples. Indeed the neck of a bird has greater freedom of motion than that of a snake. A lizard can turn his head only a little way around, and we ourselves can look only across our shoulder, but with a bird it is FIG. 50. — American Egret, showing curves into which the neck naturally falls when the bird is at rest. When striking at a fish the vertebra? straighten out. FIG. 50. — American Egret, showing curves into which the neck naturally falls when the bird is at rest. When striking at a fish the vertebra? straighten out. very different. Watch a heron or, better still, a fla- mingo and see its neck describe figures of eight as he arranges the feathers on its back. Few people would ever imagine that there are exactly twice as many neck- bones in a sparrow as in a giraffe, but such is the case, there being fourteen in the former and seven in the latter. In the neck of a swan there are twenty-three of these bones. The Bird 74 The remaining vertebrae, those of the upper and lower back, are very different from those of the neck. The flexible neck enables the bird to reach all parts of its plumage with its beak, and to pick up food from the ground or from twigs overhead, but the all-important function of flight must be provided for by means of a FIG. 51. — White-throated Sparrow, three inches tall, with fourteen neck vertebrae. (Compare with Fig. 52.) FIG. 51. — White-throated Sparrow, three inches tall, with fourteen neck vertebrae. (Compare with Fig. 52.) rigid body-frame. In reptiles and in the embryos of birds only two pelvic vertebrae are fused together, but in adult birds many dorsal and caudal vertebrae (as many as 23 in some cases) are fused into a single bone. Thus the rib-bearing tipper back vertebrae are partially fused together, and below them those of the lower back have merged until it is difficult to realize that this portion of The Framework of the Bird The Framework of the Bird The Framework of the Bird 77 hence the separate bones which unite it to the vertebrae of the lower back. The evolution of the tail will be treated of in another chapter. The Framework of the Bird 75 the skeleton was not originally one bone. Passing on for a moment to the bones of the tail, we find a number of separate pieces, ending in a curious-shaped bone, called the ploughshare. This is at the tip of the tail, or " pope's FIG. 52. — Giraffe, reaching with tongue for leaves perhaps eighteen feet above the ground, with but 7 neck bones. FIG. 52. — Giraffe, reaching with tongue for leaves perhaps eighteen feet above the ground, with but 7 neck bones. nose," of the chicken and really consists of many verte- brae fused together. It is necessary for this to be large and strong: for it supports all the feathers of the tall. But to be of efficient aid in steering, the tail, like the rudder of a ship, must have freedom of motion, and nose," of the chicken and really consists of many verte- brae fused together. It is necessary for this to be large and strong: for it supports all the feathers of the tall. But to be of efficient aid in steering, the tail, like the rudder of a ship, must have freedom of motion, and FIG. 53. — Pelvic vertebrae of young Alligator. FIG. 53. — Pelvic vertebrae of young Alligator. Fia. 54. — Pelvic vertebrae of American Flamingo. Fia. 54. — Pelvic vertebrae of American Flamingo. FIG. 55. — Pelvic vertebrae of Bald Eagle. In the reptile, where there is no need for rigidity, only two typical pelvic vertebrae are joined together; in the birds many dorsal and caudal vertebrae are joined with these to make a rigid frame for flight and for bipedal locomotion. 76 FIG. 55. — Pelvic vertebrae of Bald Eagle. In the reptile, where there is no need for rigidity, only two typical pelvic vertebrae are joined together; in the birds many dorsal and caudal vertebrae are joined with these to make a rigid frame for flight and for bipedal locomotion. 76 frame 76 The Framework of the Bird Ribs :AV7vv The ribs are the long, narrow, double-headed bones which curve out from the vertebrae of the upper back and, uniting with the breast-bone, form a barred pro- tection for the heart, lungs, liver, and other organs. These are the ribs proper, but there are other smaller ones, called false or floating ribs, which reach only part of the way around the body. Look at the largest ribs of the chicken and an added provision for making this box of bone more solid will be seen. From near the centre of the upper part of each rib a small bony projection laps across the rib next behind and thus forms a kind of lattice-work, movable but of great strength. A further interest attaches to these cross-rib pieces when we learn that every bird except the Screamer possesses them, while else- where they are found only in crocodile-like reptiles and in the Hatteria Lizard of New Zealand. FIG. 56. — Ribs of Hatteria Lizard, with uncinate processes. :AV7vv FIG. 56. — Ribs of Hatteria Lizard, with uncinate processes. FIG. 56. — Ribs of Hatteria Lizard, with uncinate processes. The similarity of the ribs — slanting one after another The Bird 7 around toward the breast-bone — hints of something which perhaps has never occurred to us. We spoke of the worm-like appearance of the lowly Amphioxus — the sand-fish with the shadow of a back-bone. When we think of a worm we think of a creature very much alike from head to tail, one in which a section across the neck is not very unlike one across the centre of the body or near the tail; indeed that is exactly what the word Am- phioxus means, — like head, like tail. This repetition of segments or similar parts is a sign of low degree in the scale of life, as it harks back to the time when the very highest form of life was worm-like. highest form of life was worm-like. The flesh of a salmon or of a trout shows such a con- dition very well, the body consisting of flake after flake of flesh. Now in birds and the higher animals this divi- sion into successive segments is hardly noticeable, and almost every inch of a man or bird, from head to toe, seems very distinct and individual. Ribs But ribs bring back the old ancestral condition very vividly, and when a peacock, strutting proudly before us, resplendent from beak to tail, picks up and swallows an unfortunate angle- worm, we may remember that, no matter what geological eras or inexplicable physical gulfs separate the two, the bird carries within his body indelible imprints which insolubly link his past with that of the lowly creature of the dust. As in various other cases throughout nature, when the many ribs of the bird's ancestors began to be reduced in number, some attained to other uses beside that of arch- ing around the whole body and protecting the heart, the The Framework of the Bi 79 lungs, and other organs. Look at the two neck-bones of the ostrich in Fig. 47, where in addition to the central aperture, through which the spinal nerve-cord passes, two other openings will be seen, one on each side. Through these the vertebral arteries carry their burden of pure blood. The outer wall of this bony canal, extending up the whole length of the neck, is formed principally by what is left of the ribs which were once long and free, like their fellows farther down the back. Though re- duced to a tiny fragmentary arc of bone, yet they still perform a protecting function. perform a protecting function. In Archceopteryx (Fig. 5) there existed well-developed abdominal ribs, exactly like those found in crocodiles and other reptiles. In no living bird, however, are these found. Breast-bone The lower portions of the true ribs of our chicken are separate pieces of bone, slanting in a forward direc- tion and attached by a movable joint to the upper parts. These end close together along the sides of the large breast-bone, or sternum as it is called. In fact the origin of the sternum can be traced to the fused ends of these ribs, and in the sternum of an immature, ostrich (Fig. 58) the line of juncture between the two lateral halves is still distinct or even open. To the edge of this bone, nearest the head, two column-like shoulder-bones are attached, and in some birds the wish-bone is also joined to it (Fig. 103). The sternum is one of the largest single bones in The Bird 8 the body of the chicken, and is very different from our own breast-bone, which is long and narrow. The pos- terior edge of the sternum is of many shapes, varying in birds of different species. Deep channels may extend into each side, leaving long slender splinters or spines FIG. 57. — Ribs and sternum of Flamingo; notice what a complex box of bone is formed by the vertebrae, scapula, ribs with their uncinate processes, and the sternum; notice large keel for the attachment of flight-muscles. FIG. 57. — Ribs and sternum of Flamingo; notice what a complex box of bone is formed by the vertebrae, scapula, ribs with their uncinate processes, and the sternum; notice large keel for the attachment of flight-muscles. of projecting bone, or this channel may be partly closed, forming a round hole quite through the bone. The sternum and its various processes are of considerable value in classification, the same configuration being found throughout allied groups, in consequence, doubtless, of the slight chance of modification resulting directly from any specialized habits in the life of the bird. The portion The Framework of the Bi 81 of this bone which is most characteristic of birds is the central ridge or keel which projects straight out from the surface of the sternum. This is of the utmost im- portance in giving firm anchorage for the great flight- muscles of the breast. FIG. 58. — Ribs and sternum of Ostrich; notice absence of keel correlated with loss of power of flight. The suture through the centre of the sternum reveals its paired origin. FIG. 58. Breast-bone — Ribs and sternum of Ostrich; notice absence of keel correlated with loss of power of flight. The suture through the centre of the sternum reveals its paired origin. The keel is of many shapes, but when well developed is generally very high at the upper end of the breast- bone and becomes lower as it slopes gradually backward. In birds which have lost the use of their wings the keel has disappeared completely, the sternum being flat, as in ourselves. This is its condition in the ostrich and cassowary, and it is the character which has given a The Bird 8 name to two great divisions of birds : Rati'tce (those with flat breast-bones, raft-like), including the ostrich, rhea, emeu, cassowary, and apteryx; and Carina'tos (birds with keeled breast-bones, boat-like), including all other living birds, whether flyers, as the thrushes, storks, and gulls, or swimmers like the penguins. But this differ- ence in breast-bones is far from being as profound as other differences existing between certain birds which are alike in having keels to their sternums. The pres- ence or absence of a keel is not of great taxonomic im- portance. portance. The size of the keel is a pretty sure criterion of the flying powers of a bird, that is, judging not the actual duration of flight, but the actual muscular power and amount of energy used in flying (Fig. 59). The alba- tross, and other birds which, trusting to the air-currents to bear them upward, flap seldom and soar much, have comparatively smaller keels than do those birds which flap their wings more frequently. Thus the pigeon has a very good-sized keel; while in the humming-bird this bone is enormous, compared to its spread of wings. Dr. Frederick A. Lucas has expressed this very graphically in a diagram, where it is supposed that the albatross, pigeon, and humming-bird have an equal spread of wings. On comparison, the keel of the first is seen to occupy but a small fraction of the surface of the same bone in a humming-bird. To account for this we must realize that the wings of the humming-bird execute from six hundred to a thousand strokes a minute; while the alba- tross may soar for miles with wings held outstretched The Framework of the Bi 83 and all but motionless. Breast-bone It is said that, comparatively, the muscular energy is greater and the wing-bones more powerful in a hummingbird than in any other animal. powerful in a hummingbird than in any other animal. Nature has a puzzling way of achieving similar results in a very similar manner in creatures wholly unrelated. We have a good example of this in bats and birds, both FIG. 59. — Comparison of the size of the keel of the Albatross, Pigeon, and Humming- bird, supposing all to have an equal spread of wing. (Courtesy of Dr. F. A. Lucas.) FIG. 59. — Comparison of the size of the keel of the Albatross, Pigeon, and Humming- bird, supposing all to have an equal spread of wing. (Courtesy of Dr. F. A. Lucas.) of which have independently learned to propel them- selves through the air by means of their front limbs. If we take the breast-bone of a common bat and that of a small bird and place them together, few persons unacquainted with the bones of the two types could tell which was that of the bat, — different as that little crea- The Bird 8 ture is in external appearance from a bird. Their keels and sternums are very much alike. This is called par- allelism, and sometimes it gives a great deal of trouble to naturalists when they are trying to find the right relationships between living animals. Shoulder-girdle It will be remembered that mention was made of the trios of bones which radiate near the juncture with the body, of each wing and each leg, — girdles or arches they are called. The pectoral, or shoulder, girdle meets the upper arm-bone of each wing at the shoulder- joint. If we run our hand along the back of our shoulders, we will feel a prominent bone, called the shoulder-blade, and in almost the same place in our chicken we notice a very long and thin bone. This is the scapula, and is one of the pectoral-girdle trio, the other two being known as coracoid and clavicle. The coracoid is a short, but stout, column of bone joined to the shoulder-blade and extending down and backward to the breast-bone. This coracoid bone is especially developed in birds as compared with other creatures. When their forefathers began to scale through the air, thus putting a great strain on the muscles of the breast, Nature seized on these coracoid bones, giving them such strength and thickness that they have become the pivots upon which, at each swift vibration through the air, turn the marvellous wings of a modern bird. In reptiles, this bone is divided into two weak, thin plates The Framework of the Bi 85 which would hardly afford strength for a single wing- flutter. Since mammals in their high evolution have found no use for this bone, it has become reduced to a small projection on the shoulder-blade. small projection on the shoulder-blade. The clavicle we will recognize instantly, when we give it another name — the wish-bone or merry-thought. FIG 60. — Pectoral girdle of bird (scapulas, coracoids, and clavicles); compared with the scapula and coracoid of a young Leopard, the latter bone in the Leopard being reduced to a tiny process. FIG 60. — Pectoral girdle of bird (scapulas, coracoids, and clavicles); compared with the scapula and coracoid of a young Leopard, the latter bone in the Leopard being reduced to a tiny process. In birds the wish-bone is generally V-shaped, the two clavicles usually meeting and fusing at their tips. Through this V-shaped opening in the neck, the resophagus and the windpipe pass from the throat into the body cavity. We too have wish-bones, although they are not placed exactly as are those of a chicken. Shoulder-girdle We call them collar- bones, but by whatever name we know them they are of importance, both in ourselves and in birds, in serving to brace out the shoulders. In creatures which, unlike The Bird 8 mankind and most birds, have less varied movements of the fore limbs, the clavicles have fallen into disuse, as in the lion and the horse. But in climbing, burrowing, and flying animals, such as the squirrel, mole, and bat, these bones have been of active use and are well devel- oped. But to keep its wish-bone a bird must continue to fly: for Nature is opposed to useless parts. So, in the flightless cassowary and ostrich, the wish-bone is very small or altogether absent. Parrots are almost alone in appearing to suffer no inconvenience in flight by the lack of clavicles, — these being greatly reduced in some species. In that anomalous bird the Hoatzin, the clavicles are fused not only at their base, but the tips are ossified firmly to a projecting spine of bone from the upper part of the breast-bone. In glancing back over the lower back-boned animals we realize that a shoulder-girdle of bones is of no use without a limb. Therefore we find the first hint of the shoulder-girdle in sharks, in which we also find the first limbs, or fins. In these fishes it is nothing but a single bar of soft cartilage. In the girdle supporting the pec- toral fin of such a fish as the trout or other bony fish, we find the adumbration of some of our bird's bones. When we remember how very wing-like is the movement of a fin in the water, we will not be surprised to learn that the girdle is almost all epiclavicle; these bones being the forerunners of clavicles, and giving place, in the higher forms, to the real wish-bones which steadily increase in size and importance. We would hardly The Framework of the Bi The Framework of the Bi 87 recognize in these primitive types the wish-bone of our Christmas turkey. In terrestrial quadrupeds and birds we usually find the front limbs near the front part of the body and the hind limbs much farther back, but it is interesting to FIG. 61.— Girdle of a bony fish. FIG. 61.— Girdle of a bony fish. FIG. 61.— Girdle of a bony fish. notice that in the fish, Fig. 61, all four limbs or fins are very far forward, almost or quite in the head region itself. This is a result of the function of balance which these structures almost wholly perform, the fin of the tail furnishing the locomotive power. It is very interesting to notice how many bones have kept to their respective places in the evolution of animals, The Bird 8 no matter how much change has occurred in their shape and size. Take, for instance, the shoulder-blades. When a tiger crouches they are very conspicuous, and whether we take a frog, a turtle, a lizard, an armadillo, a mouse, or a horse, we may always be sure of finding a scapula in the region where we have observed it in the bird. This is an important fact, and one which makes the identification of many bones an easy matter. * They were separate also in Archcenpteryx. Thigh-girdle The shoulder-girdle which we have just examined was not joined to the back-bone, but only saddled on the ribs, the scapula extending backward, just clearing them. What kept it in place in the chicken's skeleton was the fact that it was strongly attached to the sternum, and this in turn joined to the back-bone by means of the ribs. But the pelvic arch or thigh-girdle is very different. If the entire framework of the bird is to be supported on two legs, the point of attachment of these limbs must be solidly fixed to the back-bone of the body. limbs must be solidly fixed to the back-bone Although there are as many bones supporting the leg or thigh as there are bracing the shoulder, we would never know this from examining our chicken. As in other places in a bird's skeleton, the bones — six in this case — have fused together in one solid piece, and only in very young birds are they separate.* in very young birds are they separate.* The names of the bones composing the pelvic girdle, or arch, are the ilium, ischium, and pubis. The easiest The Framework of the Bi 89 way to locate these is first to find the socket in which fits the head of the thigh-bone. This is the deep cup- shaped depression on each side, and all three bones join in making the socket. The ilium lies along the back and forms a sort of roof over the portion of the back- bone in this region. If we look at the under side of this bone, we may see the fused vertebrae more distinctly — fourteen or fifteen of them. The two deep depressions in which the kidneys of the bird were located are also FIG. 62. — Pelvic girdle of a bird. FIG. 62. — Pelvic girdle of a bird. now visible. As the coracoid is the great pivot of the wing, so the ilium helps most to bear the strain of hop- ping and running. In the frog, which progresses by hops or great leaps, the ilium is also largely developed; indeed we can see it through the skin, thus giving the broken-back appearance to that creature. now visible. As the coracoid is the great pivot of the wing, so the ilium helps most to bear the strain of hop- ping and running. Thigh-girdle In the frog, which progresses by hops or great leaps, the ilium is also largely developed; indeed we can see it through the skin, thus giving the broken-back appearance to that creature. broken-back appearance to that creature. Each side of the thigh-bone box is formed by the ischium, which is closely fused with the ilium except in most of the ostrich-like birds, the tinamous, and in reptiles, where these bones are free throughout their entire length. We can readily make out the pubis as a slender bar of The Bird -JL 9° bone extending backward from the thigh-socket, sepa- rated from the ischium (except at the extreme end) by a long open slit. In other animals these bones are as different in om the thigh-socket, sepa- rated from the ischium (except at the extreme end) by a long open slit. In other animals these bones are as different in shape as can be imagined, but, almost without excep- tion, the position of each is relatively the same. The extreme extension, forward and back, of the ilium above the back-bone, thus joining with more vertebrae, is directly connected with two-legged erect locomo- tion. A parallel condition is found in some Dinosaurs — those extinct giant rep- tiles -- certain of which walked more or less on their hind legs. PuA FIG. 63 — Pelvic arch of a Dinosaur FIG. 63 — Pelvic arch of a Dinosaur FIG. 63 — Pelvic arch of a Dinosaur is found in some Dinosaurs — those extinct giant rep- tiles -- certain of which walked more or less on their hind legs. Another fundamental resemblance is found be- tween the thigh-girdles of -Js. Dinosaurs and other rep- tiles and that of a bird in the egg. As is shown in Figs. 63-65, the pubis slants slightly forward in both reptile and embryo bird ; but in FIG. 64. — Pelvic arch of an embryo bird, to show similarity of the two as contrasted with Fig. 65. Pur FIG. 65. — Pelvic arch of an adult bird. (The three figures by courtesy of Prof. H. F. Osborn.) -Js. FIG. 64. — Pelvic arch of an embryo bird, to show similarity of the two as contrasted with Fig. 65. Pur FIG. 65. — Pelvic arch of an adult bird. (The three figures by courtesy of Prof. H. F. Osborn.) FIG. 64. The Framework of the Bird the adult bird the shifting backward of this bone until it is parallel with the ischium is wholly an avian feature. FIG. 66. — Bullfrog. The bend in the back shows the great development of the ilium for bipedal locomotion in the sense of leaping ability. FIG. 66. — Bullfrog. The bend in the back shows the great development of the ilium for bipedal locomotion in the sense of leaping ability. Thigh-girdle — Pelvic arch of an embryo bird, to show similarity of the two as contrasted with Fig. 65. FIG. 64. — Pelvic arch of an embryo bird, to show similarity of the two as contrasted with Fig. 65. Another fundamental resemblance is found be- tween the thigh-girdles of Dinosaurs and other rep- tiles and that of a bird in the egg. As is shown in Figs. 63-65, the pubis slants and embryo bird ; but in Another fundamental resemblance is found be- tween the thigh-girdles of -Js. Dinosaurs and other rep- tiles and that of a bird in the egg. As is shown in Figs. 63-65, the pubis slants slightly forward in both reptile and embryo bird ; but in Pur FIG. 65. — Pelvic arch of an adult bird. (The three figures by courtesy of Prof. H. F. Osborn.) Pur FIG. 65. — Pelvic arch of an adult bird. (The three figures by courtesy of Prof. H. F. Osborn.) Pur FIG. 65. — Pelvic arch of an adult bird. (The three figures by courtesy of Prof. H. F. Osborn.) The Framework of the Bird Wings We will now consider the framework of the fore limb, or wing, of a bird, and a glance at the illustration show- ing the arm of a man and the wing of a bird will at once make plain the relation between the two. Here we again find a great help in the fact that many of the bones keep to their respective places in frogs, lizards, birds, and man. We know but little of the direct change from a fin to a hand or foot, although there are some fishes living at the present day with large finger-like bones in their pec- The Bird 9 toral fins. Even among fossil forms there have as yet been found no " missing links" in this respect. But how- ever it came about, it is certain that when the fish- amphibians of olden time, venturing into shallow water, felt more or less solid mud under them, and tried to move about upon it, their fins must have become pressed downward, and before they could safely push themselves about on dry land or lift their bodies clear of the ground, the stiff fin-rays must have become split up into a few, thick, bony rays or toes. We know that these were originally five in number on all four limbs, and when- ever, among living creatures, we find a lesser number, the reduction has been brought about by some subsequent change in the life of the animal. As yet, however, we know of no direct transitions from fins to feet. The requirements of flight demanded a fin-like stiff- ness in the wings of birds, and therefore many of the smaller bones of lizards, counterparts of which we find in our own wrists and hands, are in the bird fused together. in our own wrists and hands, are in the bird fused together. The upper arm-bone, or humerus, corresponds exactly to our bone of that name, and when we feel the two long bones of our forearm and look for them in the bird, we find both very plainly represented, the large one with notches, where the great wing-feathers are fastened, being called the ulna, and the smaller, straighter bone the radius. In our wrist there are eight little bones which are joined to each other so delicately that we can move and turn our hand in every direction. * Some morphologists homologize the fingers of a bird's wing with the second, third, and fourth digits of a pentadactyl hand. The question is still a mooted one. f In the embryos of some birds, traces of a fourth finger have been found. Wings But when a bird's wing is extended, if the wrist was at all flexible, the pres- sure of air on the great wing-feathers would turn the The Framework of the Bi The Framework of the Bi 93 f the Bird 93 FIG. 67. — Wing of Pigeon, leathered. FIG. 68. — Wing of Pigeon, bare, compared with Fig. 69. FIG. 67. — Wing of Pigeon, leathered. FIG. 67. — Wing of Pigeon, leathered. FIG. 68. — Wing of Pigeon, bare, compared with Fig. 69. FIG. 68. — Wing of Pigeon, bare, compared with Fig. 69. FIG. 69. — Arm and three fingers of a man. FIG. 69. — Arm and three fingers of a man. The Bird 9 wing-tip around and make flight impossible. So but two of these small bones are free in our chicken's wrist, although in the small chick several more (six in all) are separate. separate. If we double back our fourth and fifth fingers arid imagine that they have disappeared, extend our other three fingers and then suppose that all our wrist-bones, save two, have fused with the three long bones leading to the base of our thumb, index and middle fingers,* we will have an idea of the condition of our chicken's wing, and indeed there is very little difference between this and the wings of all other birds, f We have two separate bones in our thumb, and three in each of the next two fingers, and the bird has the same number, except in its third finger, in which there is but one. The principal value of this comparison is to show us that the bird, even in its most characteristic and specialized organ, — the wing, is not physically so unlike ourselves as we might at first glance suppose. When a bird folds its wing against its body, the joints are bent sharply, and the Z, formed by the elbow and the wrist, almost closes up. We can place our arm and hand in much the same position. position. If we move our arms slowly up and down, little by little greatly increasing the speed, we will realize how much greater strength and rigidity the whirring wings FIG. 70. — Skeleton of wing of Condor, compared with Fig. 71. FIG. 70. — Skeleton of wing of Condor, compared with Fig. 71. FIG. 71. Wings — Skeleton of a man's arm; notice close correspondence of bones in the two. (The extended thumb of the human hand is not silhouetted against the background, and hence not very distinct.) 95 FIG. 71. — Skeleton of a man's arm; notice close correspondence of bones in the two. (The extended thumb of the human hand is not silhouetted against the background, and hence not very distinct.) 95 95 95 The Bird 9 of a hummingbird or a grouse require than do the slowly flapping pinions of a gull or an albatross. When we compare the relative shortness of the upper arm-bone, or humerus, in the former groups with the long wing- bones of the sea-birds, we again realize what exquisite adaptations exist everywhere in Nature. The proportionate length of the various parts of the fore limb of a bird forms an interesting coiollary to its habits of life. For example, the hand in penguins and in hummingbirds is very long indeed; while in the os- trich the humerus is considerably longer than the fore- arm and hand combined. (Compare Figs. 269 and 272.) Before we leave the wing-bones, it will be well worth our time to consider for a moment how limbs first origi- ....., . . ...... . -^mmiim^ • • ^•^•iiVV;---- ' * FIG. 72. — Diagram showing the origin of paired fins (limbs) from a continuous fin-fold. (After Wiederscheim.) -^mmiim^ • • ^•^•iiVV;---- ' * 72. — Diagram showing the origin of paired fins (limbs) from a continuous fin-fold. (After Wiederscheim.) nated. In the lowest of fishes, such as our friend the Amphioxus and in lampreys, limbs are altogether absent, but in embryos of sharks we get a hint of what the first ^reat fish-like forms were like. Along the lower part of each side of the body there runs a continuous fin, so that nated. In the lowest of fishes, such as our friend the Amphioxus and in lampreys, limbs are altogether absent, but in embryos of sharks we get a hint of what the first ^reat fish-like forms were like. Along the lower part of each side of the body there runs a continuous fin, so that The Framework of the Bird 97 7 97 The Framework of the Bi 97 the front view of a section would be something like this, the dorsal fin being above and the lateral fins on each side. * To the lower end of this are fused, in the bird, the bones which corre- spond to our heel-bone and the small astragalus. The Bird 9 Wings Now owing to certain laws of mechanics, whenever such a creature as this moved about in the water, the stress of bal- ancing would be thrown most heavily on two points in these side fins, and gradually at these two nodes the fin became more strongly developed; while between these points it degenerated and finally disappeared. So in modern fishes we find the quartet of limbs alone left of this continuous fin or fold of skin. Look at a little embryo in the egg, taking one which has been incubated for six or seven days, and see the curious paddle or fin-like wings and feet — simply four rounded flaps projecting from the body — as unlike the limbs of the chick when it emerges from the egg as can be imagined (see Fig. 367). The ridge or fin of skin in the early, soft-backboned creatures could have been of no use whatever, except in balancing. In fact if we watch a trout carefully, we will see that it is the tail-fin which does almost all the propelling, the front- and hind- limb fins simply acting as guides and balances. So in this instance (as indeed in almost every organ in ourselves as well as in birds) we learn that the original function was entirely unlike that which the part now serves. The idea of miraculous change, which is sup- posed to be an exclusive prerogative of fairy-tales, is a common phenomenon of evolution, and the shadows of these miracles of the past are forever coming and going, over the growth of the tiny bird hidden in the egg. The Bird * These correspond in ourselves to the bones of the upper foot and the second row of carpals, this joint of the leg coming, therefore, really between the bones of the ankle, instead of, as in ourselves, between the ankle and the lower part of the leg. Legs The leg of our chicken, as we have seen, is attached to the great bone of the thigh-girdle. Being used for locomotion on land, the foot is not very different from that of a lizard, but there seems something very strange about the leg. Can it be possible that a chicken's knees bend backward? If so, it must be different from all other two-legged or four-legged creatures. Much of a bird's leg is concealed beneath its feathers, and when we see the bones as far up as the thigh-joint, we understand our mistake at once, and see that a bird has knees which bend in the same way as our own, that is, forward in an opposite direction from the elbow. The knees of a bird are usually concealed within the skin of the body, as in the short-legged ducks, and are never visible outside the plumage. Hence the wide-spread mistake concern- ing them. For this reason the femur, or thigh-bone, is, in birds, relatively very short, even in the long-shanked herons and flamingos, the extra length of limb resulting from the elongation of the next two lower joints. The thigh-bone, or femur, alone forms the upper leg, or " second joint," and two bones, as in the forearm, the next portion below. One of these, the tibia, is much the larger and is the " drumstick" of the chicken.* When we cut the dark meat from this portion, our knife some- times slits off a splinter, which is the second bone of this joint, the fibula. The Framework of the Bird The Framework of the Bird 99 In the chicken, we next come to a single long bone called the tarsus, which is, in life, covered with scales. , in life, covered with scales. FIG. 74. — Human leg and foot. FIG. 73. — Skeleton of an Ostrich leg and foot. FIG. 74. — Human leg and foot. FIG. 73. — Skeleton of an Ostrich leg and foot. In the embryo it is composed of several separate bones.* The simplest interpretation we can give to the foot of The Bird TOO the chicken, in comparing it with our own, is to imagine that our small toe has completely disappeared; the great toe (corresponding to the thumb of the forelimb) is turned backward, and the heel is lifted high from the FIG. 75. — Living Ostrich, showing entire leg; notice the knee almost within the body. FIG. 75. — Living Ostrich, showing entire leg; notice the knee almost within the body. ground, the several bones of the upper foot being greatly lengthened and fused into one. So we, like bears and raccoons, walk with our whole foot, from toe to heel, flat upon the ground, while a bird, like a cat or a horse, walks on its toes alone. FIG. 76. — Heron, standing naturally upon its eight toes. FIG. 76. — Heron, standing naturally upon its eight toes. FIG. 77. — Jaguar, showing progression upon toes alone (digitigrade). (Sanborn, photo. Courtesy of N. Y. Zoological Society.) 101 FIG. 77. — Jaguar, showing progression upon toes alone (digitigrade). (Sanborn, photo. Courtesy of N. Y. Zoological Society.) 101 FIG. 78. — Wood Ib:s, resting temporarily upon its whole foot. (Sanborn, photo. Courtesy of N. Y. Zoological Society.) FIG. 78. — Wood Ib:s, resting temporarily upon its whole foot. (Sanborn, photo. Courtesy of N. Y. Zoological Society.) FIG. 79. — Bear, walking upon the whole foot (plantigrade). Compare with Wood Ibis. 102 FIG. 79. — Bear, walking upon the whole foot (plantigrade). Compare with Wood Ibis. 102 102 102 THE SKULL BIRD'S skull has been called a "poem in bone — its architecture is the frozen music of morphol- ogy; in its mutely eloquent lines may be traced the rhythmic rhymes of the myriad amcebiform animals which constructed the noble edifice when they sang together." We should all "be able to whistle some bars of the cranial song — the ptery go-palatine bar at least." BIRD'S skull has been called a "poem in bone — its architecture is the frozen music of morphol- ogy; in its mutely eloquent lines may be traced the rhythmic rhymes of the myriad amcebiform animals which constructed the noble edifice when they sang together." We should all "be able to whistle some bars of the cranial song — the ptery go-palatine bar at least." We perhaps know that there are twenty-eight bones in our own head, and if we attempt to dissect the skull of a fish we will find many more, but at first glance the skull of our chicken seems to be composed of but one solid bone. Indeed, if we except the lower jaw and a few others, such as the two little bones which unite it to the skull, the entire cranium is soldered together, and the lines of junction obliterated. In young birds these seams are more or less visible, although the soldering process begins very early. of the cranial song — the ptery go-palatine bar at least." We perhaps know that there are twenty-eight bones in our own head, and if we attempt to dissect the skull of a fish we will find many more, but at first glance the skull of our chicken seems to be composed of but one solid bone. Indeed, if we except the lower jaw and a few others, such as the two little bones which unite it to the skull, the entire cranium is soldered together, and the lines of junction obliterated. In young birds these seams are more or less visible, although the soldering process begins very early. The origin of the skull is wrapped in obscurity, and neither the student of fossil bones, nor of those beneath the skin of living creatures, nor yet the diligent watcher of the mysterious panorama of life in the egg, can tell us very much, although many theories have been sug- 103 en s The Bird 1 gested. THE SKULL The poet Goethe thought the skull was merely a continuation of the neck-bones, very much expanded and changed, and although the division of the skull into three roughly outlined rings is possible, yet we have no direct proof of the truth of this theory. direct proof of the truth of this theory. Fortunately, in the skulls of most animals, the bones are separate, and by keeping in mind the constancy of their position, the puzzle of the skull of a chicken begins to clear up. Just as the first back-bone was a gelatinous or gristly FIG. 80. — Cranium of Dogfish, cartilaginous, generalized in structure. FIG. 80. — Cranium of Dogfish, cartilaginous, generalized in structure. one, so the old type of skull was entirely gristly or car- tilaginous. Sometimes on the seashore near the huts of the fishermen, we may pick up a strange-looking object — translucent and looking as if it were made of hard white rubber. Clinging to it is perhaps a long string of delicate beads of the same substance. This is the skull and back- bone of a dogfish or shark, and although the skull is very unlike the chicken's cranium, yet many of the parts in the latter are faintly foreshadowed in the cartilage skull washed up by the waves. Through all the long ages of geological epochs, myri- The Sk 105 ads of creatures were changing in form and structure, some growing too bulky and helpless and vanishing, others developing powers of running, burrowing, flying and leaping. But it is a very remarkable and wonder- ful thing and very fortunate for us poor mortals, striving after knowledge of the past, that in each general class of creatures, certain ones should have found a niche where they were removed from the fierce struggle for FIG. 81. — Skull of young Alligator. Bones massive and solid, adapting their owner to an active aquatic life but to sluggish terrestrial movements; eye- cavities and brain-case very small, the jaws (organs of prehension) composing by far the major part of the head. FIG. 81. — Skull of young Alligator. Bones massive and solid, adapting their owner to an active aquatic life but to sluggish terrestrial movements; eye- cavities and brain-case very small, the jaws (organs of prehension) composing by far the major part of the head. THE SKULL existence, and where for year after year, century upon century, they and their descendants changed but little. We might mention Amphioxus and sharks among fishes, Necturus among amphibians, Sphenodon among reptiles, and the duck-billed mammal and others among hair- covered creatures. These may be meaningless names, but if one will read about them and then examine their skins and skeletons in our museums, many a glance will be given into the ages of the past, compared to which the existence, and where for year after year, century upon century, they and their descendants changed but little. We might mention Amphioxus and sharks among fishes, Necturus among amphibians, Sphenodon among reptiles, and the duck-billed mammal and others among hair- covered creatures. These may be meaningless names, but if one will read about them and then examine their skins and skeletons in our museums, many a glance will be given into the ages of the past, compared to which the The Bird 1 few thousand years during which man has reigned seems but a day. When we study the early structure of some creature, say a bird, we find that before it emerges from the egg the skull is soft and cartilaginous, open and quite differ- ent in shape from what it will be eventually, and it is most startling to find a living creature — a shark — with FIG. 82.— Skull of Bald Eagle. Bones light and spongy, fitting for a very active aerial life; orbit very large and brain-case capacious, showing great advance beyond reptilian condition. FIG. 82.— Skull of Bald Eagle. Bones light and spongy, fitting for a very active aerial life; orbit very large and brain-case capacious, showing great advance beyond reptilian condition. a skull which never gets beyond this condition. It is as if the curtain of eternity had been, for a moment, drawn aside for us, and a glimpse given into the past — a past so remote and clouded that our keenest searches seem to reveal but dim, skeletal forms of weird shapes, which yet we know must have blended and imperceptibly merged, through millions of years, into the present life of the earth. a skull which never gets beyond this condition. THE SKULL It is as if the curtain of eternity had been, for a moment, drawn aside for us, and a glimpse given into the past — a past so remote and clouded that our keenest searches seem to reveal but dim, skeletal forms of weird shapes, which yet we know must have blended and imperceptibly merged, through millions of years, into the present life of the earth. Looking at the chicken's skull as a whole, we notice a number of uses which the various parts serve. The The Skull large rounded portion taking up most of the skull proper is, of course, the box of bone which protects the brain. On each side, a large cavity shows where the eyes are placed, and if we compare this skull with that of a cat or dog or with that of a human being, we will see what great importance eyes must be to a bird; the cavities for them are so much larger than in other animals. Back 3r«5u nCd.se 3r«5u nCd.se Oriit ifa.Occ. Tin* FIG. 83. — Skull of Fowl, showing orbit, brain-case, ear, lower jaw, premaxillary (Pmx.), maxillary (Mx.), vomer (Vo.), lacrymal (Lc.), jugal (Ju.), palatine (Pal.), pterygoid (.Ptg), quadrate (Qd.), and supra (Sup. occ.), ex (Ex. occ.), and basioccipital bones. (After Parker.) FIG. 83. — Skull of Fowl, showing orbit, brain-case, ear, lower jaw, premaxillary (Pmx.), maxillary (Mx.), vomer (Vo.), lacrymal (Lc.), jugal (Ju.), palatine (Pal.), pterygoid (.Ptg), quadrate (Qd.), and supra (Sup. occ.), ex (Ex. occ.), and basioccipital bones. (After Parker.) of each eye-case we see an irregular opening, the portal of the ear; and in front of the brain two apertures in the beak open toward the organ of the nose. The prominent beak and wide-spreading lower jaw are chiefly concerned in the procuring of food. We need not bother with the names of all the bones, but there are some too interest- ing and with too strange a history to be passed by. Let us glance at the back of the skull for a moment. Here we find a large round opening through which the The Bird io8 spinal chord passes into the brain, and below it is a small knob, which in the living bird fitted into the first vertebra of the neck. THE SKULL It is a very tiny projection of bone, but fraught with significance: for if we look at the skulls of a frog, a mouse, a cat, a horse or a man, we will see that the head hinges upon two bony projections, but in all birds and reptiles there is but one, — a very FIG. 84. — Rear views of bird (Hornbill) and mammal (Yaguarondi) skulls. Notice single facet (occipital condyle) in the bird and two in the mammal, connecting skull with the neck-bones. FIG. 84. — Rear views of bird (Hornbill) and mammal (Yaguarondi) skulls. Notice single facet (occipital condyle) in the bird and two in the mammal, connecting skull with the neck-bones. plain hint of the relationship of these two Classes, so dif- ferent in external appearance. The head thus pivoting upon a single point, the bird can turn its head much farther around than if there were two points of attach- ment. Before we leave this great opening, as the scien- tists call it when they speak of it as the foramen magnum, it may be worth while to mention the remarkably con- stant position of the bones around it. Whether these are all separate, or solidly fused into one, we may always plain hint of the relationship of these two Classes, so dif- ferent in external appearance. The head thus pivoting upon a single point, the bird can turn its head much farther around than if there were two points of attach- ment. Before we leave this great opening, as the scien- tists call it when they speak of it as the foramen magnum, it may be worth while to mention the remarkably con- stant position of the bones around it. Whether these are all separate, or solidly fused into one, we may always The Sk 109 know them by their position relative to the brain open- ing; the upper edge of the hole is always formed by the supraoccipital, the lower by the basioccipital, the two sides by the exoccipitals. sides by the exoccipitals. Although many bones of the skull, such as the supra- occipital, keep their names, whether found in salmon, frog, lizard, bird, or man, others identical in position have had new names given them. THE SKULL For instance, a small bone directly in front of the eye is known as the lacrymal, from its close relation to the tear-duct, but in fishes the bone is called the preorbital, as a suggestion of fish-tears would be rather absurd. We may find the dried ear-drum, or tympanum, stretched tight across the entrance of the ear-cavities, and if we break this, or even look carefully through the transparent membrane, a long thin bone may be seen beneath, extending backwards from the under surface of the drum. This is the columella, or little column of bone, and will have an interest for us later on. If we examine the way in which the upper and lower mandibles or jaws are joined to the skull, we will find a very ingenious arrangement; one very different from that in ourselves. If the beak of a bird is to serve as hand, lips, and mouth, it must be as free and movable as possible, and instead of the upper jaw being fixed im- movably to the skull, and the lower jaw swinging up and down from it, we find that the upper jaw is attached very loosely, while each side of the lower mandible hinges upon a loose irregular-shaped bone, known as the quad- rate. A long slender bone connects the quadrate with The Bird I 10 the upper mandible, which bone we may call the jugal. Indeed when we come to look closely at the quadrates we find that they are very important, and in addition to supporting the bar of bone from the upper jaw, and pivoting the lower jaw, they bear another pan1 of bones FIG. 85. — Columella in ear of Snowy Owl (magnified 2 diameters). FIG. 85. — Columella in ear of Snowy Owl (magnified 2 diameters). extending inward from them, beneath the skull, to the broad thin palate or roof of the mouth. In a chicken the individual movement of the upper jaw is not very great, but in some birds, such as parrots, it is much more noticeable. With a sharp knife we can entirely detach the upper and lower jaws of most birds, without cutting through a bone, the connection consist- ing only of exceedingly tough tendons. THE SKULL When we found our shark's skull we perhaps wondered what had become The Sk 1 1 1 of the jaws with the many rows of teeth, and we may now guess that they were attached so loosely to the skull that the action of the water washed them away with the flesh. This was the case, and in all fishes we find both jaws as separate bones. Among reptiles we find the quadrate bone free only in snakes, an admirable adaptation which enables them to swallow their prey entire. The bones forming the palate, or roof of the mouth, are of the greatest value in classification. No matter how specialized the habits or the food of a bird may be, the palate appears to be the last portion of its structure to respond to any recent outside influences. Thus while the absence or presence of a keel to the sternum is a character of little value in separating the ostriches and their allies from all other birds, yet the radical differ- ence shown by the palate bones in the two groups is reliable evidence of their early divergence from each other. These taxonomic characters may be found in any good book on systematic ornithology and need not detain us here. Although we have the skull and both jaws of our chicken, yet some very important and interesting bones are lacking, and to find them we must find the tongue of the bird. For a bird's tongue, as well as that of other creatures, is not all flesh or horn, but underneath there is a jointed framework of bone, which is called the hyoid. We may compare its shape to that of an arrow, with a central head and four barbs, two very short and blunt and two long jointed ones. The Bird 112 It may seem to us that the mandibles, the jugal, the palate, the quadrates, the ear-bones, and the hyoid are an unmeaning jumble of irregular bones, apparently bearing no relation to one another, and with absolutely no interest outside the fact that each is very well suited to its particular use. If scientists had studied only the bones of adult animals, we might have groped in vain for any an- swer to the question of how these bones came to be what they are. But the science of embryology, or egg-life, has unfolded wonderful things, and, as we shall soon see, nothing more marvellous than the strange story of these bones. The eel-like lampreys which It may seem to us that the mandibles, the jugal, the palate, the quadrates, the ear-bones, and the hyoid are an unmeaning jumble of irregular bones, apparently bearing no relation to one another, and with absolutely no interest that the mandibles, the jugal, the the ear-bones, and the hyoid are of irregular bones, apparently bearing no relation to one another, and with absolutely no interest outside the fact that each is very well suited to its particular use. If scientists had studied only the bones of adult animals, we might have groped in vain for any an- swer to the question of how these bones came to be what they are. But the science of embryology, or egg-life, has unfolded wonderful things, and, as we shall soon see, nothing more marvellous than the strange story of these bones. an unmeaning jumble FiG.86.-Gill-basket of Lamprey. The eel-like lampreys which crowd up our shallow brooks in April to spawn are curious crea- tures, and not the least remark- able thing about them is the fact that they have no jaws, although they have an elaborate cartilagi- FiG.86.-Gill-basket of Lamprey. noug net_WOrk protecting and supporting the gills. We perhaps thought that every vertebrate animal in the world had jaws of some kind, and perhaps even lampreys had them long ago, before their habit of sucking did away with any need for chewing. The Bird But the reason I have spoken of the lamprey is because The Sk 1 1 3 it brings vividly to mind the image of an animal which must have once existed — a fish-like creature with no jaws, but with a gristly mass which held up and pro- tected the delicate blood-fringes, or gills, by means of which all true fishes breathe. Our shark is a very convenient starting-point, and before going further we should mention the technical name FIG. 87.— Gill-bars of Shark. FIG. 87.— Gill-bars of Shark. of this group — Elasmobranchs, or strap-gilled fishes. of this group — Elasmobranchs, or strap-gilled fishes. There are usually five of these gills, and within each strap or fold of skin is a jointed arch of gristle. of this group — Elasmobranchs, or strap-gilled fishes. There are usually five of these gills, and within each strap or fold of skin is a jointed arch of gristle. All this may be very true, say you, but what bearing has it on the skull of the chicken? We have seen that in sharks the number of gills has been greatly reduced, and a pair of very loosely attached jaws has been acquired, — and the truth gradually dawns The Bird i upon us: the jaw of a shark is nothing but a greatly changed gill-arch, which has doubled up, bent forward and hinged to the skull. The skin has grown over the edge, and the bony scales in the skin, standing up on end, have become teeth. And now to our bird. In the embryo chick four gill-arches are at first distinguishable, but these soon begin to alter their position, to fade away, or to change in some way, and in our bony skull we may trace them as follows (see Fig. 89). The upper half of the first gill- FIG. 88. — Lower mandible, tongue, and hyoid bones of Bald Eagle. FIG. 88. — Lower mandible, tongue, and hyoid bones of Bald Eagle. arch forms the bones of the upper jaw, palate, jugal, and quadrate, and the lower jaw completes the entire arch. The central part of the second gill fades into nothing, but the top is present as the columella-bone of the ear, while the base is transformed into the head and two blunt barbs of the arrow-like bone of the tongue. The Bird The two long barbs of this bone correspond to the third gill The.S 115 and, from their rod-like jointed character, they look very much like the real gill-arches of a fish. The fourth arch vanishes. Such is the almost incredible alchemy which Nature has wrought from a plastic rod of gristle, — transforming it into beak, tongue, and ears. Few of us, when watch- FIG. 89. — Ultimate distribution of the four embryonic gill-arches in the skull of the adult bird. The dotted portions are not developed. (Adapted from Newton.) Compare with Figs. 83 and 88. ing the gently waving gills of a fish, have realized how much we indirectly owe to them. A noted German anatomist — Karl Gegenbaur — believes that we owe even our hands and arms (by way of the pectoral fins of fishes) to portions of the gill framework, but this theory is not generally accepted. ing the gently waving gills of a fish, have realized how much we indirectly owe to them. A noted German anatomist — Karl Gegenbaur — believes that we owe even our hands and arms (by way of the pectoral fins of fishes) to portions of the gill framework, but this theory is not generally accepted. ORGANS OF NUTRITION N N other pages we shall consider some of the things upon which birds feed, and shall see how surely the methods used in the search and capture of this food mould the bird's structure, modifying its form from beak to toe ; and now is it not possible to find some- thing of interest in the food after the bird swallows it? Indeed even before the swallowing takes place, if we watch carefully we may notice something which we did not before know. In the first place the bill of a bird is, of course, a primary factor, not only in procuring food, but often in killing and preparing and also holding it while it is being made ready to swallow. Less confusion will result, how- ever, if we leave the consideration of the beaks and bills to a later chapter. After the bill (which corresponds to our mouth and lips) come the glands of the mouth and here we again enter the portals of physiology, — for some unknown reason dreaded by many of us, and systematically shunned, as dry and ultra-scientific. On the contrary there are interesting facts awaiting us in all its branches. After a brief consideration of the more important, we shall we sh Organs of Nutri 117 surely return to outdoor study of the daily life of these creatures, with more balanced interest, and a "little knowledge" which, instead of being a " dangerous thing," will, in this instance, add many fold to our appreciation of the external results of these functions, whose work- ings are ever concealed from the light of day. ings are ever concealed from the light of day. The digestive apparatus of a bird, or indeed of any creature with a back-bone, is in reality a tube or canal, which begins at the mouth and extends through the body. Certain portions are contracted or expanded, and specialized to store up, moisten, grind, dissolve, digest, or absorb the food substances which pass through. The Salivary Glands The mucous membrane, or lining, of the entire diges- tive canal is very delicate and requires to be kept con- stantly moist. The lining of the mouth and throat, being so exposed to contact with the outer air, requires some special provision to lubricate it. This is accomplished by certain glands, some beneath the tongue, others situ- ated in the upper portion of the mouth. These are not found in fishes, nor in other creatures which live alto- gether in the water; but in reptiles several groups are distinguishable. In birds they vary greatly, some having scarcely a trace, while others have large well-developed glands. Salivary is the common name given to certain of these, and we will let that name represent all. will let that name represent all. In ourselves, saliva is an important aid in digestion. Besides moistening the food and softening all hard por- tions, it exerts active chemical effects, as, for example, The Bird u8 changing starch to sugar and in many other ways making ready the food, that the important changes which take place in the stomach may begin at once. In birds, how- ever, the saliva has but little chemical effect on the food, its principal use being to moisten the substances before they are swallowed. It is not often that Nature, when she has produced FIG. 90 — Nest of Chimney Swift; twigs glued together with saliva. FIG. 90 — Nest of Chimney Swift; twigs glued together with saliva. an organ or special tissue by the elaborate synthesis of evolution, confines its use to any one function. If birds were provided with salivary glands intended only for the purpose mentioned above, they soon found other uses for them. In a woodpecker we will find very large salivary glands on each side of the mouth. These secrete a sticky liquid which covers the long, many-barbed tongue and is an efficient aid in picking out insects from their holes in the bark and wood of trees. an organ or special tissue by the elaborate synthesis of evolution, confines its use to any one function. If birds were provided with salivary glands intended only for the purpose mentioned above, they soon found other uses for them. In a woodpecker we will find very large salivary glands on each side of the mouth. The Salivary Glands These secrete a sticky liquid which covers the long, many-barbed tongue and is an efficient aid in picking out insects from their holes in the bark and wood of trees. Organs of Nutri Organs of Nutri 119 Many birds carry in their beaks the grasses and twigs with which they construct their nests, and if a sticky fluid helped them to get their food, why would it not also soften the twigs and make them easy to bend? Not only this, but certain birds, such as our Chimney Swifts, are provided with saliva in such quantities, and of such tenacious consistency, that the entire nest — a mosaic of FIG. 91. — Nest of Esculent Swiftlet (edible bird's nest) composed entirely of saliva. FIG. 91. — Nest of Esculent Swiftlet (edible bird's nest) composed entirely of saliva. small twigs, each about an inch in length — is set in saliva cement, and fastened to the vertical side of a chimney or charred tree- trunk by the same means. Even this is only a step, or link, in the direction of the extreme use- fulness of saliva, for the little East Indian birds known as Swiftlets (one of which ornithologists call Collocalia fuciphaga) make their nests entirely of saliva or mucus. The second part of the scientific name, which means an eater of seaweed, refers to the idea formerly held, that small twigs, each about an inch in length — is set in saliva cement, and fastened to the vertical side of a chimney or charred tree- trunk by the same means. Even this is only a step, or link, in the direction of the extreme use- fulness of saliva, for the little East Indian birds known as Swiftlets (one of which ornithologists call Collocalia fuciphaga) make their nests entirely of saliva or mucus. The second part of the scientific name, which means an eater of seaweed, refers to the idea formerly held, that The Bird I the nests consisted entirely of half-digested seaweed, but it has been proved that this is not the case. The homes of these birds are the " edible birds'-nests " held in such esteem by Chinese gourmands. The Tongue If we had a long series of birds' tongues before us, we would be surprised at the great variety of shapes and sizes. Observing the good-sized tongues of sparrows and other small birds, we would turn to a pelican expecting to see an enormous affair to correspond with the huge bill of that bird. On the contrary we will find a tiny incon- spicuous flap not larger round than a toothpick. This reminds us of the condition of the tongue in some fishes, where it is a very simple structure indeed. The king- fisher also has a small tongue, and the same is true of many other fish-eating birds, such as pelicans and most of those which feed on large insects. The reason is ob- vious. The food, which is swallowed whole, is of such large size that a tongue of even moderate proportions would be only in the way. The tongues of many birds are provided with oblique series of teeth, either soft and fleshy or horny in struc- ture, which point backward toward the gullet and must be of great help to the bird in guiding and swallowing its food. These teeth are especially abundant around the glottis, or opening to the windpipe, guarding it from chafing or from the chance of food choking it up. In ducks and geese we find tongues large and of com- plicated appearance. The edges are often toothed or Organs of Nutri I 2 1 fringed to correspond with the serrated or otherwise indented edges of the mandibles. One which is before me as I write is very elaborate. It is that of a wild Mallard Duck. At the tip is a thin, distinct flap or lamella, horny and with smooth edges. Behind it the tongue enlarges abruptly into a thick oblong mass, deeply grooved down FIG. 92. — Bill of Brown Pelican, showing extreme reduction of tongue in a bird which swallows whole fish. FIG. 92. — Bill of Brown Pelican, showing extreme reduction of tongue in a bird which swallows whole fish. the center. The edges of the anterior half are fringed with a double line of horny hairs, while in the posterior portion the upper line is replaced with tooth-like struc- tures. The upper surface is smooth in front, but farther back two central folds arise and curve over laterally, forming tube-like grooves. The Tongue Still more posteriorly, fleshy recurved teeth are visible, singly, in groups, or in regular lines. The tongue of our common barnyard duck is the center. The edges of the anterior half are fringed with a double line of horny hairs, while in the posterior portion the upper line is replaced with tooth-like struc- tures. The upper surface is smooth in front, but farther back two central folds arise and curve over laterally, forming tube-like grooves. Still more posteriorly, fleshy recurved teeth are visible, singly, in groups, or in regular lines. The tongue of our common barnyard duck is The Bird 1 similar to this and is well worth examining. The use of such a complicated organ in a bird of so simple feeding habits as the duck is hard to explain. We will hardly find two tongues that are alike, and even the tips differ, and show as wide a range of varia- FIGS. 93 and 94. — Top and side views of the tongue of a Mallard Duck, showing complicated structure in the tongue of a bird which sifts its food from the mud. FIGS. 93 and 94. — Top and side views of the tongue of a Mallard Duck, showing complicated structure in the tongue of a bird which sifts its food from the mud. tion as the remaining portions. In many birds, such as owls, larks, and swifts, the tip is bifid, or double-pointed, bringing to mind the forked tongues of snakes and cer- tain lizards. In woodpeckers the tongue is round and exceedingly long, and can usually be thrown out some distance beyond the tip of the bill. Our common Flicker, or Golden-winged Woodpecker, Organs of Nutrition possesses a tongue of remarkable length, even for a wood- pecker, and while feeding, the bird will often shoot it FIG. 95. — Head of Flicker, showing tongue slightly protruding FIG. 95. — Head of Flicker, showing tongue slightly protruding out two or three inches beyond the tip of the beak. Easily and without a hitch it disappears again, appa- FIG. 96. — Skull of Flicker, showing rear branches of the hyoid bone, curving up over the skull and down into the right nostril. The front of the tongue is visible beyond the tip of the beak. FIG. 96. The Tongue — Skull of Flicker, showing rear branches of the hyoid bone, curving up over the skull and down into the right nostril. The front of the tongue is visible beyond the tip of the beak. rently down the very throat of the bird. If we carefully remove the skin from the skull of a dead Flicker, the magic will become plain. When we spoke of the skull rently down the very throat of the bird. If we carefully remove the skin from the skull of a dead Flicker, the magic will become plain. When we spoke of the skull The Bird I 2 of a bird, mention was made of the two long bones which branched out from the rear of the tongue and which are all that remain of the third ancestral gill-arch. In the Flicker, the slender, white tongue divides into these two branches just in front of the glottis and from here they extend backward, passing one on each side of the wind- pipe, and on upward, following the curve of the skull, then forward, lying together upon the forehead. Not even here do they end, however, but actually reach some distance into one nostril! So when this bird stretches out its tongue, the tips of the rear branches leave the opening of the nose and shoot around over the surface of the skull until they have gone as far as possible. No wonder the poor ants have but little chance when a Flicker visits their hill and sets the marvellous mechanism of his tongue rapidly to work. And no wonder the enthusiasm of an ornithologist never fails, when he thinks of the scores of similarly interesting structures still await- ing investigation. ing investigation. The tip of the tongue in the sap-sucking woodpeckers is beset with numerous hairs forming a brush-like instru- ment, but spines take the place of hairs in the species which feed exclusively on insects. It is known that the exact proportion of insects in the diet of any particular kind of woodpecker is reflected in the more or less per- fect adaptation of the minute structure of its tongue to that end. In the sapsuckers, too, the tongue is comparatively short, doubtless because the sap flows readily from the holes which these birds bore. The Tongue Hence they require no Organs of Nutrition such extension of tongue as the deep burrows of the ants necessitate in the case of the Flicker. Thus the tongue of a bird seems a very unstable character, acted upon quickly and radically by any change in the diet of the species. The entire tip of the tongue is frequently frayed out into a kind of brush, remarkably developed in the parrot-like lories. Yet this curious structure is probably only an elonga- tion of the papillae, hom- ologous with those which make the tongue of a cat or lion so rough. Cocka- toos, although first cousins to the lories, have very different tongues, thick and fleshy with club-shaped tips. Thus the tongue of a bird seems a very unstable character, acted upon quickly and radically by any change in the diet of the species. The entire tip of the tongue is frequently frayed out into a kind of brush, remarkably developed in rd seems a very unstable ly and radically by any ies. The entire tip of the out into a kind of brush, remarkably developed in the parrot-like lories. Yet this curious structure is probably only an elonga- tion of the papillae, hom- ologous with those which make the tongue of a cat or lion so rough. Cocka- toos, although first cousins to the lories, have very different tongues, thick and fleshy with club-shaped tips. FIG. 97. — Thick fleshy tongue of Cockatoo, tips. In our common gold- finch, the sides of the tongue curl inward, form- ing an admirable seed- scoop, while the same or- distinctly cleft into sev- been likened to a "four- s little owner impales the which it so industriously he great particoloured bill curious tongue — a long tips. In our common gold- finch, the sides of the tongue curl inward, form- ing an admirable seed- scoop, while the same or- gan in the chickadee, being distinctly cleft into sev- eral prongs at the tip, has been likened to a "four- tined pitchfork" on which its little owner impales the myriad grubs and insects for which it so industriously searches twigs and leaves. The great particoloured bill of a toucan conceals a very curious tongue — a long FIG. 97. — Thick fleshy tongue of Cockatoo, FIG. 97. The Tongue — Thick fleshy tongue of Cockatoo, scoop, while the same or- gan in the chickadee, being distinctly cleft into sev- eral prongs at the tip, has been likened to a "four- tined pitchfork" on which its little owner impales the myriad grubs and insects for which it so industriously searches twigs and leaves. The great particoloured bill of a toucan conceals a very curious tongue — a long The Bird 126 thin affair, narrow throughout its whole length and so thickly bordered with a deep, delicate fringe that it bears a decided resemblance to a feather. FIG 98. — Feather-like tongue of Toco Toucan. FIG 98. — Feather-like tongue of Toco Toucan. The tongue of a flamingo is thick and fleshy, filling the entire cavity of the lower mandible and in shape reflecting its crookedness. The upper edges of the man- • •» FIG. 99. — Tongue of Flamingo within lower mandible (natural position), FIG. 99. — Tongue of Flamingo within lower mandible (natural position), dible approach each other so closely that they perma- nently inclose the tongue, motion being only possible in Organs of Nutri 1 27 a forward or backward direction. Along the sides of the tongue are two series of fleshy teeth, in shape exactly like the poison-fangs of a rattlesnake. like the poison-fangs of a rattlesnake. Mention must be made of the unique and greatly specialized tongues of Hummingbirds and Honey Creepers. The outer edges of this organ are curled into two tubes, FIG. 100. — Side view of Flamingo's tongue, forcibly lifted above mandible, shopp- ing fleshy recurved teeth. FIG. 100. — Side view of Flamingo's tongue, forcibly lifted above mandible, shopp- ing fleshy recurved teeth. which are more or less split and frayed near the tip, forming delicate brushes — efficient instruments either to suck up nectar or to flick out insects from the heart of corollas. The Crop From the back of the throat to the stomach extends a tube, the gullet or oesophagus, through which the food .3 a .3 a .3 a Organs of Nutri 129 descends after it leaves the mouth. In some birds this is a simple tube of the same diameter throughout, always moist from the secretion of (mucous) glands which are found abundant in its walls, but serving merely as a passage for the food on its way to the stomach. passage for the food on its way to the stomach. In another class of birds an enlarged chamber is present, called the crop. This serves a somewhat simi- lar purpose as the external pouch of the pelican; that is, it acts as a receptacle for food. No especial digestive glands are found here, and the only agents acting on FIG. 102.— Brush, or tube-like tongue of Honey Creeper. Twice natural size. FIG. 102.— Brush, or tube-like tongue of Honey Creeper. Twice natural size. the food are water, the secretions of the salivary glands, and the heat of the bird's body. The crop exists only superficially in some birds, the dilation being hardly noticeable. From these we find a succession of more distinctly marked permanent crops, until in grain-eating birds this organ is very prominent. If we examine an English Sparrow after it has made a hearty meal in the chicken- yard, we will find the crop filled with grains of wheat, some cracked in pieces by the bill, others entire. The Bird 1 When we sought the extreme in the provision of saliva in birds, we had to refer to a swift, living in caves in islands of the Malay Archipelago, but to find the highest degree of development of crops is a much easier matter. In the ordinary pigeon the crop is of very great size and divided into two lobes. The capacity of the crop in some birds of this class is astonishing. As many as sixty-three acorns have been found in the crop of the English Wood-pigeon. If we look at the crop of a pigeon before its young leave the nest, we will discover a func- tion of this organ which would otherwise never be sus- pected. We know that herons and some other birds feed their young on fish half -digested by themselves. This process is known as regurgitation. The Crop If we have ever seen a pigeon with the beak of its young half down its throat, pumping something into the offspring's mouth, we have probably thought that a similar habit was being shown, — half -digested grain taking the place of the heron's fish. But such is not the case. At the time of the breeding season, the folds of membrane in the crops of both parent pigeons thicken and secrete or peel off in curdy cheesy masses — "pigeon's milk" some call it — and this forms the food of the young birds So in pigeons the crop not only receives food, but at times provides it. Now for a glance at some of the oddities in the struc- ture of crops. The Hoatzin — a strange bird of Brazilian swamps — which harks back to its reptilian ancestors in many ways, has a very curious crop. There are strong muscles in its walls, the use of which, it is said, is to Organs of Nutri 131 squeeze out the juice of the thick leaves of the Arum arborescens which forms its food. Thus it has a gizzard- like function, and has become so important in the life- economy of the bird that it has developed out of all proportion, and occupies so much space that the keel on the breast-bone has had to give way in part to make room for it, and even the arms of the " wish-bone" have been bent outward. In this remarkable bird the pro- ventriculus and gizzard are reduced, their functions being usurped by the crop. usurped by the crop. The facility with which most birds are able to eject the contents of their crops serves several useful pur- poses, besides the feeding of the young of herons, cor- morants, and others. When vultures have gorged them- selves to repletion on the flesh of any animal, they usually retire to some near-by retreat and sleep until digestion has taken place. But if they are suddenly approached or alarmed, they will instantly eject all they have swal- lowed and, thus lightened, take safety in flight. Pelicans and Wood Ibises also have the habit of ' unswallowing ' their fishy meals when frightened. The Crop Petrels and many fish-eating sea-birds appear to have a supply of oil always in readiness, which they shoot from the mouth to a con- siderable distance, surving as an efficient means of self- defence when taken in the hand after being wounded. In birds of prey generally, but especially in owls, another use for this habit is found. Owls always swallow their smaller prey entire, sometimes crushing the skulls of mice and plucking out the longer wing and tail-feathers of birds. Although their crops are not nearly so mus- The Bird 132 cular as that of the Hoatzin, yet there must be powerful movements of the walls, for the mice and birds are de- nuded of hair and feathers and even the bones are in some way removed from the body, and all are ejected in a neat oval ball. If we find some hollow tree where an owl has its FIG. 103. — Keel of Hoatzin, repressed by crop. FIG. 103. — Keel of Hoatzin, repressed by crop. regular sleeping-place, many of these pellets may be found on the ground below, showing the results of each night's hunting. The skulls in them are often in such perfect condition that the species of rodents may be identified. Besides allowing the bird conveniently to get rid of the indigestible portions of its food, this habit seems to be necessary to the health of the bird. In Organs of Nutrition captivity, owls and hawks are never so healthy and active when fed on fleshy meat alone, as when a dead mouse or sparrow, rat or pigeon is given occasionally. In dissecting specimens which have had nothing but a flesh diet for a year or more, I have found the throat and gullet in a very bad condition, as if the lack of some scouring process, FIG. 104. — Food-pellets ejected by Great Horned Owl, containing remains of rodents. FIG. 104. — Food-pellets ejected by Great Horned Owl, containing remains of rodents. such as is afforded by the pasage up and down of the indigestible hair and feathers, had actually resulted in the death of the bird. I have seen owls try to eat the straw on the floors of the cages, when not provided with food in the condition in which they find it when at lib- erty. The Crop In cormorants and birds of similar voracious fish-eating habits, the entire gullet serves as a receptacle for food, while the fish first swallowed are undergoing the process The Bird '34 of digestion lower down. Here, as in many other in- stances, we have a condition very similar to that in some reptiles — crocodiles in particular. These ravenous scaly creatures have such powerful organs of digestion that even bones are dissolved, but the stomach is comparatively small, and when a crocodile makes a large meal, it is at first stored away in the wide gullet. The Stomach and Gizzard In the present chapter we might easily be led into details which would strand us in the midst of dry technicalities, but we will try to avoid all this and choose only the interesting facts. FIQ. 105. — Caracara, showing crop dis- tended with food The chief organ of di- gestion, in birds as in other animals, is of course the stomach. In many fish- eating birds this organ is merely a simple, more or less enlarged chamber, rather crop-like except that it con- tains numerous digestive lands. FIQ. 105. — Caracara, showing crop dis- tended with food FIQ. 105. — Caracara, showing crop dis- tended with food glands. The typical bird-stom- ach, however, is compound, or formed of two more or less distinct parts. The first Organs of Nutrition portion — known as the proventriculus — is the smaller, and contains very active digestive glands, sometimes ar- ranged in patches, but more usually forming a band. If the lower part of the oesophagus of an English Sparrow is removed, slit open and washed, these glands can be easily seen, being more of a rose tint than the paler tissue of the portion nearer the mouth. The walls are thicker in this glandular area. This can be seen to better ad- portion — known as the proventriculus — is the smaller, and contains very active digestive glands, sometimes ar- ranged in patches, but more usually forming a band. If the lower part of the oesophagus of an English Sparrow is removed, slit open and washed, these glands can be easily seen, being more of a rose tint than the paler tissue of the portion nearer the mouth. The walls are thicker in this glandular area. This can be seen to better ad- vantage in a young chicken, where the glands take the form of conical protuber- ances which dot the entire surface. Nature has pro- duced curious modifications rtion — known as the proventriculus — is the smaller, and ntains very active digestive glands, sometimes ar- ranged in patches, but more usually forming a band. If e lower part of the oesophagus of an English Sparrow removed, slit open and washed, these glands can be ily seen, being more of a rose tint than the paler tissue the portion nearer the mouth. The walls are thicker this glandular area. The Stomach and Gizzard This can be seen to better ad- vantage in a young chicken, where the glands take the form of conical protuber- ances which dot the entire surface. Nature has pro- duced curious modifications of this typical fore-stomach, as in snake-birds, whicn have the glands of this portion en- closed in a sac, in shape not unlike a small crop. Here the food is softened and acted 106.— Glands of the stomach of a upon chemically by the secre- young chicken. ,. ,, tions from the walls. portion — known as the proventriculus — is the smaller, and contains very active digestive glands, sometimes ar- ranged in patches, but more usually forming a band. If the lower part of the oesophagus of an English Sparrow is removed, slit open and washed, these glands can be easily seen, being more of a rose tint than the paler tissue of the portion nearer the mouth. The walls are thicker in this glandular area. This can be seen to better ad- vantage in a young chicken, where the glands take the form of conical protuber- ances which dot the entire surface. Nature has pro- duced curious modifications of this typical fore-stomach, as in snake-birds, whicn have the glands of this portion en- closed in a sac, in shape not unlike a small crop. Here the food is softened and acted FIG. 106.— Glands of the stomach of a upon chemically by the secre- young chicken. ,. ,, tions from the walls. is removed, slit open and washed, these glands can be easily seen, being more of a rose tint than the paler tissue of the portion nearer the mouth. The walls are thicker in this glandular area. This can be seen to better ad- vantage in a young chicken, where the glands take the form of conical protuber- ances which dot the entire surface. Nature has pro- duced curious modifications of this typical fore-stomach, as in snake-birds, whicn have the glands of this portion en- closed in a sac, in shape not unlike a small crop. Here the food is softened and acted FIG. 106.— Glands of the stomach of a upon chemically by the secre- young chicken. ,. ,, tions from the walls. The Stomach and Gizzard The second division of the stomach is the gizzard, an organ made to perform most powerful compressing motions, thus crushing and macerating the food, so that when passed on into the intestine, every particle of nour- ishment may be extracted from it. When we think of beauty of colouring in birds, it is their plumage which at once presents itself to the mind, and yet a gizzard has a real beauty both of shape and hue. This organ, FIG. 106.— Glands of the stomach of a young chicken FIG. 106.— Glands of the stomach of a young chicken The second division of the stomach is the gizzard, an organ made to perform most powerful compressing motions, thus crushing and macerating the food, so that when passed on into the intestine, every particle of nour- ishment may be extracted from it. When we think of beauty of colouring in birds, it is their plumage which at once presents itself to the mind, and yet a gizzard has a real beauty both of shape and hue. This organ, The Bird i 3 in a chicken, is in shape like a double convex lens. The cavity in the centre is lined with a tough yellow membrane, sometimes almost as hard as bone. Two great tendons spread over the outer surface on each side, and although in life forever buried in the absolute darkness of the bird's body, yet when brought into sunlight they shine with an iridescence like the beam from a spectrum. It is hardly possible for the gizzard to grind up food in the sense of having much lateral motion, like the move- ment of the jaws in chewing, but it shuts together again and again with great force. Gravel and sharp stones are swallowed by many birds, and are of great importance in helping to grind the food. The number and size of these stones are sometimes almost beyond belief. I have known a cassowary to swallow over a quart of rubble in one day, and have given a quartz pebble twice as large as a hen's egg to one of these birds and watched it slip down the bird's throat as easily as a cube of carrot. This particular bird preferred smooth white quartz pebbles, and would search through a whole heap, picking out stones of this character. The Stomach and Gizzard The same preference was exhibited by the gigantic extinct birds of New Zealand called moas. Mr. Frederick Chapman, writing of a portion of New Zealand where the skeletons of moas were found in great abundance, says: "When we came upon the ground disturbed by the wind (the soil being shifting sand) we soon found a number of distinct groups of gigantic gizzard-stones. It was impossible to mistake them. In several cases they lay with a few fragments of the heavier bones. In all cases they were in distinct groups; even Organs of Nutri 137 where they had become scattered, each group covered only a few square yards of ground, and in that space lay thickly strewn. . . . The peculiar feature of the stones was that they were almost all opaque, white quartz pebbles. In one place I found a small group of small pebbles of different colour, more like the few brown water- worn pebbles which may be picked up hereabouts. These lay with a set of bones much smaller than the very large bones I found with most of the clusters of pebbles. "I did not gather these brown pebbles, as I thought it uncertain whether they were gizzard-stones or not, though it is possible that the species to which the smaller stones belonged was not so careful in selecting white stones. "A glance at the pebbles lying around in the sur- rounding country showed that the quartz-pebbles were not collected here. . . . Mr. Murdock and I collected three sets of pebbles, and these I can pronounce com- plete, or nearly so. It is beyond question, too, that each set belongs to a distinct bird. No. 1 weighs 3 Ib. 9 oz.; No. 2 weighs 4 Ib.; while No. 3 weighs no less than 5 Ib. 7 oz. ! This giant set contains individual stones weighing over 2 oz.; indeed, I have picked out eight stones weigh- ing almost exactly 1 pound." ing almost exactly 1 pound." The gizzard of a bird is reflective of its diet, and is very quickly affected by any change in the food. For example, a captive gull when fed exclusively on fish has but little muscular power in the gizzard, but a diet of grain will produce a change in that organ, giving it grinding power sufficient to crush the kernels of corn. 1 The Bird That this is something more than an abnormal con- dition brought about by artificial means is proved by the fact that in the Orkney Islands the wild gulls feed in winter, spring, and summer on fish, and at this time are gizzardless; but in the fall they change to a diet of corn and develop a very respectable gizzard. So we see that this organ, apparently so independent in func- tion and individual in appearance in many birds, is in reality only a physiological change from the stomach proper. proper. The history of the development of this organ may be traced in various living species, from the soft mem- branous sac of a fish-eating bird to the knot of tendons which forms the gizzard of certain Fruit-pigeons. These birds feed on nutmegs and other very hard, almost stony nuts, and to enable the bird to crush these, the lining of the gizzard is covered with several score of conical pro- jections, horny in consistence. These are probably the nearest approach to " hen's teeth "we are likely to find. nearest approach to " hen's teeth "we are likely to find. What a boon to a business man who indulges in a daily " fireman's lunch/' if his masticatory function could be an internal and unconscious one, as in a bird! A crocodile, which has so much in common with a bird, is provided with a gizzard, which, like that of a chicken, is round, muscular, and has two great side ten- dons, and no less than five pounds of grinding-stones have been found in one of these reptiles. Many interesting adaptations are found in the stom- achs of birds, made necessary by special requirements in the diet. As an instance of this, the snake-bird has Organs of Nutri I 39 a dense mat of hair at one end of the stomach, the free ends of which point outward, brush-like, and prevent the accidental entrance of any small fish-bones which otherwise might get into the small intestine. The giz- zard of a cuckoo, when opened, often gives the impression of a similar coating of hair lining the entire organ, but these are in reality only the hairs of caterpillars upon FIG. 107. — Cluster of matted hairs in the stomach of a Snake-bird. FIG. 107. — Cluster of matted hairs in the stomach of a Snake-bird. The Bird which these birds feed, which have become detached and have lodged in the folds of the gizzard lining. When considering the crops of birds we noticed the curious way in which a pigeon feeds its young, by re- gurgitating a cheesy substance which forms in its crop, and we will now speak of something still more remark- able. The strange nesting habits of the hornbills are foreign to this volume, but we cannot leave the subject of gizzards without touching on the manner in which the male birds of this group probably feed their impris- The Bird 1 oned mate and young. I say probably, because no one has seen them do this, but as in captivity the operation occurs repeatedly during the breeding season, there can be but little doubt concerning its evident significance. After walling up his mate and her egg in some hollow tree, the male hornbill takes upon himself the labour of supplying her with food throughout the period of incu- bation and the subsequent rearing of the young bird. Instead of bringing food piecemeal, — nut by nut, grape by grape, — the lining of the entire gizzard peels off at certain frequent intervals, appearing, when ejected at the mouth, like a small bag or purse, the puckered open- ing (heightening the simile) serving to retain securely the contents of the gizzard, — a dozen or score of grapes or other fruit. This, the male bird, in his native land, doubtless takes in his beak to the tiny opening of the walled-up nest and delivers into the bill of his mate. How admirable a spouse this, who not only seeks and provides sufficient food for his temporarily helpless family, but bears it to them wrapped in a packet torn from his very body — if not a "pound of flesh," at least enough to make a lunch-basket! The Intestines Beyond the gizzard is the intestinal canal, which varies greatly in length in different birds. The ostrich has forty-six feet of this digestive tube, while the nectar and tiny insects snatched by a hummingbird in its flight are digested in a delicate hair-like duct but two inches in length. Although comparatively of such great length, the Organs of Nutri 141 way in which this part of the digestive tract is coiled and twisted in the body cavity of the bird allows it to take up the least possible amount of room. up the least possible amount of room. The function of this long tube is to absorb the nutri- ment from the food after this has been moistened by the salivary glands, crushed by the gizzard, and acted on by the stomach acids, and secretions from the liver and pancreas. The digestible parts are then taken up by the blood through the walls of the intestine. In many of the lower types of birds, such as the cassowary, ostrich, and screamer, the arrangement of this long digestive tract is very simple, much like the condition to be found in alligators. THE FOOD OF BIRDS HE organs and physiological functions of a bird, as of animals generally, are so interrelated and intimately dependent on each other that it is a rather difficult matter to consider any single one by itself without being led into another's province. For example: we have for the subject of this chapter the food of birds, and unless we are very careful, we shall overstep the bounds of our theme. To limit our subject clearly we will consider only adult birds. H clearly we will consider only adult birds. We have all seen the pestiferous sparrows picking up grain in the chicken-yard; we have admired the skill which the red-breasted robin exhibits in spying and extracting earthworms on our lawns; our memory re- calls the osprey dropping upon his fish, and the wood- pecker chiselling to the wood-borer; but did we ever stop a while and attempt a "bird's-eye view" of all the classes of substances which birds find good as food? The ways in which this food is sought and caught, killed and prepared are wonderfully varied, and some idea of the remarkable variety of substances laid under contribution as food by birds of different orders may be had from a brief review of the principal divisions 142 ivisi 142 The Food of Bi 143 into which these substances are classified, and the part they take in supplying birds with food. As with all animals, certain mineral salts are very necessary to a bird's existence, such as the substances from which the calcium phosphate for the bones, and the calcium carbonate for the shells of the eggs, are de- FIG. 108. — Finch, a bird with heavy, thick bill adapted for crushing seed. FIG. 108. — Finch, a bird with heavy, thick bill adapted for crushing seed rived. The gravel and pebbles swallowed by birds in the course of their daily feeding should hardly be men- tioned in this connection, as this is only done for the mechanical assistance, derived from the hard surfaces, in triturating the food. Vegetable-feeders form a large group among birds, and they alone would offer an interesting field for study, as The Bird 1 there is such specialization for feeding on particular varieties or portions of plants. We find fruit- and grain- eaters, besides those which feed almost entirely on buds, leaves, berries and nuts, nectar, sap, and even pollen. THE FOOD OF BIRDS Lichens form a considerable item in the bill of fare of ptarmigans, the Arctic grouse. We have even dedicated certain plants to birds which show a decided partiality for them, — duckweed and partridge-berry. FIG. 109. — Vireo, an insect-hunter, with a delicate, hooked bill. FIG. 109. — Vireo, an insect-hunter, with a delicate, hooked bill. There is no doubt that a great many plants benefit from the cross-fertilization of their flowers by humming- birds carrying the pollen from blossom to blossom. Of one of the sugar-birds of South Africa it is said: "When sucking up the nectar of one of the larger protea-blossoms, the bird perches on the edge of the flower, plunges its long bill and the greater part of its head downwards among the petals, and retains it in this position until The Food of Birds satisfied. As a result the narrow, shaft-like feathers of the forehead frequently become saturated and stained with juice and dusted over with pollen, and it is probable that this bird plays an important part in the cross-fer- tilization of several species of protea." tilization of several species of protea." Desmids and diatoms, those one-celled microscopic organisms which are almost on the border-line between FOOD OF VARIOUS BIRDS. FIG. 110. — Sea-urchin. FIG. 110. — Sea-urchin. plants and animals, I have found in large numbers in the digestive tracts of ducks and other birds which are accustomed to find their food by sifting the mud at the edges of ponds and lakes. plants and animals, I have found in large numbers in the digestive tracts of ducks and other birds which are accustomed to find their food by sifting the mud at the edges of ponds and lakes. Sponges, at least in a decayng state, are devoured by crows, as I can testify from observation after dredging expeditions in the Bay of Fundy. The Bird The Bird 146 We should scarcely think that those watery creatures sea-anemones, hydroids, and jelly-fish (some of the latter FIG. 111.— Caterpillar. FIG. 111.— Caterpillar. consisting of over 95 per cent water) could afford much nourishment to any animal, and when crows and gulls are seen tearing large stranded jellies into pieces, it is consisting of over 95 per cent water) could afford much nourishment to any animal, and when crows and gulls are seen tearing large stranded jellies into pieces, it is FIG. 112.— Cocoon. FIG. 112.— Cocoon. FIG. 112.— Cocoon. probably only for the sake of the semi-parasitic shrimps which make their home in the interior canals of the The Food of Bi 147 masses of animate gelatine. But the fresh-water hydra, belonging to the same division as the hydroids, is eaten in myriads by ducks and geese. These and many other birds are remarkably fond of duckweed, which they devour with such evident pleasure that they must enjoy it as much as cats do catnip, or canaries hempseed. As the under surface of these small water plants is the fa- FIG. 113.— Butterfly with wing torn by bird. FIG. 113.— Butterfly with wing torn by bird. vourite home of the hydra, they necessarily form a por- tion of the food of these water birds. Roundworms, flatworms, and leeches are devoured by many aquatic birds, while earthworms form a staple article of diet with such different species as thrushes, woodcocks, and cranes. A favourite morsel of the curi- ous apteryx of New Zealand is a gigantic species of worm, twelve to twenty inches in length, which is highly phospho- rescent. The apteryx seeks its food by night, and when The Bird 1 devouring one of these worms, the whole bird is lighted up, and after its meal the bird's bill is illumined by the mucus which adheres to it. mucus which adheres to it. Starfish and sea-urchins are sought out by crows, ravens and gulls, and perhaps other birds. They break into them by main force, or else carry them to a height and drop them on the rocks. I have even seen a Bald Eagle, when fish and Fish- hawks were scarce, deliber- ately break into and devour a green-spined urchin. If, as is said, immense bow- head whales subsist entirely FIG. 114.-Snail. The Bird shrimps, then it is not surprising that many thousands of shore- birds are well nourished by the myriads of shrimps and prawns, large and small, which every tide leaves exposed. Starfish and sea-urchins are sought out by crows, ravens and gulls, and perhaps other birds. They break into them by main force, or else carry them to a height and drop them on the rocks. I have even seen a Bald Eagle, when fish and Fish- hawks were scarce, deliber- ately break into and devour a green-spined urchin. FIG. 114.-Snail. a green-spined urchin. If, as is said, immense bow- head whales subsist entirely FIG. 114.-Snail. shrimps, then it is not surprising that many thousands of shore- birds are well nourished by the myriads of shrimps and prawns, large and small, which every tide leaves exposed. FIG. 114.-Snail. It is a mere truism to say that insects form the sole food of scores of species of birds, and enter into the diet of many hundreds. It has been said that without birds, within a space of ten years, the earth would not be habit- able for man, owing to the unrestricted increase of nox- ious insects. There is doubtless not a single group of in- sects which does not suffer from the appetite of one or more species of bird. The eggs and larvae are dug and pried out of their burrows in the wood by woodpeckers and creepers; those underground are scratched and clawed up to view by quail, partridges, and many spar- The Food of Birds rows; warblers and vireos scan every twig and leaf; flycatchers, like the cat family, lie in watch and spring after their prey, only in the air instead of on the ground, feeding more particularly on low-flying insects; while swifts, swallows, and martins glean their harvest from the diurnal hosts of high-flying winged creatures. Many FIG. 115.— Crab. FIG. 115.— Crab. times when we think hummingbirds are taking dainty sips of nectar from the flowers, they are in reality pick- ing minute spiders and flies from the deep cups of the co- rollas. The Bird When night falls, the insects which have chosen that time as the safer to carry on their business of life are pounced upon by nocturnal feathered beings — the cavernous mouths of the whippoorwills engulf them as they rise from their hiding-places, and the bristles of times when we think hummingbirds are taking dainty sips of nectar from the flowers, they are in reality pick- ing minute spiders and flies from the deep cups of the co- rollas. When night falls, the insects which have chosen that time as the safer to carry on their business of life are pounced upon by nocturnal feathered beings — the cavernous mouths of the whippoorwills engulf them as they rise from their hiding-places, and the bristles of The Bird 150 night-hawks brush them into rapacious maws, if per- chance they have succeeded in reaching the upper air. chance they have succeeded in reaching the upper air. In tropical forests, where insects are everywhere abundant, the birds seemed to have realized the fact that to each is apportioned certain phases of insect life, and that by hunting in large flocks, instead of competition resulting between birds of different species, they play into each other's hands (or rather beaks). It is of such a flock that Hudson writes: "The larger creepers ex- FIG. 116.— Squid. FIG. 116.— Squid. plore the trunks of big trees, others run over the branches and cling to the lesser twigs, so that every tree in their route, from its roots to the topmost foliage, is thoroughly examined, and every spider and caterpillar taken, while the winged insects, driven from their lurking-places, are seized where they settle, or caught flying by the tyrant- birds.^ birds.^ The Wattled Starlings or Locust-birds of South Africa live in flocks of thousands, and so dependent are they on locusts as food, that their habitat and place of nest- The Food of Birds '5* ing is influenced by the presence or absence of sects. "When pursuing a flight of mature loc starlings perform various extraordinary and aerial evolutions with the object of intercepting rounding a portion of the swarm, and in doing movements closely resemble those of anothe destroying starling, the beautiful rose-colour of eastern Europe and Asia. Individually the t are very different; collectively and under simi tions their actions are quite similar. The Bird Starting in a dense 1 ball-like ' mass, they suddenly open out into a fan-shaped formation, then assume a semicircular arrangement, and finally end by forming a hollow cylinder in which a portion of the locusts are enclosed; as the imprisoned insects are destroyed, the starlings gradually fill up the hollow of the cylinder until they again assume t formation and proceed to follow the remainin The ground below the flock is covered with the of the birds and the snipped-off legs and wings At other times the starlings station themselve tops of bushes and trees, from which they da flying insects like flycatchers. "In Cape Colony the Locust-birds usually oc FIG. 117.— Ratt ing is influenced by the presence or absence of these in sects. "When pursuing a flight of mature locusts these starlings perform various extraordinary and beautiful aerial evolutions with the object of intercepting and sur- rounding a portion of the swarm, and in doing this their movements closely resemble those of another locust- destroying starling, the beautiful rose-coloured Pastor of eastern Europe and Asia. Individually the two species are very different; collectively and under similar condi- tions their actions are quite similar. Starting in a dense 1 ball-like ' mass, they suddenly open out into a fan-shaped formation, then assume a semicircular arrangement, and finally end by forming a hollow cylinder in which a portion of the locusts are enclosed; as the imprisoned insects are destroyed, the starlings gradually fill up the hollow of the cylinder until they again assume their 'ball' formation and proceed to follow the remaining locusts. The ground below the flock is covered with the droppings of the birds and the snipped-off legs and wings of locusts. At other times the starlings station themselves on the tops of bushes and trees, from which they dart on the flying insects like flycatchers. FIG. 117.— Rattlesnake. FIG. 117.— Rattlesnake. FIG. 117.— Rattlesnake. flying insects like flycatchers. "In Cape Colony the Locust-birds usually breed in very large colonies, in localities in which the locusts have The Bird 1 deposited their eggs. For hundreds of yards every thorny bush is packed full of cup-shaped nests, even the spaces between the nests being often filled up with sticks or rubbish, through which narrow passages are left for the ingress and egress of the birds. The Bird Many starlings that can find no room in the bushes build on the ground, or under FIG. 118. — Brown Pelicans diving for fish. (Sanborn, photographer. Courtesy N. Y. Zoological Society.) FIG. 118. — Brown Pelicans diving for fish. (Sanborn, photographer. Courtesy N. Y. Zoological Society.) stones, or in holes, and these unfortunates, together with their eggs or young, ultimately become the victims of the smaller carnivorous mammals or of snakes. It fre- quently happens also that either the young locusts are hatched in insufficient numbers or that they migrate before the young starlings are fledged. In either case large numbers of birds perish of hunger, the majority of the stones, or in holes, and these unfortunates, together with their eggs or young, ultimately become the victims of the smaller carnivorous mammals or of snakes. It fre- quently happens also that either the young locusts are hatched in insufficient numbers or that they migrate before the young starlings are fledged. In either case large numbers of birds perish of hunger, the majority of the The Food of Bi 153 old birds and the more advanced young following the locusts." locusts." Slugs and snails are eaten by thrushes and many other small birds with avidity, and the name " Snail-hawk" has been given on account of one bird's fondness for these mollusks. The Oyster-catcher feeds on clams and oysters and derives its name from the facility with which it in- serts its bill and pries open the shells. The Courlan, a near relative of the rails, feeds on clams and mussels and has a most ingenious method of obtaining its food. In shallow water it feels about with its feet for these mollusks, and when they are found the bird inserts its bill between the valves with a sudden quick stroke, and, thus suspended, the heavy shell and its occupant are carried to the shore, where the shell is forced open and the animal eaten. Crows treat shell-fish in the same way that they do sea-urchins and crabs, carrying them aloft and, after dropping them, descending to feed on the nutritious flesh exposed by the shattered shells. Squids, the "head-footed" leaders of the division of mollusks, are eaten by penguins at least, and so numerous and at times so conspicuous a feature of marine life are they that probably many other birds also feed upon them. The Bird Even deep-water snails and crabs are not safe, as the sturdy sea-ducks will sometimes dive to a depth of one hundred and fifty feet to feed upon them. Fish count many enemies among birds, which have numerous ways of obtaining their victims from ocean or lake. Some of these are so ingenious that they well The Bird 1 deserve notice. In their variety they rival the methods of man himself, and we find many analogies between the two. Penguins earn their food with perhaps the hardest work, as they follow the fast-swimming fish of the open ocean in their own icy element and capture them not- withstanding their speed and quick turns. withstanding their speed and quick turns. We must not forget the slim, evil-looking snake- birds of the tropical swamps, which also dart through the water, but impale their victims on their needle-pointed beaks, suggesting the fish-spears of mankind. Cormo- rants and sheldrakes also dive after the fish on which they feed. they feed. Next in the list of strenuous seekers after fish we must mention the osprey, which hovers on slowly vibrat- ing wings, treading the air, as it were, over some favourite spot, until a finny back shows itself near the surface, when, giving itself to gravitation, the bird drops like a plummet. It seizes its prey in its talons, while our com- mon kingfisher, after watching patiently from some branch overhanging the water, uses its bill to capture the fish. Terns dive for their fish, gulls usually snatch them from the surface, and skuas and jaegers get theirs at second hand, stealing fish from the more skilful fishers of the sea. When schools of mullet leap in frantic fear from the water to escape the attacks of porpoises, or when the dolphins force the flying-fish above the surface, the merciless Frigate-bird has but to pick and choose. Certain cormorants are the analogues of man's gill-nets, a flock of these birds surrounding a school of fish in a half-circle and driving them ashore or into shallow water. The Bird i56 Herons are the 'still-fishers' of the bird world, and stand in the shallows, silent and motionless as the reeds around them, with their lance-like beaks in rest and their necks IG. 120.— Great Blue Heron, a still hunter. (Sanborn, photographer. Cou N. Y. Zoological Society.) FIG. 120.— Great Blue Heron, a still hunter. (Sanborn, photographer. Courtesy N. The Bird Y. Zoological Society.) at a hair-trigger poise. So we see that few kinds of fish, from the lowly lamprey to the jewelled brook trout, escape the sharp eyes of birds, and even when decayed at a hair-trigger poise. So we see that few kinds of fish, from the lowly lamprey to the jewelled brook trout, escape the sharp eyes of birds, and even when decayed The Food of Bi 157 masses of fish are thrown ashore, feathered scavengers are always alert. masses of fish are thrown ashore, feathered scavengers are always alert. Frogs always suggest storks to our minds, the rela- tion being of course solely a gastronomic one, and indeed most of the near relatives of the frog pay their tithe to birds in a similar way. V. r FIG. 121. — Wild Mouse, the most frequent victim of birds. FIG. 121. — Wild Mouse, the most frequent victim of birds. Turtles, lizards, and snakes enter largely into the food of certain birds, some of which, such as the Secretary- bird and our native Road-runner, are adepts in the cap- ture and killing of members of the latter division of reptiles. Certain sea-eagles subsist chiefly upon sea- snakes. 1 The Bird The most unpleasant items in the bill of fare of the bird kingdom are birds themselves, although few, if any, hawks or owls feed exclusively on members of their own Class. The most systematic cannibal among birds is the Peregrine Falcon or Duck Hawk, and, where birds are abundant, this fastidious gourmand merely eats the flesh of the head and neck and the eyes of each victim, leaving the remainder of the body untouched. Occa- sionally, as among other animals, a bird of strictly vege- tarian habits will attack another bird, even one of its own kind, and kill and eat it in the most matter-of-fact way. Owls are the terror of many birds, from the tiny Elf- Owl which sometimes finds a sparrow too great a match for him, to the great Strenuous Owl of Australia, which snatches full-grown Lyre-birds from their perches. But these birds of the night are ever ready to vary their diet; as we read of certain owls in India feeding chiefly on fish and crabs which they snatch from the water. In that same country, too, bats form a large part of the Barn Owl's diet. Barn Owl's diet. The Bird Every class of living beings appears, at certain phases of its existence, to check or come into intimate contact with other unrelated groups, radically affect- ing the most isolated, in ways too subtle for our observation. A little green flycatcher snatch- ing a tiny gnat from its hiding- place beneath a leaf seems a trivial incident, and yet ^— FI the effects of accumulated ^^^ events no more important than this a world, so delicate is the balance of Natu surprising in its numbers and extent. Every class of living beings appears, at certain phases of its existence, to check or come into intimate contact with other unrelated groups, radically affect- ing the most isolated, in ways too subtle for our observation. A little green flycatcher snatch- ing a tiny gnat from its hiding- place beneath a leaf seems a trivial incident, and yet ^— FIG 122._Red.tailed Hawk the effects of accumulated ^^^ (thunteTatcher) an aetive events no more important than this are felt around the world, so delicate is the balance of Nature. surprising in its numbers and extent. Every class of living beings appears, at certain phases of its existence, to check or come into intimate contact with other unrelated groups, radically affect- ing the most isolated, in ways too subtle for our observation. A little green flycatcher snatch- ing a tiny gnat from its hiding- place beneath a leaf seems a trivial incident, and yet ^— FIG 122._Red.tailed Hawk the effects of accumulated ^^^ (thunteTatcher) an aetive events no more important than this are felt around the world, so delicate is the balance of Nature. FIG 122._Red.tailed Hawk (thunteTatcher) an aetive his are felt around the f Nature. FIG 122._Red.tailed Hawk (thunteTatcher) an aetive are felt around the The Bird The eggs of birds are delicacies which many feathered robbers, such as jays and crows, can never resist. There are two birds, however, one a raven and one a hawk, which well deserve the eggs which they steal, — so inge- nious is their method of obtaining them. In South Africa, on an ostrich-farm, when a female bird has left her nest for a few minutes, a black form will often appear and hasten toward the great white eggs. Hovering over them the raven will let fall a stone into their midst, The Food of Bi 159 instantly swooping down and regaling himself on the yolk pouring out through the crack in the shell. His beak being too weak to break the shell, he has learned to adopt this effective method. A similar remarkable habit is related of the Black-breasted Buzzard of Aus- tralia, but in this case it is an emeu which is the victim. After breaking a hole in the thick shell, this bird inserts its foot and carries the egg to its nest. Perhaps every Order of the higher warm-blooded animals may be included in our list, from the sloth which mutely resigns itself to the terrible grip of a Harpy Eagle to the human child which is powerless before the attack of some bird of prey frenzied with hunger. In certain districts eagles and hawks have been shot smelling strongly of skunk, but whether that fearless animal really figured in their diet remains to be proved ! If any entire group of mammals is to be excepted from the birds' bill of fare, it is only that of the whales, although indeed, when one of these leviathans dies from any cause, his blubber and oil furnish food for sea-birds of many kinds. The small gnawers of wood, the rodents, suffer most heavily, and untold thousands are devoured by hawks and owls, while cranes, shrikes, and ducks make away with their share. This brief and very imperfect review of the vast variety of substances eaten by birds is at least instruc- tive in revealing vividly the complex interrelations of all organic life on the earth. A counter-list of animate creatures which cause the death of birds would be as The Bird i6o surprising in its numbers and extent. Oddities of Birds' Diet To give any adequate idea of the vagaries of the diet of birds would require a volume by itself, but certain changes in feeding habits, due to some increased pressure in the struggle for existence, are too interesting to be To give any adequate idea of the vagaries of the diet of birds would require a volume by itself, but certain changes in feeding habits, due to some increased pressure in the struggle for existence, are too interesting to be passed by unnoticed. They show us how plastic and adaptive birds as a whole are, — how, often, instead FIG. 123.-Red Squirrel (the watched), of giving Up and becoming food of hawks and owls. . . (R. H. Beebe, photographer.) extinct, a Certain race Will FIG. 123.-Red Squirrel (the watched), food of hawks and owls. (R. H. Beebe, photographer.) FIG. 123.-Red Squirrel (the watched), food of hawks and owls. (R. H. Beebe, photographer.) The Food of Birds 161 instantly accept changed conditions and flourish under the new regime. the new regime. As the range of diet of the whole Class of birds is so vast, doubtless the food of the individual species varies more than we should ever suppose, but many instances are recorded of birds regularly feeding on food for whose capture they seem very ill adapt- ed. Insects form the staple food of all flycatchers and tyrant- birds, but the Sulphur Tyrant and several others readily devour snakes. They dash down at one of these reptiles, catch it up in their beak, and, flying back to a branch or stone, hammer the snake flail-like, until its life is battered out. Certain small king- fishers living in New Zealand have deserted the habits of their group, and subsist on the remarkable diet of " flies, young birds, and cherries"! FIG. 124.— Texas Kingfisher fishing for insects. As the range of diet of the whole Class of birds is so vast, doubtless the food of the individual species varies more than we should ever suppose, but many instances are recorded of birds regularly feeding on food for whose capture they seem very ill adapt- ed. Insects form the staple food of all flycatchers and tyrant- birds, but the Sulphur Tyrant and several others readily devour snakes. They dash down at one FIG. 124.— Texas Kingfisher fishing for insects. FIG. 124.— Texas Kingfisher fishing for insects. Oddities of Birds' Diet remarkable diet of " flies, young birds, and cherries"! The change in habits of the Kea Parrot is only too well known, especially to the sheep-raisers in New Zea- land, the home of these birds. Originally exclusive fruit-eaters, they have lately become so fond of the fat from the backs of living sheep that they have developed into ravenous birds of prey, vivisecting their victims and rejecting all but the choicest morsels. Gulls have long been known to enjoy an insect diet, and on the pampas in the vicinity of Buenos Ayres the people look and pray for flocks of gulls as the only relief from the hordes of The Bird 1 6 grasshoppers which occasionally devastate that region. In the antipodes we find a gull with crepuscular habits, whose entire food consists of night-flying moths. whose entire food consists of night-flying moths. Birds in captivity may sometimes be induced to eat food which they would never touch when in a state of freedom, but there are three species of birds the variety of whose natural diet will challenge that of any living creature. The first is a Burrowing Owl. This bird will not disdain vegetable food, and in its underground dining- chambers have been found remains of ducklings, spar- rows, mice, and many other small birds and rodents ; snakes and frogs, besides spiders, beetles, and apparently all small forms of life which these little birds are able to catch and kill. But leaving even the Burrowing Owl far behind in this respect is the Chimango Carrion-hawk of southern South America. Hudson tells us that noth- ing comes amiss to these birds. The vulture habit is per- haps strongest, and all offal and decaying meat is pounced upon with eagerness. All wounded and sickly creatures are closely watched until they die, or, if the opportunity offers, are despatched at once. When a large extent of grass is burned, bountiful repasts are ready for these birds in the shape of roasted snakes and small mammals. Eggs and young birds are especial dainties for the Chi- mango, and young sheep are often attacked, bringing to mind the Kea Parrot. The bird is, at times, a vegetable- feeder, and in fact it would be hard to find any organic object near its home, the edibility of which it has not tested. whose entire food consists of night-flying moths. Oddities of Birds' Diet Birds in captivity may sometimes be induced to eat food which they would never touch when in a state of freedom, but there are three species of birds the variety of whose natural diet will challenge that of any living creature. The first is a Burrowing Owl. This bird will not disdain vegetable food, and in its underground dining- chambers have been found remains of ducklings, spar- rows, mice, and many other small birds and rodents ; snakes and frogs, besides spiders, beetles, and apparently all small forms of life which these little birds are able to catch and kill. But leaving even the Burrowing Owl far behind in this respect is the Chimango Carrion-hawk of southern South America. Hudson tells us that noth- ing comes amiss to these birds. The vulture habit is per- haps strongest, and all offal and decaying meat is pounced upon with eagerness. All wounded and sickly creatures are closely watched until they die, or, if the opportunity offers, are despatched at once. When a large extent of grass is burned, bountiful repasts are ready for these birds in the shape of roasted snakes and small mammals. Eggs and young birds are especial dainties for the Chi- mango, and young sheep are often attacked, bringing to mind the Kea Parrot. The bird is, at times, a vegetable- feeder, and in fact it would be hard to find any organic object near its home, the edibility of which it has not tested. The Red-winged Starlings of South Africa during The Food of Birds the greater part of the year feed upon larvae and insects, but grapes, figs, and other soft fruits are eagerly devoured. They catch locusts and flying ants and occasionally devour the young of small birds. When their travels take them near the seashore they search the seaweed for snails and shrimps, and one of the greatest delicacies is the FIG. 125.— Moth and Hummingbird. Both half natural size. FIG. 125.— Moth and Hummingbird. Both half natural size. fruit of the syringa-tree, "on which they sometimes gorge themselves until they are no longer capable of flight, . . . affected by some narcotic property of the berry itself." affected by some narcotic property of the berry itself." This state of semi-intoxication is by no means rare among fruit-eating birds, when over-ripe or fermented fruit is abundant. Oddities of Birds' Diet 1 The Bird The great extent to which all the external organs and parts of birds are adapted to facilitate the obtain- ing of food is evident in every species; but in humming- birds this adaptation is especially apparent, because we can compare these feathered mites with other creatures far beneath them structurally, but with feeding habits and general environment so similar that such a com- parison is fraught with interest. These other creatures to which I refer are hummingbird moths. Again and again collectors have shot the moths, mistaking them for hummingbirds, as the manner of flight is the same in both, and the way in which each species poises before a flower, probing it with proboscis or bill, is identical. Of the way this wonderful resemblance is carried out even in details of the body Bates writes: "It is certainly very curious, and strikes me even when both are in the hand. Holding them sideways, the shape of the head and position of the eyes in the moth are seen to be nearly the same as in the bird, the extended proboscis represent- ing the long beak. At the tip of the moth's body there is a brush of long hair-scales resembling feathers, which being expanded look very much like a bird's tail." being expanded look very much like a bird's tail." It seems very improbable that this resemblance can be attributed to mimicry, as neither has many danger- ous enemies, their marvellous powers of flight being an all-sufficient protection. So we are left to conclude that it is solely to similarity in method of seeking their food that the likeness is due. THE BREATH OF A BIRD HINK of a mite of a hummingbird shooting southward mile after mile; his singing wings beginning their throbbing in the cool damp air of an Alaskan fall, whirring through the dry heat of des- erts and around the wind-eddied spurs of mountain-ranges, until they hum in the warm atmosphere of Mexico or Brazil, where tiny insects are never lacking throughout the winter! How exquisite an adjustment must exist in his organs; how mankind's engines of locomotion are put to shame! The only comparison of which we can think is with an insect, —a sphinx-moth or a beetle, whose wings of gauze lift and carry their owners so easily, so steadily. It will be interesting to keep this similarity in mind, superficial though it is. H Birds require, comparatively, a vastly greater strength and "wind" in traversing such a thin, unsupporting medium as air than animals need for terrestrial locomo- tion. Even more wonderful than mere flight is the per- formance of a bird when it springs from the ground, and goes circling upward higher and higher on rapidly beating wings, all the while pouring forth a continuous series of musical notes, the strength of the utterance of which 165 whi The Bird i66 is attested by their distinctness in our ears after the bird has passed beyond the range of vision. A human singer is compelled to put forth all his energy in his vocal ef- forts, and if, while singing, he should start on a run even on level ground, he would become exhausted at once. The apparatus which gives to a duck the "wind" to out- strip an express train, and to a Mockingbird notes which hold us spellbound as by a motif of grand opera, is most interesting, and as easy to understand in its general scheme as it is effective in operation. The Trachea, or Windpipe Look into the beak of a sparrow or back of the tongue, on the floor of the mouth, a narrow slit is visible —the glottis, or opening of the windpipe. In the gaping yellow mouth of a nestling robin this may be seen to excellent advan- tage, and watched as it widens and narrows with each breath. But give the young bird a mouthful of food, and this air-passage closes instantly and remains so until all danger of an intruding substance is past. No matter how suddenly you may eject a stream of water from a medicine-dropper into the bird's mouth, reflex action wi anticipate the danger of choking an Look into the beak of a sparrow or pigeon and directly back of the tongue, on the floor of the mouth, a narrow slit is visible —the glottis, or opening of the windpipe. In the gaping yellow mouth of a nestling robin this may be seen to excellent advan- tage, and watched as it widens and narrows with each breath. But give the young bird a mouthful of food, and this air-passage closes instantly and remains so until all danger of an intruding substance is past. No matter how suddenly you may eject a stream of water from a medicine-dropper into the FIG. 126— Open glottis of bird's mouth, reflex action will a Pelican, anticipate the danger of choking and close the aperture. FIG. 126— Open glottis of ll a Pelican, FIG. 126— Open glottis of ll a Pelican, The Breath of a Bird The swollen rim of this opening suffices to close it, and there is no elaborate trap-door arrangement as in mam- mals, only a few backwardly directed fleshy points. Birds have no trace of an "Adam's apple." The vocal chords and other adjuncts to the voice of mammals are entirely absent in birds, not a single note or song being produced in the upper throat. ,, FIG. 127. — Windpipe and oesophagus of bird compared; the former always dis- tended; the latter soft and collapsed. Passing down the neck from this orifice is the wind- pipe, which follows the course of the oesophagus, or food canal, passes to one side of the crop and be- tween the two branches of the wish-bone, and finally divides into two equal parts called bronchi, which carry the air directly to the lungs. ,, FIG. 127. The Trachea, or Windpipe — Windpipe and oesophagus of bird compared; the former always dis- tended; the latter soft and collapsed. FIG. 127. — Windpipe and oesophagus of Comparison Of the tWO bird compared; the former always dis- tended; the latter soft and collapsed. tubes which traverse the throat and neck of birds shows them to be very different in appearance and structure, and consideration of their respective functions gives us the key to this dissimilarity. The only occasion for the oesophagus to open is to permit the passage of food, and thus a limp, fleshy canal answers all requirements. The windpipe, on the contrary, must always be wide open, and not only this, but it must be kept open no matter what the pressure upon it. In addition, it must be flexible, yielding to every motion of the neck, The Bird 1 and elastic, in order to stretch and contract as the bird reaches out or draws back its head. We find a most ingenious arrangement fulfilling all these requirements. A series of bony rings is imbedded in the wall of the trachea, beginning with that portion FIG. 128. FIG. 129. FIG. 128. — Windpipe of Flamingo, extended and contracted, showing delicate mechanism of supporting rings. FIG. 129. — Syrinx-drum of Mallard Drake; the windpipe above; the bronchi below leading to the lungs. . FIG. 129. FIG. 128 FIG. 129. FIG. 1 FIG. 128. — Windpipe of Flamingo, extended and contracted, showing delicate mechanism of supporting rings. FIG. 129. — Syrinx-drum of Mallard Drake; the windpipe above; the bronchi below leading to the lungs. immediately back of the glottis, and extending through- out its entire length. The membrane which connects these rings is so elastic that a section of trachea can be drawn out until it is twice as long as when contracted. When in the latter condition (I have in my hand an inch of the windpipe of a flamingo, but the general structure immediately back of the glottis, and extending through- out its entire length. The membrane which connects these rings is so elastic that a section of trachea can be drawn out until it is twice as long as when contracted. The Trachea, or Windpipe When in the latter condition (I have in my hand an inch of the windpipe of a flamingo, but the general structure The Breath of a Bi 169 is common to all birds) the trachea appears to be com- posed of alternating half-rings, but when elongated these are seen to be complete, the illusion being pro- duced by the overlapping of half of each ring by an equal part of the rings on each side. When the trachea is ex- tended, the only hint of this clever device is a small notch on the sides of every ring. The illustration demonstrates the working better than any description. Nature is ingenious but not perfect, as is seen even in the inch of Flamingo's trachea which I have utilized for illustration and description. Two of the rings do not "jibe" on one of their sides, and overlap the wrong way, but the loss in motion is infinitesimal, the defect being hardly noticeable even when the rings are bent into a semicircle. In a very young English Sparrow there are about fifty rings around the trachea, appearing to be of a more cartilaginous nature than those of the flamingo. This latter long-necked bird has no less than four hundred and fifty rings. In some members of the Class of amphibians (frogs, toads, and newts) the trachea is supported by small ir- regular pieces of cartilage, tending in the higher forms toward ringed areas. Among reptiles an intermediate condition exists, complete rings being present, but of cartilage instead of bone. The wonderful music of birds is produced in a rela- tively small area, known as the syrinx. This organ is situated at the point where the trachea divides into the two bronchi. The latter arise as if by a splitting of the The Bird 170 windpipe, and the effect is heightened by the rings which extend as far as the lungs, which are half-rings or semi- circles, the inner halves being replaced by membrane. This organ is peculiarly characteristic of birds, there being not a trace of it in any reptile. being not a trace of it in any reptile. The Bird The Bird 7 The Trachea, or Windpipe But though the syrinx alone is concerned in the pro- duction of sound, this may be modified, made resonant, or given a reverberating quality by a special structure or by windings of the trachea before it reaches the syrinx, and which are perfect analogies of human musical instru- ments. Many species of ducks have an enlarged box of bone, a kind of drum, on the lower portion of the trachea, sometimes of one shape, sometimes of another, serving, doubtless, to give power to the bird's voice. Cranes and swans have veritable French horns in their breast-bones. The windpipe enters between the arms of the clavicles or wish-bone, and describes an S or even a more intricate figure before passing out and dividing into the two bronchial tubes. When a Trumpeter Swan stretches out its neck and utters a musical clang, most maligned by comparing it to a whoop, we should remember the cause of its mellow- ness. In the majestic Whooping Crane of our Western States, which in a few years will have vanished from the earth, the windings of the trachea reach their maximum. The entire windpipe of this bird is four feet in length, and of this, one-half is coiled within the sternum, or breast- bone, giving remarkable volume and resonancy to the voice. The Breath of a Bird 171 FIG. 130.— Breast-bone of Sandhill Crane. FIG. 130.— Breast-bone of Sandhill Crane. FIG. 131. — Breast-bone of Whooping Crane, showing convolutions of trachea within the keel. FIG. 131. — Breast-bone of Whooping Crane, showing convolutions of trachea within the keel. The Bird The Syrinx This organ is peculiar to birds and, as stated before, is alone concerned in the production of the voice, although the tongue in parrots may be of some aid in distinctness of articulation. But this is not true of any other Order of birds, and the operation of splitting the tongue of a magpie or crow to "make it talk" is as unnecessary as it is inhumanly cruel. The syrinx is singularly uniform among birds, and this seems the more remarkable when we consider the great variety of vocal sounds which are produced. The position and the structure of this organ vary within nar- row limits, but in general it is composed of several modi- fied rings of the lower trachea or upper bronchial tubes. The membranes which cover the inner half of each bron- chial tube unite at their juncture with the windpipe and extend some little way into it as a thin median fold of tissue, supported by a bony framework. The tense- ness or looseness of this membrane is governed by special muscles, of which there are from one to seven pairs. It is by the action of these muscles that the varying tones of croak, scream, warble, or trill are produced, the air from the lungs rushing out through the bronchial tubes and past the varying aperture controlled by the syringeal membrane. We may dissect out every muscle and study trachea, syrinx, and bronchi with all the apparatus and instru- ments afforded by modern science, and yet the mystery The Breath of a Bi 173 of song is not solved. The marvel of the Canyon Wren's melody becomes but the more wonderful; the voice of the Seriema, carrying over a mile, and the never-to-be- forgotten evening song of the Solitaire only impress us with the failure of the scalpel and microscope to explain more than superficially the varied expressions of life. Lungs and Air-sacs At the beginning of this chapter a bird was compared to an insect, and the reason will now be apparent. The body of an insect is aerated by means of an intricate sys- tem of tubes ramifying throughout the body, which in many instances are connected with air-sacs. The com- parison with a bird is not to its lungs, which are small and compact, but to a series of nine air-sacs, distributed through much of the body, — four pairs, and two which have coalesced into one. When a bird is dissected, the thin membranous walls of these air-cavities are collapsed and rather difficult to make out, being very similar in appearance to other connective tissues of the body. But if we insert a small blowpipe into the trachea of a dead bird, tie it tightly about with a piece of string and blow into it, all the air- sacs will become distended and bladder-like and can easily be made out. It is remarkable how closely these sacs fit around the viscera and muscles, occupying every crevice and filling the whole body of the bird with air, thus reducing its specific gravity, and making it a crea- ture literally "of the air." There is sometimes a layer of The Bird 1 air between the muscles and the skin, and when we handle a bird thus aerated the skin crackles under our touch. The lungs and air-sacs send off tiny membranous tubes which enter the bones of the limbs and skull and sometimes even the small bones of the wings and toes, which are hollow and thus filled with air. It seems in- credible, but nevertheless it is true that the connection between the lungs and the upper arm-bone of a bird is so substantial that a bird which has had its wing broken with shot is able to breathe through the splintered end of this hollow bone when its windpipe is completely choked with blood. We may compare the body of a bird to a submarine boat with many water-tight compartments, and as such a vessel is made buoyant by admitting air to these bulk- heads, so a swimming bird may float high out of water by inflating its sacs and filling its bone-cavities with air. Conversely, when we see a grebe slowly and mysteriously submerge its body, we conclude that it has but emptied its lung auxiliaries. Lungs and Air-sacs We now come to the most important part of the re- spiratory system, where the blood and the air come into closest contact and exchange gases, the oxygen of the air vitalizing the entire body. If we follow the two bronchial tubes after they leave the syrinx, we shall find that each enters a lung, and passes through it, giving off a number of side branches which open into the vari- ous air-sacs. The lungs are not elastic and, instead of lying freely in .the body, are flattened against the back- The Breath of a Bird bone and ribs, and when carefully removed show fur- rows made by these latter bones. There is still much to be learned of the manner of a bird's breathing, but it is probable that there is a sidewise or dorsal expan- sion of the ribs, rather than of that portion corresponding to our chest. In a bird the latter region is chiefly an im- mense flight-muscle, which could hardly yield to the action of breathing while carrying on the tremendous work of keeping the wings in motion, and when a FIG. 132. — Cross-section of wing bone of Ostrich and Black Swan. In life the bone of the Ostrich is filled with marrow; that of the Swan with air. FIG. 132. — Cross-section of wing bone of Ostrich and Black Swan. In life the bone of the Ostrich is filled with marrow; that of the Swan with air. bird squats on a branch with its breast pressed close to the perch, " chest expansion" must be all but impossible. We cannot help being surprised at first when we see how small are the lungs of a bird in comparison with the size of its body. The first thought that occurs to us is that the air- sacs in birds, and the hollow cavities of the bones, must function chiefly as aids to flight, and we should expect to find as best flyers those birds in which the air-cavities The Bird 1 7 are most numerous, but there are many exceptions. The bones of storks and vultures (birds of great powers of flight) are extremely pneumatic, while the bones of the flightless ostriches are filled with marrow, and in the aquatic penguins even this is reduced to a thread, the bones being almost wholly osseous tissue. Lungs and Air-sacs A swan, although a heavy bird, flies remarkably well when once on the wing, and is highly aerated, but, on the other hand, terns and swifts — past-masters both in aerial evolutions — have solid bones! Now an athlete who is trained in running has always a very large lung capacity. Two persons of equal health and strength, one of whom has run many races or who has the power of keeping up a dog-trot for hour after hour, while the other has led a more sedentary life, may show a remarkable difference in the amount of air which they can draw into their lungs — perhaps one hundred or one hundred and fifty cubic inches more in the case of the runner. The average person uses only about one sev- enth of his lung capacity in ordinary breathing, the rest of the air remaining at the bottom of the lung, being termed "residual." As this is vitiated by its stay in the lung, it does harm rather than good by its presence. When great exertion is required, as in running, the person who can admit the largest amount of fresh air to his lungs in each breath has command of an equally great power of action. As we have seen, the lungs of a bird are small and non-elastic, but this is more than compensated by the continuous passage of fresh air, passing not only into The Breath of a Bi 177 but entirely through the lungs into the air-sacs, giving, therefore, the very best chance for oxygenation to take place in every portion of the lungs. When we compare the estimated number of breaths which birds and men take in a minute — thirteen to sixteen in the latter, twenty to sixty in birds — we realize better how birds can per- form such wonderful feats of song and flight. Birds, having no sweat-glands in the skin, and the action of the capillaries being impeded by the feathers, would have no way of regulating the temperature of the body, much as this is necessary in flight, if it were not that the great quantity of air exhaled with each breath relieves the body of any excess of heat. Lungs and Air-sacs However directly or indirectly the air-sacs are con- cerned with flight, a bird which sings uninterruptedly as it flies upward must be immeasurably aided by the great quantity of air at its command. And again, when a Prairie Hen inflates the orange-hued air-sacs on both sides of its neck, there is only one explanation as to their use, at least at the time of courtship, namely, an added decoration, and as an aid in the "booming" — factors both of which, for aught we know, may help to soften the hearts of the coquettish females. Looking down the scale of life we find an animal among the reptiles with a lung which at once suggests that of a bird. The lungs of a chameleon are spongy and com- pact in front, but farther back they are hollow, and give off a dozen or more finger-like tubes or lobes, thus foreshadowing, at least in appearance, the air-sacs of birds. i78 The The Bird i78 i78 The Bird We have learned that the chick through a stage when it possess veloped gills. This proves that in t the ancestors o aquatic and fish- lungs? Fishes ha in their aquatic l be useless. Neve see, the lungs of mammals are leg tures of the sea. We have learned that the chick in the egg passes through a stage when it possesses several well-de- veloped gills. This proves that in the dim, distant past the ancestors of birds were once aquatic and fish-like. But how about lungs? Fishes have none, and indeed in their aquatic life such organs would be useless. Nevertheless, as we shall see, the lungs of reptiles, birds, and mammals are legacies from the crea- tures of the sea. Many fishes have within their bodies a thin-walled sac, known as the swim-bladder. This is filled with gas, and as the fish ascends to the surface, or dives to where the pres- sure of the water is very great, the amount of gas varies; so that the specific gravity of the fish changes with that of the water. This swim- bladder is generally connected with the throat by a delicate tube; and FIG. 133. — Lung of Chame- , , i_ xi Icon, foreshadowing con- in these tWO Structures W6 have the dition in bird. , , /• ,LI i • i » i i homologues 01 the birds lungs and trachea. Proof of this is to be found in the growth of the lungs in all young chicks. A tiny bud appears upon the primitive oesophagus, just behind the little gill-clefts, and We have learned that the chick in the egg passes through a stage when it possesses several well-de- veloped gills. This proves that in the dim, distant past the ancestors of birds were once aquatic and fish-like. But how about lungs? Fishes have none, and indeed in their aquatic life such organs would be useless. Nevertheless, as we shall see, the lungs of reptiles, birds, and mammals are legacies from the crea- tures of the sea. FIG. 133. — Lung of Chame Icon, foreshadowing con- dition in bird. Many fishes have within their bodies a thin-walled sac, known as the swim-bladder. The Bird This is filled with gas, and as the fish ascends to the surface, or dives to where the pres- sure of the water is very great, the amount of gas varies; so that the specific gravity of the fish changes with that of the water. This swim- bladder is generally connected with the throat by a delicate tube; and e- , , i_ xi in these tWO Structures W6 have the , , /• ,LI i • i » i i homologues 01 the birds lungs and his is to be found in the growth of the chicks. A tiny bud appears upon the , just behind the little gill-clefts, and l it is larger than the food-canal itself. es into two equal parts which become r canals — the beginnings of the lungs. Many fishes have within their bodies a thin-walled sac, known as the swim-bladder. This is filled with gas, and as the fish ascends to the surface, or dives to where the pres- sure of the water is very great, the amount of gas varies; so that the specific gravity of the fish changes with that of the water. This swim- bladder is generally connected with the throat by a delicate tube; and FIG. 133. — Lung of Chame- , , i_ xi Icon, foreshadowing con- in these tWO Structures W6 have the dition in bird. , , /• ,LI i • i » i i homologues 01 the birds lungs and trachea. Proof of this is to be found in the growth of the lungs in all young chicks. A tiny bud appears upon the primitive oesophagus, just behind the little gill-clefts, and increases in size until it is larger than the food-canal itself. It then in turn divides into two equal parts which become diminutive flaps, or canals — the beginnings of the lungs. FIG. 133. — Lung of Chame Icon, foreshadowing con- dition in bird. FIG. 133. — Lung of Chame Icon, foreshadowing con- dition in bird. The Breath of a Bi The Breath of a Bi 179 A simple experiment will show what fishes have a canal, or duct, leading from the throat to the swim-blad- der and what have not. If a goldfish and a perch or sunfish be placed in a bowl of water and the air exhausted, the two latter will be forced to the surface, while the gold- fish will soon eject a few bubbles of air, or gas, from its mouth and stay at the bottom. Thus we can see the ad- FIG. 134. — Diagram of growth of lungs. X, the lower part of the primitive diges- tive tract, divides into two parts, XX, the lungs. 4. — Diagram of growth of lungs. X, the lower part of the primitive diges- tive tract, divides into two parts, XX, the lungs. vantage of such a canal in enabling the fish to regulate the amount of gas in the bladder. When the fish-like creatures of old took to living on land, the change from swim-bladder and gas to lung and air was a remarkable example of change of function of an organ, and the more we learn of the lungs of living creatures the more marvellous does this transformation The Bird i seem to us. In changing, Nature seems to have tried numberless experiments, only a few of which have sur- vived. For example, we know that fish breathe by a sort of swallowing, the water being taken in at the mouth and poured out through the gill-clefts. So in frogs and salamanders we find that, although they possess lungs, yet they still employ a swallowing process to get the air down their throats. This is the reason why a frog will suffocate if its mouth is held open. There are certain salamanders which are wholly without lungs, their moist skin being so vascular that the blood is purified through it. But strange to say, these amphibians still swallow and swallow, as did their ancestors, although no air passes down their throats, and indeed there is no place to which it could go! As we have seen elsewhere, birds exhale air largely by the action of certain abdominal muscles. The Breath of a Bi Watch a goldfish rise to the top of the water and eject or gulp down a bubble of air, and observe the rapid breath- ing of a bird, and you have the two extremes before you —the swim-bladder of ages ago and the wonderful lungs of a bird of to-day. The Heart and the Life-blood Perhaps the most wonderful organ in a bird's body is its heart. In the very lowest of back-boned animals the heart is merely a long tube, in fact a simple artery or vein, which contracts at certain intervals and so pro- pels in a forward direction the fluid which it contains. A fish may almost be said to have its heart in its head, so far forward in its body is it placed; nevertheless, as The Breath of a Bi 181 is the case in all the warm-blooded creatures above it, the heart is nearer the under side of the body — the breast — than near the back. And herein lies an important difference between the two great divisions of the Ani- mal Kingdom, vertebrates and invertebrates, — the former always having the heart near the breast, while in the back- boneless organisms it is near the back. boneless organisms it is near the back. The heart of a fish is fairly concentrated and muscu- lar, but the blood which passes through it is but an im- pure and sluggish stream. In reptiles both pure and impure blood is found in the heart, but they mingle, and thus half destroy the purifying action of the lungs. This explains why these animals are cold-blooded, and also accounts for their usual lethargic disposition and low mental plane of life. In crocodiles we find a significant condition. There are four chambers in the heart, as in mammals and in birds, but this avails nothing; for, leading from the heart are two arteries instead of one, and where these cross each other there is a tiny aperture — a small opening in the partition which allows the impure blood to leak into the stream of pure, red blood, and so a crocodile is only a crocodile, although evolution has lifted his heart al- most to a level with birds and the warm-blooded ani- mals. If this tiny hole could become closed, and the two streams of blood be kept separate, the eyes of the crocodile would brighten, his activity increase many fold, and in fact his entire plane of life would be changed. I have thus briefly reviewed the heart in the lower vertebrates in order to give a more vivid idea of this organ The Bird 1 8 in birds. The Heart and the Life-blood Here we find an organ remarkably large in proportion to the size of the bird's body — a conical knot of muscle, the power of which is almost beyond belief. The heart of a bird is said to beat a " hundred and twenty times a minute when the bird is at rest. The first flap of the wings doubles the pulsations, and when the bird is frightened or exhausted the number of beats are too many to be counted." many to be counted." There are four separate chambers, known as right and left ventricles and auricles, and the partition which di- vides the heart in the middle is blood-tight so that not a particle of "bad" blood can get through and vitiate the life-giving stream which has just come from the lungs. life-giving stream which has just come from the lungs. A Bluebird is perched on a twig near its nest mur- muring its sweet warble; a Wood Pewee, half hidden in the shadows of some dense, moist forest, speaks to us in its sad dreamy phrase; how calmly, how quietly they sit! It seems impossible to believe that every drop of blood in their bodies is rushing back and forth with inconceivable rapidity — from heart to head, from body to wings and legs, and back again ! Let us take the blood as it is just leaving the heart in the breast in one of these little feathered beings, and trace its course through the body and back again to the starting-point. The left ventricle opens into the aorta, the greatest artery, or blood-tube leading from the heart, in the body. The clean oxygen-food-bearing stream rushes through this channel, which we may com- pare to the trunk of a tree, and is carried into branch arteries, dividing finer and finer, just as the trunk of FIG. 135. — Circulatory system of Pigeon (injected), showing blood-vessels rami- fying from the heart to every part of the body. 183 FIG. 135. — Circulatory system of Pigeon (injected), showing blood-vessels rami- fying from the heart to every part of the body. The Bird 1 8 the tree merges into limbs, and these into branches, twigs, stems, and at last into the delicate foliage. The Heart and the Life-blood This last we may liken to the capillaries or hair-tubes in which the blood does its real work of supplying nourishment directly to the tissues, and where it receives the waste matters, carrying them away in its current. When we have followed the divisions of a tree out to the foliage, we may find that they touch and interlace with the foliage of another tree, and this is very much like what occurs in the course of the blood. The capil- laries run together and form larger vessels, these in turn coalesce, and soon the blood — dark now and filled with the waste matters of the body-cells — is flowing through only two large veins (veins always lead toward the heart). These enter the right auricle, which opens into the right ventricle. From here the blood rushes to the lungs to be purified and back again to the left auricle and ventricle, and its cycle is complete. If we look at a drop of bird's blood (or that of any kind of warm-blooded creature) under the microscope, we shall see thousands upon thousands of oval discs, or corpuscles, like tiny platters floating in a fluid. These flow about under the cover-glass through little channels, mechanic- ally and very slowly of course, and giving but a faint idea of the way they must tumble and rush after each other through the veins and arteries of the bird. Scat- tered among these oval bodies will occasionally be seen others of indefinite shape and white in colour. As we watch one of these tiny cells, the thought suddenly comes over us, — what are birds indeed but collections of untold The Breath of a Bird millions of one-celled animals! For here before us we have what is almost exactly like the little flowing drops of jelly called Amcebse which we may find in quiet ponds and watch as they move about in search of food ; flowing around a bit of nutriment, digesting it and flowing away from the waste matter which is left. This is just what the FIG. 136. — Blood-corpuscles of bird. FIG. 136. — Blood-corpuscles of bird. white corpuscles do; they flow around the food which is absorbed by the walls of the digestive canal, and in fact act like tiny independent animals, parts though they are of the great whole. The Heart and the Life-blood The oval corpuscles carry and dis- tribute the oxygen, and here we have in a sentence the inner 'living' of a bird: the food-canal bringing in food and preparing it; the windpipe and lungs admitting white corpuscles do; they flow around the food which is absorbed by the walls of the digestive canal, and in fact act like tiny independent animals, parts though they are of the great whole. The oval corpuscles carry and dis- tribute the oxygen, and here we have in a sentence the inner 'living' of a bird: the food-canal bringing in food and preparing it; the windpipe and lungs admitting The Bird i86 oxygen; and the blood taking up and transporting both to every part of the body. The normal temperature of our body is about 98£°; if it rose to 106°, we should soon succumb to the burning fever, while the little bird before us is healthy and com- fortable with a temperature of 110° to 112°! FIG 137. — Amoeba, greatly magnified. (Courtesy of Dr. G. L. Calkins.) FIG 137. — Amoeba, greatly magnified. (Courtesy of Dr. G. L. Calkins.) The next time you see a wee chickadee, calling con- tentedly and happily while the air makes you shiver from head to foot, think of the hard-shelled frozen in- sects passing down his throat, the icy air entering lungs and air-sacs, and ponder a moment on the wondrous little laboratory concealed in his mite of a body; which his wings bear up with so little effort, which his tiny legs sup- The Breath of a Bi 187 port, now hopping along a branch, now suspended from some wormy twig. Can we do aught but silently marvel at this alchemy? A little bundle of muscle and blood, which in this freez- ing weather can transmute frozen beetles and zero air into a happy, cheery little Black-capped Chickadee, as he names himself, whose bravery shames us, whose trust- fulness warms our hearts ! And the next time you raise your gun to needlessly take a feathered life, think of the marvellous little en- gine which your lead will stifle forever; lower your weap- on and look into the clear bright eyes of the bird whose body equals yours in physical perfection, and whose tiny brain can generate a sympathy, a love for its mate, which in sincerity and unselfishness suffers little when compared with human affection. The Heart and the Life-blood FIG. 138. — Chickadee in the snow. FIG. 138. — Chickadee in the snow. I IRDS exhibit probably a greater degree of activity than any other class of animals. Some seem never to be still, and, whether soaring, fluttering, running, hopping, climbing, dancing, or swimming, every motion is the result of the action of one or more muscles. I The entire flesh of a bird is divided up into layers or bundles of distinct muscles, each having its function, — raising, lowering, or in some way moving feathers, eye- lids, legs, wings, tail, and other portions of the body. The number and intricacy of these muscles can be imagined when it is stated that in a goose there are more than twelve thousand muscles or parts of muscles immediately be- neath the skin, which serve to raise or otherwise move the feathers. In a penguin the muscles immediately beneath the skin are unusually well developed, and for an excellent reason. By means of them the water "may be readily expelled from the interstices of the plumage so soon as the bird quits the water. Were it otherwise, in the low temperature of the Antarctic region, which the majority of these birds inhabit, their plumage would soon be frozen 1 88 e fro 1 88 Muscles and Ner 189 into an icy mass, the high temperature of the bird being of itself insufficient to obviate this, although assisted by the great development of the subcutaneous fatty layer, which far exceeds in thickness that of the corre- sponding structure in the member of any other group of birds, and recalls to mind the fatty deposit of 'blub- ber7 of the seals and cetaceans." ber7 of the seals and cetaceans." When we looked at the blood of a bird, we saw the tiny white corpuscles, which in life flow and move in every direction, constantly changing their outline; and now if we take a piece of a bird's muscle or flesh and examine it carefully, after "teasing" it out into shreds with a needle, we shall see another kind of cell-animal. These are long and generally pointed, each a single cell with a tiny spot or nucleus in it, differing from the white- blood animals in being able to stretch out and contract in only one direction. When we will our arm to close together, bringing our hand close to our shoulder, a thick colony or bunch of these muscle-animals shortens, be- comes stouter, and bulges up under the skin on our upper arm. In our own body the bones of the spinal column are movable, and we can bend in almost every direction, and so we are provided with many important back-muscles. But if we have ever carved a chicken, we shall remem- ber that the ribs and shoulder-bones are close to the sur- face, and but poor pickings are to be had from them. The breast and chest, on the contrary, are hidden in a thick mass of muscles, most of which are concerned with moving the wings in flight. The immense pectoral or The Bird I 90 breast muscle, which makes possible the all-important downward sweep of the wings, weighs one-fifth as much as the entire bird, bones and all. This arrangement of a great weight of muscle hung below the point of attach- FIG. 139. — Wing and breast of Pigeon, showing immense pectoral muscles, and tendons of wing used in flight. FIG. 139. — Wing and breast of Pigeon, showing immense pectoral muscles, and tendons of wing used in flight. ment of the wings is, for mechanical reasons, the only one possible in a bird of flight; since any excess of weight above the wings would instantly overbalance the bird. If we remove the skin from the upper arm of a bird, Muscles and Nerv 191 we shall see a tangle of bundles of red flesh — the muscles which unite to make the arm of a bird such an exquisite flying-machine. Where a muscle narrows and is fastened to a bone, its fibres merge into a thin, tough white cord — FIG. 140. — Model of bird's foot, showing perching tendons; toes extended. FIG. 140. — Model of bird's foot, showing perching tendons; toes extended. a tendon. This is not elastic like the main portion of the muscle, but is much more tough. In the slender legs and feet of birds there is little more than bone, tendon, and skin. The tendons which clasp and unclasp the toes are very interesting, and if we will The Bird 1 bend the tarsus back and forth in the leg of a dead chicken, the workings of these strands of tissue may be traced beneath the scales. Reference to the photograph, where catgut replaces these tendons, will make their workings still more plain. FIG. 141. — Same as Fig. 140; toes contracted. FIG. 141. — Same as Fig. 140; toes contracted. Many birds cannot flex the leg without drawing the toes up, and we may be sure that these birds are safe when they perch; the closer they sit to the branch the tighter becomes their grip. But this safety mechanism is not found in all perching birds by any means (Fig. 143). A strange thing about muscles is that there are fine Muscles and Nerves wavy cross-lines, or striations, on those which are moved voluntarily, such as the muscles of the neck or wing; but those which are moved involuntarily, as the gizzard, are smooth and without the cross-lines. Amazon Parrot in sleeping position, hanging by its toes; the wonderful strength of the tendons. ng birds new muscles have appeared, or o FIG. 142, — Amazon Parrot in sleeping position, hanging by its toes; illustrating the wonderful strength of the tendons. FIG. 142, — Amazon Parrot in sleeping position, hanging by its toes; illustrating the wonderful strength of the tendons. Among birds new muscles have appeared, or old ones have split up or so changed in position that it is all but impossible to compare them, muscle for muscle, with other The Bird 1 animals. There are so many resemblances between birds and reptiles that we naturally turn to the latter for com- parison, but even here we find a great unlikeness. We learned, when we reflected on the number of ribs of a bird, that the repetition of so many similar structures was merely the last remaining vestige of ancestral body segments, which reach their extreme development (in number and similarity) among the worms; but in regard to muscles birds show little or nothing of this. In liz- ards we may count dozens upon dozens of bands of muscles succeeding one another, all more or less alike, from head to tail, but it is only in the neck of a bird that we shall find anything like this. In order to give to muscles a firm anchorage, they must of course be attached to the bones. At these points of attachment deep furrows or cavities are often found in the surface of the bones, and in still other ways we are reminded, even in fossil bones, of the flesh and muscle which once moved them. These muscle impres- sions are often a valuable source of identification in naming the bones of creatures which, many thousands of years ago, disappeared from the earth. And indeed so great variety exists in the muscles of living birds that many of them, those of the upper arm for example, are of considerable value in classification. Nerves The last great system of internal organs which we shall consider, and perhaps the most mysterious of all, is that of the nerves. We have learned that the back- The Bird 1 bone supports the entire body and gives a point of at- tachment for the limbs, but long before limbs were found among animals on the earth, in fact long before bone existed, a sheath of cartilage surrounded and supported the primitive spinal cord of creatures which lived long ago in earlier epochs of the earth's history. So we may say this protection to the nerve-trunk is the most im- portant, as it was the original, function of our vertebrae. When "brainy" creatures appeared, that is, when the front end of the nerve-cord became enlarged, it needed some special protection, so a box — the skull, — first of cartilage, then of bone, was evolved. One more fact which may hark back to old, old times, and then we shall leave the past as perhaps trespassing too much on the province of the chick while he is yet within the egg. Birds (and all the higher classes of ani- mals) have what we may call two separate systems of nerves, although in some ways they are insolubly con- nected with each other. The brain and spinal cord send numerous branches which subdivide into countless nerve- lets, permeating every portion of the body, as we can easily prove by the feeling, on pricking our skin anywhere with a needle. This is the principal nervous system of back-boned animals, and it is by this that birds, and all creatures with well-developed nerves, see, hear, taste, smell, and by which they send messages to the muscles when they desire to move them. Below the vertebral column is another lesser system which sends nerves to the digestive tract and other organs, the movements and functions of which are not under control of the will, and . 144. — Nervous system of Pigeon, show leading to wings FIG. 144. — Nervous system of Pigeon, showing large eyes and brain, and nerves leading to wings and legs. The Bird 1 this is the sympathetic or reflex system. It is a very wonderful thing, this not having to think about the heart beating or the lungs expanding. We can understand how a muscle (such as the heart) can pump the blood through the body, but we know little or nothing of the action of nerves. An eagle soars high above the clouds; a rabbit is discovered crouching in a field far below; the eye of the eagle telegraphs this discovery to the brain; a message is sent along the spinal cord, switches off to the wings, repeats to the muscles, which half close and set the great pinions firmly; the eye is the pilot, never leaving the mark; a triple message now goes out, to the wings to hold back, to the legs to reach forward, to the talons to open and clutch! All is done without a break or hesitation, so quickly that one's eye can hardly register the act, and all by means of impulses sent through the finest of white, hair chan- nels, consisting of a substance so unstable that it tears and falls apart, like wet tissue-paper, when we examine it. And if the sending and receiving of impulses seems wonderful to us, what can we say of the brain, the master of all, where instinct, mind, soul, — no matter what we call it, — directs the whole life? It is here that fact upon fact, experience upon experience, is stored from the mo- ment the bird breaks its shell throughout its whole life- time, and it is from the brain that the benefit derived from this perception of experience, failures and successes, causes and effects, is intelligently brought into play and made to redound to the bettering of the subsequent life. Muscles and Ner Muscles and Ner 199 When we carefully remove the upper part of a bird's skull, we find that the brain occupies the whole interior, FIG. 145. — Comparison of skulls of Heron and Hawk, showing unlikeness caused by difference in manner of procuring food. FIG. 145. — Comparison of skulls of Heron and Hawk, showing unlikeness caused by difference in manner of procuring food. the shell or box of bone which protects it being very thin, although strong. It would be very interesting if we could compare the short and thick bullet-shaped skull and brain the shell or box of bone which protects it being very thin, although strong. It would be very interesting if we could compare the short and thick bullet-shaped skull and brain The Bird 2 of a rapacious hawk with the thin-templed head of a timid heron and say, "phrenologically," in the first we have the bump of combativeness well developed, analo- gous to a prize-fighter; in the second case, timidity is prominent! But unfortunately, characteristics such as these are compound, and made up of many simple fac- tors, the synthesis of which is not confined to any par- ticular "bump." ticular "bump." At the first sight of the bird's brain we are struck with the very great size of the two larger masses of brain- matter — cerebral hemispheres these are called. It is in these that the higher faculties reside, and when these are destroyed, all knowledge, all power of voluntary move- ment passes from the bird. These great brain-halves are much larger than in the brain of a reptile, in fact the cerebral hemispheres, set deep in the great buttressed skull of a full-grown crocodile, are no larger than those of the duck which he snaps up. Not only this, but in the days of the Archceopteryx (which had a typical bird- brain), the monster Dinosaur, Triceratops, 25 feet long, had, in its 6 feet of skull, a brain proportionately only one tenth as large as that of a modern crocodile! When compared with a mammal there is seen to be a conspicu- ous difference, since the outer surface is perfectly smooth in birds, but is wound about in convolutions in the higher four-footed animals. Muscles and Ner This latter condition is said to indi- cate a greater degree of intelligence, but when we look at the brain of a young musk-ox or walrus and find convo- lutions as deep as those of a five-year-old child, and when we compare the wonderfully varied life of birds, and Muscles and Nerves 201 realize what resource and intelligence they frequently display in adapting themselves to new untried con- ditions, a smooth brain does not seem such an inferior organ as is often inferred by writers on the subject. I would willingly match a crow against a walrus any day, in a test of intelligent behaviour! Between the hemispheres is a small projection which FIG. 146. — Vertical section through skull of bird, showing great size of brain. FIG. 146. — Vertical section through skull of bird, showing great size of brain. is called the pineal body. It is very tiny, and we know little of its function at present, but its history is one of the most interesting chapters in the evolution of the bird, which we shall leave to the chapter on the senses. The other most conspicuous part of the brain is the cerebellum, or "little brain," a section of which shows a most remarkable tree-like appearance. This has been called the arbor vitce — the tree of life. It is in this portion of the brain that a few tiny drops The Bird 2 of blood are found when a bird dies of fright, which oc- curs more often than in any other class of animals. Sports- men have fired at a bird, missed it completely, and yet have seen it drop dead as suddenly as if it had received the full charge. In captivity, herons succumb more frequently to fright apoplexy than other birds. When we assume the care of any creature, bird or beast, we should treat it as a timid child, and the person who moves quietly but unhesitatingly will win the confidence of wild creatures much sooner than when he alarms them' by sudden motions, or arouses their suspicions by jerky half-hearted approaches. half-hearted approaches. There are twenty-four nerves given off in pairs from the brain, which pass out through minute holes in the skull, and energize eye, ear, tongue, and other organs. THE SENSES E E have seen that the brain is the storehouse of facts and experiences, but whence come these and how do they gain admittance to that soft gray matter which is one of the wonders of the world? There are five channels (and sometimes there seems the shadow of a metaphysical sixth) which are cognizant of and receptive to environmental influences. These are the nostrils, eyes, ears and tongue, and the tactile nerves of the surface of the body; or in other words the bird is in direct connection with his surroundings on land or water or in the air, by means of the senses of smelling, seeing, hearing, tasting, and feeling. Muscles and Ner Each of these has an individual name, and as they are homologous with similar nerves in ourselves, the same name is retained, such as the olfactory, or that leading to the nostril; and the pathetic, the function of which is to control the obliquely raising eye-muscle, producing a pathetic expression, although it must be confessed that the effect of this in the immobile face of a bird is not especially affecting. Back of the cerebellum is a thickening of the spinal cord, and after again narrowing it enters the bones of the neck and back, as the true spinal cord. At the base of the neck and near the thigh- joints this cord increases in size, large nerves being given off at these places to the wings and legs. It terminates in a fine white thread. The Senses In all animals the mucous membrane which lines the nasal cavity is very delicate and filled with nervelets. These nervelets unite and form a single nerve on each side which passes to the brain and transmits the impres- sions derived from the odours in the air. The thin bones within the nostril, which, in dogs and deer, curl and re- curl in delicate lines and scrolls and thus expose such a large surface to the odour-bearing air, are but poorly represented in birds. The simple curve of the bone in the nos- trils of birds is very simi- lar in structure to that found in reptiles. The question whether vultures perceive their prey by sight or smell has been decided in favour of the former sense. Lacking the ability readily to distinguish delicate odours, we find among birds none of the glands which are so common among hairy-coated creatures: the oil-gland is the only one on the body, and this is practically odourless. But slight as is the scent which diffuses from birds, it is FIG. 149. — Turbinal scrolls of dog. In a bird these bones are far more simple. In all animals the mucous membrane nasal cavity is very delicate and fille These nervelets unite and form a singl side which passes to the brain and trans sions derived from the odours in the air. within the nostril, which, in dogs and d curl in delicate lines and scrolls and thus expose such a large surface to the odour-bearing air, are but poorly represented in birds. The simple curve of the bone in the nos- trils of birds is very simi- lar in structure to that found in reptiles. In all animals the mucous membrane which lines the nasal cavity is very delicate and filled with nervelets. These nervelets unite and form a single nerve on each side which passes to the brain and transmits the impres- sions derived from the odours in the air. The thin bones within the nostril, which, in dogs and deer, curl and re- curl in delicate lines and scrolls and thus expose such a large surface to the odour-bearing air, are but poorly represented in birds. The simple curve of the bone in the nos- trils of birds is very simi- lar in structure to that found in reptiles. The Sense of Smell The sense of smell is dependent upon the diffusion in the air of minute particles of objects, and naturally is effective at very short distances compared to the senses of sight and hearing, which require only vibrations in the atmosphere. When we remember that the nostrils of birds are usually encased in horn and that there is no exposed moist surface, as in the nose of a dog, we shall see how it is that this sense is but little developed among feathered creatures. 203 The Bird 204 FIG. 147. — Nostrils of bird encased in horn. FIG. 147. — Nostrils of bird encased in horn. FIG. 148.— Nostrils of deer encased in moist flesh. FIG. 148.— Nostrils of deer encased in moist flesh. The Senses The Senses The question whether In all animals the muco nasal cavity is very deli These nervelets unite and side which passes to the b sions derived from the odou within the nostril, which, curl in delicate lines and scrolls and thus expose such a large surface to the odour-bearing air, are but poorly represented in birds. The simple curve of the bone in the nos- trils of birds is very simi- lar in structure to that found in reptiles. FIG. 149. — Turbinal scrolls of dog. In a bird these bones are far more simple. The question whether vultures perceive their prey by sight or smell has been decided in favour of the former sense. Lacking the ability readily to distinguish delicate odours, we find among birds none of the glands which are so common among hairy-coated creatures: the oil-gland is the only one on the body, and this is practically odourless. But slight as is the scent which diffuses from birds, it is sufficient to enable a dog, with his wonderful keenness of smell, to detect a crouching bird some distance away. FIG. 149. — Turbinal scrolls of dog. In a bird these bones are far more simple. FIG. 149. — Turbinal scrolls of dog. In a bird these bones are far more simple. The woodcock of our inland swamps and marshes, and the apteryx of New Zealand, probably have the The Bird 2o6 sense of smell most acutely developed, although in both cases it is the delicate nerves of touch in the bill which are most helpful in detecting the presence of the earth- worms which constitute the food of these birds. To whatever degree the nostrils of land birds aid their owners in procuring food, it is certain that those FIG. 150. — Head of Apteryx, showing tactile hair-like feathers, nostrils at tip of beak, and small eyes. FIG. 150. — Head of Apteryx, showing tactile hair-like feathers, nostrils at tip of beak, and small eyes. species which feed entirely on fish, which they swallow whole, have little use for nostrils, except for breathing. Thus Nature, ever on the watch to economize, has re- duced these organs, in such birds as pelicans and cor- morants, and, at least in the adults, the nostrils are com- pletely filled up with bone and horn. The Sen 207 The Sense of Sight The Sense of Sight Birds, so wonderful and interesting in all their structure and life, have that most treasured of all the senses — sight — so highly developed that there is nothing with which we can compare it among living creatures. With our great telescopes we can see to a greater distance than any bird; with the high-power lenses of our microscopes we can distinguish infinitely smaller objects than any feathered creature is capable of perceiving, but where else on the earth is there an organ of vision which in a fraction of time can change itself from telescope to micro- scope; where is the eye that, seeing with wonderful clear- ness in the atmosphere, suddenly adapts itself to the re- fraction of water, or (less slowly, although no less surely) to the darkness of night? Next to our powers of reasoning, we value sight above all things, and fortunate indeed should we be could we but exchange our imperfect vision for sight like that of an eagle! Little need of spectacles or binoculars has he, for the perfection of his eye enables him to become near- sighted or far-sighted at will. sighted or far-sighted at will. "The eye," says Professor Coues, "is an exquisitely perfect optical instrument, like an automatic camera which adjusts its own focus, photographs a picture upon its sensitized retinal plate, and telegraphs the molecu- lar movements of the nervous sheet to the optic 'twins' of the brain, where the result is translated from the phys- ical terms of motion in matter to the mental terms of consciousness. But no part of the nervous tract, from The Bird 2 the surface of the retina to the optic centre, sees or knows anything about it, being simply the apparatus through which the bird looks, sees, and knows. In this Class of vertebrates the optic organs, both cerebral and ocular, are of great size, power, and effect; their vision far tran- scends that of man, unaided by artificial instruments, in scope and delicacy. The faculty of accommodation, that is of adjusting the focus of vision, is developed to a marvel- lous degree; rapid, almost instantaneous changes of the visual angle being required for distinct perception of objects that must rush into the focal field with the velocity at least of the bird's flight. Observe an eagle soaring aloft until he seems to us but a speck in the blue sky expanse. The Sense of Sight He is far-sighted, and, scouring the earth below, descries an object much smaller than himself, which would be invisible to us at that distance. He prepares to pounce upon his quarry; in the moment re- quired for the deadly plunge he becomes at once near- sighted, seizes his victim with unerring aim, and sees well how to complete the bloody work begun. A humming- bird darts so quickly that our eyes cannot follow him, yet he instantaneously settles as lightly as a feather upon a tiny twig. How far off it was when first perceived we do not know; but in the intervening fraction of a second the twig has rushed into the focus of distinct vision, from many yards away. A woodcock tears through the thickest cover as if it were clear space, avoid- ing every obstacle. The only things to the accurate per- The Sen 209 thousands of birds are annually hurled against these objects to their destruction." objects to their destruction." A bird's eye is very large in proportion to the size of its head, and is correspondingly perfect and delicate in its workings. It rests in a deep cavity hollowed out of the skull, and is protected by soft cushions of fat and controlled by bands and pulleys of muscle which control its motions. Looking closely at the eye of a live bird, we at once remark its brightness — that alertness of expression which so truly reflects the virile life of these creatures. The eye, more than any other part of a living organism, is an index to the relative power of its intelligence — more surely than all the other facial features taken together. The eyes of a sloth are expressionless black spots, and even those of an orang-utan are bleary and watery. But a crow or magpie, or any other bird you may choose, though with horny, shapeless lips, nose, and mouth, looks at us through eyes so expressive, so human, that no won- der man's love has gone out to feathered creatures through- out all his life on the earth. A dog is a four-legged, hairy animal with the eyes of a bird. The eye of a bird appears perfectly round, and is composed of a central area of black, encircled by a ring, sometimes hardly distinguishable from the inner divi- sion, or again it may be highly coloured. The Sense of Sight The circular centre or pupil is always of a uniform black, and no won- der, for "it is not a thing — it is the hole in a thing." As when we look through the lens of a camera, only the blackened inside of the bellows is reflected to us, so in the eye of a bird, the delicate living lens, itself invisible, The Bird 2 reflects the black pigmented tissue at the back of the eye- ball. The image passes through this lens and is thrown upon the curtain of jet, and here the brain nerves find it and know it — how, we cannot even guess. and know it — how, we cannot even guess. If the eye-camera of the bird has no long bellows to focus out and back, it has something infinitely better — the coloured ring or iris which surrounds the pupil. We FIG. 151.— Head of living Sloth. FIG. 151.— Head of living Sloth. are all familiar with the way the oval iris of a cat nar- rows to a slit in bright sunlight and broadens at night to let in all the light possible. Look closely at the eye of an owl or parrot, even in broad daylight, and the circle of the iris will be seen to contract and enlarge at the will of the bird. We have always been inclined to pity the poor "blind" owl during the daytime, but the truth is that, because of this power of adjustment, almost The Senses 21 I all owls can see very well, even in sunshine, although of course their eyes are especially adapted for use in the dim light of the evening and of the stars. In Nova Scotia I have noticed Barred Owls flying about and feeding at noonday. FIG. 152.— Duck Hawk. (Courtesy of N. Y. Zoological Society.) Compare the alert expression with the sloth in Fig. 151. FIG. 152.— Duck Hawk. (Courtesy of N. Y. Zoological Society.) Compare the alert expression with the sloth in Fig. 151. Birds have well-developed lachrymal glands, although it is seldom that they actually shed tears. Still I have seen a flamingo in a flying-cage weeping copiously from terror, anticipating all sorts of torture from a harmless The Bird 2 condor which was playfully galloping around the fright- ened bird. The Sense of Sight Millions of years ago, in the geological period of time known as the Jurassic, there existed gigantic sea-lizards, which we call Ichthyosauri. All we know of them we have learned by study of their fossil bones which, through the ages, have been preserved in rocks. One notable FIG. 153. — Brown Thrasher with eyes wide open. FIG. 153. — Brown Thrasher with eyes wide open. thing about them was the great size of their eyes — meas- uring as much as twelve and fourteen inches across. These orbits were surrounded by a series of bony plates, and in certain birds of to-day we find a similar circle of small overlapping bones. To make the simile between a camera and an eye hold good, we must show that the latter is provided with a The Senses FIG. 154.— Brown Thrasher with nictitating membrane drawn. FIG. 154.— Brown Thrasher with nictitating membrane drawn. FIG. 155. — Same with eyelids closed. FIG. 155. — Same with eyelids closed. The Bird 2 shutter, and in fact our bird has not one, but three — eyelids we call them. So "between winks" all day our bird is taking snapshots, inconceivably more perfect and continuous than any cinematograph ever produced. We have but two eyelids, and every time we wink these shoot toward each other, moisten the surface of the eye- ball, clear it of dust, and are back in their places so quickly that we are not aware of any interruption of our vision. The upper lid has most to do with covering the eye. In almost all birds this condition is unusual and the lower lid comes far upward over the eyeball. Perhaps the most notable exception to this is among the Great Horned Owls, where the action of the two lids is like that of our own. When birds are sleepy these lids close, but usually in winking, the third eyelid, or nictitating membrane, alone is drawn across the eye. This lid is a delicate, semi- transparent sheet of tissue, which, when not in use, lies snugly packed away in folds at the inner corner of the eye, held back out of sight by its own elasticity. It is drawn across the front of the eye by a slender thread of tendon which is suspended, pulley-like, from a muscle which keeps it from pushing against the optic nerve. The Sense of Sight When you see an owl in the daytime with eyes dull and glazed, this third eyelid is drawn partly across them, diluting the strong glare of light and yet enabling the bird to distinguish much that is going on. When an eagle turns his head upward and looks full at the sun, it is not " unwinkingly, " but with the help of this eyelid shield. " unwinkingly, " but with the help of this eyelid shield. It is interesting to know that this membranous lid is found in many other creatures, from sharks to monkeys, The Senses although usually much less perfectly developed than it is in birds. Alligators, however, have it fully functional. In the inner corner of our own eyes we may detect a trace of it, useless to us, but showing that far back in dimly imaginable geological epochs our forebears had need of a third eyelid. FIG. 156. — Vestige of nictitating membrane in a human eye. FIG. 156. — Vestige of nictitating membrane in a human eye. The Sense of Hearing "The Gauls," says Livy, " having discovered that the rock Carmentalis was accessible, one night when it was pretty clear, sent a man to examine the way, without his arms which were afterward handed to him. Others followed, lifting and assisting each other, according to the difficulties which they encountered in the ascent, till they reached the summit. They proceeded with so much silence that neither the sentinels nor even the dogs, ani- mals usually so vigilant as to be aroused by the slightest noise, took any alarm. They did not, however, escape the notice of the geese, which, being sacred to Juno, had been fed by the Romans notwithstanding the famine caused by the siege. This saved the capitol; for, by The Bird 2 their cackling and beating their wings, they roused Mar- cus Manlius, a brave soldier and formerly consul, who, snatching up his arms and giving the alarm, flew to the ramparts, set upon the Gauls, and by precipitating one of them over the rocks terrified the rest so much that they threw down their arms." So also Pliny, ^Elian, and Columella vaunt the hearing of Geese. But leaving leg- endary lore, it is certain that birds would not have the power of producing the most varied as well as the sweet- est sounds in all Nature, had they not been provided with powers of hearing, correspondingly acute and dis- criminating. The organ of hearing is complicated and there are many points about it which are still mysteries to scientists. The flap of skin to which we give the name of ear is entirely absent in birds, and indeed in ourselves is a very unimportant part of the auditory apparatus, serving only as a collector of sound-waves. The opening of the inner ear on each side ol the head, in birds, is usually protected by a cover of feathers which are bristle-like, partly denuded of barbicels, doubtless to avoid any muf- fling of sound-waves. In owls this opening is of very large size and protected by a movable flap of skin which may serve to aid in focussing the sounds from below — a very useful function to an owl at night, silently wing- ing its way over field and meadow in search of mice and other terrestrial prey. The Sense of Hearing A rather singular fact is that in many owls the two ear-openings are unlike, one being larger and of a different shape from the other, and this asymmetry extends even to the form of the skull itself. The Senses The Senses Two membranes are stretched across the ear-tube, and between these a tiny bone, the columella, is sus- pended, taking the place of the chain of three bones in the ear of a mammal. When sound-waves strike against the outermost membrane, or drum of the ear, vibrations are transmitted by the little bony suspension bridge to the inner membrane, and this in turn troubles the fluid FIG. 157. — External ear of Barred Owl. FIG. 157. — External ear of Barred Owl. which fills the inner ear. The hair-like endings of the nerve of hearing are affected by the vibrations of the fluid and thus is hearing accomplished. Rather say, thus the disposition of the physical components of the ear may be explained; but how anything more than the monotone of a sea-shell's cavity is translated to the brain, no one can say. The fluid contained in three semicircular canals, situ- The Bird 2i 8 ated in the inner ear — which occupy the three planes of space, — exercises a most important function, that of equi- libration. They have been compared to the glass tube filled with water and a shifting bubble of air, by centring which a surveyor knows his instrument is perfectly level. which a surveyor knows his instrument is perfectly level. If these canals be injured or cut, the bird loses all control of his actions ; if a certain one of the three canals suffers, the bird moves its head rapidly sideways and spins around in a circle; if another of the trio is by an accident severed, the motion of the head is back and forth, and the bird is compelled to execute forward som- ersaults; when the third of these canals is cut the bird continually falls backward. In reptiles and mammals the same thing occurs, so the wisdom of Nature in pro- tecting these delicate organs by a sheafh of hard bone is very apparent. The Sense of Taste and Touch "The hands of birds being hidden in the feathers which envelop the whole body, — their feet and their lips and usually much, if not all, of their tongue, being sheathed in horn, — these two faculties would appear to be enjoyed in but small degree." be enjoyed in but small degree." The sense of taste is probably the least developed of all. The nerves which find their way through the pores of the bill and tongue are more properly those of touch than of taste, and this seems the more credible when we consider the food of many birds, which is swal- lowed entire, besides being so hard that nerves of taste would be useless. Parrots and ducks, with their fleshy The Sen 219 tongues and ample membranes of the mouth, doubtless possess this sense to a considerable degree, while in birds which are exclusively fish-eaters we may expect to find taste least developed, the character of their food pre- cluding all need for this faculty. But from no bird is taste entirely absent, as we may easily see by presenting some nauseous insect, which will be instantly rejected with very evident signs of dis- gust, the bird wiping its bill on a branch and shaking its head violently. The sense of feeling, although much deadened by the feathery and horny character of a bird's integument, is most active at the tip of the tongue and the beak. At the base of the feathers, especially those of the wings and tail, tactile nerves are found, so that even a touch on the tips of the feathers awakens a response in the nervous system. system. The delicacy of the tactile touch is remarkable in those long-billed birds which seek their food in the muddy bottom of shallow water, detecting by means of their sensitive bills the presence of worms and snails, — aided little or not at all by eyesight. In the woodcock and apteryx this dependence on the senses of touch and smell has even wrought a change in the position and character of the eyes. The upper mandible of the woodcock is probably unique in being so sensitive and mobile that the distal third can be curved some distance upward, the base of the two mandibles remaining close together. The Sense of Taste and Touch This is an admirable provision by which, when the bird has driven its beak deep down into the moist soil, it may feel about I O I g 2 I O I g 2 The Senses 221 and seize the earthworm for which it is seeking. The eyes have become unusually large in consequence of its nocturnal habits and in addition are placed far back upon its head, permitting a clear lookout for danger, above and even behind, while probing with its head held close to the ground. FIG. 160 FIG. 159. FIG. 160 FIG. 159. — Photograph of living Woodcock with bill closed. FIG. 160 — Same with bill open, showing mobility of upper mandible. The bird is thus enabled to feel about and seize the earthworms deep down in the mud. FIG. 159. FIG. 159. FIG. 159. FIG. 160 FIG. 159. — Photograph of living Woodcock with bill closed. FIG. 160 — Same with bill open, showing mobility of upper mandible. The bird is thus enabled to feel about and seize the earthworms deep down in the mud. FIG. 159. — Photograph of living Woodcock with bill closed. FIG. 160 — Same with bill open, showing mobility of upper mandible. The bird is thus enabled to feel about and seize the earthworms deep down in the mud. Thus ends our brief survey of the five senses; that of smell taking note of minute particles of matter diffused in the air; sight and hearing depending on vibrations of the atmosphere; the sense of taste detecting matter which is dissolved in water, and that of feeling making the bird cognizant of the qualities of bodies by actual contact. o> O> T) o & £ o> O> T) o & £ o> O> T) o & £ BEAKS AND BILLS I IE a man's hands and arms tightly behind his back, stand him on his feet, and tell him that he must hereafter find and prepare his food, build his house, defend himself from his enemies and perform all the business of life in such a position, and what a pitiable object he would present! Yet this is not unlike what birds have to do. As we have seen, almost every form of vegetable and animal life is used as food by one or another of the species. Birds have most in- tricately built homes, and their methods of defence are to be numbered by the score; the care of their delicate plumage alone would seem to necessitate many and varied instruments: yet all this is made possible, and chiefly executed, by one small portion of the bird — its bill or beak. If one will spend an afternoon at a zoological park, or with any good collection of live birds, watching the ways in which the bills of the various species are used, one will not boast of his own accomplishments, when it is realized how much more, comparatively, the bird is able to achieve with the aid of two projecting pieces of horn. 223 The Bird 224 More than a single volume could be filled with in- teresting facts about the bills of birds and the uses to which they are put, — hardly any two species using their beaks in a similar manner. The general way in which the vast subject of the adaptation of the bird to its needs and to its surroundings is treated in this volume will, it is hoped, be a stimulus to the reader to observe for himself, — to discover the thousand and one facts to FIG. 162. — Beak of Snapping-turtle. (Courtesy of N. Y. Zoological Society.) FIG. 162. — Beak of Snapping-turtle. (Courtesy of N. Y. Zoological Society.) which Nature has not yet given us the key. Our lan- guage is too often lacking in phrases expressing delicate shades of meaning, and thus we are compelled to identify structures among the creatures which rank below us with portions of our own anatomy corresponding only in relative position or a general vague likeness of function. We are accustomed to speak of the mouth of a starfish, the arms of a sea-anemone, the foot of a snail: in these respective cases, structures specialized for receiving food, Beaks and Bi 225 reaching about, or for progression being understood. But no one would think of alluding to a bird's lips or nose ; both are included in the terms beak, or bill, and nostrils. The finding and securing of food being the most im- portant problem which birds have to solve for themselves, it is for these purposes, and especially the last mentioned, that we find bills most adapted. This is so universally FIG. 163. — Bill ot American Raven. FIG. 163. — Bill ot American Raven. the case that we may often judge accurately of the kind of food of a certain bird from a glance at its beak. As is the case with so many other avian structures, the horny, toothless beak or bill is duplicated elsewhere in Nature only in a group of reptiles, the turtles and tor- toises, whose mandibles furnish a splendid example of parallel evolution. In certain of those long-extinct Dinosaurs, such as The Bird 2 Triceratops, an interesting transitional condition is found. The front of the mouth was beak-like and horny, while farther back were the masticatory teeth. The Bird Starting with the generalized beak of the Archce- opteryx, which, we remember, was furnished with teeth, we are almost at a loss in which direction to turn, so many and so varied are the beaks of modern birds. No trace of teeth, however, is to be found in the adults of any of them. The bill of a crow or raven and, to a lesser extent, that of his near relatives, the jay and the blackbird, is perhaps in shape most like that of the 'bird of old,' and is suited to the many purposes which the varied life of these intelligent birds requires. The crow or the raven is an excellent example of a modern bird with a remarkably generalized diet, in striking contrast to those birds whose bills show them to be fitted for feeding only on some strictly defined food. With his strong, ample beak the crow can dig up recently planted corn, or crack the hard shells of acorns; he en- joys stealing the eggs and the young birds of thrushes, orioles, sparrows, warblers, and quail, and I have seen a crow chase, capture, and carry off a half-dozen wild Mal- lard ducklings in one morning ! These birds are, in ad- dition, able to capture insects of all kinds, besides pick- ing berries, and ducking their heads under water in quest of the shrimps which live in tide-pools. In short, their bill serves them well in procuring many kinds of food1, from earth, water, or tree; as well as in carrying great quantities of sticks, which they use in the construction of their nests. These birds are so skilful with their Beaks and Bills FIG. 164.— Beak of Gannet. FIG. 164.— Beak of Gannet. FIG. 165. — Beak of Cormorant. Birds closely related, but procuring food in different ways. FIG. 165. — Beak of Cormorant. Birds closely related, but procuring food in different ways. The Bird 2 beaks that a new trick is learned in a very short time. In captivity a crow, when it thinks no one is watching, will often take a morsel of food, thrust it beneath a piece of sod, and cover it up with grass, almost with one motion of the beak. The Bird Functional or adaptive radiation is beautifully il- lustrated by the beak of a gannet, cormorant, snake- bird, and pelican — birds which are closely related to one another structurally, also having in common a fish diet, swallowing their prey whole. The gannet's beak is thick and very strong, and along the inner edge is a series of fine serrations pointing backward. The bird dives, from a great height, into the water and seizes the fish in a grip of steel. The upper mandible of the cor- morant is furnished with a large, sharp hook, with which the bird gaffs its prey, pursuing it under water. The snake-bird, or darter, has a bill like a needle, with which it spears the fish, impaling it through and through; while the pelican, because of its great pouch least vicious of all in its methods, simply engulfs the fish, the water in which it is swimming and all, then straining out the liquid, tosses the unfortunate into the air and swallows it head first. The under mandibles of this bird are long and pliable and so arranged that they can bend far apart, thus making of the great bag of skin beneath the bill and throat an admirable fish-trap. and throat an admirable fish-trap. This is one of the many instances where several closely related species, with needs so similar that there is danger of fatal competition, are able to exist in great numbers and to avoid all undue struggle for existence by having Beaks and Bills Beaks and Bills FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. FIG. 166.— Beak of Snake-bird. FIG. 166.— Beak of Snake-bird. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. The Bird 23° each an individual method — a niche into which it fits perfectly in the great scheme of Earth's hungry creat- ures. The snake-bird's prey is in the water of dense FIG. 168. — Brown Pelican catching fish, showing bag-like distension of lower mandible. (Sanborn: photographer. Courtesy of N. Y. Zoological Society.) FIG. 168. — Brown Pelican catching fish, showing bag-like distension of lower mandible. (Sanborn: photographer. Courtesy of N. Y. Zoological Society.) swamps and bayous; cormorants and pelicans amicably share inland lakes and tidal waters; while the haunt of the gannet is the high seas. swamps and bayous; cormorants and pelicans amicably share inland lakes and tidal waters; while the haunt of the gannet is the high seas. Even more closely related to each other are terns Beaks and Bills 231 and Black Skimmers. Except in their bills these birds are almost identical in structure, but the bill makes a and Black Skimmers. Except in their bills these birds are almost identical in structure, but the bill makes a FIG. 169.— Bill of Tern. FIG. 169.— Bill of Tern. FIG 170.— Bill of Skimmer Closely related birds which differ in their feeding habits. FIG 170.— Bill of Skimmer Closely related birds which differ in their feeding habits. vast difference in the appearance of a bird, as is very apparent when these two species are seen flying about The Bird 232 • together on their breeding-grounds, — low, sandy islands along our coast. The small, delicately pointed beak of the tern finishes off its neat appearance, and the entire bird is the personification of grace, as it dashes through the air, or plunges headlong into the sea, — to rise almost immediately with a small fish in its beak. • the air, or plunges headlong into the sea, — to rise almost immediately with a small fish in its beak. The beak of the skimmer lends a heavy aspect to the whole bird. It is long and high, and the lower man- FIG. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. 172.— Two-year-old Skim- mer, reared in captivity, showing abnormal growth of mandible, due to lack of friction against water. PIG. 171. — Bill of young Skimmer, showing under mandible already slightly the longer. The beak of the skimmer lends a heavy aspect to the whole bird. It is long and high, and the lower man- F PIG. 171. — Bill of young Skimmer, showing under mandible already slightly the longer. FIG. 172.— Two-year-old Skim- mer, reared in captivity, showing abnormal growth of mandible, due to lack of friction against water. PIG. 171. — Bill of young Skimmer, showing under mandible already slightly the longer. dible extends a full inch beyond the upper. Both are as thin and as pliable as paper-knives. A unique method of obtaining food is the secret of this apparent deformity: the strong wings of the bird enable it to fly very close to dible extends a full inch beyond the upper. Both are as thin and as pliable as paper-knives. A unique method of obtaining food is the secret of this apparent deformity: the strong wings of the bird enable it to fly very close to Beaks and Bills. the surface of the water, so close in fact that the lower mandible dips below the surface, thus ploughing a zig- zag furrow and catching up any organisms, shrimps or fish, which chance to be floating on the water. FIG. 173. — Bill of Merganser, a fish-eating duck. FIG. 173. — Bill of Merganser, a fish-eating duck. FIG. 174. — Bill of Shoveller Duck, a bird which strains its food from the mud. FIG. 174. — Bill of Shoveller Duck, a bird which strains its food from the mud. Among ducks, we find those which feed on fish, and those which sift their food from the mud at the bottom of ponds, and these differ radically in respect to their beaks. The fish-eating merganser has perhaps, of all living birds, the nearest resemblance to a toothed beak. The Bird 2 The deep serrations, however, are but indentations in the substance of the strong, narrow bill of the bird. When once in this saw-like grasp, the most slippery fish is helpless. The beak of the Shoveller Duck shows how well Nature has provided for its wants. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. The beak is arched and spatulate, while the sensitive epidermis is pro- longed at the edges into a series of comb-like teeth,— analogous to the whalebone in the mouth of a whale. Through this sieve the water is drained out, leaving entangled the edible worms and insects. If we should elevate our Shoveller Duck, placing him on long, slender legs and providing him with a corre- spondingly long neck, he would indeed be in a predica- ment, since only the tip of his beak could be brought to bear in feeding. Now a flamingo is really a long- legged duck, which feeds in much the same way as the Shoveller, and the difficulty mentioned is overcome in a most ingenious way. The mandibles are bent down- ward, almost at right angles, so that, when the head reaches the ground, not the tip but the whole inverted bill is in a position to sift out food. To meet the reversed condition, the lower mandible is deeply arched, instead of the upper as in the Shoveller Duck. We are able to follow the probable evolution of such remarkable beaks as those of the flamingo and skimmer by observing the growth of this organ in any individual from the time when the bird hatches from the egg until it is full-grown. In the very young flamingo chick there is no sign of the subsequent deflection, the mandible being short, perfectly straight, and rather slender. As the FOUR STAGES IN THE DEVELOPMENT OF THE BILL OF THE FLAMINGO. FIG. 175. — Young bird in down. FIG. 176. — Young in gray plumage. FIG. 175. — Young bird in down. FIG. 175. — Young bird in down. FIG. 175. — Young bird in down. FIG. 175. — Young bird in down. FIG. 176. — Young in gray plumage. FIG. 176. — Young in gray plumage. FIG. 177. — Young in gray plumage, later stage. FIG. 177. — Young in gray plumage, later stage. FIG. 178. — Adult living bird. 235 FIG. 178. — Adult living bird. FIG. 178. — Adult living bird. FIG. 178. — Adult living bird. The Bird 2 bird at first feeds upon regurgitated food, taking it drop by drop from the bill of the old bird, it of course has no need of the curved beak of its parents. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. Later, when its bill has increased in length and has begun to be marked by the ultimately sharp angle, the birds begin to sift from the coral mud the small mollusks of which their food consists. Until its wings are full-feathered the young skimmer is compelled to limit its wanderings to the sand-dunes along the shore near its nest. Thus, although at birth the lower mandible is a trifle longer than the upper, yet even when the birds are half-grown the disparity in length between the two mandibles is but slight. Later, when the young bird is able to join its parents in their skimming of the seas, the lower mandible quickly attains its full development. The friction of the water upon the bill must be considerable, as in a skimmer which I have had for years in captivity, the lower mandible grew remark- ably fast, measuring 6f inches from base to tip when the bird was eighteen months old. Herons and ibises, through all the years, sought their food in much the same places as have ducks ; the straight- billed herons seizing their living prey with a single light- ning dart, as it swims past them; the spoonbills spatter- ing in the shallows; and the curved-beaked ibises prob- ing every crevice along shore. The spoonbills swing their necks and heads from side to side, as they walk slowly through the water, gleaning their food with the motion of a mower wielding his scythe. Two of the herons are interesting enough to hold our attention for Beaks and Bills a moment. The common Black-crowned Night Heron is abundant throughout most of North America, and he fishes in legitimate heron fashion; but his near relative, the Boat-billed Heron, is a more tropical species. In voice, appearance, and structure there is little to choose between the two birds, — except that the latter has a broad, scoop-like beak, — a pelican's fish-trap in miniature, which seems to answer every requirement of this strange-look- ing bird. From the muddiness of the water in the tropical swamps from which I have flushed these birds, it seems probable that much of their food may be lesser fry than fish. FIG. 179.— Bill of Great Blue Heron. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. Pebbles and shells, which shelter so many toothsome morsels along the shallows of our sea- shore, offer sumptuous feasts to birds furnished with beaks adapted to prying and probing, and we find all sorts of sizes and shapes. A collection of bills of various wading-birds would look like a complete set- of surgical tools ! There is the stilt, whose bill is almost straight; the ibis, with mandibles curved downward to probe the crevices between the pebbles on which he stands; the avocet has a pair of recurved pliers, which search out the worm or snail in the deepest fissures ahead of him. At the slightest touch FIG. 179.— Bill of Great Blue Heron. FIG. 179.— Bill of Great Blue Heron. The Bird 238 of such a beak, the oysters and other large bivalves close with a snap, defying these birds to penetrate their living armour. Indeed, more than once a gull or wader has rashly pecked at the sweet flesh, when the two tight- fitting doors have suddenly closed, pinning the bird help- FIG. 180. — Boat-billed Heron. Figs. 179 and 180 represent birds with slightly different feeding habits. FIG. 180. — Boat-billed Heron. Figs. 179 and 180 represent birds with slightly different feeding habits. less and holding it captive despite its struggles, until the rising tide has ended its life. But along comes a bird, well named Oyster-catcher, and woe to the mollusks now. It allows them to close tightly upon its bill, the mandibles of which are thin like blades, many years antedating man's oyster-knives. The mollusk is wrenched free by the sturdy bird, car- ried from the water still gripping the bird's bill, and is Beaks and Bills FIG. 182.— White Ibis, showing curved bill. FIG. 181. — Spoonbill, with spatulate mandibles FIG. 182.— White Ibis, showing curved bill. FIG. 181. — Spoonbill, with spatulate mandibles FIG. 182.— White Ibis, showing curved bill. FIG. 181. — Spoonbill, with spatulate mandibles FIG. 183.— Bill of Avocet, recurved for probing. FIG. 183.— Bill of Avocet, recurved for probing. The Bird 240 then pried open and eaten. The bill of this bird shows the wear and tear of forcing apart the shells, and it is sometimes slightly bent to one side. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. The short-billed gulls are denied the power of opening these oysters and mussels, but they sometimes get an unlawful feast by fol- lowing up and robbing the Oyster-catchers of the shells which the latter have opened. The bill of the Shell Ibis of India may be likened to an ordinary lemon-squeezer, having a cavity in which FIG. 184. — Bill of Oyster-catcher; used for prying open the shells of mollusks. FIG. 184. — Bill of Oyster-catcher; used for prying open the shells of mollusks. the half -lemon rests before it is compressed. When the mandibles of this bird are closely opposed the central portion of the beak gapes slightly. In this cavity the bird firmly holds the shells of the land-snails upon which it feeds, until it can bring the pressure of both mandibles to bear and so crush the shell of the mollusk. the half -lemon rests before it is compressed. When the mandibles of this bird are closely opposed the central portion of the beak gapes slightly. In this cavity the bird firmly holds the shells of the land-snails upon which it feeds, until it can bring the pressure of both mandibles to bear and so crush the shell of the mollusk. The asymmetry of the bill — as seen in the Oyster- catcher — is not accidental, but constant, in the Crook- billed Plover of New Zealand. In this bird the bill is Beaks and Bi 241 permanently bent to the right, a beautiful adaptation to help the bird in its search for insects, which, in the dry country that it inhabits, are found almost entirely under stones. As a rule, beaks are rather immovable throughout their length, but in the woodcock, and to a less extent FIG. 185. — Bill of Crook-billed Plover, for probing under stones. FIG. 185. — Bill of Crook-billed Plover, for probing under stones. in the Dowitcher Snipe, the extremity of the upper man- dible can be raised some distance (Figs. 159, 160). This extreme sensitiveness is especially necessary, as the eyes of the woodcock are placed very far back on the top of its head, and are of little or no use in seeking food. What an interesting study the various beaks of land birds would offer, were we able to devote to them the The Bird 242 space which they deserve! FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. They defy classification and refuse to be arranged in any linear sequence. The ma- jority of those birds which have their beaks armed with a strong hook feed upon living prey, — from the great mandible of the Golden Eagle to the tiny vireo, which snaps up the dancing gnats. sna up the da gn The owls and the parrots, which, by the way, are much more closely related than most of our classifications would indicate, have bills very much alike, and afford FIG. 186. — Bill of Golden Eagle, hooked for tearing prey. FIG. 186. — Bill of Golden Eagle, hooked for tearing prey. a striking example of two large related groups of birds whose diet has become radically unlike, although even in this case "blood will tell" and the Kea Parrot slips back into carnivorous habits with ease. Owls tear their prey apart with their beaks, or swallow it entire, but parrots gnaw and gnaw upon their nuts and seeds, reducing their food to powder. This grind- ing and rasping is aided by several file-like ridges which many parrots have within their beaks. The hinging of the upper mandible with the skull is more evident in a Beaks and Bi 243 parrot than in any other bird. This arrangement allows much freedom of motion. parrot than in any other bird. This arrangement allows much freedom of motion. It is not clearly known what use the immense beaks of toucans may serve, although there seems little excuse for this ignorance in those who know the birds in their native haunts. The delicate, spongy texture renders the FIG. 187. — Toucan, showing enormous bill used perhaps for reaching fruit on the tips of branches. FIG. 187. — Toucan, showing enormous bill used perhaps for reaching fruit on the tips of branches. clumsy-looking appendages exceedingly light, and they are usually banded or marked with brilliant hues, — blue, yellow, red, brown, green, or black. But light as the beaks are in these birds, in the unrelated but similarly monstrous-beaked hornbills the weight must be con- siderable, for the first two vertebrae of the neck in these The Bird 244 latter birds are fused together, to yield a firmer support for the muscles of the neck. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. Chimney Swifts and hummingbirds both feed upon insects and are rather closely related to each other, but here again the most decided difference is to be found in their bills. The broad, flattened mandibles of the swifts FIG. 188. — Bill of Toucan; cut open to show its light, spongy structure. FIG. 188. — Bill of Toucan; cut open to show its light, spongy structure. open wide, as the birds dash through the air, engulfing gnats and flies with wonderful skill; while the humming- birds, as we all know, probe the deepest calyxes. Could two bills more unlike be imagined? In very young hum- mingbirds the bill is short and broad, very like the swift type, and later its long and slender shape is acquired Beaks and Bi 245 very rapidly, as we can see in Figs. 190 and 191. There are many resemblances between hummingbirds and in- sects, due entirely to the similarity in their feeding habits. Certain flowers are especially adapted in structure to attract certain bees or moths, which in return unconsciously cross-fertilize the blossoms; and certain of the various bills of hummingbirds reflect the exact contour of the corollas in which the birds seek their food. Among hummingbirds the various shapes of bills of other groups are reproduced again. Humming through the air about us in the tropics speed miniature avocets, ibises, stilts, mergansers, and we realize, as never before, the never- ending devices of Nature, providing for the needs of all, from the greatest to the least; endless patterns paral- leling each other, but never identical. Indeed, in the great family of South American birds known as Wood- hewers the diversity in shape, size, and direction of bills is so great that it seems as if not a niche, or crack, or hollow in the bark of any tree in the forest where these birds abound would afford a safe retreat to an insect! It remains to mention the woodpecker's bill, which is used chisel-like, for excavating his home as well as in boring for grubs. With his beak the nuthatch ham- mers his acorns, and the tailor-bird sews his nest. The thick conical beaks of all sparrows and finches are for cracking seeds; while the weaker, more slender beaks of warblers, thrushes, and wrens reflect a diet of insects. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. Among the finches is a group of several species which, by a thrust of the bill, have at their command a new source of food, one which there are none to dispute with The Bird 246 FIG. 189. — Bills of adult Hummingbird and Chimney Swift, showing great dis- similarity in form, due to different methods of procuring food. FIG. 189. — Bills of adult Hummingbird and Chimney Swift, showing great dis- similarity in form, due to different methods of procuring food. FIG. 190. — Bills of young Rufous Hummingbirds, showing swift-like character. (Photograph by Finley & Bohlman.) FIG. 190. — Bills of young Rufous Hummingbirds, showing swift-like character. (Photograph by Finley & Bohlman.) 191. — Slightly older Hummingbirds, with bills half as long as the adults. (Photograph by Finley & Bohlman.) 247 FIG. 191. — Slightly older Hummingbirds, with bills half as long as the adults. (Photograph by Finley & Bohlman.) The Bird 2 them. Both mandibles of the crossbill are curved into sharp hooks which cross one another, either to the right or left, thus forming a unique pair of pliers, with which the bird pries out the seeds shut tight behind the over- lapping scales of pine-cones. FIG. 192.— Two extreme types of Hummingbirds' bills adapted for insertion in flowers with shallow and with deep calyxes. FIG. 192.— Two extreme types of Hummingbirds' bills adapted for insertion in flowers with shallow and with deep calyxes. The beak of a bird is always growing, and in captivity, from lack of proper use, the mandibles sometimes grow to a great length, and, if not trimmed, will often inter- fere with the bird's feeding. fere with the bird's feeding. Perhaps the most remarkably adapted beaks in the world are those of the male and female Huia birds — Beaks and Bi 249 natives of New Zealand — in which not only is the bill of the species designed for a special method of procuring food, but the bills of the two sexes are very different in form and use, and complement each other's methods. Concerning the peculiar use of the bill in the Huia birds, FIG. 193. — Bill of Purple Finch and Crossbill compared; the latter specialized for extracting seeds from pine-cones. FIG. 193. — Bill of Purple Finch and Crossbill compared; the latter specialized for extracting seeds from pine-cones. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. Professor Newton writes: "Its favourite food is the grub of a timber-boring beetle, and the male bird with his short stout bill attacks the more decayed portions of the wood, and chisels out his prey, while the female with her long slender bill probes the holes in the sounder part, the hard- ness of which resists his weapon; or when he, having removed the decayed portion, is unable to reach the grub, The Bird 25° the female comes to his aid and accomplishes what he has failed to do." has failed to do." The bill of a bird, besides serving in so many other ways, is invaluable in preening the plumage, arranging disordered feathers, drying them, and, most important of all, in pressing out the oil from the gland on the lower back, and with it carefully dressing all the feathers, giv- FIG. 194. — Bill of male and female Huia Birds, showing difference of the bill in the two sexes. FIG. 194. — Bill of male and female Huia Birds, showing difference of the bill in the two sexes. ing to them that brightness and gloss and also the water- proof quality — so surely a sign of perfect health in a bird. When, after the bath of a caged bird, you see the drops roll from its feathers, literally like " water off a duck's back," then the good health of the bird is certain. duck's back," then the good health of the bird is certain. The all-important use of the bill as a needle, shuttle, pick and shovel, auger, or trowel in nest-building does not concern us here, nor does its function in expressing emo- tion, or in taking the place of the voice or of the foot. Beaks and Bi 251 All this is expressive rather of the mental than the phys- ical life of the bird. All this is expressive rather of the mental than the phys- ical life of the bird. Within a period of five minutes I have observed the following uses of the beak of a parrot perching in my study. FIG. 166.— Beak of Snake-bird. FIG. 167.— Beak of Pelican. Birds related to each other and to Figs. 164 and 165, but with different feeding habits. With its mandibles it picked up a sunflower seed and comminuted it; it then hooked the upper man- dible into a wire and swung itself along; gnawed at a nest- ing-hole it had begun to excavate; nibbled gently at my finger, showing affection; bit fiercely in anger and fear at a dead snake which I presented; preened several feathers of one wing, smoothing out all the dislodged barbs; rattled its beak along the wires to make a sound to attract my attention; and finally seized its water-pan and turned it over in pure playfulness! HEADS AND NECKS H HE head of a bird is indeed a wonderful object, when we consider its comparatively small size and yet realize that it contains the brain, as well as being the seat of the five senses. It also sup- ports that most important organ the beak, which, as we have seen, takes the place of hands and tools in the life of its owner. Of expression, with the exception of that caused by raising its feathers, the bird has but little; although fear, that emotion which must needs be expressed all too often in the life of these timid and comparatively de- fenceless creatures, is made apparent by the dilating eyes and the open, panting beak. The only exception which comes to mind is the Crowned Crane, Fig. 206, the suffusing of whose bare, white cheeks indicates the changing emotions. Perhaps the best index is to be found in the crest, which we will find to be developed to a very remarkable degree. a very remarkable degree. The eyes of most birds are placed at the sides of the head, in such a position that the bird cannot bring both to bear simultaneously upon the same object, but is com- pelled to turn its head and look sideways. As birds 252 s bi Heads and Necks spend so much of their time in the air, or in trees, where danger may threaten from all sides, above or below, this arrangement is most useful to them, giving them com- mand of almost their whole surroundings, whereas, with- out turning the head, we can see only ahead of us. In much the same relative position, the two ears are placed, FIG. 195. FIG. 196. FIG. 195. — Head of Dove, with eyes at side FIG. 196.— Head of Owl, with eyes in front Showing difference in position of eyes in a pursued and a pursuer in Life's race FIG. 196. FIG. 195. FIG. 195. FIG. 196. FIG. 195. — Head of Dove, with eyes at side FIG. 196.— Head of Owl, with eyes in front Showing difference in position of eyes in a pursued and a pursuer in Life's race FIG. 195. — Head of Dove, with eyes at side FIG. HEADS AND NECKS 196.— Head of Owl, with eyes in front Showing difference in position of eyes in a pursued and a pursuer in Life's race and the absence of a directive outer ear renders the bird susceptible to sounds coming from every direction. Owls, for very obvious reasons, are interesting excep- tions to the above statements. Living most of their active life at night, playing always the role of pursuers, these raptorial birds have few enemies to fear; and their subsistence depends upon the keenness of their senses when focussed in one direction — downward. When its strong, soft-feathered pinions carry a mousing owl over The Bird 2 field and stubble, the head, like the nose of a hound, is held low, and, that not a rustle nor a motion of the little field-mice may be lost, the ear-openings are turned down- ward and the eyes look full upon the ground. Look a Barn Owl in the face and you will see the entire cir- cumference of both eyes, but a dove — one of the pursued in life's race — shows in the front view only the profile of the eyeballs. The same story is told in the eyes of the fox and the rabbit — examples of Nature's parallels, which are never repetitions. It is interesting to compare the eyes of owls with those of mammals in general. With the exception of man, and of some of the monkeys, we find that when the eyes show but slight divergence the animal is invariably a lover of the dusk, or is wholly nocturnal. We know that when we are asleep, or are under the effects of ether, our eyes tend to roll upward and outward, and now we realize that the cause of this is the old ancestral pulling outward, toward monocular vision, as in the fish or rabbit or dove. Our distant ancestors, far from having books or work which focussed their attention directly in front, had most vital need of looking out for dangers in all direc- tions. Another adaption found in the eyes of almost all noc- turnal birds is the great size of the orbit, fully one half of the skull being hollowed out to receive the eyeballs. HEADS AND NECKS No degeneration of the eyes, as a result of nocturnal habits, is recorded among birds, such as exists in moles and bats, except in the case of the apteryx, the diminutive New Zealand representative of the ostrich-like birds. The Heads and Necks Heads and Necks FIG. 197.— Skull of Owl. FIG. 197.— Skull of Owl. FIG. 198.— Skull of Apteryx. Showing opposite effects of nocturnal habits on the size of the eyes. FIG. 198.— Skull of Apteryx. Showing opposite effects of nocturnal habits on the size of the eyes. FIG. 198.— Skull of Apteryx. FIG. 198.— Skull of Apteryx. Showing opposite effects of nocturnal habits on the size of the eyes. Showing opposite effects of nocturnal habits on the size of the eyes. The Bird 2 small eyes of this bird become dazzled by strong light, its food being detected by the senses of touch and smell. its food being detected by the senses of touch and smell. The eyes of the woodcock show an interesting adapta- tion to its habits. The bird feeds at night in marshes, probing the mud for worms and, being in frequent danger of attack from owls or other foes, it has need of constant vigilance. So we find that its eyes, which are large and lustrous, are placed far back on its head and also up near the top of the skull. Useless in guiding the bird in its search for food, they have become altered in size and posi- tion and so best fulfil their function of aiding their owner to all but look through the back of its head. Even the iris of a bird's eye may share in the won- derful colour scheme of its feathers, although the most common hue is a hazel-brown. And in birds of two related species or races, there is sometimes a marked dif- ference in the colour of the iris; such, for example, as be- tween the Red-eyed and White-eyed Vireos, or the simi- larly named Towhees. Puffins have blue irides, pigeons pink ones, while young Bald Eagles have brown eyes which, in the adult, turn yellow. The eyes of Barred Owls seem to be a deep, lustrous black, but they are really dark brown; while the great yellow eyes of Snowy and Horned Owls are the most brilliant bits of colour about these birds. In cormorants the irides are a glittering emerald-green. emerald-green. HEADS AND NECKS It might be thought that "making eyes" was con- fined to the more frivolous of our own race, but certain it is that, whether or not it plays a part in charming the females, the irides of the males of a number of species Heads and Necks of birds change, at the season of courtship, from a dull hue to some bright tint, either red, green, or yellow. Although eyelashes, as we understand them, are merely stiffened hairs which have been inherited from hairy-coated ancestors, yet among birds we sometimes find lashes similar in appearance and function, but struc- turally derived from feathers. The ostrich has well- FIG. 199. — Head of Seriema, showing eyelashes. FIG. 199. — Head of Seriema, showing eyelashes. developed eyelashes, which must be of value in helping to exclude the dust of the desert; but why such birds as hornbills and the Seriema should possess them we cannot say. A savage thrusts feathers into his hair, warriors of old bedecked their helmets with flowing plumes, the opera hat of milady is by way of wonderful and strange creations; but withal feathers are really beautiful only The Bird 258 where they by rights belong — upon a bird. Among birds we find a more remarkable development of crests than in any other class of animals. Indeed nearly all birds have the power of slightly raising the feathers on the head. FIG. 200.— Crest of Java Peacock. FIG. 200.— Crest of Java Peacock. Most, if not all, plumes and crests are probably orna- mental, and, since many are more highly developed in the male sex and at breeding-time, we must conclude that they are of value in attracting and holding the at- tention of the females during the period of courtship. What a list of these crests we may compile in a walk through a zoological park! The photographs show the grace and delicacy of these feathers, to which words can Heads and Nec 259 add nothing. Note the slender shafts which rise from the head of the Indian Peacock, each one tipped with a dainty feather tuft; and the variation in the crest of its splendid cousin from Java. In one of the Birds of Para- dise, six long, fan-tipped shafts extend backward from the head, much longer than, but similar to, the crest of the Indian Peacock. The California Partridge has a tiny, FIG. 201. HEADS AND NECKS — California Partridges, showing difference in crest of male and female birds. FIG. 201. — California Partridges, showing difference in crest of male and female birds. club-shaped crest which points in a forward direction, and, when the bird is excited, the feathers which com- pose it spread out, breaking into a fan. The ornament of the Plumed Partridge is a long, sweeping plume. The crest of the curassow is most peculiar, being composed of curly, recurved feathers, resembling in texture and appearance jet-black or parti-coloured shavings. club-shaped crest which points in a forward direction, and, when the bird is excited, the feathers which com- pose it spread out, breaking into a fan. The ornament of the Plumed Partridge is a long, sweeping plume. The crest of the curassow is most peculiar, being composed of curly, recurved feathers, resembling in texture and appearance jet-black or parti-coloured shavings. appearance jet-black or parti-coloured shavings. The nuptial plumes of the Night Heron hang far down upon its shoulders, and the soft barbs are curved inward, The Bird 2 forming a slender tube. The glory of the Great Crowned Pigeon is a maze of lavender lacework, — one of the most beautiful of all crests; while the most graceful, perhaps, is the mist of filmy whiteness which, at the slightest breath of air, floats about the neck of the Snowy Egret, like the mantilla of a senorita. Cockatoos are decorated with a profusion of beautiful crests, each characteristic. These are under the complete control of the birds, and take an important part in expressing changing moods and emotions. The crests may lie so flat as to be ordi- narily invisible, when, in a flash, the whole head is sur- mounted by an auricle of colour or whiteness. An ex- cited Leadbeater Cockatoo is a wonderful sight. Before the crest is raised, all that is visible is a single, rather elongated white feather, but a wealth of colour is hid- den, which flares out, showing a band of scarlet close to the head, next a streak of bright yellow, then a second band of red, and finally the white tips of the crest feathers. The nod or jerk of the head in spreading wide the crest reminds one of the sudden flick with which a fan is thrown open. open. HEADS AND NECKS Concealed crests bring to mind the Kingbird and the Ruby-crowned Kinglet, both of which derive their names from their crowns of ruby. It is said that the former bird is aided in its search for food by the bright spot of colour which, flower-like when exposed, attracts in- sects. This, however, should be confirmed before being accepted as a fact; although in a tropical flycatcher, which has a beautiful red and purple transverse crest, the evidence of this novel use seems fairly well corroborated. FIG. 202.— Crest of Banded Curassow (female). FIG. 202.— Crest of Banded Curassow (female). l" :>-" e II FIG. 203. — Crest of Victoria Crowned Pigeon. 261 FIG. 203. — Crest of Victoria Crowned Pigeon. 261 FIG. 203. — Crest of Victoria Crowned Pigeon. 261 FIG. 204. — Harpy Eagle. (Courtesy of Dr. Frank Baker.) 262 FIG. 204. — Harpy Eagle. (Courtesy of Dr. Frank Baker.) 26 FIG. 204. — Harpy Eagle. (Courtesy of Dr. Frank Baker.) Heads and Necks Heads and Necks The Laughing Thrush of the Himalaya Mountains has every feather upon its head lengthened and perma- nently erect, forming a soft, spreading halo. IG. 205. — Hooded Merganser. (From a photograph provided by the America Museum of Natural History.) FIG. 205. — Hooded Merganser. (From a photograph provided by the American Museum of Natural History.) In almost every Family of birds we find certain spe- cies with long, well-developed crests. Among the ducks, the Hooded Merganser has a compressed, semicircular halo of delicate feathers, while the Mandarin Duck has a broad, many-coloured, erectile crown, which is con- The Bird 2 spicuous even in contrast with the gorgeous ornamenta- tion of the body of this feathered harlequin. Of birds of prey, the Harpy Eagle has perhaps the most imposing crown of feathers. Sometimes the crest is sharply set off from the rest of the bird's plumage, as in the scarlet-plumed wood- peckers, whose crests give them the appearance of having long hair, which is gracefully brushed straight backward and upward. and upward. We must not discuss the subject of crests without men- tion of two birds of extraordinary appearance, the Crowned Crane and the Umbrella-bird. The former illustrates admirably what strange and unfeather-like forms, feathers may assume in the course of evolution. The illustration shows better than words can describe the dense, velvety cap of plush-like feathers, and the glorious crown of a myriad radiating points — a decoration unrivalled, even among birds, in exquisite colour and delicacy. In addi- tion to this, the cheeks are entirely bare of feathers, and the lower half suffused with blood, which shows through the skin, — an ever-changing blush of deep pink. the skin, — an ever-changing blush of deep pink. The decorations of the Umbrella-bird are as beautiful as they are bizarre; while if shorn of its crest and streamers, this bird would resemble a small crow in appearance. The crest really bears a resemblance to the article which has given the bird its name— a high, arching mass of feathers, overshadowing the entire head and beak, con- tinually spreading and partly closing again, as the bird's emotions change. From the neck of the bird dangles a streamer of black feathers, as long as the bird's entire Heads and Necks Heads and Necks FIG. 206 —Crowned Crane. FIG 207. — Demoiselle Crane. FIG. 206 —Crowned Crane. FIG. 206 —Crowned Crane. FIG. Heads and Necks 206 —Crowned Crane. FIG. 206 —Crowned Crane. FIG 207. — Demoiselle Crane. FIG 207. — Demoiselle Crane. The Bird 266 body and which, when it flies, blows back between its feet. The filament of feathers looks for all the world as if a strip of the bird's plumage had caught on a thorn and torn loose. The core of the streamer is a very slender FIG. 208.— Umbrella-bird. FIG. 208.— Umbrella-bird. FIG. 208.— Umbrella-bird. ribbon of skin which hangs from the neck. Would that we could state the causes and the manner of the devel- opment of these curious structures which our fancy likens to an umbrella and a feathery handle! ribbon of skin which hangs from the neck. Would that we could state the causes and the manner of the devel- opment of these curious structures which our fancy likens to an umbrella and a feathery handle! One or two small tufts of feathers may spring from Heads and Nec 267 some part of the head of a bird, such as the feather " horns " of owls, motmots, and larks. In Screech Owls these prominent "ears" certainly play a useful part in breaking up the outline of the bird, rendering it very difficult of detection when it is perched upon some jagged limb or stub. Or again, tufts or pencils of feathers may arise from near the ear, or over the eye; as shown by the Dem- oiselle Crane (Fig. 207), some of the Puffins, and the Man- churian Pheasants (Fig. 209). The Great Bustard has long tufts of chin-feathers which, like wide-spreading whiskers, spread to each side, and the Bearded Vulture has a simi- lar goatee of stiff, black bristles. lar goatee of stiff, black bristles. Of the wonderful crests, frills, ruffs, breastplates, and cloaks of hummingbirds there is no room to speak, and indeed no words or pictures can aught but parody them. The eye alone can record their marvels, in the collection of a museum, or, better still, in the living birds, as the little creatures hover over, their favourite flowers, or vibrate before us, fanning the air in our very faces with their invisible wings. Brief mention should be made of two Birds of Para- dise, those beautiful creatures inhabiting a region where the eye of man seldom sees them. The Six-shafted Bird of Paradise is found only in New Guinea. Heads and Necks "The plumage appears at first sight black, but it glows in certain lights with bronze and deep pur- ple. The throat and breast are scaled with broad, flat feathers of an intense golden hue, changing to green and blue tints and certain lights. On the back of the head is a broad recurved band of feathers, whose brilliancv FIG. 209.— Head of Eared Pheasant. FIG. 209.— Head of Eared Pheasant. FIG. 210.— Head of Great Horned Owl. 268 FIG. 210.— Head of Great Horned Owl. 268 Heads and Necks is indescribable, resembling the sheen of emeralds and to- paz, rather than any organic substance. Over the fore, head is a patch of pure white feathers, which shine like satin; and from the sides of the head spring the six won- derful feathers." IG. 211. — Six-shafted Bird of Paradise (a mounted bird). (From a phot graph provided by the American Museum of Natural History.) FIG. 211. — Six-shafted Bird of Paradise (a mounted bird). (From a photo- graph provided by the American Museum of Natural History.) Head decorations reach the acme of strangeness in the King of Saxony's Bird of Paradise. The bird itself is sombre-hued and small, about the size of our robin, with nothing unusual about its appearance, except for the two streamers springing from opposite sides of the The Bird 270 head. They are twice (or more) the length of the body, and, far from being feather-like, they are best described as a series of thirty or forty tiny flags of blue enamel, each separate, each hanging pendent from the main shaft (Fig. 212). It would seem as if Nature herself could go no farther in unusual decoration than this. FIG. 212. — King of Saxony Bird of Paradise. (From a photograph provided by the American Museum of Natural History.) FIG. 212. — King of Saxony Bird of Paradise. (From a photograph provided by the American Museum of Natural History.) In the Double-crested Pigeon of Australia the core or fleshy covering of the beak is completely feathered; while some of the birds known as plantain-eaters are feathered to the very tip of the short beak with plumes of delicate green, tipped with white. Heads and Necks The extreme of feathering is shown by the Cock-of-the-Rock, in which Heads and Necks 271 the whole beak, in fact every part of the head except the eyes, is buried in a maze of soft, orange plumes. the whole beak, in fact every part of the head except the eyes, is buried in a maze of soft, orange plumes. eyes, is buried in a maze of soft, orange plumes. As the antithesis to this condition, we find many birds which have the head partly or entirely bare of feathers, such as the vultures and some of the waders. In the former group this lack of feathers is doubtless FIG. 213. — Head of male Condor FIG. 213. — Head of male Condor of value in enabling the birds to avoid soiling their plu- mage, when engaged in their scavenger work. The great Condor of South America has, just below this naked area, a necklace of the whitest of fluffy down, and in addition the male has a large wattle of skin upon the front of the head. The Caracara of Mexico is partly vulturine in its habits, and the feathers have disappeared from part of its face. Wherever the skin of the head and neck is even of value in enabling the birds to avoid soiling their plu- mage, when engaged in their scavenger work. The great Condor of South America has, just below this naked area, a necklace of the whitest of fluffy down, and in addition the male has a large wattle of skin upon the front of the head. The Caracara of Mexico is partly vulturine in its habits, and the feathers have disappeared from part of its face. Wherever the skin of the head and neck is even The Bird 272 FIG. 214. — Caracara. partly vulturine in habits. FIG. 214. — Caracara. partly vulturine in habits. FIG. 215.— Young King Vulture. FIG. 215.— Young King Vulture. Heads and Nec 273 partly bare, ornamentation often takes the form of many- shaped and often highly coloured wattles, such as we see highly developed in a King Vulture. The most common example of this is seen in a domestic rooster or a turkey, but in many other birds these wat- tles of skin are very brilliant in hue. Indeed the casso- waries are resplendent in their gorgeous hues of blue, yel- low, red, and many other intermediate shades. Heads and Necks Turkeys too, at the breeding season, develop bright colours. The Yucatan Wild Turkey, which has thus far resisted all attempts at domestication, has the bright blue naked skin of the head, dotted with tubercles of the most bril- liant orange, while a long tube-like wattle, also tipped with orange, dangles down over the beak. The wattles, or caruncles, of the Bell Bird are interesting as being con- nected with the windpipe in such a way that they become inflated with air when the bird utters its wonderful note. In the White Ibis the face only is bare, in the spoon- bill the head and face, and the whole head and neck in the Marabou Stork; the effect of this condition in the latter bird being heightened by the enormous pouch which hangs suspended from the neck. The same is true of the Adjutant. A close inspection of the neck of one of these storks will show that, while ordinary feathers are absent, there is a scanty covering, here and there, of what looks like soft, curling ringlets of chestnut hair. The resemblance is absolutely perfect, and no naturalist in the world, if shown one of these locks, would say that it came from a bird and not from one of the hair-covered mammals ! FIG. 216. — Head of domestic cock. Extreme development of comb. FIG. 216. — Head of domestic cock. Extreme development of comb. FIG. 217. — Head of domestic cock. Extreme development of crest. 27 FIG. 217. — Head of domestic cock. Extreme development of crest. Heads and Necks Heads and Necks We must pass by all the strange ornaments of horn upon the heads of birds, such as the scarlet plate of the gallinules, the immense recurved casques of the hornbills, the use of which is as yet unknown, unless it be purety ornamental. But the impressive helmets of the cassowaries, FIG. 218.— Head of Wild Turkey. FIG. 218.— Head of Wild Turkey. Fig. 228, demand especial notice in this volume, as being very useful adaptations to life in a dense forest. These great running birds are the only members of their Sub-class which inhabit thickly forested regions, and in speeding with great leaps and bounds through the undergrowth, the tall, smooth helmet of horn protects the head of the bird The Bird 2/6 FIG. 219.— Head of Adjutant. FIG. 219.— Head of Adjutant. FIG. 219.— Head of Adjutant. FIG. 220. — Ringlet of hair from the neck of an Adjutant. FIG. 220. — Ringlet of hair from the neck of an Adjutant. Heads and Nec 277 and shunts off the hanging lianas and vines which would otherwise impede its progress. In our hasty paragraphs we have seen to what a re- markable extent the ornamentation of the heads of birds is carried, and as many extraordinary examples could be given of decoration of the neck. The Loon has a speckled black and white throat with a long colour band- age of black feathers wound about its neck; the cervical ruffs of our Ruffed Grouse are like wings in miniature; in the Golden and Amherst Pheasants this form of orna- ment is extended into a circular ruff of black and gold and black and white respectively; while in the Superb Bird of Paradise a shoulder-cape flares back, large enough to cover almost the entire body of the bird, giving it the appearance of being clothed in two distinct sets of plu- mage! The nuptial attire of the Ruff, a species of sand- piper, is as greatly developed, except that it forms a double cloak over the breast. This cloak or shield of feathers in the Ruff plays a vital pa*rt in the life of the bird. We must first notice the remarkable variation in the pattern of this cloak of battle, — for such it really is. Heads and Necks If we could see fifty Ruffs standing side by side, some would be seen to have ruffs of pure white, others of gray, black, orange, buff, or chest- nut, while the waving ear-plumes are also independent in colour, varying from white to purple, green, or blue. Then there is a type of Ruff with barred cloaks, another with spotted patterns, and so on in almost endless variation. This condition of affairs is wholly unlike the uniform pattern of colouring of other wild birds. The Bird 278 FIG 221. — Lady Amherst Pheasant. FIG 221. — Lady Amherst Pheasant. FIG. 222.— Casque of Hornbill. FIG. 222.— Casque of Hornbill. Heads and Necks We can only compare these little Joseph-coated birds with the unnatural sports among domestic poultry and pigeons. pigeons. But whatever their colour, these Fighting Snipe find their ruffs of service in their encounters at the breeding season. Four male birds which I observed in captivity FIG. 223.— Ruff with battle-cloak partly moulted. FIG. 223.— Ruff with battle-cloak partly moulted. were adorned respectively, — plain gray, dotted gray, chestnut barred with black, and a rich golden rufous. Though no females were present, yet their fighting instinct often cropped out and a pair of them would dart and side- step about each other, bills held low and far advanced, ruff spread out from the breast and trailing low, hiding almost the whole body. Now and then one of the fencers would make a vicious dash, sending his bill through the were adorned respectively, — plain gray, dotted gray, chestnut barred with black, and a rich golden rufous. Though no females were present, yet their fighting instinct often cropped out and a pair of them would dart and side- step about each other, bills held low and far advanced, ruff spread out from the breast and trailing low, hiding almost the whole body. Now and then one of the fencers would make a vicious dash, sending his bill through the The Bird 280 feather shield of his opponent. But the force of the blow would spend itself on the inch of space between the shield and the feathers of the bird's breast. When, in his native haunts, the Ruff has conquered his rival, his triumphant dances before the female are most elaborate. While these facts are not exactly pertinent to the physical FIG. 224. — Breast ornament of a Wild Turkeycock. FIG. Heads and Necks 224. — Breast ornament of a Wild Turkeycock. life of the bird, yet I mention them to show to what prac- tical, as well as aesthetic, uses the development of some portion of the bird's plumage may be devoted. portion of the bird's plumage may be devoted. What a contrast to the cloak of the Ruff is the pectoral decoration of the Wild Turkeycock: a great tuft of coarse, black hair-like feathers, like the tail of a horse Heads and Necks 281 in miniature, growing almost a foot in length from the centre of the breast ! The length of the neck of birds is often correlated with that of the legs, — a long-legged bird of necessity re- quiring a long neck to permit its bill to reach the ground. Geese and swans are an exception, and in their case we FIG. 225. — Flamingoes Correlation of long neck with long legs. FIG. 225. — Flamingoes Correlation of long neck with long legs. find that the long, mobile neck is of great use in making up for the awkwardness of their waddle when on land, and in allowing them to reach beneath them while floating in shallow water, thus feeding along the bottom. Herons are uniformly so light of body that they would have difficulty in steadying themselves in the air, were it not that, when in flight, their necks become compressed to an incredible thinness, thus acting as does the cut- The Bird 282 water of a ship's prow. The perpetual crook in the necks of these birds is significant of their method of fishing — a patient watch until the prey comes within striking distance- In the snake-bird this crook, or Z-shape, has, by the it FIG. 226. — Swan. Correlation of long neck and short legs due to feeding habits. (Sanborn, photographer.) FIG. 226. — Swan. Correlation of long neck and short legs due to feeding habits. (Sanborn, photographer.) adaptation of three of the neck-bones, become a veritable trigger, by the springing of which the bird literally spears the fish. Heads and Necks If the mention in this chapter of a few examples of crests and other decorations has seemed in the least to Heads and Necks verge upon the monotony of a mere catalogue, my plea is that they have been cited with the intention of empha- sizing the fact of the remarkable degree which decoration, pure and simple, plays in courtship. Viewed from such a standpoint, these facts and comparisons become im- portant data in the observation of the courtship of birds, FIG. 227. — Snake-bird, showing crook in neck. FIG. 227. — Snake-bird, showing crook in neck. which in its turn is one of the most important and interest- ing corollaries of the psychology of these beings. Whether female birds have highly developed aesthetic feelings, or whether the songs and dances and colour masses act more along the line of the passes of a hypnotist, is yet to be ascertained. It is also hoped that a realization of the more immedi- The Bird 2 ately practical uses of such structures as the cassowary's horny helmet, the feather shield of the Ruff, perhaps the crest of the kingbird, and many others as yet unknown, will impel amateur observers to further efforts in the investigation of the life-habits of birds. THE BODY OF A BIRD N , N experimenting with balloons and flying-ma- chines, weight is a question of prime import- , ance, and among birds there seem to be certain limits to the bulk of the body, beyond which flight is impossible. The tiny hummingbirds, with bodies weigh- ing less than some insects, have remarkable powers of flight, and throughout all the groups of larger birds we find certain species with exceptional flight ability, until in the birds of widest extent of wing, such as the condor and the albatross, flight seems to reach the acme of perfec- tion. But the flying birds of actual heaviest bulk are perhaps the Wild Turkey, the Great Bustard, and the Trumpeter Swan, the two latter reaching weights of thirty- two and twenty-five pounds respectively. Even the gigantic Pterodactyls, those flying reptiles of olden time, some of which had heads a yard long, and an expanse of eighteen feet or more of bat-like wings, are estimated to have weighed but twenty pounds or thereabouts. 285 But when the necessity for flight ceases, a bird may begin to assume larger . proportions and greater weight without detriment; just as a mammal which adopts a life in the dense medium of water may attain a much more 285 The Bird 2 gigantic size than one which has to support its body in the thinner atmosphere: a whale is to a horse as an ostrich is to a dove. The ostrich is the largest of all living birds, a full- grown male being able to reach to a height of nine feet and weighing as much as three hundred pounds; but even these figures were exceeded by its extinct relative of Madagascar, the moa, whose height is variously esti- mated at from ten to eleven feet, and whose massive leg- bones show that its weight must have been much greater than that of the ostrich. There is a great difference in the relative condition of the body in various birds. Herons, even when fish are abundant, with opportunities of feeding from morn- ing to night, are thin to emaciation. Truly they belong to the "lean kine." A fat heron would be an anomaly. On the other hand, the flesh of many sea-birds seems as constantly encased in thick, oily layers of fat. THE BODY OF A BIRD Petrels are used by the inhabitants of some islands as candles, simply by threading the body of the dead bird with a wick, the excess of fat burning steadily until the whole is consumed. Penguins are well protected against the icy waters of their Antarctic home by a layer of fat under the skin, so thick in proportion to their size as to remind one of the blubber of whales. If we were writing of the bodies of the fur-bearers instead of birds, we would have much to say concerning the various kinds of scent-glands and secreted odours; but in birds the only gland is that above the tail, which furnishes the oil with which the bird preens its plumage, The Body of a Bi 287 thus both cleansing it and rendering it water-proof. That birds, and especially those which, like quail, are found in flocks, possess odours is borne witness to by the ability of dogs to point successfully the hidden game; but that this is of much use in enabling the birds to find one another is doubtful, both from the fact of the slight development of the sense of smell, and because of the loud call-notes which are so characteristic of these birds. One exception, however, may be noted, that of the apteryx, which is said to have a strong and persistent odour, with corre- spondingly well-developed nostrils. spondingly well-developed nostrils. Again, among fur-covered animals we find usually a poor development of the sense of sight and but few of them exhibit bright colours, while, as we have seen, birds excel in the power of seeing, and, correlated with this, possess an unparalleled array of colours upon the body. There are many ways in which the body or its feathers are adapted to aid the bird in some special way. For example, the Puff-back Shrike of Africa has a habit of suddenly puffing out and erecting a patch of long, loose, white feathers on its back, giving the appearance of a large powder-puff, an act so startling and unexpected being well calculated to make any attacking hawk or other bird hesitate. The general texture of the body feathers is usually an accurate index to the bird's power of flight. THE BODY OF A BIRD Although the feathers of the breast and back are never as compact or as stiff as those of the wings and tail, yet in birds of good flight their barbs are quite firmly connected. In a 288 The Bird small African bird, called from its habits the Rock-jumper, the wings are so small that the power of flight is almost nil, and we find an interesting corollary in the plumage, FIG. 228. — Cassowary, showing the loose plumage of a flightless bird. (Sanborn, photographer. Courtesy of N. Y. Zoological Society.) FIG. 228. — Cassowary, showing the loose plumage of a flightless bird. (Sanborn, photographer. Courtesy of N. Y. Zoological Society.) which is so loose and fluffy that it blows about in the least wind. In the ostrich and rhea this down-like char- acter is still more noticeable and extends even to the feathers of the wings and tail. The extreme is to be found which is so loose and fluffy that it blows about in the least wind. In the ostrich and rhea this down-like char- acter is still more noticeable and extends even to the feathers of the wings and tail. The extreme is to be found The Body of a Bi 289 in the apteryx and emeu (Fig. 23). Compare a feather of the latter with one of a condor and the difference is remarkable. So unfeatherlike is the emeu's plume and so loose are its barbs that it brings to mind the much- divided leaflets of an Acacia. The plumage of the snake-bird is inexplicable. This bird is so emphatically aquatic that we would expect a dense, compact covering of the body; but in reality it more nearly resembles hair or fur, soaking through so quickly and thoroughly that, after immersion for some time, the bird becomes waterlogged and has to hang itself out to dry by seeking some sunlit perch, opening wide its wings and waving them to and fro. The feathers of the penguin are small, flat, and rigid, approaching in these respects the scales of fishes — an interesting reacquinng of characters consequent upon an all but wholly aquatic life. It is interesting to com- pare the colouring of such a bird as the Scaled Partridge with a fish like the Carp, the dark margins of the feathers and scales bringing about a remarkable resemblance. THE BODY OF A BIRD Taking up the subject of colour in general, we realize, after even a superficial glance at a collection of birds, that in gorgeousness of hue and diversity of shade and pat- tern, they are to be compared only with insects. In a former chapter we have briefly considered the chemical and optical causes of colour in feathers; but the causes due to environment (using that word in its widest sense) cover a vastly greater field and one as yet comparatively unexplored. Advancement of actual knowledge of any subject in The Bird 290 science depends upon two things: first, the accumula- tion of facts; and secondly, a philosophical spirit capable of generalizing and bringing order out of the chaos of of generalizing and bringing order out of the chaos of FIG. 229.— Breast of Scaled Partridge. FIG. 230. — Carp, a fish with distinctly marked scales. (Keller, photographei.) these myriad observations. A knowledge of museum facts is of but slight use in such a subject as the one under consideration, which requires more adequate knowledge FIG. 229.— Breast of Scaled Partridge. FIG. 229.— Breast of Scaled Partridge. FIG. 229.— Breast of Scaled Partridge. FIG. 230. — Carp, a fish with distinctly marked scales. (Keller, photographei.) FIG. 230. — Carp, a fish with distinctly marked scales. (Keller, photographei.) these myriad observations. A knowledge of museum facts is of but slight use in such a subject as the one under consideration, which requires more adequate knowledge these myriad observations. A knowledge of museum facts is of but slight use in such a subject as the one under consideration, which requires more adequate knowledge The Body of a Bird 291 than we now possess of the life-habits and the psychology of birds. This is especially true of the great number of cases which we can explain only by calling them orna- mental and decorative. Hence we find one ornithologist FIG. 231. — White-throated Sparrows. The light-coloured bird is in normal plu- mage; the dark bird was subjected to moisture-laden air through two moults. FIG. 231. — White-throated Sparrows. The light-coloured bird is in normal plu- mage; the dark bird was subjected to moisture-laden air through two moults. explaining a certain colour as due to one cause, while another scientist gives an entirely different interpretation of the same fact. THE BODY OF A BIRD From personal observation among the birds of the The Bird 292 New York Zoological Park, I have had opportunity to record many cases of the effect of food upon colour. An experiment very commonly known is that of feeding canaries on red pepper, thus causing their plumage, after FIG. 232. — Variation due to climate, etc., in races of North American Song Sparrows (From a photograph provided by the American Museum of Natural History.) FIG. 232. — Variation due to climate, etc., in races of North American Song Sparrows (From a photograph provided by the American Museum of Natural History.) successive moults, to become of an intense orange colour. This is the more remarkable since the actual red pigment, or capsicin, of red pepper is not the direct cause of the canaries' changed hue, but a fatty substance known as triolein, which is a constituent of the pepper. The Body of a Bird The Body of a Bird It is generally thought that the fact that, in captivity, Purple Finches and orioles frequently moult into yellow- ish hues, instead of their rightful tints, is due to some change in food. Indeed in many species the bright colours are wholly lacking after a year or two in captivity. But I have transferred a male Purple Finch, which had for several years moulted yellow, from a dark cage to one which was exposed to bright sunlight, and in one moult the bird as- sumed his original and normal colour. FIG. 233.— Effect of environment on Bob- white, shown by specimens from Min- nesota, Florida, and Cuba. (From a photograph provided by the American Museum of Natural History.) A more probable ex- ample of the effect of food upon colour is seen in our American Flamin- goes. In captivity these birds fade out moult by moult, until they become almost white, like the European species. By mixing with their food a quantity of some strong but harmless dye, I have had them either retain their original colour for years, or at least the fading process has been appreciably lessened. FIG. 233.— Effect of environment on Bob- white, shown by specimens from Min- nesota, Florida, and Cuba. (From a photograph provided by the American Museum of Natural History.) FIG. 233.— Effect of environment on Bob- white, shown by specimens from Min- nesota, Florida, and Cuba. (From a photograph provided by the American Museum of Natural History.) FIG. 233.— Effect of environment on Bob- white, shown by specimens from Min- nesota, Florida, and Cuba. (From a photograph provided by the American Museum of Natural History.) The effect of climate upon colour is even more readily proved, and may be noticed in wild birds as well as in those in captivity. In regions which have a very dry The Bird 294 climate, the birds, and in fact all of the animals, are of a much lighter hue than those living in an atmosphere of great humidity, where moisture does not readily evapo- FIG. 234. — Male Scarlet Tanagers, showing moult from the scarlet summer dress, (a), through the parti— coloured garb (6), into the green winter plumage (c). FIG. 234. — Male Scarlet Tanagers, showing moult from the scarlet summer dress, (a), through the parti— coloured garb (6), into the green winter plumage (c). rate. The Body of a Bird In such a place birds tend to be very dark-coloured. In the case of captive birds, I have seen White-throated Sparrows and Wood Thrushes become almost like black- rate. In such a place birds tend to be very dark-coloured. In the case of captive birds, I have seen White-throated Sparrows and Wood Thrushes become almost like black- The Body of a Bird birds in colour when confined in a bird-house where the air was constantly moist. Correlated with the effect upon colour is often a difference in size, and in many instances among birds the more northerly individuals are larger, those inhabiting warmer regions being less in stature. Among wild birds, the Quail, or Bob-white, shows an almost unbroken series from the northern, light-coloured variety, ten inches in length, to the Cuban bird, very FIG. 235. — Siberian Black Lark, male bird in the spring. FIG. 235. — Siberian Black Lark, male bird in the spring. much darker in shade and measuring only eight inches from beak to tip of tail. The race of Bob-whites seems very susceptible to climatic influence; as in Mexico there are nearly a dozen different geographical races, each in- habiting a distinct portion of the country. Many other wide-spread groups of birds, such as the Song Sparrows, vary in a similar manner. It is strange what a marked effect this greater or less amount of moisture has upon birds, even in very limited districts. A South Ameri- can pipit, the individuals of which spend their lives on very circumscribed plots of earth, exhibits two colour much darker in shade and measuring only eight inches from beak to tip of tail. The race of Bob-whites seems very susceptible to climatic influence; as in Mexico there are nearly a dozen different geographical races, each in- habiting a distinct portion of the country. Many other wide-spread groups of birds, such as the Song Sparrows, vary in a similar manner. It is strange what a marked effect this greater or less amount of moisture has upon birds, even in very limited districts. A South Ameri- can pipit, the individuals of which spend their lives on very circumscribed plots of earth, exhibits two colour The Bird 2 forms entirely different, and thought to be due solely to the amount of moisture in the ground on which it lives. The Body of a Bird Very dark-coloured and very pale individuals live within a few hundred yards of each other, in dry and swampy situations respectively, each, it is said, keeping entirely to its own little beat. We are all fairiliar with the changes of colour due to FIG. 236. — Nighthawk perching lengthwise on a fallen branch. FIG. 236. — Nighthawk perching lengthwise on a fallen branch. age, as, for instance, in the young Rose-breasted Gros- beaks, which are very different from the male parent, and the young Bald Eagles, which lack the white colour of the feathers of head and tail. Certain wild pigeons show marked differences in colour patterns between the young birds and the adults, and very good evidence of the gradual evolution which must have preceded these changes is The Body of a Bi 297 to be found by plucking out a few of the feathers of the young bird. Those which replace the ones pulled out will show intermediate stages, which have long since been dropped from the sequence of patterns, as observed in the regular moults of the birds. Another important phenomenon is the seasonal moult, which was spoken of in the chapter treating of feathers- In the fall of the year the brilliant Scarlet Tanager assumes the olive-green dress of the female, and the Indigo Bunt- ing and the Bobolink likewise don the dull garb of their mates. There is another very interesting cause of change in colour, namely, the wearing off of the brittle tips of the feather-vane. An excellent example of this is seen in the Snowflakes, which come south in the depth of severe winters, flying in small flocks about our fields, like an animated flurry of the actual crystals. When we see the birds at this time they are brownish and brownish white. In the spring, in their northern home, they change to a clear-cut black and white, not by shedding the entire plumage, but merely by the breaking off of the brown feather-tips. By a similar process the Bobolink changes from the buffy female dress to his rich black-and-white spring suit, and, as we saw in Chapter II, Fig. 35, the English Sparrow gains his cravat of jet. English Sparrow gains his cravat of jet. The Body of a Bird Another excellent example is found in the Black Larks of Siberia, the males of which, in winter, are of an almost uniform sandy colour, like a Skylark, but by the wearing off of the buff tips of the feathers, the birds become jet-black in the summer — a most remarkable and radical change. The Body of a Bi The Body of a Bi 299 The relation of a bird's colours to its haunts and its habits of life is a subject of intense interest. This is, of course, not in the same category as the subjects of the foregoing paragraphs, but indeed includes them all. The most common class of colours is known as protective. These are such that the bird resembles its environment or surroundings and is thus given a better chance of escap- ing the observation of its enemies. It is evident that, in a study of this nature, observation of the bird in its natural haunts is of far greater value than any other method. We find that the majority of sparrows, sandpipers, and quail are gray or brown, like the grasses, sedges, and leaves among which they live; while the birds which spend their lives higher up among the branches of trees are greenish, or at least more brightly coloured. are greenish, or at least more brightly coloured. Many birds which are protectively coloured are dark above and white or whitish beneath. The significance of this pattern of coloration has been beautifully demon- strated by an American artist, Mr. Abbott Thayer. His experiment, which is as follows, may be repeated by any one: Take two wooden decoy ducks, and place them against a sand-bank. Colour one the exact tint of the sand, or even coat it with that substance. Repeat this with the upper parts of the second decoy, making its back darker than the surrounding sand, but grade the under part of this one to pure white below. At a little distance away, decoy number one will still be distinctly seen; while number two will absolutely disappear, merg- ing perfectly into its background. The reason for this is The Bird 00 300 that the conspicuous white of the under surface of the second bird is, when normally lighted up by the sun, neutralized by the shadow of the bird, and the darker upper parts are softened and toned down by the strong direct light; while if the entire bird be unshaded, although FIG. 238.— Sooty Tern on her nest. FIG. 238.— Sooty Tern on her nest. coloured like the environment, the dark shadow beneath will reveal it clearly. The Body of a Bi Whether or not birds really appreciate the value of the protective colour of their plumage, it is certain that a quail or ptarmigan will remain crouching on a brown bit of turf, until all hope of evading danger is gone; while birds which are very evidently not protectively coloured are invariably more wary and difficult of approach. When FIG. 239. — Seven young Flickers clinging to a tree. (R. H. Beebe, photographer.) FIG. 239. — Seven young Flickers clinging to a tree. (R. H. Beebe, photographer.) The Bird 302 a ptarmigan, while yet in the brown garb of summer, is exposed against a hillside of snow, it becomes very wary. It is interesting, in this connection, to observe how a Nighthawk carries out its colour resemblance to a knot or a rough piece of bark, by perchiyg, not crosswise, but lengthwise, along a branch or fallen tree-trunk. lengthwise, along a branch or fallen tree-trunk. A volume might easily be written of the various ways in which protective coloration works out among birds, but there is so great a difference of opinion, and indeed so many exceptions to every theory which may be ad- vanced, that it is better, for the most part, to go to Nature without a priori theories, and putting ourselves as nearly as possible in the position of the creatures themselves, to hope for better ability to see with their eyes. And it is right along this line that we most need fresh data and experiments, namely, the actual ability of birds and insects to distinguish shades, colours, forms, and motion, — whether efficient in certain ways or not. We know that many men cannot distinguish a scarlet ball lying upon green grass; that is, they are partly colour- blind. If this were the case with certain hawks, a male Scarlet Tanager would be forever safe from them among the green foliage. An important fact, which for years had been appar- ent to me, but unexpressed until Mr. Abbott Thayer put it into words, is that colours which we would ordinarily term conspicuous are often exactly the opposite when found in the plumage of a bird. The Body of a Bi Writing of the Motmot in my volume "Two Bird-lovers in Mexico," I say: "I have often The Body of a Bird 3°3 wondered, when I saw mounted specimens in museums, with what special immunity from danger these birds were blessed, their beautiful colouring would seem to be such a startling advertisement of the bird's whereabouts. But in reality the very diversity in hue is their protec- tion, and they merge per- fectly into their environ- ment of green foliage and bright sunlight." FIG. 240. — Brown Creeper circling up the trunk of a spruce. bright sunlight." Indeed absolute uniform- ity of coloration instantly reveals the outline of the bird entire, and renders it very conspicuous. Birds which have but few ene- mies are often thus mono- crome in hue. But look at the photographs and see how a broken colora- tion baffles the eye. If the Sooty Tern, Fig. 238, were totally black, it would be conspicuous even against a patch of dark-coloured mottled shingle. But the transverse lines of white across the back totally destroy the symmetry of form, while the white wing-edges fairly force the eye to call them, not part of a bird sheltering her eggs, but only two among a myriad irregular edges of coral rock! FIG. 240. — Brown Creeper circling up the trunk of a spruce. FIG. 240. — Brown Creeper circling up the trunk of a spruce. Observe closely the seven young Flickers clinging to their natal stump. As the warriors of Jason sprang forth from the ground fully armed, so the very bark, mottled The Bird 3°4 with spots of lichen and sunlight, seems to have gendered these baby birds. Yet they were hatched in a dark hole from the whitest of white eggs. Is this and a thousand of other resemblances to be termed accidental? Then is all Nature one great accident! When the Flicker flies with swift wing-beat from tree to tree, then the white FIG. 241. — Laughing Gull on nest. FIG. 241. — Laughing Gull on nest. rump blazes forth. At such moment no protection is needed; but in these young Flickers upon the tree-trunk, how exquisitely do their spots deceive the eye! The Body of a Bi They are, we say, perhaps sunlight splashes, — nothing more- Yet others which, like the Brown Creeper, haunt the tree-trunks of the forest, seem veritably to be but stray bits of roughened bark creeping here and there. tt tt tt The Bird 3 Let us glance at one more bird upon her nest, — a Laugh- ing Gull. At a distance we see a shapeless blotch of white sand among the reeds, that is all. We walk over a hundred other similar patches; but when near enough, we at last are able to distinguish the dark head and wing-tips, all but invisible among the shadows, and even through the centre of the head we can see two spots of light be- yond,— or no, it is the little subtle ring of white about the eye! Two majestic Black-necked Swans may swim closely along in full view near the opposite bank of a pond, and yet be totally unrecognizable; showing to the eye as bodiless necks or neckless bodies, according to the chang- ing conditions of light and shade around them. ing conditions of light and shade around them. We see a troop of ostriches rushing past. Surely nothing could hide birds such as these! Again we see one of these birds prone upon the ground, and a mighty creature towering eight feet or more above the earth, becomes naught but a dark ant-hill, which the photo- graph picks out clearly, but which in the desert, dotted with ant-hills, would seldom be noticed even by the hungri- est of lions. Of course, like most other theories, this of protective coloration can easily be carried too far, but there are hundreds of instances where it seems to answer every requirement of the case. Few fields offer such opportuni- ties for original work of the most delightful character. As one example out of untold numbers, what explanation can we give of the Blood-breasted Pigeon or Bleeding- heart Pigeon, which, as its name denotes, has a splash The Body of a Bird 3°7 FIG. 243. — Group of Ostriches on the run. (Cawston, photographer.) FIG. 243. — Group of Ostriches on the run. (Cawston, photographer.) FIG. 244. — Ostrich as it hides from an enemy. FIG. 244. — Ostrich as it hides from an enemy. The Bird 8 3o8 of blood-like scarlet in the centre of its breast? The Body of a Bi The re- markable and inexplicable resemblance is heightened by the stiffened vanes of the centre feathers, causing them to appear bedraggled and clotted, as if by an actual wound ! The photograph does but little justice to the bird's real appearance. appearance. Another class of colours, while still protective, is so for a purpose very different from those cases which we FIG. 245. — Bleeding-heart Pigeon. FIG. 245. — Bleeding-heart Pigeon. have been considering. The colours which we are now to mention have been aptly called aggressive colours, as, by their means, a bird of prey is enabled to approach its victim more easily. So, throughout the entire animal world we find two phases of phenomena constantly pres- ent: on the one hand the pursued ones, striving to escape by all means in their power; and on the other hand the pursuers, ever trying to outwit those upon which they prey. If a duck acquires great speed of flight, the Duck have been considering. The colours which we are now to mention have been aptly called aggressive colours, as, by their means, a bird of prey is enabled to approach its victim more easily. So, throughout the entire animal world we find two phases of phenomena constantly pres- ent: on the one hand the pursued ones, striving to escape by all means in their power; and on the other hand the pursuers, ever trying to outwit those upon which they prey. If a duck acquires great speed of flight, the Duck The Body of a Bi 309 Hawk must learn to fly still faster. If the duck learns to crouch close to the reeds when his flight-feathers are moulted and he is helpless, the hawk must develop ever sharper eyesight. We may puzzle and puzzle over a characteristic habit or a colour of some bird, finding no solution, until we discover some special enemy or other factor in its life which makes all clear. So, among aggressive colours we may mention the garb of the penguin, which is steel-gray on the back and silvery white below; not to protect it from danger, but to enable it the better to approach fish without alarming them. The Body of a Bi It is curious how fish-like the coloration of these birds really is, and they are said frequently to lay feet and tail together and, drawing their flipper-like wings to their sides, spring clear of the water again and again, by a single motion of the back muscles, exactly as the mammalian dolphins leap ahead of a vessel's bow. mammalian dolphins leap ahead of a vessel's bow. Again, while we find the ptarmigan mimicking the snow in colour, we find the Arctic Fox, the Snowy Owl, and the Gyrfalcon, all of which are enemies of this bird, also garbed in white. The ptarmigan may crouch upon a drift, but it must ever be on the alert, lest from amid the snowflakes a white death come suddenly upon it. Nature is terribly just in her plan of life's battles. Nature is terribly just in her plan of life's battles. In the same region with these lives the Ivory Gull, immaculate as the ice-floe over which it flies, and in its whiteness we can perhaps read two purposes: a better chance to elude the fierce Gyrfalcon, and a better chance to float cloud-like unperceived over the unsuspecting fish which it seeks for food. FIG. 246.— Black footed Penguin. FIG. 246.— Black footed Penguin. FIG. 247. — Pickerel. (Keller, photographer. From life, swimming.) PREDACIOUS, AQUATIC ANIMALS, SHOWING AGGRESSIVE COLORATION. FIG. 247. — Pickerel. (Keller, photographer. From life, swimming.) PREDACIOUS, AQUATIC ANIMALS, SHOWING AGGRESSIVE COLORATION. FIG. 252.— Young Robin. FIG. 252.— Young Robin. FIG. 253. — Fallow Deer fawn one day old. BOTH SHOWING SPOTS WHICH ARE ABSENT IN THE ADULTS. FIG. 253. — Fallow Deer fawn one day old. BOTH SHOWING SPOTS WHICH ARE ABSENT IN THE ADULTS. The Body of a Bi 3 1 7 thickly spotted. This gives a clue to the coloration of its ancestors, — birds probably resembling our Wood Thrush, and lacking the rufous, immaculate breast of the parents. We find a similar condition existing among many deer, whose young are spotted, entirely unlike the brown coats of their parents. FIG. 254. — Nestling Turkey Vulture. (T. H. Jackson, photographer.) FIG. 254. — Nestling Turkey Vulture. (T. H. The Body of a Bi Jackson, photographer.) In many cases the colouring of the downy young is the opposite of the adult, as in the Turkey Vulture, the nestling being clad in down of purest white, and ultimately moulting into the blackish plumage of the parent birds. It would be out of place in this volume to speak further of the wonderful colours which the Class of birds, as a The Bird 3 1 8 whole, exhibits, or of the beautiful plumes which, as in the case of the Snowy Egret, are assumed only during the season of courtship. The great majority are now explained either as decorations to charm the female, or as mere by-products of the vitality of the bird, according as to whether one believes in a greater or less degree of aBsthetic appreciation among birds. When we consider the nervous, high-strung natures of birds and realize with what ease they are thrown into what seems a kind of trance, it seems unnecessary to credit them with too great an appreciation of pure beauty. The repetition of many similar bright spots, as, for example, the eyes of a pea- cock's train, may well serve to attract and hold the atten- tion of the female; while the antics and sounds which many birds bring into play in courtship may appeal in some more directly psychic way than we know. That birds do have a certain appreciation of beauty and har- mony there can be little doubt. When we remember the jarring discords and clashing tints in which a human savage takes delight under the name of music and beauty, we should be very willing to admit some degree of appreciation to the demure Impeyan Pheasant hen which chooses among her suitors, clad each in hues such as artist could never imitate; or the fair Hermit Thrush, which selects a singer from the incomparable choir of her serenaders. I believe that future field study and experi- ments with caged birds will reveal much that we do not suspect in regard to the causes of coloration. * Extremes in regard to the number of primaries are the three flightless groups, penguins with approximately 36, ostriches with 16, and cassowaries with perhaps but 2 feathers which can be called primaries. WINGS E EFORE the front limbs of any creature had be- come adapted to flight through the air, they served to assist the hind legs in locomotion on the ground, and, ages before this, a many-rayed mem- brane stretched across the primitive fin, aided its owner in cleaving a way through the water. So, like a palimp- sest, if we look beneath the outer covering of feathers, we see, in the wing of the modern bird, the three fingers hinting of widely different ancestral habits. hinting of widely different ancestral habits. The general structure and appearance of the bills, the feet, and wings of various birds is the result of a function characteristic of each. The bills are used to procure food, the feet to walk or perch, and the wings to propel the bird through the air. But, as we have seen in the case of the bill, these organs are put to many other uses besides the one for which they were primarily adapted. This is only what we should expect when we consider the relative high position which avian intelligence holds, and the remarkable extremes of environment with which these structures — bill, feet, and wings — are brought into close touch. The photograph of the young heron's wing shows sho The Bird 320 320 the two principal divisions into which the flight-feathers are divided: the primary feathers, or those growing on the fingers and wrist-bones, and the secondaries which sprout from the bone of the forearm. The several feathers FIG. 255. — Young Green Heron, showing various divisions of wing-feathers. FIG. 255. — Young Green Heron, showing various divisions of wing-feathers. supported by the thumb are also very distinctly shown. When a wing is greatly elongated it is the secondary feathers which are increased in number, the two extremes being represented by the hummingbird and the albatross, Wings 32 Wi 321 each of which has ten primaries, but the one has six and the other forty secondaries (Figs. 259, 260).* the other forty secondaries (Figs. 259, 260).* Let us observe the wings of living birds in the woods and fields or in a zoological park and see what of interest we can discover. We have all noticed how well adapted FIG. 256. — Great White Heron stretching its wing. (E. R. Sanborn, photographer.) FIG. 256. — Great White Heron stretching its wing. (E. R. WINGS Sanborn, photographer.) to its owner's many uses is the foot of a parrot — how hand-like it is, — and now if we again watch one of these birds we will see that, as we should expect from its being to its owner's many uses is the foot of a parrot — how hand-like it is, — and now if we again watch one of these birds we will see that, as we should expect from its being The Bird 2 322 so much like a human hand, it is not a good walking foot. When a parrot is in great haste to reach some object on the ground without flying, it waddles awkwardly, " toe- ing in" and frequently tripping up. When this happens, out fly the wings, and, as if reverting to some clouded memory of the habits of its pre-Jurassic forefathers, it walks on all fours. A young Canada Goose, when climb- ing about its nest, or a Fish Hawk in the downy nestling FIG. 257. — Nestling Catbird, supporting itself, lizard-like, on all four limbs. 257. — Nestling Catbird, supporting itself, lizard-like, on all four limbs. plumage, does the same thing, and young birds of many species, when too young to stand, push themselves along the ground with feet and wings; a young grebe doubtless being the most accomplished in this motion. In certain adult birds, such as the swan, Osprey, Turkey Vulture, and the various ostrich-like birds, there are perfect claws at the tips of one or more of the skin-bound wing-fingers. These are true relics of a lizard-handed ancestry. plumage, does the same thing, and young birds of many species, when too young to stand, push themselves along the ground with feet and wings; a young grebe doubtless being the most accomplished in this motion. In certain adult birds, such as the swan, Osprey, Turkey Vulture, and the various ostrich-like birds, there are perfect claws at the tips of one or more of the skin-bound wing-fingers. These are true relics of a lizard-handed ancestry. These are true relics of a lizard-handed ancestry. Before going on to find the more curious uses to which Wings 323 wings are put, we will look at certain birds whose flight can teach us something interesting. WINGS If a pheasant in captivity becomes suddenly alarmed, or its spacious aviary tempts it to rise from the ground, we hear a great whirr, — broad, round-curved wings buzz in a half -circle of haze around the bird and it is off like a shot to the farther FIG. 258. — Young Green Heron, reaching out with its wing toward a branch which it hooked with the sprouting feathers, and steadied itself for a new foothold. FIG. 258. — Young Green Heron, reaching out with its wing toward a branch which it hooked with the sprouting feathers, and steadied itself for a new foothold. end of the runway. It may go right through the sash and pane of glass — such is the impetus gained in this mad rush. Fortunate it is for these birds, and for their cousins, tne grouse and quail, that they can thus spring up and escape from foxes and other enemies to whom their scent so often betrays them. If the pheasant were at liberty, end of the runway. It may go right through the sash and pane of glass — such is the impetus gained in this mad rush. Fortunate it is for these birds, and for their cousins, tne grouse and quail, that they can thus spring up and escape from foxes and other enemies to whom their scent so often betrays them. If the pheasant were at liberty, The Bird 3 we should see that this burst of speed would end in a long, slowly descending sail, and with wings held mo- tionless the bird would sink into the nearest cover. It is most interesting and exciting to walk through a field of tall grass where many pheasants are feeding, and see them shoot up to the right and to the left; a hen with her brood waiting until one's foot is almost upon her before booming away. In a zoological park we may observe another extreme of bird flight by watching a condor take wing. He waits until a breeze is blowing and then, facing the direction from which it comes, he runs with all his might, flapping awkwardly until sufficient headway is gained, when strong downward strokes carry him to the perch he has selected. WINGS We may, at first thought, pity him, but if we could see him soaring for hours high among the cloud-peaks of his native Andes, we should instead pity the low-flying pheas- ant. These two examples — the pheasant and the condor — show what differences may be found in flying birds, and as we examine the wings of other species, we find that each is perfectly adapted to the wants of its owner. A wing is a most delicately adjusted organ; its feathers being just strong enough to lift the body of its owner into the air, and, like evenly balanced scales, the least excess or lack of use is quickly met by a reaction. Com- pare the Black Skimmer of the seas, which is only eighteen inches in length, but whose long wings expand four feet, with a stubby-winged quail or grouse. with a stubby-winged quail or grouse. There are some species of flycatchers with wonderful Wings 325 FIG. 259. — Whig of Hummingbird with 16 flight-feathers. 1/2 natural size. FIG. 259. — Whig of Hummingbird with 16 flight-feathers. 1/2 natural size. FIG. 260.— Wing of Albatross with 50 flight-feathers. 1/28 natural size. FIG. 260.— Wing of Albatross with 50 flight-feathers. 1/28 natural size. The Bird 326 326 powers of flight. When perched on a branch, they can evade the shot from a shot-gun. It is said that one will sometimes "chase another for three or four minutes, doubling, turning, twisting, and shooting, now brushing the grass, now rising to a height of at least two or three FIG. 261. — Condor about to take flight. FIG. 261. — Condor about to take flight. hundred feet, and all the movements so rapid that the eye can scarcely follow them; and at the end of it would go back to his own chosen weed-stalk, apparently without a feather ruffled." a feather ruffled." Any attempt to explain the mechanics of the way of a bird in the air would at most be imperfect in the present Wi 327 state of our knowledge. Suffice it to say that if we will think of a bird flying through the air when we ourselves are swimming in the water, we can realize the achievement more vividly than from any amount of descriptions and diagrams. The under surface of a bird's wing is concave; and FIG. 262. WINGS — Wing of living Golden Pheasant; rounded and curved for short, sudden flight. FIG. 262. — Wing of living Golden Pheasant; rounded and curved for short, sudden flight. while the front edge is rather straight and firm, the hinder rim of the feathers is soft and yielding; thus a downward stroke both raises the bird or holds it sustained at the height already reached and urges it in a forward direction. Similarly we push our hollowed palms backward and pro- pel ourselves through the denser medium of water. The manner of flight varies greatly in different birds The Bird 328 328 and is often so characteristic that when too far off to dis- tinguish the colour of its plumage, or for its notes to reach our ears, the bird may be recognized by the undu- lations or the directness of its flight. No one who has ever visited the tropics can have failed to admire the Fro. 263. — Wing of living Herring-gull; long and narrow for slow, continuous flight. Fro. 263. — Wing of living Herring-gull; long and narrow for slow, continuous flight. soaring vultures, — spots of black swinging across the heavens or swooping low in grand arcs over the palms. Gulls and their kindred fly steadily with continuous wing- beats, which, however, are much less rapid than in the flight of a duck or a parrot. Many sparrows have an abrupt jerking motion, hitching themselves over trees soaring vultures, — spots of black swinging across the heavens or swooping low in grand arcs over the palms. Gulls and their kindred fly steadily with continuous wing- beats, which, however, are much less rapid than in the flight of a duck or a parrot. Many sparrows have an abrupt jerking motion, hitching themselves over trees Wi 329- and bushes; while goldfinches and woodpeckers swing past in long undulations, a loop and a catch, a loop and a catch, — with wings wide extended, then quickly closed. Hummingbirds have a remarkably insect-like flight; the rapid reflex whirr of the wings holding them perfectly still, poised in mid-air. still, poised in mid-air. When ornithologists think that they have formed a correct theory of flight and that, given such and such conditions, certain results must follow, such a bird as the Crested Screamer soars into their mental atmosphere and upsets every calculation. WINGS Such a bulky and short-winged bird, by all good "rules" of flight, should confine itself to short laboured efforts, barely skimming the low bushes of its South American haunts! But it refuses to be thus limited. Of this species it is said : " The Screamer is a very heavy bird, and rises from the ground laboriously, the wings, as in the case of the swan, making a loud noise. Nevertheless it loves soaring, and will rise in an immense spiral until it wholly disappears from sight in the zenith, even in the brightest weather; and considering its great bulk and dark colour, the height it ultimately attains must be very great. On sunny windless days, especially in winter and spring, they often spend hours at a time in these sublime aerial exercises, slowly floating round and round in vast circles, and singing at intervals. How so heavy and comparatively short-winged a bird can sus- tain itself for such long periods in the thin upper air to which it rises has not yet been explained." which it rises has not yet been explained." I find in my journal the following account of a flight of vultures which we saw in a desolate alkali desert in The Bird 33° 33° western Mexico: "One of the most wonderful exhibitions of bird-flight came to us to-day as we left the alkali plain and rode among the mesquite scrub. A confused mass of black appeared in the air which, as we advanced, re- solved itself into hundreds of individual black specks. FIG. 264. — Crested Screamers. FIG. 264. — Crested Screamers. The atmosphere was so deceptive that what at first seemed to be a vast cloud of gnats close at hand, was soon seen to be a multitude of birds, and when a quarter of a mile away we knew them to be vultures. Three burros lay dead upon the plain. This we knew yesterday, and here were the scavengers. Never had we seen Vultures more Wings 33 numerous or in more orderly array. A careful scrutiny through our glasses showed many scores of Black and Turkey Vultures walking about and feeding upon the carcasses of the animals, and from this point there ex- tended upward into the air a vast inverted cone of birds, all circling in the same direction. WINGS From where we sat upon our horses there see ed not one out of place, the FKJ. 265. — Turkey Vulture soaring. FKJ. 265. — Turkey Vulture soaring. outline of the cone was as smooth and distinct as though the birds were limited in their flight to that particular area. It was a rare sight, the sun lighting up every bird on the farther side and shadowing black as night those nearest us. Through one's partly closed eyes the whole mass appeared composed of a myriad slowly revolving wheels, intersecting, crossing each others' orbits, but never breaking their circular outline. The thousands of soaring outline of the cone was as smooth and distinct as though the birds were limited in their flight to that particular area. It was a rare sight, the sun lighting up every bird on the farther side and shadowing black as night those nearest us. Through one's partly closed eyes the whole mass appeared composed of a myriad slowly revolving wheels, intersecting, crossing each others' orbits, but never breaking their circular outline. The thousands of soaring The Bird 3 forms held us spellbound for minutes before we rode closer. Now a change took place, as gradual but as sure as the shifting clouds of a sunset. Until this moment there was a tendency to concentrate at the base of the cone, that portion becoming more and more black until it seemed a solid mass of rapidly revolving forms. But, at our nearer approach, this concentration ceased, and there was perfect equilibrium for a time; then, as we rode up a gentle slope into clearer view, a wonderful ascent began. WINGS Slowly the oblique spirals swing upward; the gigantic cone, still perfect in shape, lifts clear of the ground and drifts away, the summit rises in a curve which, little by little, frays out into ragged lines, all drifting in the same direction, and before our very eyes the thousands of birds merge into a shapeless undulating cloud which rises and rises, spreading out more and more until the eye can no longer distinguish the birds which from vultures dwindle to motes, floating and lost among the clouds." the clouds." Concerning the greatest extent of wing which any bird possesses, there are records of a Wandering Albatross which measured fourteen feet from tip to tip, but the condor of South America exceeds this, certain individuals having an expanse of fifteen feet. Having considered the finest flyers among the birds, we may now begin to go down the scale and see what has happened when certain species have deliberately dis- carded the wonderful power of flight with which Nature has provided them and for which human inventors are so earnestly striving. But always we must remember Wi 333 that this restriction and disuse have been to subserve some good and useful purpose, — food perhaps being more easily obtained, or enemies avoided by terrestrial or aquatic locomotion. Functional radiation, working always for the good of the race, once gave to all birds the power of traversing the globe, passing high over sea and land; but later this was withdrawn, until in some cases their wings have become a mockery. The wings of the Owl Parrot of New Zealand are of full size, but the muscles are so en- cased in fat that they are useless for flight. These par- rots feed on ground-mosses, and being nocturnal and tnerefore having few enemies, their only use for wings is occasionally to sail gently to earth, like a Flying Squir- rel, from the trees in the hollows of which they some- times roost. For this purpose their flabby muscles are perfectly suited. perfectly suited. The Spotted Tinamou of South America is one of a number of birds which have not quite lost the power of flight, but in which, as in the first attempts of a young bird, almost no control is possessed over the direction or height of their flight. WINGS In fact, the condition is much the same as that of a man in an ordinary balloon, who is at the mercy of the wind and the sustaining power of the gas. Hudson gives the following interesting account of this bird: "It is an exceedingly rare thing to see this bird rise except when compelled. I believe the power of flight is used chiefly, if not exclusively, as a means of escape from danger. The bird rises up when almost trod- den upon, rushing into the air with a noise and violence that fill one with astonishment. It continues to rise The Bird 3 at a decreasing angle for fifty or sixty yards, then gradu- ally nears the earth, till, when it has got to a distance of two or three hundred yards, the violent action of the wing ceases, and the bird glides along close to the earth for some distance, and either drops down or renews its flight. I suppose many birds fly in much the same way; only FIG. 266. — South American Tinamou. FIG. 266. — South American Tinamou. this tinamou starts forward with such amazing energy that, until this is expended and the moment of gliding comes, the flight is just as ungovernable to the bird as the motion of a brakeless engine, rushing along at full speed, would be to the driver. The bird knows the danger to which this peculiar character of its flight exposes it Wi 335 so well that it is careful to fly only to that side where it sees a clear course It is sometimes, however, compelled to take wing suddenly, without considering the obstacles in its path; it also often miscalculates the height of an obstacle, so that for tinamous to meet with accidents when flying is very common. In the course of a short ride of two miles, during which several birds sprang up before me, I have seen three of these tinamous dash themselves to death against a fence close to the path, the height of which they had evidently misjudged. I have also seen a bird fly blindly against the wall of a house, killing itself instantly. A brother of mine told me of a very curious thing he once witnessed. He was galloping over the pampas, with a very violent wind blow- ing in his face, when a tinamou started up before his horse. WINGS The bird flew up in the air vertically, and, beat- ing its wings violently, and with a swiftness far exceeding that of its ordinary flight, continued to ascend until it reached a vast height, then came down again, whirling round and round, striking the earth a very few yards from the spot where it rose, and crushing itself to a pulp with the tremendous force of the fall. It is very easy to guess the cause of such an accident: while the tinamou struggled blindly to go forward, the violent wind, catch- ing the under surface of the wings, forced it upward, until the bird, becoming hopelessly confused, fell back to earth. I have often seen a swallow, gull, or hawk, soaring about in a high wind, suddenly turn the under surface of its wings to the wind and instantly shoot straight up, appar- ently without an effort, to a vast height, then recover The Bird The Bird 3 itself and start off in a fresh direction. The tinamou, when launched on the atmosphere, is at the mercy of itself and start off in a fresh direction. The tinamou, when launched on the atmosphere, is at the mercy of FIG. 267.— Feathers of Ostrich and Condor. FIG. 267.— Feathers of Ostrich and Condor. chance; nevertheless, had this incident been related to me by a stranger, I should not have recorded it." me by a stranger, I should not have recorded it." So in this bird we have a most rare and suggestive Wi 337 instance of a condition where an important organ is actu- ally in process of losing its primary function, and in so doing becomes a source of danger to the bird. doing becomes a source of danger to the bird. In the waters of the sea near the Falkland Islands is a duck known as the Steamer or Side-wheel Duck. The young birds of this species are good flyers and whistle through the air on strong pinions. But maturity, instead of bringing, as in most birds, a fully perfected power of flight, takes from them what they have, and after the first moult they are helpless to rise above the great waves of their haunts. However, this duck finds another use for its wings, and the stiffness which forbids their being used in the air makes of them bladed paddles which are all the better for their lack of flying power, and with wings and feet these birds make remarkable speed through the water — "twelve or fifteen miles an hour" — and they are thus able to live out their lives in safety. Thus the study of the flight of these birds carries us a step farther than the tinamou, with the all-important difference that, in this case, loss of the primary function is compensated by a direct adaptation of the wing to tlie new conditions of life. In the ostriches and their near allies the extreme reduc- tion of wings is to be found, and yet in the true ostriches and rheas the great expanse of soft feathers is a consid- erable help to the birds when running at full speed, acting as a sail or aeroplane to assist in the onward motion. The Bird But the contrast between a loose, open-work feather from the wing of one of these birds and a compact, firmly vaned plume from a condor's wing is very striking. The casso- wary has from four to six flight-feathers, but, far from The Bird 3 being of any use in supporting his great frame, they are so vestigial that they look exactly like black slate-pencils projecting in a row from the little fleshy flap which con- tains the evidence of his full-winged ancestors tains the evidence of his full-winged ancestors A full-grown ostrich was once imported to this country from Abyssinia. When the native keepers learned that the bird was to be sent away, they surreptitiously plucked the FIG. 268. — Wing of Cassowary, showing degenerate flight-feathers. FIG. 268. — Wing of Cassowary, showing degenerate flight-feathers. poor creature, until but few feathers were left on its body. The bird was tame, and, by keeping its attention busy with a basket of carrots, I inserted a piece of white card- board beneath one of its skinny, denuded wings and se- cured an excellent photograph (Fig. 269). This clearly shows the black, curved claws on the first two fingers. In this same bird I noticed that occasionally the crooked forearm would be raised, the claw at the end of the wing poor creature, until but few feathers were left on its body. The bird was tame, and, by keeping its attention busy with a basket of carrots, I inserted a piece of white card- board beneath one of its skinny, denuded wings and se- cured an excellent photograph (Fig. 269). This clearly shows the black, curved claws on the first two fingers. In this same bird I noticed that occasionally the crooked forearm would be raised, the claw at the end of the wing Wi 339 drawn up, and the ostrich would scratch its body or head with this interesting finger relic ! When the plume feathers of the wing are full grown, the foot or leg is thus used, FIG. 269. — Wing of Ostrich, showing reptile-like claws. FIG. 269. — Wing of Ostrich, showing reptile-like claws. the head or neck being rubbed against its roughened scales. The Bird The Great Auk — a sea-bird which has become extinct within the last sixty years — was without the power of flight, and its living allies, the Razor-billed Auks and Murrelets, have very small wings and are rather weak The Bird 3 flyers. The latter, in fact, use their wings, the feathers of which have very stiff and long quills, as much in diving under water as in flying in the air, and, strangely enough, they are said to swim breast upward, propelling themselves by means of both wings and feet. Grebes, too, are very weak of wing, and these birds cannot rise from level ground, no matter how much of a fluttering run is taken, and even in the water much splashing and headway are needed. in the water much splashing and headway are needed. Perhaps the most wonderful birds in the world are penguins, and the strangest part of these strange birds is the wing. There is no doubt that they are descended from birds which possessed the power of flight; but the penguins have discarded this gift and have returned to a life in the sea, whence in long ages past their forebears had crawled out upon land. As in the ostriches, the relics of flight-feathers have increased greatly in number, but have become small and scaly, and the wings have virtually become flippers or fins. Instead of a given num- ber of feathers, divided into well-marked series, the pad- dles of a penguin are covered thickly with small feather- scales, and the rigidity of the wings, together with the rotary movement at the shoulder- joint, make the propeller of a ship an apt simile. The colour of the feather-scales on the upper side of the wmg is dark, like the back of the bird, but those on the under side have run rampant, the white and black being mixed irregularly, not corre- sponding even in the two wings of an individual bird. The outline of the wing is exactly like that of a shark's fin, the flatness and breadth including even the bones, while (also like a fin) all of the bending quality of a wing The Bird 2 342 is lost, — all the flexibility of wrist and elbow. With these propellers the penguins fly through the water, with almost the identical motion of a bird in the air. The Bird Though it is usually asserted that the wings move alternately, this was never the case with a pair of Black-footed Pen- guins which I carefully observed. As regards the speed of swimming, I found that one of these birds, though in bad health at the time and so weak that it could take but a few steps on land, was able to progress under water FIG. 271. — Penguin swimming with its wings. FIG. 271. — Penguin swimming with its wings. considerably faster than a man could walk an equal dis- tance on land. The greatest speed was about seven miles an hour; but I have no doubt that when in full health this rate can be far surpassed. Surely no fairy-tale can match the marvellous evo- lution of a penguin's wing : fin becoming hand, hand evolving into wing, and wing reacting to the environ- ment of long ago and again taking on all the outward characteristics of a fin! Wings 343 FIG. 272. — Wing of Black-footed Penguin, top view. FIG. 273.— Wing of Black-footed Penguin, side view. FIG. 272. — Wing of Black-footed Penguin, top view. FIG. 272. — Wing of Black-footed Penguin, top view. FIG. 273.— Wing of Black-footed Penguin, side view. FIG. 27-4. — Wing of Black-footed Penguin, under surface of wing. ALL FROM THE LIVING BIRD. FIG. 272. — Wing of Black-footed Penguin, top view. FIG. 272. — Wing of Black-footed Penguin, top view. FIG. 273.— Wing of Black-footed Penguin, side view. FIG. 273.— Wing of Black-footed Penguin, side view. FIG. 27-4. — Wing of Black-footed Penguin, under surface of wing. ALL FROM THE LIVING BIRD. FIG. 27-4. — Wing of Black-footed Penguin, under surface of wing. ALL FROM THE LIVING BIRD. The Bird The Bird 3 We have seen how wings guard their owners from the risk of sudden surprises from enemies, and now let us observe how, in a sleeping bird, the tender nostrils and eyes are protected against cold and other dangers. Birds do not put their heads under their wings, but behind them, often using the shoulder-feathers as cover. It is inter- FIG. 275. — Green Heron with head behind wing. FIG. 275. — Green Heron with head behind wing. esting to see how many birds, from all quarters of the earth, have this same habit. The pelican, however, de- parts from this custom and snuggles his tremendous bill between the feathers in the centre of his back, and flops both wings up so as completely to cover it. Even the cassowary vainly tries to tuck his bill behind his absurd wing. His smaller wing-coverts are mere soft, loose hair- esting to see how many birds, from all quarters of the earth, have this same habit. The pelican, however, de- parts from this custom and snuggles his tremendous bill between the feathers in the centre of his back, and flops both wings up so as completely to cover it. Even the cassowary vainly tries to tuck his bill behind his absurd wing. His smaller wing-coverts are mere soft, loose hair- Wi 345 like shafts, while the larger quills, as mentioned before, are reduced to four or six horny sticks. If we watch an owl flying about its cage at night, or if, in the woods, an owl passes near, his shadow in the moonlight is all that warns us of his presence. The feathers of an owl's wing are soft and downy, and the bird moves as lightly as a falling leaf. Little warning, except by FIG. 276.— Trumpeter Swan asleep. FIG. 276.— Trumpeter Swan asleep. sight, the mice and birds have of its deadly presence. Few birds have a flight as noiseless as that of owls, and in some species the motion of the wrings makes, as we noticed in the pheasant, a very audible sound. When a widgeon rises from the water, the whistling of its quills, so dear to the ears of the sportsman, is quite shrill. A dove claps its wings together above its back while gain- ing impetus for flight. The characteristic sound from which a hummingbird takes its name is well known. The Bird 3 When wild geese and swans nest in captivity, their wings are put to most excellent use as weapons of de- fence, and of course this use must come into play fre- quently when nesting in their native haunts. I have seen a man knocked breathless by a Canada gander who thought his nest in danger. When preparing for attack, the bird approaches hissing, with head stretched low along the ground, and suddenly, without warning, launches FIG 277.— Trumpeter Swan preparing to attack an intruder with its wings. FIG 277.— Trumpeter Swan preparing to attack an intruder with its wings. itself straight at one's breast and, clinging with bill and claws, beats a tattoo with the hard bend of its wings. One is not likely to forget such a drubbing for a long time. The wings of certain birds are armed with weapons of offence, such as the Spur-winged Goose, Jacana, Plover, and Screamer. The Spur-winged Goose is a really danger- ous antagonist and can strike incredibly strong blows, bringing the sharp spur to bear with telling effect. These itself straight at one's breast and, clinging with bill and claws, beats a tattoo with the hard bend of its wings. One is not likely to forget such a drubbing for a long time. The wings of certain birds are armed with weapons of offence, such as the Spur-winged Goose, Jacana, Plover, and Screamer. The Spur-winged Goose is a really danger- ous antagonist and can strike incredibly strong blows, bringing the sharp spur to bear with telling effect. These Wings 347 spurs are not claws, but correspond in structure to the ordinary spurs on the legs of a rooster. The great heavy-headed and heavy-bodied hornbills fly with great effort, and it is said upon good authority that when passing low overhead they make a noise like a steam-engine. Although not strictly within the prov- FIG. 278. — Spur-winged Goose. FIG. 278. — Spur-winged Goose. ince of this volume, mention should be made of the inten- tional use of the wings as instruments of sound, — to at- tract the females, as in our Ruffed Grouse and other birds. A little Bush Warbler of Africa has indeed never been heard to utter a note, seeming to depend upon an occasional whirr of wings, in lieu even of the usual call-note or chirp. The Bird ince of this volume, mention should be made of the inten- tional use of the wings as instruments of sound, — to at- tract the females, as in our Ruffed Grouse and other birds. A little Bush Warbler of Africa has indeed never been heard to utter a note, seeming to depend upon an occasional whirr of wings, in lieu even of the usual call-note or chirp. 3 The Bird In the woodcock we find the vane of the three outer primaries of the wing remarkably narrowed and stiffened; probably a direct adaptation for the production of the high, whistling sound which plays so important a part in its aerial courtship performance. FIG. 279. — Wing ornaments of Twelve-wired Bird of Paradise. FIG. 279. — Wing ornaments of Twelve-wired Bird of Paradise. As upon all other parts of the bird's body, we find beautiful decorations upon the wings — inexplicable unless we are willing to credit the females with appreciation of, or at least a reaction to, these beauties. Otherwise we know not the uses of the brilliant wing-mirrors of ducks, or the scarlet wax-like tips of the Cedar-bird's feathers, Wings 349 349 or the bizarre decoration of the Twelve-wired Bird of Paradise. A strange appendage is found in the wing of the West African Goatsucker. Conspicuous enough when the bird is flying, it is wonderfully protected when the bird rests, FIG. 280.— Wing of Woodcock. FIG. 280.— Wing of Woodcock. as is its wont, upon the ground among tall, feathery- topped grasses. From each wing a single long feather extends in an upward direction, almost bare of barbs for most of its length, but tipped with a mottled, loose- vaned tuft which corresponds very perfectly with the flower-heads of the grasses among which it lives. As The Bird 3 this decoration, so protective and yet so beautiful, is assumed only during the breeding season, its use is doubt- less to aid in attracting the attention of the females. Herons and other birds make still another use of their wings and the long, tough flight-feathers: as shields for parrying the blows of a rival, or to catch the poison of a snake when it strikes and thus give an opportunity to seize and despatch the reptile. Two Snowy Egrets will sometimes fence with each other in play, and use beak and wing as a soldier would use sword and shield. beak and wing as a soldier would use sword and shield. I once saw the wing of a bird used in an entirely original manner — a use peculiar, doubtless, to this individual. The Bird Several spoonbills suffered severely from the frozen ground upon which they were forced to stand, and no method of relief was found, except by one of their num- ber, who every night stretched one wing beneath him, drew up one foot deep into his plumage, and with the other stood upon the tips of the primaries. Much might be written concerning the swiftness of birds' flight, but so much of exaggeration has entered into estimates of this kind that it would be difficult to select facts and figures of indisputable verity. However, it may be asserted as at least within the actual facts that ducks can attain a speed of ninety miles an hour. An apparently well-authenticated record of a swallow's flight at Antwerp is as follows: A gentleman arranged a flight of homing pigeons from Compiegne to Antwerp, — a dis- tance of one hundred and forty-eight miles, — and with the pigeons he liberated a swallow captured on her nest under the eaves of his house in Antwerp. The swallow, which 03 I a O r 0, 03 I a O r 0, O r 0, The Bird The Bird 3 was marked for identification, covered the distance in one hour and eight minutes, or at the extraordinary speed of about two miles and three hundred yards per minute. The first pigeon to arrive took four hours and a quarter to make the journey. \ FIG. 282. — Terns in flight. (Photograph provided by the American Museum of Natural History.) FIG. 282. — Terns in flight. (Photograph provided by the American Museum of Natural History.) FEET AND LEGS ARRIED far and wide by the power of flight, no two species of birds have exactly similar environments. When the wings cease their la- bour and are folded close to the sides, the bird must depend upon its feet to carry it to its food and to keep it out of danger, whether its footing be in a tree-top or on a cliff; in shallow water or on the deep; in mud, sand, or snow. Thus we realize the need for many varied adaptations in the way of feet and legs. A Although birds are descended from five-toed ancestors, yet no living wild bird, and none of those which we know only as fossils, has more than four toes on each foot. The disposition of these toes — four, three, or two, as the case may be — is always in accordance with the habits of the bird. The most common type of avian foot is that in which the arrangement is of three toes in front, with the fourth, corresponding to our great toe, pointing backward. This was the arrangement in our first bird, the Archceopteryx, and for perching birds, as well as for many others with very different habits, it has stood the test of six millions 353 The Bird 354 of years, or thereabouts, since the days of its venerable prototype. prototype. This is the kind of generalized organ which, we should think, would be able to cope with changes in the bird's surroundings more successfully than any other; but that this theory fails when put to the test is proved by the variety of specialized toes and legs which we may observe FIG. 283.— Foot of Alligator. FIG. 283.— Foot of Alligator. among the birds on the earth to-day. Indeed, in the variety of uses which they subserve, the feet and legs of birds are second only to the bills. A classification of birds, generally accepted for many years, was based on the uses of the feet, or mode of loco- motion. In this scheme birds were divided into runners, scratchers, climbers, swimmers, perchers, etc. Although Feet and Legs 355 these, as exact divisions, have long since been abandoned, yet it is worthy of note that even in the most modern classifications many of these groups hold good in the main, although based on other and more fundamental FIG. 284.— Foot of Brown Pelican. FIG. FEET AND LEGS 284.— Foot of Brown Pelican. characters. Examples of these are the ostrich-like birds, or runners; the fowl-like birds, or scratchers; and the Passeres, or perching birds. But there is no doubt that several unrelated groups have independently acquired the The Bird 3 specialized type of foot which is adapted to climbing or to swimming, so that any classification based on such similarity of locomotion is obviously false. similarity of locomotion is obviously false. From the tiny limbs of a hummingbird to the gigantic shanks of an ostrich, the legs of birds, with a very few exceptions, are covered with scales, most emphatic re- minders of the reptilian ancestry of both these extreme forms of feathered life. The real foot of a bird, as the term is used in speaking of other animals, extends to the backward-bending joint, or heel. Part of the lower leg FIG. 285.— Foot of Raven. FIG. 285.— Foot of Raven. is thus concealed by the feathers and skin, while the upper leg, or thigh, is generally wholly within the body, as we saw in the chapter treating of the framework. As before, we must call on the crow, in many respects standing very near the top of the scale of bird life, yet which has found it good to hold to the typical bird's foot. And indeed it serves him well, for with it he can walk on snow or ice; w&de in shallow water; perch in trees; scratch or claw the ground and hold down a crab's carapace, while he extracts the edible portion. Not only this, but he can hop like a sparrow or walk like a lark at will. Feet and Le 357 We have hardly to leave the group of birds to which the crow belongs to find dozens of interesting and unex- pected adaptations of the feet to unusual habits. For example, the Rhinoceros-birds of Africa attach them- selves to some of the larger mammals, such as buffalos, rhinoceri, or antelopes, and spend much of their time in freeing these animals from troublesome ticks and other parasites. The power which these birds possess in their feet and legs is remarkable. FEET AND LEGS Millais says of them: "The prehensile power of the claws is, as I found by experience, so great that when a dead bird which had grown stiff was thrown on to the back or sides of an ox, so that the feet touched the animal's hide, the claws held fast at once and could not be withdrawn. It is most interesting to note the way in which a party of these birds will move about on the body of a horse or ox, searching every part of him as they run or hop over it in the most lively fashion. At the risk of being accused of telling a traveller's yarn, I must state the fact that they can hop backward quite as well as forward, and they often make long drops down- ward from the shoulders to the foreleg, or down the side of the animal whose coat they are engaged upon. It is quite immaterial to them how or in what direction they move." they move." No hard and fast laws can be laid down, but it is gener- ally the rule that birds which are especially at home in the trees usually hop with both feet simultaneously when on the ground. Ground nesters and feeders, such as the Meadow Lark, Bob-white, and Vesper Sparrow, usually walk or run. The Bird 3 The great Order of perching birds (Passeres) shows to what varied uses the typical foot can be put. All birds of this Order have three toes in front and one behind, and there is scarcely a place on the globe to which these birds have not adapted themselves; and recently too, as would seem probable from the similarity of the foot- type running through all. type running through all. This very foot holds much of interest too, if we con- sider it from another point of view. Many apes and monkeys, and we ourselves, still have the five fingers and toes which we suppose was the number originally devel- oped upon the limbs of the vertebrate prototype; while horses and deer — animals much lower in the scale of life — have had the five original digits reduced to one or two. FEET AND LEGS So among birds the ostriches and some other low forms have become extremely specialized in the same respect, possessing but two or three toes, while those birds which in mental and physical attributes excel all others of their Class are still more reptilian, and thus more primitive — more Archseopteryx-like — in possessing a larger number of digits — four. Thus when we speak of an animal as high or low in the scale of life, we must carefully distinguish between mere specialization and actual upward progress, mentally or physically, toward some ideal goal. The branch of a tree, which stretches horizontally farthest from the parent trunk, is not likely to be the one which reaches upward high enough to catch the first rays of the morning sun. The majority of the Passeres are arboreal and the strength of the tiny tendons which run down the leg and 359 through each toe is sufficient to clasp and unclasp a thou- sand times a day, and to hold and balance the bird on whatever bending twigs or wind-blown foliage it chances to alight. In this matter of perching the hind toe plays an important part, so much so that when the necessity for grasping ceases, this digit begins to wax flabby and weak and often becomes reduced in size. FIG. 286.— Nuthatch on tree, clinging upside down. FIG. 287. — Nuthatch clinging to a gloved hand. (Bowdish, photographer.) FIG. 287. — Nuthatch clinging to a gloved hand. (Bowdish, photographer.) FIG. 286.— Nuthatch on tree, clinging upside down. The creepers, Fig. 240, are passerine woodpeckers in habit and forever wind their spiral paths about the tree-trunks. But the nuthatch is the marvel of the whole Class of birds in this climbing ability. With no support whatever from the tail, and without special adaptation of toes, it defies all laws of gravitation and creeps up and down or around the vertical trunks, as if on a level surface. Never a misstep, never a slip, but The Bird 36o 36o each foothold as secure as if its feet were vacuum- cupped. In the swallows the feet are very small, having fallen into disuse with the great increase of the power of flight. FEET AND LEGS Orioles and weaver-birds make occasional use of their feet to hold a strand of grass or string which they are weaving with their beaks into their elaborate nests, and certain flycatchers pounce upon and hold their insect FIG. 288. — Swallow, showing small size of feet. FIG. 288. — Swallow, showing small size of feet. prey as an owl grips a bird, or a jay clings to a nut; but with the exception of a few such cases, the feet of perching birds serve principally the function of locomotion. As variation in habitat or haunt depends so much upon the power of locomotion, it will not be out of place to mention here, in rather more detail than usual, a splendid example of adaptive radiation which we can all verify for ourselves. There is no more wonderful fact in Nature than the way Feet and Le 361 in which birds have inherited the earth. When we realize the immense advantage which the power of flight gives to them, we do not marvel at this remarkable distribu- tion, but the more we think about it the more wonders appear. The utmost efforts which man has made to reach the North Pole have shown flocks of birds winging their way still farther to the North, heedless of the ter- rible cold. In the heat of deserts and the . sweltering jungles of the tropics, birds find congenial haunts and abundant food. Thousands of miles out at sea, on the highest mountains, and even in dark underground tun- nels; the whole day — twilight, midnight, and dawn, — all have been conquered by these tireless, energetic feath- ered ones. When we see a large collection of birds, we can appre- ciate how they are adapted to such varying conditions of temperature, of moisture, of light, and of altitude. Their bodies, wings, legs, feet, and tails — in fact every organ and member is of all sizes and shapes, and shows to what condition of life the individual is suited. FEET AND LEGS But when we come to know birds better, and we realize that there are wheels within wheels, that behind these very evident divisions into Families and Orders there are lesser groups, among the members of which the competition is no less keen, we look for and find gentler gradations and adapta- tions which, in their way, are more to be wondered at than the larger, more radical differences; for these birds have changed their habits and haunts without waiting for Nature to adjust their wings or their feet. They have taken the initiative as it were, and, like a man of letters The Bird 3 who is suddenly forced to work at some arduous manual labour, they have entered on new ways of life — ways to which their structure seems but ill adapted, and yet, by the very daring of their efforts, they have won success. by the very daring of their efforts, they have won success. The great-grandfathers, many times removed, of the modern Families of birds lived lives which were much broader and more generalized than those of their descend- ants of to-day, and it is this variety, this seeking of new opportunities and overcoming of new difficulties by the feathered sons, which makes the study of birds so fascinat- ing a pursuit. Let us follow the diverging paths of the later gen- erations of some of our own birds. Take the wood- warblers of our own country. The only way we can imagine what the earlier ancestors of the warblers were like is to make a composite of the whole Family. All its members are tiny, delicate birds which feed on the smallest insects, their bills are slender and pointed, and their feet and toes like the finest wire. Yet, far from waiting for Nature to alter these delicate organs, they have struck out boldly for themselves and, to avoid a fatal competition with one another, have varied their methods of hunting and the limits of their preserves so successfully that a dozen may live in close proximity and yet never poach on each other's domains. and yet never poach on each other's domains. FEET AND LEGS Our well-known little Maryland or Northern Yellow- throat has chosen the low bushes of a marsh as his sphere in life, and, although he has hidden his face behind a black mask, yet he is a true warbler, and the blood of his fathers forces him up now and then into some exposed position, Feet and Le 363 where he bursts into a joyous bubbling and warbling, calling to his brethren of the tree-tops that, though his haunts are changed, his heart is true to the clan. His cousin, the Worm-eating Warbler, is tending in his direc- tion, living in low bushes and in his habits drifting ever marshward, where there may not be sufficient competition to prevent his eventually sharing it with his more original kinsman. The Yellow Palm Warblers, although more conventional in their ordinary tree-top haunts, have de- parted from ancient customs in their feeding habits. They dine on the ground, then fly back to the trees; ob- serving, like some humans, the traditions of their family in the spirit, if not in the letter. The brilliant Redstart clings even more closely to the ancestral ideas of high trees, and cares little what kinds he may find himself in; but he has a failing for water, and if he may not descend, as have his two cousins men- tioned above, yet he overlooks them and often swings low through the air toward them. For in his feeding habits he is one of the most radical of warblers. Has he not seen the little green flycatchers in the woods, sit- ting so lazily upon some favourite perch, and with an occa- sional swoop snapping up an unfortunate insect? Why, indeed, search all day for the tiny mouthfuls? Why not wait for them to appear? So Redstart attempts fly- catching and with perfect success. But the active blood which surges through his veins will not allow him to assume the patient waiting tactics of the genuine flycatchers. He may imitate their meth- ods of actual capture, bagging his game on the wing, but The Bird 3 he is still ever on the move, from twig to twig, from tree to tree. FEET AND LEGS Nevertheless, he has gained an advantage which ensures to his race a long life; for in a tree whose foliage and twiglets are being scanned with the microscopic glances of his relatives, he gets more than the others by watch- ing for the many insects which are alarmed at the dis- turbance of the tiny hunters, and which flutter out in the bright sunshine only to flutter straight down his throat. Mother Nature has seen his efforts in the new field with satisfaction, and has given to him a little re- ward; for from either side of his mouth several stiff bristles project, and many times, when he has misjudged the dis- tance or the dodging powers of his prey, these little hairs shunt the gnat or fly into his mouth. The Myrtle Warbler is an expert catcher of flies, and has in addition another string to his bow, which bids fair to place him at the head of the list of new departures in warblers. He has learned that bay berries are not only an occasional welcome variety to the everlasting diet of insects, but that a warbler can comfortably live upon them when the cold has benumbed the little winged and crawling creatures. So, instead of migrating south at the first hint of winter, these hardy little Myrtle Warblers sometimes remain with us throughout the whole season of cold and snow. A most daring departure from old-established prin- ciples of the warbler clans is that of the Water Thrushes. Ages ago, perhaps, we may imagine that some member of this group, while drinking at a stream or pond, watched the little bobbing sandpipers as they scurried past along Feet and Le 365 the brim, now wading in a short distance, then leaping to a soft rim of clay, everywhere finding the most delicious morsels abundant. A strange fascination took hold of the tree-haunting warbler, and although perhaps you and I would have said he was a very silly bird and that such a thing as a warbler turning into a sandpiper was utterly absurd, yet the little fellow and his descendants persisted. Sandpipers and sandpipers only they wished to be, and Nature has given them their wish. Study the Water Thrushes of to-day. Their whole life is spent along some stream or pond, searching for worms and snails in true sandpiper fashion. FEET AND LEGS Not only this, but even the dipping gait of the pipers has been copied, and though we cannot give a reason for this char- acteristic, yet the warblers have learned it by heart, and many an amateur bird-lover do they confuse! But the heart of the old clan instinct can never be entirely eliminated, and even if a warbler should attempt to hum away his life on the wing like a hummingbird, or to run with the speed of the wind through dry deserts like an ostrich, yet, like the Water Thrushes, he would occasion- ally drift back to the old tree-tops and there sing of the happiness which is within his heart. A 'strange whim of evolution in one member of the warbler tribe results in his mimicking the sandpiper as far as terrestrial locomotion, a walking gait, and the peculiar tilt- ing habit go, but the fondness for water did not accompany these changes, and so we find the Oven-bird content with the deep woods where he builds his home upon the ground. He often returns for a time to the trees, but, like a college The Bird 3 boy whose whole ideas of life have been changed by ab- sence from his rural home, the Oven-bird carries aloft with him the mincing gait of the littoral sandpipers, walk- ing sedately along the limbs among his agile, hopping, creeping cousins. creeping cousins. Of the conventional aristocracy of the warblers there would be much to say had we the space. The Black- and-white Creeping Warbler has been transformed into a woodpecker, as far as mode of progression goes; and lucky for him too, for he never fails to find cocoons and small edible things among the cracks and crevices of the bark, no matter how vainly the others may be searching the overworked twigs and leaves. And Nature has helped him, too. She has dipped him in a bath of the essence of these very same crevices and cracks, and out he has come, covered with the semblance of the rough surface and the long, dark shadows which may shield and hide him from many enemies. Of the typical tree-loving species, the Pine Warbler haunts the growths which have given him his name; the Black-throated Green also loves the evergreens, and the beautiful Magnolia delights in thick forests of spruces. FEET AND LEGS Thus we have taken a brief survey of the recent branch- ing of the warbler's genealogical tree. Each has found a niche in which to live, and the food and safety which permit him to rear a nestful of young each year. So far so good, but we must not forget to give a thought to the untold thousands and tens of thousands of generations which have failed in their attempts. Nature has removed all traces from view and in the general advancement of Feet and Le 367 the race as a whole they are forgotten, but it is well for us to think of them occasionally: their birth, the chance which came, which seemed so full of promise, which they so eagerly accepted and which betrayed them; the myriad little dead forms which gave up their lives in ages past, and upon whose bodies and whose efforts the birds of to-day have risen to their present high place in the scale of the creatures of the world. We might have used this same illustration, or many others like it, in connection with almost any other portion of the bird's body. Although, indeed, it pertains more strictly to the mental characters, and so is in a way out- side the province of this volume, yet its application to physical adaptations is so evident that its omission would leave incomplete a most interesting phase of the possi- bilities of the adaptation of bird structure. Although among perching birds the bill is the important organ for procuring food, yet such birds as the Chewink, the White-throated Sparrow, and the jays, in search of small insects use their feet to scratch away dead leaves and rubbish, kicking backward with both feet at once. There are many curious things about toes to which we have not yet found the key. Who can tell why the Horned Lark, Pipit, and some other birds have such elongated claws on their rear toes? Perhaps the fact that these birds live almost entirely on the ground may have something to do with this peculiarity. Any one who has kept a cage full of small birds will soon have learned the fact that the claws of birds are continually growing. FEET AND LEGS In a remarkably short time their claws become long and The Bird 8 368 curved, and in a neglected aviary I have seen birds which were prisoners on their perch, unable to untwist their claws from it. When wild, birds wear down these struc- tures by constant rubbing, and if given plenty of rough bark and wood in their cages, their claws will remain of usual length. or that portion of the foot which, is, in almost all birds, covered with horny scales, yet these vary con- siderably in different groups. la many the scales are small, six- sided or oblong, as in plovers. In some of the higher song-birds these scales have become joined together until, as in our robin, the front of the leg is covered with a long "boot" of horn. The cause of this coalescence yet remains to be discovered. Although the tarsus, or that portion of the foot which, we usually call the leg, is, in almost all birds, covered with horny scales, yet these vary con- siderably in different groups. la many the scales are small, six- Although the tarsus, or that portion of the foot which, we usually call the leg, is, in almost all birds, covered with horny scales, yet these vary con- siderably in different groups. la many the scales are small, six- sided or oblong, as in plovers. In some of the higher song-birds these scales have become joined together until, as in our robin, the front of the leg is covered with a long "boot" of horn. The cause of this coalescence yet remains to be discovered. FIG. 289. — Foot of American FIG. 289. — Foot of American Pipit. FIG. 289. — Foot of American Pipit. A considerable degeneration of the legs and feet is found among goatsuckers, humming- birds, and chimney swifts; but, small as are the feet of the latter birds, they make frequent use of them to break off the short twigs which are used in the construction of their nests. One may take a young swift and place it against the vertical surface of an ordinary brick (Fig. 326), and the bird will hold fast without slipping a fraction, of an inch. The slender nails fasten in the slightest irregularity of the surface and hold the bird safely. FIG. 289. — Foot of American Pipit. FEET AND LEGS Some species of swifts have all four toes pointing Feet and Legs 369 forward, forming a four-tined grapple by which they hang themselves up in their hollow nesting-trees. Whip- poorwills and some other birds have a curious comb, or pecten, along the edge of the middle claw, which is perhaps of use in cleaning the long bristles about their mouths, or in arranging their very delicate, soft plumage. Kingfishers and several related groups of birds make so little use of their feet, except in motionless perching, that FIG. 290. — Comb on toe of Chuck-wilPs-widow. FIG. 290. — Comb on toe of Chuck-wilPs-widow. not only are the toes small and weak, but two of the front ones have grown together for over half their length. Perhaps the most interesting condition of toe struc- ture is found among the woodpeckers, parrots, cuckoos, and owls. In these groups we find a similar plan of gen- eral arrangement: two toes in front and two behind. With few exceptions it is the great, or first, toe and the fourth, or outer, toe which are reversed. This arrange- ment of toes is known as yoke-toed, or zygodactyl. The Bird 0 We have seen that in perching birds the arrangement is three toes in front and one behind; and now turning to the woodpeckers we are struck with the excellent toe arrangement of these climbing birds,— their claws spreading so that they point almost to the four points of the compass, thus forming an admirable grapple or vise, which makes a vertical position as safe for a wood- pecker as a horizontal one for a percher. pecker as a horizontal one for a percher. Woodpeckers, the world over, have feet and toes which are remarkably alike; but in Canada and the most northern parts of our own country, and in certain por- tions of the Old World, there are several woodpeckers which are unique among the birds of this Order in pos- sessing but three toes. For some unknown reason their first, or great, toe, which in all other woodpeckers points backward, has disappeared, leaving but a vestigial trace beneath the skin, while the outer toe is reversed to take its place. We may see one of these hardy three-toed fel- lows sliding and hitching up a pine-tree, pounding and hammering vigorously, the loss of an entire toe evidently not handicapping him in the least. In such fashion does Nature occasionally upset our hard-worked-out theories, leaving us confused and baffled before her inexplicable surprises. us confused and baffled before her inexplicable surprises. Is it not rather disconcerting to find that this same arrangement of two toes in front and two behind also holds good for the other Orders of birds mentioned above, the parrots, cuckoos, and owls, — their toes all arranged in pairs, fore-and-aft? This is an excellent example of what is called parallelism, or the independent develop- ment of similar structures. Feet and Le 371 Parrots use their feet for more different purposes than do any other birds : they are the monkeys of the feathered IG. 291. — Cockatoo perching with one foot and holding food with the other FIG. 291. — Cockatoo perching with one foot and holding food with the other. world. They climb wires or branches one step after the other, their beaks taking the place of a third foot in this style of locomotion. They pick up food, such as a banana The Bird 3 or a nut, and, holding it in the foot while eating, turn it from side to side as we revolve an apple in our hand. With their claws they preen their plumage, and push each other aside when too closely crowded. In fact the functions of the feet and toes of parrots approach nearer to those of a human hand than the limb of any other Order of birds. PIG. 292. — Foot of Cuckoo, perching, and with toes outstretched. PIG. 292. — Foot of Cuckoo, perching, and with toes outstretched. Cuckoos are perching birds, and when we see the skil- ful way in which they creep through a dense thicket, never missing their hold, we wonder why all perching birds do not have this arrangement of two toes in front and two behind. So completely are our theories set at naught that we should hardly be surprised to see a bird with one toe in front and three behind cheerfully hopping from branch to branch! In the deserts of the south- Feet and Le 373 western part of the United States much of the vegetation consists of prickly cacti and thorny mesquite, most un- pleasant to perch upon, and here we find the Road-runner, a kind of ground cuckoo, who has the fore-and-aft toe arrangement of his arboreal relations, but whose terres- trial life has developed remarkable powers of running and leaping. One of these birds can outstrip a horse for a hundred yards or more and, almost without effort, can leap upward ten or twelve feet, to all appearances unaided by its wings. The owls can move their outer toes backward or for- ward at will, thus being able to assume the arrangement of toes both of a crow and of a parrot. However the yoke, or two-and-two, plan is the one most commonly seen among these birds. Feet and Le With such an automatic vise-trap ready to descend silently and with deadly swiftness upon him, the little mouse in the grass has indeed need to be ever on the alert. The talons of owls are curved and under the control of tendons of great strength. Their chief use is to capture living prey and then to hold it firmly while it is torn to pieces by the beak. The deserts and plains where the Road-runner dwells are also the home of the Burrowing Owl, Fig. 351, which finds in its sharp little talons admirable picks and shovels, certainly a novel use for yoked toes. The feet and toes of birds are, in zero weather, their most vulnerable points (except their eyes), and they are most liable to be frozen. In the black wastes of the frozen boreal regions, the Arctic Owl is able to defy the intense cold, by means of a furry covering of hair-like feathers, which extends to the very The Bird 374 claws, and even the soles of the feet are thickly covered , so that the skin of the bird is never in contact with the snow and ice on which it roosts. The osprey, or fish-hawk, can, like the owls, reverse its outer toe, but all typical hawks and eagles have the FIG. 293. — Owl gripping a piece of meat. FIG. 293. — Owl gripping a piece of meat. perching-bird arrangement. The talons of the osprey are immensely strong, and the scales on the soles of its feet and toes are hardened and roughened to such a degree that they are almost spike-like. A more efficient fish- trap cannot be imagined. The Golden Eagle has a splendid foot, with great curved talons, which, when they have perching-bird arrangement. The talons of the osprey are immensely strong, and the scales on the soles of its feet and toes are hardened and roughened to such a degree that they are almost spike-like. A more efficient fish- trap cannot be imagined. The Golden Eagle has a splendid foot, with great curved talons, which, when they have Feet and Le 375 once clasped an object, never let go. It required two men and two pairs of the thickest buckskin gloves to obtain Fig. 295, and even then the foot could be held still for only a moment. As the photograph shows, the FIG. 294.— Foot of Snowy Owl. Feet and Le FIG. 294.— Foot of Snowy Owl. leg is feathered all the way down to the toes in this eagle, for some unexplained reason, while in almost all its rela- tives, as in the Bald Eagle, the legs are covered with scales. The feet and toes of the Harpy Eagle, Fig. 204, are probably the most terrible of their kind in the world : The Bird 76 376 certainly they are the strongest. When once they have closed on an object, and remain clutched, nothing short of severing the bird's leg will avail to loosen the fearful FIG. 295.— Foot of Golden Eagle. FIG. 295.— Foot of Golden Eagle. FIG. 295.— Foot of Golden Eagle. grip. Besides capturing their food, birds of prey carry the sticks for their nests in their talons. grip. Besides capturing their food, birds of prey carry the sticks for their nests in their talons. When, instead of killing its prey, a species of bird feeds upon carrion, the change in its habits is reflected Feet and Legs 377 clearly in the appearance of its feet. Compare the feet of a vulture (Fig. 296) with those of one of the true birds of prey (Fig. 295). The muscles are weaker and the claws are shorter, more blunt, and, as a result, the toes have lost their clasping power, while the hind toe is higher and so FIG. 296. — Feet of Vulture. (E. R. Sanborn, photographer.) FIG. 296. — Feet of Vulture. (E. R. Sanborn, photographer.) small that it is of no use even in perching. Such is the condition in the condor of South America. When in captivity an eagle is given a piece of meat, it seizes the food in its talons and flies to some favourite spot to devour it, but a condor transports its meal in The Bird 3 its beak, then holding it down firmly with one of its feet, it pulls upward and so tears the meat. it pulls upward and so tears the meat. So exactly correlated are these changes of habit and of feet that in the Caracara, a Mexican bird of mixed habits, partly rapacious and partly vulturine, the toes and claws are correspondingly midway between the two groups of birds. Feet and Le This bird lacks sufficient grasping power to enable it to lift its prey from the ground after the manner of a true Hawk; but it will overcome this difficulty by carrying up the object in its beak, and then reaching for- ward with its feet, while in full flight, and taking a careful grip with its talons. In South Africa is a bird known as the Secretary, which is really a terrestrial hawk, rarely flying, but spend- ing most of its time stalking about in search of food. Any one who has seen an eagle progressing upon the ground by means of its awkward ga41op, can realize the impos- sibility of such a short-legged bird preferring terrestrial life, but the legs of the Secretary are as long as those of a crane, although in other respects the bird would pass for a very long-tailed species of hawk; it is really a hawk on stilts. However, there are reasons for supposing that the Secretary Bird may be, not a more or less recent off- shoot from the hawks, but a surviving type of old, old days when there were no hawks and cranes and herons, but instead, a few strange birds which combined the characteristics of all these groups. of all these groups. The skilful way in which the Secretary Bird brings its feet into play in the capture of serpents, of which it is very fond, has been described as follows: Feet and Legs Feet and Legs 379 "When the snake strikes, the bird either evades the blow, by skipping to one side or the other, jumping back- ward, or springing into the air, or else, as frequently hap- pens, he simply receives the venomous thrusts of his antagonist on the broad stiff feathers of the outer half \ FIG. 297. — Secretary Bird. FIG. 297. — Secretary Bird. of the long wing, with which he knocks the reptile down, following up the fall with a vigorous kick. His extreme agility enables him in a very short time to baffle and overcome a snake of four or five feet in length, whereupon he finally seizes it near the head with his bill, and hold- The Bird o 38o ing the body down with one foot, proceeds to swallow it. In case a snake proves unusually hard to manage on the ground the dauntless bird watches his opportunity, seizes his adversary close to the head, and, flying aloft to a considerable height, lets it drop on the hard ground, which is usually sufficient to prepare it for the final ceremony of swallowing." eremony FIG. 298. — Feet of Ruffed Grouse, showing snowshoes of horn. FIG. 298. — Feet of Ruffed Grouse, showing snowshoes of horn. Quail, grouse, pheasants, turkeys, and all the fowl- like birds are scratchers, according to the old classification, and they well deserve the name; for scratching first with one foot and then the other among the leaves and soft dirt for insects is a very pronounced habit of them all. The arrangement of toes is the same as in the perching birds, but the claws are very different. These birds are Feet and Le 381 true horny-handed sons of the soil : their claws are stubby, short, and blunt. Sharp edges would soon be dulled by scratching, and elongated ones would sliver and break. So, with his blunt claws, our chicken and his kind are well provided for. The most interesting feet among these birds are those of the grouse. The ruffed drummer of our woods walks about, in summer, on slender toes over moss and logs, but, when soft deep snows come, his weight would make it difficult to keep from being buried at each step. So Nature provides him with snowshoes. Feet and Legs From each side of each toe a broad, horny comb-like fringe grows out; not a web of skin which might soon freeze, but rows of horny projections, as of a myriad extra claws. This distributes his weight so that he trots merrily over snow through which a fox sinks deep and flounders awkwardly at every step. But what of the ptarmigan, that snow-white grouse of the far North, whose home is amid those frigid barren regions? This bird is much more of a walker than the Snowy Owl, and its feet would surely freeze during the long winters if they were bare of feathers. So we find indeed that scarcely a claw is visible beyond the thick feathers which cover legs, toes, and soles. Such a provision against cold is evident and reasonable enough, but how are we to account for the feet and toes of the House Martin of Europe, which are densely feathered to the very claws? It breeds in Iceland and Lapland, but only in summer, when it would need no such protection against cold, and it is also true that it breeds upon the cliffs of Persia and southern India. The Bird 2 382 If we watch a duck as it settles itself for the night upon the snow, we will see it squat down, snuggle its beak deep among the feathers of the back, and finally draw up each foot from the frozen surface and tuck them up out of sight. Thus they are protected from freezing during the long, cold night. The pugnacity of the males of the Order of game- birds has become proverbial; almost all are "fighting cocks" and yet their beaks are not fitted for defence or FIG. 299. — Mallard asleep on the snow, with its feet drawn up to avoid freezi FIG. 299. — Mallard asleep on the snow, with its feet drawn up to avoid freezing. offence, nor can they clutch and tear with their claws. But we find spurs developed on the tarsus, or upper foot, in fowls, turkeys, pheasants, and peacocks, which are used with remarkable skill in their battles. In structure these outgrowths are identical with the horns of antelopes and cows, consisting of a bony projection over which grows a sheath of horn. The spurs of the peacock are long and sharp and are occasionally used with such effect that the results are fatal to each of the contestants. A diminutive relative of Pavo, the Pea- Feet and Le 383 cock Pheasant of the East Indies, has two, three, or even four spurs of full size on the legs. The bird photographed on page 419 had two on the right leg and three on the left. Yet these birds are not as correspondingly pug- nacious as we should imagine from their increased arma- ture. There is a small group of peculiar birds, known as Sand-grouse, which in many respects stand midway FIG. 300. — Spur of Java Peacock. FIG. 300. — Spur of Java Peacock. between the true grouse and the pigeons. In certain of these the toes, to their very tips, are encased in the skin of the foot, the effect being of a mitten with only the claws free. The reason for this is yet to be found. We now come to the water-loving birds, and we find that their varying associations with this element have wrought many interesting changes in their feet and legs. The Bird Those birds which are content to wade along the shallow margins of ponds and streams require long legs and long The Bird 3 toes, the latter to distribute their weight as they walk over the soft muddy bottom, the former to lift their bodies above the surface of the water. Such, broadly speaking, are the plovers and sandpipers and herons. Let us see how the feet of these birds reflect theh1 habits. With the exception of the tribe of plovers, almost all have four toes. The plovers have but three, and these are slender and not webbed, for although they usually feed on aquatic forms of life, yet their food is gleaned from the upper part of beaches, or from the sand-flats when the tide is out, and they therefore seldom have occasion to swim. The sandpipers venture into the shallows and are some- times lifted from their feet by a small inrushing wave. But the majority even of these go through life unwebbed. One, the Semipalmated Sandpiper, shows a beginning of this in the half-webbed condition of the toes, but the group of phalaropes are actually sandpipers of the sea. I have seen them in flocks of thousands, resting upon the surface of the ocean, scores of miles from land. Yet when ashore they have need to be as active as other mem- bers of their Order in order to find sufficient food; so, in- stead of being hampered with a confining web, each toe has a series of broad scalloped lobes, serving admirably as water propellers, yet allowing the toes freedom of motion when the owner is scurrying over the sand. I have observed Great Blue Herons almost hip-deep I have observed Great Blue Herons almost hip-deep in the breakers along the Florida beaches, yet this is not a usual haunt for members of this group of birds. They usually prefer quiet inland waters, where they wade and watch — ever striving to satisfy their insatiable hunger. Feet and Le 385 So, in the case of herons, webs would be superfluous, length of limb being their only requirement. length of limb being their only requirement. The Wood Ibises (or more properly Storks), which are more active searchers after food than the herons, make use of their toes to stir up the bottom mud of shal- FIG. 301.— Toes of Gallinule outstretched. FIG. 301.— Toes of Gallinule outstretched. The Bird low water, keeping the bill ready to snap up any small creatures thus disturbed. When one sees a flock of gal- linules or jacanas feeding quietly in their haunts they appear to be walking on the water, and we find an in- teresting connection between the structure of their feet and toes and certain tropical plants. Such are the The Bird 6 386 great pads of water-lilies, which in places cover miles of water, over whose trembling surfaces the birds are able to run or walk. To enable them to do this without sink- ing! both the toes and claws are remarkably long and slender, so that in a bird which stands but ten or eleven inches in height the weight is distributed over an area of some fifty square inches. This makes it possible for them to feed in places too deep for wading birds and too FIG. 302.- Gallftmle holding food in its foot. FIG. 302.- Gallftmle holding food in its foot. tangled with aquatic vegetation for swimmers readily to make their way. This is but another forceful example of the successful adaptive radiation of birds. Gallinules have found that their long toes can be made useful in other ways besides locomotion, and we find that they are well-nigh as skilful as a parrot in grasping and holding. One of these birds perhaps spies a tuft of water-soaked reeds. He clasps it firmly, draws it up, and, holding it in the air near his bill, picks the small worms and snails from among the stems, finally discarding Feet and Legs it for another footful. forming such an action. are so long and slender, they can swim quite rapi their feet with such effo concert. Their cousins, the coots, resemble the phalaropes in having broad lobes of skin along each toe, so that, although they and the gallinules are often seen feeding in the same locality, yet the nata- tory ability of the coot allows it to venture beyond the reserves of the other species. The toe-lobes also serve an- other important func- tion in permitting the coots to feed upon soft mud, thus keeping them from sinking below the surface, just as the horn ports it on the snow. Herons are furnished it for another footful. We cannot imagine a forming such an action. The Bird Although the toes of are so long and slender, yet, when the necess they can swim quite rapidly for a short distanc their feet with such effort that the whole bod concert. Their cousins, the coots, resemble the phalaropes in having broad lobes of skin along each toe, so that, although they and the gallinules are often seen feeding in the same locality, yet the nata- tory ability of the coot allows it to venture beyond the reserves of the other species. The toe-lobes also serve an- other important func- tion in permitting the coots to feed upon soft FIG. 303.— Foot of mud, thus keeping them from sinking below the surface, just as the horny "snowshoe" of the ports it on the snow. Herons are furnished with a comb-like edge FIG. 303.— Foot of Coot. FIG. 303.— Foot of Coot. FIG. 303.— Foot of Coot. surface, just as the horny "snowshoe" of the grouse sup- ports it on the snow. Herons are furnished with a comb-like edge to one of the claws, similar to that on the claw of the whippoor- will, but as yet we have no clue to its use. Although differing so greatly from hawks in their method of feeding, The Bird 388 388 yet, when put upon the defensive, herons resort to much the same tactics as do the birds of prey. When cornered or wounded, so that escape by flight is impossible, the bird throws itself upon its back and, with uplifted claws and levelled beak, awaits the attack of its assailant. The talons and mandibles of a hawk offer ten sharp points which can all do severe damage; but the heron depends only on the grasping power of its toes to hold fast, while it strikes savage, spear-like blows with its beak. it strikes savage, spear-like blows with its beak. On the borderland of the fully webbed aquatic birds FIG. 304. — Comb on the toe of Heron. FIG. 304. — Comb on the toe of Heron. we find the flamingo, combining characters of the herons and ducks. Its haunts are the exposed coral-flats of tropical keys, where at any time a high tide or a severe storm may sweep all, old and young, from their feet. Then it is lucky indeed that the youngsters have webs between their toes in addition to their long legs. The Bird It is a case of swim or be drowned. In the great Orders of sea-birds, and in the ducks and their allies, the three front toes are joined together by Feet and Legs 389 389 a web of skin which, when swimming, offers a large area of resistance to the water when the foot is pushed back- ward. The chick in the egg has a shadow-membrane of his fish-like ancestors between his toes, and in these water-birds the web of skin continues throughout life. In the terns or sea-swallows, which swim much less than they fly, the web is excised, or scalloped out deeply, a return to an almost semipalmated condition. FIG. 305. — Rough-legged Hawk in position of defence. FIG. 305. — Rough-legged Hawk in position of defence. A duck or swan out of sheer laziness will often hold one foot up out of the water and propel itself with the other, slightly altering the angle at which the web meets the water, so as to maintain a perfectly direct course. There is a little-known habit which I have frequently observed in captive ducks and several times in wild ones, of swimming thus with one foot when both eyes are shut and the bird is apparently fast asleep. But, in such a The Bird 9° 39° case, no attempt is made to proceed in a straight line. In a pool only thirty feet square I have seen a duck revolving thus for an hour or more at a time, impelled with slow, rhythmical (and apparently reflex) strokes. We FIG. 306.— Foot of Black-necked Swan. FIG. 306.— Foot of Black-necked Swan. can imagine that such a habit would sometimes be of much advantage to a wild bird, enabling it to keep away from enemies on the shore and yet at the same time secure rest. The name Steganopodes is applied to the gannets, pelicans, snake-birds, tropic-birds, and cormorants, be- Feet and Le 391 cause the toes of these birds are all bound together with a single web. The hind toe points almost in a forward direction when the foot is in action, and, to complete the adaptation for a perfect swimming foot, the outer toe is the longest, a rare condition among birds. The Bird If one will watch the snake-birds in a zoological park, as they swim about their glass tank, the extreme delicacy of the foot mechanism becomes apparent at once. Not only is the flat side of the leg used as a cutwater, but the toes curl and uncurl with a slight oblique revolving motion like the blades of a propeller. When drawn for- ward through the water they are rolled up into a very small compass and then instantly spread out as widely as possible on the return stroke. To the eye it seems as if the bird was constantly grasping something tangible in the water and thrusting it behind. This propeller motion may be observed even better in a captive grebe. If the bird's head is placed in a glass of water, its feet will move back and forth in the air with all the motion of swimming. The adaptation for swimming in these birds is so fundamental and thorough that even the claws are broadened and flattened until they resemble finger-nails. On land, grebes are absurdly awkward, although they can walk upright even up a slight incline. But they are powerless to rise from the ground, even with the aid of the wind,— needing the greater speed which a swimming take-off from the water will give them. a swimming take-off from the water will give them. The most aquatic of all birds, the penguins, make much more use of their wings than of their feet in swim- ming and diving. The toes are webbed, however, and The Bird 92 392 are doubtless of considerable use when the bird is emerg- ing from the water, which it generally does with a sudden spurt of speed and a strong leap which lands it on its feet. In landbirds which have either lost or are losing the power of flight there is often an interesting correlation to be observed between the lapsing of this mode of loco- FIG. 307.— Feet of Penguin. FIG. 307.— Feet of Penguin. motion and an increased use and consequent greater de- velopment of the legs and feet. The Bird Of a South African bird, about the size of our American Robin, known as the Rock- jumper, it is said: "These curious birds are only to be found on the rock-strewn slopes and summits of mountain- ranges where they are able to hop from rock to rock for a Feet and Le 393 distance without having to cross level or open ground; ... at the slightest alarm they either drop into a crevice or bound from rock to rock with extraordinary speed, look- ing more like india-rubber balls than birds, for there is no perceptible interval between the end of one leap and the beginning of the next, and the distance they can clear at a single hop must be seen to be believed. Should they have to cross a piece of level ground between two rocks which they cannot clear with a single bound, they run across it with great speed and usually with outspread wings. So feeble are their powers of flight that they seldom attempt to fly, and never when in a hurry or alarmed; at the most they flutter feebly for a few hundred yards down hill. I have occasionally amused myself by trying to drive these birds across a piece of open ground, but I have never succeeded in getting them to quit the shelter of the rocks, where they easily avoid one by leaping over the stones or hiding in the crevices. In spite of his loose, fluffy plumage, which blows about in the slightest breeze and gives him a rather untidy appearance, the cock is an ex- ceedingly handsome bird." So we have here an isolated case of direct relation between two organs, the balance of power changing from wing to feet and affecting much of the bird's structure, even the plumage losing its cohe- siveness. The weak-flying Tinamou have unusually sturdy legs, and many other instances might be mentioned. any reasons the most interesting of all birds' legs, and many other instances might be mentioned. For many reasons the most interesting of all birds' feet are those of the ostriches and their allies, and among them the most extreme examples of this same cause and effect are to be found. The Bird The Bird 394 When one trains in college for a long-distance race, one rule to observe is, never touch your heels to the ground ; run wholly on the ball of the foot. Untold centuries ago, wise old Nature whispered the very same direction to those of her children who had most need to run for their lives in life's great race, and down through the ages some of them have never broken training. When an animal acquires great speed in running or leaping, there is a tendency for one toe to become greatly en- larged at the expense of the others, as is seen in the case of the horse, the kangaroo, and the ostrich. In the horse only When one trains in college for a long-distance race, one rule to observe is, never touch your heels to the ground ; run wholly on the ball of the foot. Untold centuries ago, wise old Nature whispered the very same direction to those of her children who had most need to run for their lives in life's great race, and down through the ages some of them have never broken training. When FIG. 308. — Feet and legs of Cassowary. In the horse only the middle toe is functional, the second and fourth having de- generated into the small splint-bones at FIG. 308. — Feet and legs of Cassowary. the side of the leg. The kangaroo progresses upon the fourth and fifth toes, the second and third being small and skin-bound. The ostrich has but two toes, one of which, the third, as in the case of the horse, is very large and armed with a thick claw, which, hoof- like, grows close to the toe. This toe supports most of the bird's weight, while the fourth or outer toe is only FIG. 308. — Feet and legs of Cassowary. small splint-bones at the side of the leg. The kangaroo progresses upon the fourth and fifth toes, the second and third being small and skin-bound. The ostrich has but two toes, one of which, the third, as in the case of the horse, is very large and armed with a thick claw, which, hoof- like, grows close to the toe. This toe supports most of the bird's weight, while the fourth or outer toe is only Feet and Legs 395 mm FIG. 309. The Bird — (a) Front and (b) side view of foot of Ostrich. Feet and Legs Feet and Legs 395 FIG. 309. — (a) Front and (b) side view of foot of Ostrich. FIG. 310. — Feet of Donkey. FIG. 311. — Feet of young Kangaroo. FIG. 310. — Feet of Donkey. FIG. 311. — Feet of young Kangaroo. The Bird 9^ 39^ one quarter as large; and indeed it bids fair to disappear altogether in the course of time, and even now the dimin- utive nail which is often present is only as large as the claw of a chicken. claw of a chicken. The power of the ostrich to defend itself by kicking is proverbial, but the claw on the large toe is blunt and the ability to inflict injury lies in the terrible force of the blow. Its ally, the cassowary, has three good-sized toes, and on the innermost one a specially adapt- ed weapon in the shape of a strong, pointed, talon-like claw, four inches in length. The two photographs (Figs. 312 and 313) show how similar the tracks which the modern cassowary makes in walking over moist clay, are to those made by the bipedal reptilian Dinosaurs millions of years ago, which have been found in the Connecticut valley. Thus in our brief review we have seen how the feet and legs of birds serve them well in walking, hopping, running, perch- ing, scratching, climbing, burrowing, swimming, diving, in addition to the finding of their food, fighting, FIG. 312.— Tracks of Casso- wary in soft clay. The power of the ostrich to defend itself by kicking is proverbial, but the claw on the large toe is blunt and the ability to inflict injury lies in the terrible force of the blow. Its ally, the cassowary, has three good-sized toes, and on The power of the ostrich to defend itself by kicking is proverbial, but the claw on the large toe is blunt and the ability to inflict injury lies in the terrible force of the blow. Its ally, the cassowary, has three good-sized toes, and on the innermost one a specially adapt- ed weapon in the shape of a strong, pointed, talon-like claw, four inches he ostrich to defend itself by kicking is proverbial, but the claw on the large toe is blunt and the ability to inflict injury lies in the terrible force of the blow. The Bird Its ally, the cassowary, has three good-sized toes, and on the innermost one a specially adapt- ed weapon in the shape of a strong, pointed, talon-like claw, four inches in length. FIG. 312.— Tracks of Casso- wary in soft clay. The two photographs (Figs. 312 and 313) show how similar the tracks which the modern cassowary makes in walking over moist clay, are to those made by the bipedal reptilian Dinosaurs millions of years ago, which have been found in the Connecticut valley. Thus in our brief review we have seen how the feet and legs of birds serve them well in walking, hopping, running, perch- ing, scratching, climbing, burrowing, swimming, diving, in addition to the finding of their food, fighting, preening their feathers, and in countless other ways. The story of the bird's foot has not half been told, FIG. 312.— Tracks of Casso- wary in soft clay. FIG. 312.— Tracks of Casso- wary in soft clay. Feet and Legs 397 but enough has been said to arouse our interest in this member and to put us on the watch for new facts. FIG. 313.— Fossil Dinosaur tracks, found at Middletown, New York. (Courtesy of Prof. R. S. Lull.) FIG. 313.— Fossil Dinosaur tracks, found at Middletown, New York. (Courtesy of Prof. R. S. Lull.) TAILS E have found that almost every organ of a bird's body may be compared directly with the corre- sponding structure in the body of a lizard or of some reptile, and the tail is no exception: although a lizard with a fan-shaped group of feathers sprouting from the root of his tail would certainly be an anomaly; and even if we substitute scales for the feathers, the result would be ridiculous and unmeaning. But glance at the photograph of the tail of our ancient, original-bird ac- quaintance, the Archseopteryx, Fig. 315, which was taken expressly for this purpose. E Take twenty feathers and arrange them as in Fig. 314 a, representing the tail of Archa3opteryx ; then rearrange them as in 314 6, corresponding to the tail of modern birds, and the whole matter will be clear. Archaopteryx had twenty bones in its tail, all separate, long and slender, and arranged end to end, just as are the bones of a lizard's tail to-day. But in the case of the bird of olden time a pair of feathers grew out, one on each side of the tail- bone, making forty tail-feathers in all. As we have seen, this bird was rather weak-winged and probably more mo a) Arrangement of 20 feathers, as in Archceopteryx ; (6) t of a Sparrow in place. FIG. 314. — (a) Arrangement of 20 feathers, as in Archceopteryx ; (6) tail-feathers of a Sparrow in place. 399 399 The Bird 4 of a flutterer, or sealer, than a true flier, but as time went on, and birds became more and more expert on the wing, their wings grew stronger and their tails shorter and more compact. We can readily see the reason for this, if we imagine a ship which has been built with a rudder as long as its whole deck. What an awkward thing such a rudder would be! The waves would beat against it and great force would be necessary to turn it and to steer the ship. As long as a bird was content to climb a tree with its hands and feet, and then scale, like a flying squirrel, to the base of the next, a lizard-like tail would be all-sufficient. TAILS So conspicuous and so unbirdlike was the long appendage of the Archseopteryx that Saururce — lizard-tailed — has been given as the name of the Sub-class which it occu- pies all to itself. pies all to itself. When we look at the bones of the tail of a modern bird, we find that many interesting changes have taken place since the days of the lizard-tailed ancestors. Thus in the common duck, for example, we find eight free bones followed by a large upturned bone, which, from its shape, is known as the ploughshare. It is this terminal bone which supports all the tail-feathers of modern birds, and in the duck it represents ten of the lizard-tail bones all telescoped and fused into one. Some of the feathers have been lost, as there are but sixteen in this bird's tail. This loss of tail-feathers is of no value in classifica- tion, as it may vary within narrow limits. For example, one species of cormorant has seven pairs of tail-feathers, while a closely related species has but six. Not only this, but the variation may be merely sexual, as in the • FIG. 315. — Tail of Archceopteryx in British Museum. • FIG. 315. — Tail of Archceopteryx in British Museum. The Bird 4 peacock, which has ten pairs, while the peahen has one pak less. pak less. The fusing together of these bones has resulted in the drawing together of the feathers, so that, instead of the long, unwieldy, paired affair, they are arranged in fan shape, although still in pairs, and usually showing a slight graduation reminiscent of the old-style tail. Some birds have as few as four pairs of tail-feathers, while others FIG. 316.— Tail-hones of Ostrich. FIG. 316.— Tail-hones of Ostrich. have as many as twelve. In the abnormal domestic breed of pigeons known as fantails, as many as forty tail-feathers are sometimes found. The cassowary and the emeu have none at all, while the ostrich seems to have an in- definite number; the tails of these two unrelated groups of birds seeming, like their wing-feathers, to have lost uniformity from little use. Besides these true tail-feathers there are others, usually smaller, which grow from above and below the tail, being known as upper and under tail- have as many as twelve. TAILS In the abnormal domestic breed of pigeons known as fantails, as many as forty tail-feathers are sometimes found. The cassowary and the emeu have none at all, while the ostrich seems to have an in- definite number; the tails of these two unrelated groups of birds seeming, like their wing-feathers, to have lost uniformity from little use. Besides these true tail-feathers there are others, usually smaller, which grow from above and below the tail, being known as upper and under tail- Ta 403 coverts. Mention is here made of these because of the important part they take in certain sham tails which will soon be described. In the embryos of most birds of true flight the tail- tip of the back-bone is represented by six or ten separate pieces, which, before the chick hatches from the egg, fuse into the ploughshare bone. In the ostrich-like birds FIG. 317. — Tail-bones of Bald Eagle, showing greater fusion and more specialization than in Fig. 316. FIG. 317. — Tail-bones of Bald Eagle, showing greater fusion and more specialization than in Fig. 316. these small bones never fuse, but remain separate through- out life — a reptilian character persistent in these strange birds (Fig. 3 16). The ploughshare bone is seen splendidly developed in such a bird of strong flight as the Bald Eagle. Now that we have explained the origin of the tail, let us consider what part it plays in the lives of the birds about us. So diverse are the modes of life, and so varied are the surroundings of this class of creatures, that we The Bird 404 FIG. 318. — Fan-tailed Pigeon, showing extreme development of tail. FIG. 318. — Fan-tailed Pigeon, showing extreme development of tail. Fio. 319. — Emeu, a tailless bird. (Courtesy of N. Y. Zoological Society.) Fio. 319. — Emeu, a tailless bird. (Courtesy of N. Y. Zoological Society.) Tails shall find many unexpected uses to which the tail is put, and yet those which have been explained are a mere frac- tion of the problems which still await solution. ch still await solution. TAILS The principal use of the tail-feathers in birds is, of course, to perform the function of a rudder, and we find that the arrangement of the bones perfectly carries out the simile of a tail to the rudder of a ship; namely, a broad, expanded surface which is closely hinged to the FIG. 320. FIG. 321. Tail of Barn Swallow, closed (320) and spread (321). 320. FIG. 321. Tail of Barn Swallow, closed (320) and spread (321). 320. FIG. 321. Tail of Barn Swallow, closed (320) and spread (321). body by several movable joints. The real tail of a bird is the small, fleshy protuberance which in our roast chicken we call the "pope's nose"; but in common par- lance the word tail has come to be applied to the large feathers which sprout from this structure. Thus, although not comparable to the appendages of mammals, the so- called tail of a bird is superficially more like the correspond- ing organ of a whale than the tail-fin of a fish, since it is expanded horizontally instead of vertically. The Bird 06 406 One interesting analogy to the fin of a fish is found in the tail of the Blue Duck of New Zealand. This bird lives in swift mountain streams and when swimming carries its long tail entirely submerged. By vigorous sidewise flicks of these tail-feathers it can turn around, as if on a pivot, without being carried down-stream, even when in the centre of a rapid, swirling current. PIG. 322. — Murre showing tail. (Compare with Fig. 246.) PIG. 322. — Murre showing tail. (Compare with Fig. 246.) It is interesting to compare this use of the tail-feathers with the function of the tail in the flightless penguins. In the Black-footed species, at least, the tail-feathers are stiff and short, but the bones of the tail are unusually elongated and the flesh which covers them is flattened into a kind of vertical rudder. Strong muscles control Ta 407 this, and by it the extremely quick dives and turns are made possible. No feather would be stiff or rigid enough to offer to the water the resistance which these feathered seals require. Exceptions to the rudder use in flying birds are found in the murres — sea-birds which share the cliffs of our north- ern coast with cormorants and gulls. The tail-feathers of a murre are so short as to be useless for steering pur- poses, so in flight the bird uses its webbed feet instead, stretching them out behind, opening, turning, and twist- ing them in harmony with the wings, with as satisfactory results as could be desired. The shape of the tip of the tail varies greatly in birds. It may be square or rounded, or cuneate, or indented in the centre, or swallow-tailed, as we appropriately call the latter deeply forked condition. These conditions may be paralleled or duplicated in many different Families of birds. For example, the forked type is seen in our com- mon Barn Swallow, in those dainty relatives of the gulls, the terns — " Swallows of the Sea," — and again in the Forked-tailed Kite and the Scissor-tailed Flycatcher. By closely watching a swallow as it courses swiftly over a meadow, or shoots upward, buoying itself against the breeze, we can appreciate the delicate adjustment of the muscles which govern the tail-feathers. The Bird Each feather seems vital with life, now sliding one over the other until all are in a narrow line, then expanding, with less friction than ever a fan opened, into a wide-spreading, gently graduated fork. The quartet of forked-tailed birds men- tioned above are splendid fliers, but we shall see that skill The Bird 08 408 in flight depends but little upon the shape of the tip, when we consider certain birds with cuneate tails, or those in which the central feathers, soft and not rigid, are elongated, instead of the outer ones. The Undulated Grass Parrakeet shows a condition almost the opposite of the swallow. The Mexican long- tailed jays, the magpies, and the tropic-birds are also all of this type, the latter being especially fine fliers and capable of remarkable aerial evolutions. Again, some FIG. 323.— Tail of Grass Parrakeet. FIG. 323.— Tail of Grass Parrakeet. of the flycatchers with moderate, rounded tails can exe- cute most wonderful flight movements, steering in erratic darts through the air, or darting aside at right angles while at full speed, this being accomplished principally by means of the tail. of the flycatchers with moderate, rounded tails can exe- cute most wonderful flight movements, steering in erratic darts through the air, or darting aside at right angles while at full speed, this being accomplished principally by means of the tail. A tail serves also an important use as a brake. When a great pelican settles gradually toward the surface of the water, or a duck momentarily hovers before alighting, the tail, wide-spread and brought downward, gives effi- cient aid in retarding the impetus. Tails 409 We notice that birds which have very short tails are unable to turn quickly and that their flight is very direct, or even where there is a long tail, if it is principally for ornament and not well muscled, it is of little use in help- ing its owner to change the direction of flight. The partridge-like tinamous of South America are good exam- ples of the first-mentioned group. Their tails are small and useless, and when once the bird launches itself into FIG. 324. — Tail of Pelican alighting. FIG. 325.— Tail of Tern in flight. FIG. 324. — Tail of Pelican alighting. FIG. 325.— Tail of Tern in flight. the air, it can keep on only in a straight line and is at the mercy of every cross-current of air. A more familiar case, which any one may observe, is a Song Sparrow, or other small bird, which, from accident or from some irregu- larity of moult, has lost all or most of its tail-feathers. Instead of rising with the strong, darting flight with which such a bird is accustomed to make its escape from our path, its flight under such conditions is weak and direct, like the trial efforts of a young bird. The Bird 41 o Reserving the mention of partly ornamental tails until the last, we may now consider the use of this member as a prop or support to the bird as it clings to or makes its way up vertical surfaces. Four groups of birds which are thus distinguished are the woodhewers — a tropical FIG. 326.— Chimney Swift clinging to wall, resting upon tail. FIG. 326.— Chimney Swift clinging to wall, resting upon tail. family, — the creepers, woodpeckers, and swifts. These birds really sit upon their tails, the feathers of which are adapted for this special use, while retaining perfectly the rudder function in flight. The tail-feathers of the Chim- ney Swift are peculiar in having the ends, for a short dis- Ta 411 tance, free of barbs, the tips being thus composed of a num- ber of bare spines which are admirably adapted to catch in the irregularities of hollow trees, or, as now in their recently adopted homes, in the roughness of chimney- bricks. I one day caught a Chimney Swift and placed it against a varnished wall composed of composition bricks; and, smooth though the surface was, the bird's tail and toes held it firmly, not slipping even a quarter of an inch. After photographing it, I watched it for some minutes and saw the bird shift its position several times, moving always with a certainty and surety of grasp most inexplicable. The tails of woodpeckers and creepers are not thus denuded at the tip, but they are stiffened throughout and are very elastic (Fig. 240). When a woodpecker brings up against a comparatively smooth tree-trunk, its certainty of hold is a perfect bit of magic. Then when it braces itself and sets to work to hammer a hole into the wood, or to excavate its nest, how the tail-feathers bend and spread, buttressing themselves against every roughness, the elasticity of the feather-tips allowing them to slip into every crevice! In many birds the tail is a perfect index of the emotions, doing much to compensate for the lack of facial expression. Especially is this true of the wrens, those feathered bundles of tireless energy and curiosity, whose tails, upturned so high that they fairly tilt forward over the back, twitch and jerk with every passing mood. The Bird Even the genetic individuality of a species may be hinted at in the way it carries its tail; quiet, soft-mannered birds holding it The Bird 412 412 low, beneath the wing-tips, while active, nervous species carry it more or less raised. FIG. 327.— Tail of Chimney Swift. In certain of the flycatchers the tail, which hangs demurely downward, reacts with a jerk to every note of the bird, as if connected with the bird's vo- cal apparatus, as in our com- mon Least Flycatcher at every " Che-bee'!" " Che-bee'!" The jerking motion of the FIG. 327.— Tail of Chimney Swift. tail seems to have become a regular habit with many birds, and, curiously enough, FIG. 327.— Tail of Chimney Swift. FIG. 328.— Tail of Flicker. FIG. 328.— Tail of Flicker. especially with those which spend their lives chiefly along Ta 4 1 3 the borders of streams. We are all familiar with the tip- ping of the tail in sandpipers, and, including the Green Heron, we will see much the same motion in birds which haunt the stream borders ; even in the Water Thrush the same habit prevailing, although, as we saw in the pre- ceding chapter, this bird is closely related to the bright- coloured warblers of our tree-tops. The wagtails have received their name from this same habit, of which no explanation has yet been offered. The Road-runner, a ground cuckoo of the Western plains, has a tail as long as its entire body, which is as expressive as the gestures of a Frenchman. When sitting quietly in the shade of a mesquite-bush in Mexico, I have seen one of these birds dash into sight and drop, like an arrow, upon a luckless lizard. At the moment of attack all ten tail-feathers of the bird were wide-spread and a-tremor, indicative of the extreme excitement attendant upon the capture of the reptile. While eating what choice parts were desired, the tail was folded and lifted out of the way. Soon the bird spied some motion of mine, and with the suspicion came the high extended neck, while the tail turned up and forward, until almost touching the bird's head. A second motion on my part, and the tail manoeuvred to a line and trailed limply after the bird, as it half-flew, half-leaped to a high rock and on out of sight. sight. The Bird The white under sides of the tails of the wild rabbit and the white-tailed deer have been explained as warning signals to others of the family or herd: white guides which the less experienced members may follow and so The Bird 414 escape from danger. Again, the theory has been advanced that these white patches merge with the sky when the rab- bit or deer makes the first high frantic leap to escape an assailant, the white spots thus tending to confuse the creature making the attack. We are, however, far from certain whether any such interpretations can be applied to those birds, such as the Junco, the Meadowlark, and the Vesper Sparrow, which have the lateral feathers of escape from danger. Again, the theory has been advanced that these white patches merge with the sky when the rab- bit or deer makes the first high frantic leap to escape an assailant, the white spots thus tending to confuse the creature making the attack. We are, however, far from certain whether any such interpretations can be applied to those birds, such as the Junco, the Meadowlark, and the Vesper Sparrow, which have the lateral feathers of the tail white; but in these cases the first theory seems at least more probable, as these birds live in flocks and in a more or less open en- vironment, where such a sig- nal would have the greatest chance for use. When a Junco is upon the ground, its black and gray plumage renders it very inconspicu- , FIG. 329.— Tail of Junco. ous> but the instant it takes to wing, out flashes the white V in its tail. , FIG. 329.— Tail of Junco. FIG. 329.— Tail of Junc We have seen that not a portion of the external parts of the bird has escaped, in one species or another, being utilized for ornament; generally, as well as we can tell? as some decoration to attract or charm the female. Tails bear even more than their share of adornment, which we cannot pass by without mention, although, as dealing with the psychological side of bird life, any discussion of this question is outside the province of this volume. Some- times it is only some slight addition to the feathers of Ta 415 the tail proper, as the elongated middle feathers of the male Pintail Duck and the Sharp-tailed Grouse. The Bird male Pintail Duck and the Sharp-tailed Grouse. Turning to a few of the more decorative tails in the world of birds, we find a small Australian bird, known as the Emeu-wren, bearing aloft a half-dozen long feathers, so scantily clothed with barbs as to resemble somewhat the plumage of the Emeu itself. These skeleton plumes, for they are little else, while giving a striking appearance FIG. 330.— Tail of Emeu-wren. (Cf. with Fig. 23.) FIG. 330.— Tail of Emeu-wren. (Cf. with Fig. 23.) to the owner, must radically weaken its flight, as regards steering capacity; since the open-work mesh of the vanes can offer no resistance to the air. Indeed it is said of this bird that it is such a poor flier that it is seldom seen on the wing, but it runs rapidly and is able to leap into the lower branches of trees. The penalty of danger from weakened flight which the Emeu-wren must pay for his caudal decoration is paralleled by certain little whydah- finches of Africa, the males of which at the breeding season are decorated with several tail-feathers over five to the owner, must radically weaken its flight, as regards steering capacity; since the open-work mesh of the vanes can offer no resistance to the air. Indeed it is said of this bird that it is such a poor flier that it is seldom seen on the wing, but it runs rapidly and is able to leap into the lower branches of trees. The penalty of danger from weakened flight which the Emeu-wren must pay for his caudal decoration is paralleled by certain little whydah- finches of Africa, the males of which at the breeding season are decorated with several tail-feathers over five The Bird 4i 6 times as long as their tiny bodies. When a heavy dew falls during the night, drenching the plumage of these birds, they become helpless and quite unable to fly. At such times many are killed by their natural enemies; and such indeed is their helplessness, brought about by FIG. 331. — Male Paradise Whydah-bird showing tail. FIG. 331. — Male Paradise Whydah-bird showing tail. this excess of nuptial dress, that a person can pick them up in the hand without difficulty. this excess of nuptial dress, that a person can pick them up in the hand without difficulty. The Bird The beautiful tails of pheasants are in harmony with the wealth of colour which many of these birds display upon other parts of the body; the long graceful tail of the Reeves being especially striking. he folded, roof-shaped tail of the common rooster, Japanese Long-tailed Fowls. (From a photograph provided by the American Museum of Natural History.) 417 Fio. 332.— Japanese Long-tailed Fowls. (From a photograph provided by the American Museum of Natural History.) 417 417 The Bird 4i8 4i8 and of his wild ancestors the Jungle-fowl, with the graceful overarching feathers, is a type of tail found elsewhere FIG. 333.— Roof-like tail of Jungle-fowl. FIG. 333.— Roof-like tail of Jungle-fowl. only in certain pheasants. In the Boat-tailed Grackle the arrangement is reversed, the apex of the slope being only in certain pheasants. In the Boat-tailed Grackle the arrangement is reversed, the apex of the slope being FIG. 334. — Decorative tail of Reeves Pheasant. FIG. 334. — Decorative tail of Reeves Pheasant. FIG. 334. — Decorative tail of Reeves Pheasant. beneath instead of above. The possibilities of abnormal feather growth are well shown in the tails of the Japanese Tails 419 Long-tailed Fowl — a breed of birds in which, by artificial stimulation, such perhaps as periodical pulling of the feathers or else retardation of moult, has produced, in the cocks, tails from twelve to twenty feet in length. This process dates back, in Corea at least, to A.D. 1000, and necessitates keeping the birds continually upon high perches, or else wrapping the feathers carefully in paper. The arrangement of feathers in this artificially induced character is duplicated in nature in the Paradise Why- dah-finch mentioned above. Long-tailed Fowl — a breed of birds in which, by artificial stimulation, such perhaps as periodical pulling of the feathers or else retardation of moult, has produced, in the cocks, tails from twelve to twenty feet in length. This process dates back, in Corea at least, to A.D. 1000, and necessitates keeping the birds continually upon high perches, or else wrapping the feathers carefully in paper. The arrangement of feathers in this artificially induced character is duplicated in nature in the Paradise Why- dah-finch mentioned above. FIG. 335.— Useful tail of Peacock Pheasant. dah-finch mentioned above. The Bird In both the male and fe- male Peacock Pheasant the tail is quite long and the feathers are decorated with beautiful iridescent " eyes." But in this bird usefulness ex- ists as a corollary of beauty. When the young chicks are reared under a bantam hen, they invariably keep close be- hind their foster-mother, for no apparent reason; indeed this position often results in their death, a kick from the bird's foot generally being fatal. The reason for this strange instinctive act is at once clear when we see the chicks with their rightful mother. They spend much of their time hidden beneath the shelter of her long, sloping tail, coming out now FIG. 335.— Useful tail of Peacock Pheasant. FIG. 335.— Useful tail of Peacock Pheasant. The Bird 420 420 and then to feed when she calls them, then hurrying back to their snug shelter. Thus when she walks from place to place, the tiny feet of the chicks may be seen scurrying along beneath the beautiful tail-feathers, all FIG. 336.— Tail of Lyre-bird. FIG. 336.— Tail of Lyre-bird. but their legs concealed from view, giving a most re- markable appearance to the mother bird. markable appearance to the mot Among ornamental tails assumed for show during the breeding season, that of the Lyre-bird of Australia is unequalled. The name is well given, since the outer tail-feathers carry out the graceful, curving outline of Tails 421 the classic form of a lyre; while twelve of the central feathers, so scantily barbed that their stems are plainly visible, hold positions corresponding to the strings of that ancient instrument. The two elongated middle feathers cross each other and curve outward, adding still more to the decorative effect of this strangely beautiful member. Naturally we find that these birds are better runners than fliers. The females lack the or- namental tail. FIG. 337.— Tail-coverts of Quezal. If we judge from analogy with the human race, when an inordinate amount of ostenta- tious show is noticeable among birds, we occasionally find that it is, in a sense, a sham display ; although the analogy ceases when we find that such a case among birds is no less interesting than where the phenomenon is really what it appears to be. Upon seeing a specimen of the beau- tiful trogon commonly called j.i rv i , i , FIG. 337.— Tail-coverts of Quezal. The Bird the (Quezal, the involuntary ex- clamation is, " What a magnificent tail!" And no wonder; for, while the bird is only about the size of a small dove, behind it, for three and a half feet, there stream long, iridescent green plumes, soft as down, brilliant as emeralds. Yet the true tail is a short, squarish affair, completely If we judge from analogy with the human race, when an inordinate amount of ostenta- tious show is noticeable among birds, we occasionally find that it is, in a sense, a sham display ; although the analogy ceases when we find that such a case among birds is no less interesting than where the phenomenon is really what it appears to be. Upon seeing a specimen of the beau- tiful trogon commonly called j.i rv i , i , the (Quezal, the involuntary ex- clamation is, " What a magnificent for, while the bird is only about t behind it, for three and a half iridescent green plumes, soft as dow Yet the true tail is a short, squ FIG. 337.— Tail-coverts of Quezal. The Bird The Bird 2 422 hidden by the overhanging train of gorgeous plumes, which are in reality the upper tail-coverts. which are in reality the upper tail-coverts. The same thing is true of the peacock, whose real tail, while it has the power of spreading, consists solely of FIG. 338. — Train of Peacock spread. FIG. 338. — Train of Peacock spread. short, dull, brownish feathers, acting as a support to the glorious train of ocellated plumes which springs from the lower back. Indeed the tail-feathers of a turkey-cock are far more beautiful than the real tail of a peacock. This is especially evident when, after a peacock has moulted Ta 423 his long train, he sometimes spreads the real, incon- spicuous tail. Large and heavy as this decoration of the peacock is, the birds fly with remarkable ease. In such places as the New York Zoological Park, after roost- FIG. 339. — Rear view of train of Peacock, showing real tail. FIG. 339. — Rear view of train of Peacock, showing real tail. ing all night in the tallest trees, they sail down in the early morning, the long train waving gracefully behind — a sight which, once seen, is never forgotten. The Bird ing all night in the tallest trees, they sail down in the early morning, the long train waving gracefully behind — a sight which, once seen, is never forgotten. We must leave unmentioned scores of beautiful and The Bird 424 interesting types of tail-feathers — those of hummingbirds, birds of paradise, and many others; but there is one which deserves especial mention. These birds, of which there are a number of species, are the motmots, abundant in many parts of Mexico and southward. The tail-feathers of the Mexican motmot, which are bluish green in colour, FIG. 340. — Tails of Motmot: (a) young male; (6) adult female. FIG. 340. — Tails of Motmot: (a) young male; (6) adult female. have nothing peculiar about them, except the middle pair, which are two inches longer than the others. Of this extra length one inch is bare shaft, while at the tip the barbs are normal, forming a racket-shaped extremity. The fact which places this slight decoration above all other more elaborate examples in point of interest is that the birds themselves voluntarily produce the racket condition. Even the youngest birds, of both sexes, when the long central have nothing peculiar about them, except the middle pair, which are two inches longer than the others. Of this extra length one inch is bare shaft, while at the tip the barbs are normal, forming a racket-shaped extremity. The fact which places this slight decoration above all other more elaborate examples in point of interest is that the birds themselves voluntarily produce the racket condition. Even the youngest birds, of both sexes, when the long central Tails 425 tail-feathers have grown beyond the others, instinctively begin to pick at the vane, soon denuding the shaft so symmetrically that the rackets are equal in size. The FIG. 341. — Motmot swinging its tail. FIG. 341. — Motmot swinging its tail. photographs show this perfectly. Figure a is the tail of a young male where the operation of ornamental de- nudation has just begun; while b shows the condition in an adult female. The photograph of the entire liv- ing bird also shows the rackets, as well as the peculiar pendulum motion of the tail from side to side, although The Bird 4 the motion is more abrupt than is the motion of a pendu- lum. The Bird The Mexican motmot is brilliantly coloured, yet in a densely foliaged tree, among the bright spots of sunlight, it becomes almost invisible. It is the motion of the tail which most often betrays the bird. In the tail marked a in Fig. 340 it will be noticed that, where the barbs have not yet been picked off, the unmu- tilated vane is considerably narrowed — an interesting fact for the consideration of evolutionists, as it offers strong circumstantial evidence, but by no means absolute proof, of a case of the inheritance of acquired characters, a much- rr ooted question not many years ago among scientists. If we choose to accept the evidence thus, we may presume that if this habit is continued through a sufficient number of generations, the vane will, at the point of continued denudation, ultimately become naturally bare. But, in any case, it is a fact which must hold the inter- est of the most superficial bird student that here is a bird which voluntarily tears away a portion of its plu- mage. To the best of our present knowledge this is solely to ornament itself, but the fact that both sexes equally possess this habit makes such an explanation the merest theory. The interest which this has for us here is not the ultimate psychological significance of the habit, but the fact that there is a bird which thus voluntarily mutilates its plumage. As in so many other cases, we must depend on future study of live birds in their natural haunts to clear up the difficulty. It is this very com- plexity of Nature's problems which makes a naturalist's life ever one of enthusiasm and zest. THE EGGS OF BIRDS ERHAPS the most fascinating phase of Nature is the way in which she cares for her chil- dren during the early part of their lives. The story of seeds and eggs has not been half told. Think of the tiny thistle-fluff which soars away, borne on the lightest breath of air; of the great cocoanuts in their husks, so hard that they will turn the edge of a knife; of the burrs which ever patiently reach out for some passing creature to carry them to a distant home; of the cones of the forest, whose seeds may be transported by birds, or dropped to the ground only to smother in the shadow of the parent tree. E In that "mother of life" the sea, the wonder of the first beginnings holds us spellbound. We see the tiny hydroids, those animal plants, flowering and budding on their waving stalks, and presently setting free their "seeds" — jelly-fish, — throbbing with life, drifting away on the ocean currents. Again observe these jellies scatter- ing behind them an untold host of eggs, as a rocket marks its path with a myriad sparks. Think of the salmon seeking her spawning-grounds in the uppermost reaches of rivers, or the cod boldly playing for her offspring the 427 The Bird 4 chance in the lottery of life in the open ocean. Of her nine millions of eggs, will one survive? How strange is the four-tendriled, purse-like cradle of the baby shark; how delicate the forms and patterns of butterflies' eggs ! and was there ever a more model parent than that frog which holds its eggs in its mouth until the tadpoles grow up? The white leathery eggs of turtles and lizards bring us to our subject. Leading all in beauty and interest are the eggs of birds. Precious stones have always exerted a great fascination over mankind, and in appearance birds' eggs may be compared with gems; indeed the shell itself is almost wholly composed of mineral matter. But, far from being an inanimate crystal, an egg shelters one of the marvels of the world — an embryo bird. The gaudy sea-shell cloaks a slimy snail, but from the beautiful egg of a bird emerges a greater beauty. Reptiles lay white eggs whose shells are not brittle, but, when broken, curl up like a celluloid film. THE EGGS OF BIRDS Some of these reptilian eggs are oblong in shape, but most are spherical and the great majority are deposited in the ground, or under bark, and are hatched by the heat of the decaying vegetation or by the direct rays of the sun. Thus we see that there is little need for variation in shape or colour. Among birds, however, we find very different conditions. As we know that birds have evolved from reptiles, we have a right to suppose that the early forms of birds laid white, leathery eggs, perhaps in hollow trees; but the power of flight has taken birds entirely out of the FIG. 342.— Comparison of eg (a) Egg of Hen. (b) Egg of Skate, Egg of Alligator. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turtl Egg of Alligator. 429 FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turtle. (e) Egg of Alligator. 429 429 The Bird 43° reptilian horizon, and greatly altered all the conditions of their life. The history of the egg of a bird, from the time it is laid until it hatches, has an all-important effect on its form, colour, and even upon the number of eggs laid. This is not strange when we consider that every minute of the bird's life is open to many dangers, and that the egg stage — that bridging over of generations — is a most precarious period. That which adds the greatest interest to anything is the why of it, and a vast collection of eggs, beautiful though they are, yet, if ignorantly looked at, is worse than useless. Why one bird lays twenty eggs and another but two ; why one bird's eggs are white, another's of varied colours, we will never learn from blown museum speci- mens. Not until we have the patience and skill to watch and to find the most deadly enemies which threaten the nests and eggs of birds, their number and modes of attack, can we hope for successful solutions to the thousand and one problems which offer themselves. What we know in respect to eggs is fragmentary and rests on so slight a degree of proof that every theory is attacked and re- attacked in turn. Supposing that the eggs of the early forms bf birds were round, — that being the most typical form of a single cell, — we find many variations in shape among the eggs of living species. Many of the eggs which are laid in hollow trees still retain the primitive spherical form, per- haps an advantage in keeping the eggs in a close group in the centre of the floor of the cavity. So characteristic of the eggs of birds is the pear-shape The Eggs of Bir 4^1 — one end blunt and narrowing to the other — that they have given to it its name: oval. In the eggs of certain sea-birds which breed on the narrow ledges of perpendicular cliffs this oval shape is carried to an extreme, and ap- parently for an excellent reason, mechanical, but of ines- timable value to the birds. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 Eggs laid in such positions FIG. 343.— Egg of Murre. FIG. 343.— Egg of Murre. are of course especially exposed to danger from the wind or from some sudden movement of the birds, which gener- ally nest very close together. Were it not that the eggs, on account of their peculiar shape, describe an arc of very small diameter when they roll, doubtless a far greater number wrould roll off and be dashed down upon the rocks below. Among the plovers, sandpipers, and phal- aropes we again find a peculiarly pronounced pyriform are of course especially exposed to danger from the wind or from some sudden movement of the birds, which gener- ally nest very close together. Were it not that the eggs, on account of their peculiar shape, describe an arc of very small diameter when they roll, doubtless a far greater number wrould roll off and be dashed down upon the rocks below. Among the plovers, sandpipers, and phal- aropes we again find a peculiarly pronounced pyriform The Bird 432 432 shape of egg, serving in these instances a very apparent and useful end. These birds almost invariably lay four eggs, which are of large size in comparison with the birds, and their shape allows them to be fitted closely together, each forming one of the four segments, their points all but meeting in the centre. Thus the little body of the FIG. 344.— Eggs of Killdeer. FIG. 344.— Eggs of Killdeer. parent is large enough to cover them all, which would be impossible were the eggs arranged at random. The eggs of grebes are peculiar in having both ends alike. The number of eggs which a bird lays has been found to bear a definite relation to the amount of danger to which the species is exposed — a fact which holds good The Eggs of Bir 433 in the young of many, if not all, other Phyla of animals, and which is one of the most interesting provisions brought about by the slow but sure working of evolution. We may instance the few eggs of the voracious and masterful sharks and the millions of spawn necessary to enable the halibut and the cod to continue in existence. Mr. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 Ernest Ingersoll has so admirably summed up the matter of this relation of the number of eggs to the corresponding danger that I cannot do better than to follow his argument, quoting his words with a slight change here and there. This phase of the study of eggs being so clearly understood, it is well worth a little detail as an illustration of how interesting all the other problems will become when we once get on the right road to their solution. Among the majority of birds the average number of eggs in a nest is from three to six; we may take five as a typical average. "Any considerable departure from this normal number in a species or Family must then be ac- counted for by some specific or tribal peculiarity in cir- cumstances. " Beginning with the ostrichlike group at the bottom of the list, we find ourselves face to face with an inter- esting state of things, to which the number of eggs is an index. Ostriches, rheas, and emeus incubate large clutches — a dozen or more, — those inhabiting the conti- nents of Africa and South America, however, producing twice as many eggs annually as their relatives of Australia and the neighbouring smaller islands. " Immediately following and contrasting with them are The Bird 4 the three groups characterized by the curious elephant- footed, often gigantic moas, and similar birds of Mada- gascar, Mauritius, New Zealand, and the Papuan region, which have become extinct within the historic period, except the kiwis, to be spoken of later. All of these, so far as we know, laid only one egg at a time, which^ plainly enough, was sufficient to keep the race going in the limited space afforded to each species by its island, but which did not suffice to prevent an almost immediate extinction of these species as soon as mankind discovered that the birds and their eggs were serviceable. But Provi- dence, or Nature, or natural selection, or whatever has been the ruling influence in determining means and limits for animal life, seems never to have taken man into ac- count. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 " Turning now to the sea-birds — penguins, gannets, murres, puffins, auks, petrels, guillemots, tropic-birds, and the like, — we find that none of them is in the habit of laying more than one egg, as all breed on such remote and inaccessible rocks, often in holes, that harm can rarely happen to their young, and therefore a very high percentage comes to maturity. Many of these breed in companies, and are so unacquainted with danger that they make no attempt to hide their eggs or to leave the nest when the place is visited by some wandering natu- ralist or egging party. "The habit of the King Penguin deserves a note to itself. This big Antarctic bird guards its one white egg from harm by carrying it somewhat as a marsupial does its young, in a pouch formed by a fold of the skin of the The Bird 6 436 body between the thighs. Both sexes are provided with this contrivance during the breeding season, and relieve each other of the burden at intervals. " The gull tribe, however, are far more exposed to acci- dent and to enemies, both in adult life and as to their eggs and young, than are the penguins, petrels, and others mentioned above; and here the rule is from two (skuas) to four (gulls and terns) eggs in a nest. When we come FIG. 346. — Eggs of Ostrich, Cassowary, Hummingbird, and Hen, showing comparative size. FIG. 346. — Eggs of Ostrich, Cassowary, Hummingbird, and Hen, showing comparative size. to the shore- and marsh-birds — the plovers, snipe, sand- pipers, jacanas, all of which nestle on the ground, usually near the shore of the sea or lakes — we judge them to be exposed to about the average of dangers, since their nest complement is from four to six. The northern, tundra- loving cranes need raise few young, and hatch only two eggs; but when we come to the water-birds — the rails, gallinules, ducks, and geese — we find an extensive group to the shore- and marsh-birds — the plovers, snipe, sand- pipers, jacanas, all of which nestle on the ground, usually near the shore of the sea or lakes — we judge them to be exposed to about the average of dangers, since their nest complement is from four to six. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 The northern, tundra- loving cranes need raise few young, and hatch only two eggs; but when we come to the water-birds — the rails, gallinules, ducks, and geese — we find an extensive group The Eggs of Birds 437 whose nests average a dozen eggs in each set. Explana- tions are ready for this: the birds themselves are exposed to unusual peril, from weather as well as from active enemies, since they mostly emigrate to the extreme North and nest in the edges of marshes, where the sitting birds, FIG. 347.— Nest of Laughing Gull. FIG. 347.— Nest of Laughing Gull. eggs, and young are all subjected to freezings, floods, and countless marauders that depend largely upon them for food during the Arctic summer, so that a heavy annual recruiting must be made to repair losses. Few birds are liable to so many misfortunes and mishaps as the water- eggs, and young are all subjected to freezings, floods, and countless marauders that depend largely upon them for food during the Arctic summer, so that a heavy annual recruiting must be made to repair losses. Few birds are liable to so many misfortunes and mishaps as the water- The Bird 438 438 fowl, except perhaps the big and pugnacious swans, who can take better care of themselves, and lay only five eggs or fewer. The long-legged wading birds also, such as the storks, ibises, herons, and the like, are fairly safe in the breeding season, because they nest in trees, as a rule, (Fig. 356,) and consequently we here find only two FIG. 348. — Nest and eggs of California Partridge. FIG. 348. — Nest and eggs of California Partridge. to four young in the annual brood; so with the snake- birds. "This brings us to the game-birds — the world-wide tribes of partridges, pheasants, grouse, turkeys, jungle fowls, peacocks, and the like — which are of large size, run about on the ground, and are of interest to sportsmen and epi- cures. With few exceptions, these must put forth a The Bird 4 large complement of eggs (eight to twenty) in order to bring to maturity enough young to replace the yearly mortality, for the ground-built homes and huddling chicks encounter a multitude of dangers to which birds in trees, or even the small-sized ground-nesters, are not exposed. One exception here singularly favours the rule. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 The Thibetan Peacock Pheasant inhabits the heights of the Himalayas, where it has to contend with only three or four nest- robbers, instead of the countless foes that infest the lower jungles; hence its ample breast warms but two eggs. " The doves and pigeons lay only two eggs, and a few lay but one; but this seems to be due to the fact that their extraordinary powers of flight render them, as adults, unusual immunity from capture and famine, rather than to any special safety pertaining to their method of nidifi- cation. " Hawks and owls in general have four or five eggs, and as this is about the average number of the small birds on which they largely prey, it seems evident that their chances of life and the difficulty of sustaining it are, on the whole, no less than are met with by their victims. The owls, however, vary much among them- selves in this respect; the Snowy Owls, whose home is in the snowy north, where a nest in the tundra moss is acces- sible to every marauder, and the Burrowing Owls, whose underground homes are constantly robbed, being obliged to lay twice as many eggs as the remainder of the family in order to overcome the high percentage of casualties due to these unfortunate situations. An odd feature in the nidification of some of the The Eggs of Bi 441 Arctic-breeding owls, where the nesting must take place at an unreasonably early and cold date in order to give the fledglings time to reach mature strength before the suc- ceeding winter assails them, is that these birds deposit their eggs at intervals of a week or ten days. In this way the mother can envelop in her plumage and keep thoroughly warm one egg and a callow fledgling at a FIG. 350.— Eggs of Screech Owl. FIG. 350.— Eggs of Screech Owl. time, and is assisted, in respect to the later eggs and fledglings, by the warmth of the older young in the nest. fledglings, by the warmth of the older young in the nest. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 ' The parrots are a wide-spread and numerous tribe, and none of the larger species need lay more than two or three eggs, for they protect them in deep holes in the earth or in trees, and are able to defend them; but some of the smaller parrakeets lay as many as twelve eggs, reflecting the greater dangers with which they have to The Bird 442 442 contend. Toucans are able to get along with a pair of eggs; while a hornbill, by sealing its mate up in its little arboreal cavern during nidification, is so adequately protected that one to three eggs in each family suffice to keep the race going, since practically every young FIG. 351. — Burrowing Owl at nesting hole. FIG. 351. — Burrowing Owl at nesting hole. bird is brought to maturity. Of the host of smaller and weaker birds nesting in cavities, two to five eggs are the usual quota. This brings us to the tribes of little singing birds with which we started, whose average is about five; but a few interesting exceptions may be noted. Our whippoorwills and night-hawks, for instance, lay only two eggs. These are placed on the ground in the The Eggs of Bi 443 woods, surrounded by no nest, and are so precisely the colour of the dead leaves that nothing but the merest accident would lead to their discovery by the eye alone. The same is eminently true of the bird itself. None of the almost uncatchable hummingbirds needs to lay more than two eggs in order to recruit the ranks of its FIG. 352. — Nest and eggs of the Anna Hummingbird. FIG. 352. — Nest and eggs of the Anna Hummingbird. species to the full quota permitted it in the numerical adjustment of bird life. " I have gone into this matter somewhat at length, though by no means exhaustively, because I am not aware that the matter has ever been exploited, and be- cause it embodies a general law or principle. Thus we see that the nest complement of eggs of any bird is in exact proportion to the average danger to which that " I have gone into this matter somewhat at length, though by no means exhaustively, because I am not aware that the matter has ever been exploited, and be- cause it embodies a general law or principle. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 Thus we see that the nest complement of eggs of any bird is in exact proportion to the average danger to which that Bird 444 species is exposed. I believe that this factor is fairly constant for species or tribes of similar habits, and that exceptions indicate peculiarities of circumstances which in many cases we can easily perceive, because I believe that Nature is strictly economical of energy, allowing no more eggs to be laid, and consequently young to be produced, than the conditions justify in each case. Thus the uniformity of avine population — the balance of bird- life — is maintained." life — is maintained." When a bird's nest and eggs are destroyed, she will often lay another setting, and some birds raise two and even three broods in a season under normal conditions. If the eggs of a bird are removed as fast as they are laid, the bird will sometimes continue to lay, one of the most remarkable instances of this in an uncaged bird being a Flicker which laid seventy-one eggs during the space of three-and-seventy days. A tiny African Waxbill in captivity has been known to rear fifty-four young in the course of a year, during the same period laying an addi- tional sixty-seven eggs! The domestic hen has become a veritable egg-laying machine, thanks to careful breed- ing in the past, since the wild Red Jungle Fowl from which all varieties of poultry are descended, lays only one nestful of seven to twelve eggs once a year. Many birds still hold to the old style of nesting in hollow trees and such concealed places. Whether they hunt around until they find a cavity ready-made by the elements, or whether, like the woodpeckers, they pro- ceed to excavate a home in a dead branch, or, kingfisher- like, to tunnel deep into a sand-bank, their eggs are almost The Eggs of Bi 445 invariably white. Many indeed have such glossy, highly polished shells that, were they laid in exposed situations, their shining surface would be a sure guide to hungry FIG. 353. — White eggs of Hairy Woodpecker in hollow tree. (Bowdish, photographer.) FIG. 353. — White eggs of Hairy Woodpecker in hollow tree. (Bowdish, photographer.) egg-eaters. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 Among such birds may be mentioned the owls, woodpeckers and parrots, trogons, motmots, king- fishers and puffins, besides many others which hide their The Bird 4 eggs in domed nests. On the other hand we find a num- ber of birds laying spotted eggs in concealed nests, and white eggs in open places; so that no universal law can be framed to account for the varied colouring. This is not surprising when we think of the great difference of conditions under which each species lives. Take for ex- ample the two species of marsh wrens which live so happily among the reeds of the marshes of our Eastern States. Both birds build globular mouse-like nests, both hide their treasures deep in the interior, but the eggs of the Long- billed species are dark chocolate-brown, while the Short- bill's eggs are like pearls. We do not know why this difference exists, but that need not deter us from accept- ing the facts to which the majority of eggs seem to point: that eggs which are concealed, having no need for colour- ing, are white like those of reptiles. If, as many writers have suggested, the colours of eggs are only meaningless by-products, there is no reason why these hues should not run riot upon each egg or nestful of eggs, as is the case in one or two interesting isolated cases to be men- tioned shortly. Perhaps the most marked exceptions to the theory of the protective coloration of eggs is to be found in doves and pigeons, which lay white eggs in open nests (Fig. 349) ; with the exception, curiously enough, of the Rock Dove, the wild progenitor of our domestic birds, which places its nest in inaccessible caverns in the face of cliffs. The almost total extermination of the Passenger Pigeon has been instanced as an example of a "mistake" of Nature in allotting to it white eggs; the absurdity of The Eggs of Bir 447 which statement is apparent when we consider that the havoc was wrought upon the adult birds and by manl Wallace has suggested that the nests of doves are so loosely and so flimsily built — being in reality mere plat- forms of sticks — that, looking up at them, the eggs simu- lated the colour of the sky beyond and so became incon- spicuous; but unfortunately that argument is so decidedly FIG. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 354. — Nest and eggs of Mallard Duck. FIG. 354. — Nest and eggs of Mallard Duck. suggestive of human presence that it loses much of its value when we remember that egg-hunters among the mammals and birds do not stand on the ground to take observations, but either climb the trees in search of nests or fly low above the branches. The eggs of ducks and grouse are white or very light- coloured, and are laid in open nests upon the ground. The mother duck's plumage is the very essence of the mottled lights and shadows among the reeds, and when The Bird 4 she leaves her eggs she backs carefully away, drawing over them, at the same time, a coverlet of beautiful down, the protective colouring of which is ample to shield the eggs. Ordinarily this coverlet is rolled up at the edge of the nest. It is to such a habit that the eider-down hunters owe their supply. A grouse does not pluck the down from her breast, but in devotion and ability to remain close upon her eggs she has few equals. It is rare indeed to find the nest of a grouse unguarded, and the mother bird will all but wait until your hand is upon her before leaving her eggs exposed. The many species of hummingbirds lay the whitest of eggs, but here it is the nest which is protected, — fash- ioned of dull-hued plant-down, with beams and rafters of cobweb, covered outside in our Eastern species with lichens exactly like those which are growing upon the limb to which the tiny air-castle is attached. The nests of vireos, also, are much like their surroundings. of vireos, also, are much like their surroundings. Herons and egrets, pelicans, cormorants, storks, swans and geese, all lay white or whitish eggs in open nests; but obviously these birds require little protection, all being able to defend themselves with beak or wing. Some of them nest, too, in large colonies, adding the advantage of numbers. The constant need of vigilance in protect- ing eggs thus exposed is at once evident when mankind —that disturber of Nature for whose intrusion she seems never prepared — comes upon the scene. FIG. 342.— Comparison of eggs of reptiles and birds. (a) Egg of Hen. (b) Egg of Skate, (c) Egg of Snake, (d) Egg of Turt ) Egg of Alligator. 429 If we make our way into the heart of a Florida rookery of herons, ibises, or cormorants, many of the birds will be frightened from their nests and the Fish Crows take instant advantage, The Bird The Bird 45° 45° swooping down one after another upon the nests and each impaling an egg upon its beak and flying off with it. They would never dare such open villainy were the herons undisturbed. FIG. 356. — Colony of Great Blue Herons. Many of the more isolated cases of exposed whi FIG. 356. — Colony of Great Blue Herons. Many of the more isolated cases of exposed white eggs are to be explained, I think, by the fact that the habits of birds often change rapidly, while their structural The Eggs of Bi 45 1 adaptation follows more slowly. For example, let us take the group of owls. The majority of these birds nest in hollow trees, but even these occasionally make use of an open hollow or a very shallow one, and individual, radical departures from the conventional owl-habitation are doubtless not uncommon. But these exposed eggs are soon destroyed; for no crow, jay, or squirrel could ever resist any opportunity to avenge himself for the wrongs inflicted by his ancestral enemy, the owl. But when, urged on by that impulse which ever tends to make birds vary their habits in all directions, some owl, such as the Short-eared, finds good feeding on marshes and open, treeless plains, it naturally takes to nesting on the ground, in nests but partly concealed by the overhanging grasses. in nests but partly concealed by the overhanging grasses. Three things might now happen. If sufficient varia- tion occurred and the conditions demanded it, natural selection might bring about a protective colour on the shells of the eggs; if enemies were few and easily over- awed, the eggs might remain white; while, on the other hand, the enterprising race might be wiped out of exist- ence for no more reason than the colour of the egg-shells. The second result seems to be the good fortune of the Short-eared Owls. All of these fates have undoubtedly overtaken birds again and again, and it is by the inter- action of such condition , combined with an ever-chang- ing environment, that many phenomena are brought about. ing environment, that many phenomena are brought about. It was by reason of the general similarity in colour which the eggs of related groups of birds tend to show to each other that oology, or the science of egg-shells, was able to initiate an important scientific discovery. The Bird The Bird 452 452 At one time the sandpipers and plovers were classed as wading birds, and the gulls and terns in an Order placed at a remote distance in the scheme of classification from the former birds; no one suspecting that the two groups were in any way related. The striking resemblance which their eggs showed, however, suggested an affinity FIG. 357. — (a) Egg of common Tern compared with (6) egg of Black-necked St FIG. 357. — (a) Egg of common Tern compared with (6) egg of Black-necked Stilt which was later perfectly confirmed by anatomists and embryologists. The few thousands of years during which our race has risen to inheritance of the earth is all too short a time, geologically speaking, for us to flatter ourselves that any of the protective colours of animals were de- veloped on our account; but in many instances we, sharing the same five senses of animals, may put our- selves in their position. Imagining ourselves egg-hunting FIG. 358. — Eggs of Cassowary. FIG. 358. — Eggs of Cassowary. FIG. 359.— Eggs of Ostrich. 453 FIG. 359.— Eggs of Ostrich. Bird 454 animals, let us consider some of the more patent cases where eggs are coloured for protection — where they mimic their surroundings so perfectly that only the most careful search reveals their whereabouts. Ostriches and Cassowaries are two interesting examples, the former bird laying its white eggs upon the white sands of the desert; while the cassowary, in the depths of its jungle home, incubates a nestful of eggs of the most exquisite emerald hue, matching perfectly the green moss upon which they rest. I knew of one of these birds confined in a small paddock of green grass, whose splendid eggs, measuring three by six inches, once remained undiscov- ered for weeks, although laid openly upon the ground. Special search was necessary to find even these great eggs. Special search was necessary to find even these great eggs. If we walk in the woods in June and happen to flush a night-hawk from the ground, the most careful scrutiny of the place where the bird rose will often fail to reveal to our sight what at last our fingers detect — two eggs, their shells imbued with the colours of the forest floor. The Bird I have led persons to a spot on a beach of shells and sand, told them that there were twenty-one good-sized eggs within a radius of fifteen feet, and seen them utterly baffled. The olive-gray, blotched shell of a tern's egg rests among dark pebbles, or more often upon a wisp of seaweed, into whose irregularities the hues of the eggs melt and mingle perfectly. The Black Skimmer, that most interesting bird of our coast, lays its eggs upon the bare sand among, or sometimes in, the large clam-shells which the storms throw up in windrows. Against man's systematic search their wonderful assimilative colouring is of course often useless, The Eggs of Birds 455 but sharp as is the eye of passing crow or beach-patrolling bear, the eggs to them would appear but bits of sand and shadow. And thus we might go on with many other examples of protection derived from the pigment on the shells — protection which in a hundred instances might prove FIG. 360.— Eggs of Night-hawk. FIG. 360.— Eggs of Night-hawk. futile, but which in the great summing up and balancing of Nature's profit and loss is of inestimable value to the race. We find an unusual condition in the colouring of the eggs of sea-birds, — of certain of those species which nest on inaccessible cliffs. If pigment was developed in the The Bird 56 456 eggs of the ancestors of these birds for the sake of protec- tion, all need for it is now lacking, and as an apparent result the various hues seem to have run riot. One may place a hundred murres' eggs side by side and find no two alike, while the extremes would never be recognized as belonging to the same species of bird. FIG. 361. — Nests of Tern and Skimmer. FIG. 361. — Nests of Tern and Skimmer. Another instance of extreme variability in the colour of eggs and an instance of intensely interesting import is found in the English Cuckoo, which may be taken as an example of species which are parasitical, — in the sense that the females make no nest of their own, but deposit their eggs in the nests of other birds, the young being thus The Bird 4 hatched and reared by foster-parents. Such an unusual, almost unique habit has brought about a considerable modification of the eggs. The Bird Anything which would tend to deceive the greatest number of intended victims would, of course, greatly redound to the advantage of parasitical birds. The remarkable similarity of the English Cuckoo's egg to those in the nest in which it is laid has been explained as due to each individual bird being accustomed to lay its egg in the nest of the same species favoured by its parents and its more distant ancestors ; its eggs in course of time, by natural selection, thus coming to resemble the eggs of that particular species. Other adaptations are the extremely small size of the egg in comparison with the parent bird, and also the unusual strength and weight of the shell. This last is doubtless of great value; for, strange as it may seem, the bird first deposits its egg upon the ground and then picks it up in its beak and places it in the nest selected. Thus a strong shell is a very necessary require- ment. The colours of eggs have been carefully examined with the spectroscope and are found to consist, chemically, of seven pigments: a brownish red, two delicate blues, two clear yellows, a peculiar brown hue, while the seventh is a rather indefinite shade, known as lichenixanthine — most interesting of all as being identical with a colour substance common in plants and especially in lichens and fungi. These substances somewhat resemble those found in the blood and the bile. They are deposited on the shell while the egg is passing down the oviduct, and it is to the circular The Eggs of Birds 459 or erratic motion of the egg that the curious scrawls and blotches upon some eggs are due. The shell is deposited in successive layers, and from the dim, clouded appearance of many colours we judge that the pigment is often partly concealed by the outermost layers of the shell. FIG. 363. — Nest and eggs of Skimmer, showing the remarkable variation in colour of the eggs in a single nest, heightening their resemblance to pebbles or sea- shells. FIG. 363. — Nest and eggs of Skimmer, showing the remarkable variation in colour of the eggs in a single nest, heightening their resemblance to pebbles or sea- shells. The Bird Occasionally, in the eggs of birds which number only two in a nest, one egg will be almost white and the other coated with an abnormal density of pigment. In certain species of small birds which lay four or five eggs, one egg always differs remarkably from the rest. Can we not The Bird 4 account for this latter condition on the hypothesis that an actual change — an increase — is slowly taking place in the number of eggs of this species, the abnormal shell reflect- ing the as yet only partial readjustment of the pigment- gland to meet the extra demand? gland to meet the extra demand? The carbonate of lime, of which the shell is chiefly composed, varies in its composition, being sometimes so fine that the surface has a high gloss, the eggs of wood- peckers being a good example, or again loose and chalky, as in cormorants. In tinamous the glossiness is carried to an extreme, their eggs resembling ovals of highly burnished metal, green and purple in colour. The shells of ducks' eggs are impregnated with an oily substance, which must be of great use in resisting the dampness and moisture of their surroundings. The eggs of some entire Families of birds are easily recognized by the resemblance of the grain of the shell; while, on the other hand, this microscopic appearance in the eggs of individual species may differ considerably, as in the case of the eggs of the Mute and Whooping Swans. The eggs of the North African Ostrich have a surface smooth as ivory, while the eggs laid by the South African birds are deeply pitted. The beautiful eggs of the casso- wary show an extreme condition, the light green surface of the egg being covered with raised irregularities of a darker green colour. The thickness of the shells of ostrich eggs is remarkable, and their strength permits their use as water-bottles — an invaluable boon to the Arabs of the desert. With the relative size of the egg and the bird which The Eggs of Bi 46 1 lays it we will not here concern ourselves, except to remark that the largest egg in proportion to the size of the bird is that of the apteryx. The Bird If we imagine a rather smallish hen laying an egg 3X5 inches in size, we will get a vivid idea of this bird's ability, and it lays two at a setting! The smallest of all eggs is that of the hummingbird, while the largest is the egg of the extinct giant jfipyornis of Madagascar, the shell of which measures 9X13 inches. In some cases the fossil egg is all that is left to us to hint of the existence of these great feathered creatures. Many of these shells have been found buried with some old native chief, the whole egg placed beside him to furnish food for the long journey after death. Whether we look at eggs from the standpoint of an artist's delight in harmonious and delicate colouring, or from the wonder of their scientific composition, or even from the point of view of a hungry man sitting down to breakfast, we must admit that they deserve all the appre- ciation which their beauty and their utility demand. THE BIRD IN THE EGG ]HE embryology, or life of the bird in the egg, is the most mysterious and wonderful part of the entire physical aspect. Many of the lesser de- tails of growth are very difficult to study without the use of microscopic sections and wax models; but a little knowledge of the subject is more interesting and simple than one would imagine. ] The very best way to begin our study of the life in the egg will be to go to the nearest pond or marsh, if it is spring- time, and bring home a pailful of freshly laid frog's eggs — those queer, gelatinous masses filled with black dots. Place them in a flat, white basin, and into a smaller saucer near by break a fresh hen's egg, being careful not to injure the yolk. Separate one of the frog's eggs with a spoon and put it beside that of the fowl. Now examine them carefully with a good dissecting-microscope or even with a hand-lens. We see a large, round, yellow yolk in the case of one egg, and a tiny speck of black and white in the other, — both apparently inanimate bits of matter, but which, merely by the application of heat in the one instance insta The Bird in the Egg 463 and the presence of water in the other, will slowly take on the semblance of living creatures; the one eventually to swim forth, live the life of a fish for a time, then to leap upon the land and croak among the reeds. The other yolk would have evolved into a downy, yellow chick. We cannot hope to solve the mystery of life, but there is a fascination in seeing how near its beginnings we can approach. FIG. 364. — Egg of Hen, opened to show a 3-day embryo in position on the yolk. (Slightly enlarged.) FIG. 364. — Egg of Hen, opened to show a 3-day embryo in position on the yolk. THE BIRD IN THE EGG (Slightly enlarged.) If we have ever watched under the microscope the strange little creatures which live in the mud at the bottom of ponds, we will have realized the wonderful possibilities of a single drop of living matter, — a single cell, — from the amceba with its ever-changing shape to the swiftly moving slipper paiamecium and the beautiful animal vases, — the The Bird 4 vorticella, on their queer little corkscrew stems. All these are made up of but a single cell, and in the beginning all seeds of plants and all eggs of animals likewise consist of one cell. If we examine a chicken while it is being dressed for the table, we can easily find the ovary, a mass of hundreds of tiny golden spheres, — eggs which would have been laid during the coming years. So we realize that the most essential part, in fact the real egg, is only the yolk; all else being merely protective. The shell protects the yolk while the chick is developing during incubation, and although formed of crystals of lime, yet it is so porous that oxygen can enter and carbonic acid gas escape. The viscid white, or albumen, is nutritious as well as protective, while the yolk itself is the real food of the embryo and also acts as a support to the developing chick. If we look carefully, we will see two whitish, twisted strands which extend from the yolk through the white. These two strands have whitish opaque knots strung along them, and from a fancied resemblance to hailstones they are called chalazce. These act as pads to protect the yolk from sudden jars, but they do not act as suspensories. A hen never turns her eggs, as many people imagine, to warm the different sides equally, for the germ-dot — the position of the future embryo (of which we will speak presently) — is always on the lightest side of the yolk, and whichever way the egg is turned it always swings uppermost, nearest the heat from the body of the sitting hen. The turning, however, may be of advantage in allowing moisture to act upon a greater surface of shell. The Bird in the Eg The Bird in the Eg 465 Now let us examine closely the egg of the frog. It, too, has a protective gelatinous outer coating. Before the egg was laid it was enveloped with several very delicate membranes, which were sponge-like in their property of absorbing water, and when deposited in a pond they immediately swelled up to the present gelatinous con- sistency. If the egg has been deposited but an hour or two, it will show a perfectly smooth surface under the lens, but look at it intermittently for a half-hour, or even longer, and you will be well repaid. Slowly but surely, as the shadow of an eclipse darkens the face of the sun, a tiny furrow ploughs its way over the surface of the dark end of the egg. It lengthens and deepens and soon divides the egg into two equal halves. Let us stop a minute and realize what we have seen. It is all but the beginning of life, the first hint of a higher order of things than those one-celled creatures which we dredged from the mud, — than the life which, untold ages ago, was all that the earth boasted. The original cell of the egg has, before our eyes, divided into two! But while we have been lost in wonder and awe, — for the lover of Nature must indeed be stolid if the first sight of such a happening does not stir his deepest emotions, — the life has ceased its progress never an instant. A new furrow appears, crossing the first at right angles, dividing the egg into quarters; then other furrows dividing it into eighths, then cross-furrows, and the count is lost; the multitude of cells repeating themselves hour after hour, day and night, arranging themselves, each in its right position, obeying some inscrutable law, until at the end of about 300 hours The Bird 4 the tadpole wriggles his way through the cloudy mass of gelatine and swims into the water. The first steps of this dividing or cleaving of the original single cell is similar in all eggs. The deep significance of the equality of the first two cells may be better appre- ciated when we know that if one of these be destroyed by a touch from a red-hot needle, a perfect half tadpole will develop from the other unharmed twin cell. The Bird in the Eg If we observe the cleavage of the whiter portion of the frog's egg, we will notice that the furrows, though ultimately extending all the way around, yet grow very slowly in that portion. This is because much of the white part consists of yolk, or true food-matter, the more active formative material being confined to the black portion. If we follow this segmentation of the cells for some time, the egg of the frog will come to look like a diminutive blackberry — a single layer of cells thickly covering its entire surface, like the rounded protuberances of the berry. Now a curious thing happens. A tiny nick appears in one side, which gradually deepens and widens until it extends deep into the egg, pressing two rows of cells into close proximity to each other. This will be perfectly clear if we take a small rubber ball and squeeze it until one hollow hemisphere is pressed into the other. This stage of em- bryological life is called the gastrula, and is of the greatest significance, as we shall soon see. Without further comment at present, let us now leave the frog's egg and consider that of the fowl. When the yolk or egg has but just left the ovary a tiny dot is visible on one side, — the germinal vesicle, which after fertilization FIG. 365. — Stages in the development of frog's egg, from first division into twe cells up to well-formed larval tadpole. (From original drawings by the author.) 467 FIG. 365. — Stages in the development of frog's egg, from first division into twe cells up to well-formed larval tadpole. (From original drawings by the author.) 467 The Bird 4 immediately begins to divide into numerous cells, as in the case of the frog's egg. This goes on until the egg is laid, and when we break the shell, we see at the uppermost part of the sphere of yellow yolk a well-defined portion, in appearance a tiny ring of cloudy, opaque matter enclos- ing a transparent circle. So now we see the use of begin- ning our investigation with the frog's egg, that of the fowl having reached quite an advanced stage before it is laid. having reached quite an advanced stage before it is laid. The Bird in the Eg The ring and circle of the embryonic spot on the yolk consists of a layer of small, even cells, like cobblestones. These are spread over the top of the yolk, while just be- neath is a jumbled mass of many larger cells. The opaque ring is caused by a thicker, denser concentric layer of these lower cells. When heat is applied, this outer layer begins to segment rapidly, the new cells spreading down over the surface of the great ball of yolk; a curving depression dimples the surface of the little transparent circle, pushing in deeper and deeper; and behold! we have the very same condition — the gastrula stage — which we saw in the frog's egg. To make this stage in the egg of the hen more real, squeeze the rubber ball into a hemisphere and clap it upon an orange so that the two layers of rubber fit, cap- like, upon the fruit. This is all very wonderful, but what special significance has it? What particular point upon which we may sus- pend it in our memory, so that it will always return to us with a thrill of interest and wonder whenever we see an egg ? Just this. When we first examined the frog's egg, and when the egg of the chick was still attached to the ovary, they were comparable to the one-celled creatures The Bird in the Egg 469 living in the mud of the pond, which are the most lowly organized beings in the world. The gastrula stage — the double-walled cup, into which the real egg-part of each double-walled cup, into which the real egg-part of each j&cain Ear FlG. 366.— Third-day stage of embryo chick. (See Fig. 364.) Greatly enlarged- M, Muscle-plates (false vertebrae). j&cain FlG. 366.— Third-day stage of embryo chick. (See Fig. 364.) Greatly enlarged- M, Muscle-plates (false vertebrae). yolk forms itself, is comparable with the next higher class of living creatures, the sponges. For the simplest of these are nothing more than a cup of cells, two layers deep The Bird 4 (these layers being known as the ectoderm and endoderm, or outer skin and inner skin). The name gastrula, or little stomach, is certainly most applicable, for an animal of this kind consists of hardly more than stomach and mouth. The Bird in the Eg But the embryo of the frog's egg does not long remain in this sponge-like condition; for almost immediately a third layer, the mesoderm, or middle skin, appears between the other two. From these three layers of cells all the parts of the body of the future chick arise, by the continued dividing of the cells. The details are far too involved to be followed without going into technicalities. Suffice it to say that in the development of the embryo chick we have one of the surest proofs of the truth of the theory of evolution, — of the gradual evolving of each of the higher groups of animals from some lower, more generalized form, until all are originally derived from an organism consisting of a single cell, with its tiny germ- spot. The dividing of this germ-spot in the dawn of creation was the beginning of that wonderful unrolling of life which to-day culminates in birds and the higher mammals, — even in man himself. mammals, — even in man himself. It would be too much to expect that the growing embryo chick distinctly reflects in its successive stages of growth, during a short three weeks, the embryonic states of all its unnumbered generations of ancestors. The record, like that of palaeontology, is imperfect. Many important phases are slurred over or apparently entirely omitted; in order, evidently, to give freer play to the development of organs which will be of vital importance in the future active life of the bird. Now and then, however, a gleam — The Bird in the Eg 471 a spark of life reflected from the far- distant past shines forth so vividly as to hold us spellbound, almost instantly to fade out forever, having no part in the actual life of the chick. Like the finding of the Archseopteryx, these dim reflections seem to have been preserved by some kind Providence, especially to aid our groping efforts to find the truth of ages that are past. Were it not for these we should never dare to voice such an incredible theory as the story of evolution would be, were it not supported by unanswerable proofs. The question which interests scientists to-day is not whether evolution is true, but how its processes and changes have been brought about. The Bird in the Eg The difficulty of seizing upon these evanescent bits of realism of the past will be appreciated when we know that while, in the case of the hen's egg, three weeks are required before the chick is ready to break the shell, yet when incubation has proceeded but eighteen hours, a tiny rod of cells shows where the notochord will be formed — that gelatinous foreshadowing of the back-bone. Thus a character, found first in living organisms as high in the scale of life as fish and primitive fish-like creatures, makes its appearance in a few hours, giving but the scantest opportunity for the passing in review of embryonic features of the great group of invertebrates, or those animals, like starfishes, crabs, worms, and insects, which lack a back- bone. The simplest way to study the growing embryo is to put a number of eggs in an incubator, or under a hen, and examine one on each successive day. If the egg is held firmly, by pressing it down into a box of loose sand, the The Bird 4 upper part of the shell may be carefully picked away with a pin and the little embryo exposed to view. a pin and the little embryo exposed to view. When thirty-six hours old it measures almost one quarter of an inch in length and shows many interesting things. The embryo is set off from the rest of the yolk, much as one's hand is if placed under a piece of cloth, the latter then being tucked in beneath the palm in all direc- tions, until the gathered portion is closely constricted. We are able with a good lens to make out which is the head and which the tail end of the future chick, the former being broader and showing the beginning of the two tiny swellings — the future eyes. Behind these, four faintly outlined enlargements along the central line show the anlagen of the various parts of the brain. These take up about one third of the entire length of the embryo, showing the importance of the organs of the head. Still farther back are two rows of little segments strung along the centre line — the false back-bone, hinting of the worm-like series of muscles, of which we have already spoken (page 69). A heart is even now hinted at, but is seen better in a later stage. The Bird in the Eg An interesting thing about it, however, is that, at this stage, it is really in the head region, vividly recalling the condition existing in fishes, where it is very far forward in the body, in fact only just behind the gills. At this period in the chick embryo the heart, instead of being a complicated organ, divided into four complete cavities, is very similar to that organ in our old friend Amphioxus, that lowliest of all fishes, where it is nothing but a slightly enlarged, contractile blood-vessel. In this The Bird in the Eg 473 latter creature there have been found as many as a hun- dred and eighty pairs of gill-clefts, such a remarkable number aerating the blood with but little necessary pro- pulsion, but when in the higher fishes the number of gills in many species is reduced to four, we realize at once the need for a stronger engine to force the blood through the lessened number, this accounting for the increased com- plexity of the heart. plexity of the heart. Up to about the twelfth day the tiny foreshadowings of bones are cartilaginous, like those of the shark, but at this time real osseous, or bony, tissue begins to be deposited in spots which spread rapidly. In the various portions of the skull these bony centres spread until the bones are separated only by narrow sutures, and in the adult bird even these are obliterated, unlike the condition in the skull of a cat or a dog. The bones of the adult bird are so neatly joined together, and are so mutually dependent, that we might easily imagine that they were formed in the order of size or importance, or in a regular series, following their connection with one another; but this is not true. The ribs, for example, are formed between the segments of the primitive sheets of muscle, independently of the back-bone, and only later become attached to it. There is no trace of the great keel-bone, or even of the sternum of the adult fowl, until after the ends of the ribs have met in the middle line of the body, when they grow together and give rise to the sternum — a structure not found in fishes. The Bird in the Eg We have learned that the repetition of similar structures (as the ribs) is a sign of a low degree of organization, and the truth The Bird 474 of this is emphasized in the development of the embryo, during which process a number of additional ribs dis- appear. The abortive ribs of the neck-bones are especially noticeable during the egg-life of the bird, so that in some species we can make out traces of as many as fifteen ribs all told. age 97 a short account was given of the origin of the wings and feet, in the case of the chick — from a primi- tive fin-fold in some general- ized aquatic ancestor. About the fourth day of incuba- tion, sections of our embryo chick will show a low, round- ed ridge, extending the whole length from the neck to the tail. While we can never be absolutely certain that perfect homology exists between the two, yet it is very significant that soon after its develop- ment it dwindles away, leav- ing four conical, isolated he beginnings of the limbs of the bird. Within hree days after the appearance of the limbs, faint become visible upon the tips of the extremities, e hints of the bones of fingers and toes, for such , soon push out beyond the edge, still bound by their transparent membrane, and for some ey present the appearance of webbed paws or arly embryo of Canada howing fin-like limbs. . page 97 a short account was given of the origin of the wings and feet, in the case of the chick — from a primi- tive fin-fold in some general- ized aquatic ancestor. About the fourth day of incuba- tion, sections of our embryo chick will show a low, round- ed ridge, extending the whole length from the neck to the tail. While we can never be absolutely certain that perfect homology exists between the two, yet it is very significant that soon after its develop- ment it dwindles away, leav- ing four conical, isolated the beginnings of the limbs of the bird. * This process is quite pronounced in the case of the Ostrich. The Bird in the Eg Within three days after the appearance of the limbs, faint become visible upon the tips of the extremities, ese hints of the bones of fingers and toes, for such are, soon push out beyond the edge, still bound er by their transparent membrane, and for some they present the appearance of webbed paws or — Early embryo of Canada , showing fin-like limbs. ount was given of the origin of the wings and feet, in the case of the chick — from a primi- tive fin-fold in some general- ized aquatic ancestor. About the fourth day of incuba- tion, sections of our embryo chick will show a low, round- ed ridge, extending the whole length from the neck to the tail. While we can never be absolutely certain that perfect homology exists between the two, yet it is very significant that soon after its develop- ment it dwindles away, leav- ing four conical, isolated the limbs of the bird. Within appearance of the limbs, faint on the tips of the extremities, es of fingers and toes, for such beyond the edge, still bound rent membrane, and for some ppearance of webbed paws or On page 97 a short account was given of the origin of the wings and feet, in the case of the chick — from a primi- tive fin-fold in some general- ized aquatic ancestor. About FIG. 367. — Early embryo of Canada Goose, showing fin-like limbs. FIG. 367. — Early embryo of Canada Goose, showing fin-like limbs. The Bird in the Eg The Bird in the Eg 475 radiate fins. But as early as the tenth day, except for the absence of feathers and claws, the limbs are, in appear- ance, very perfect wings and feet. The most interesting fact in connection with the limbs is that their develop- ment begins superficially and works inward, not, as would be thought, starting at the shoulder and ending at the digits. Even the deep-seated shoulder- and thigh-girdles of bone (pp. 85 and 89) are not derived from the axial skeleton. The former, in the long ago, was gradually pushed inward from the surface by the deep-reaching rays of the fin-like fore limbs, and it is believed that the pelvic girdle had its origin in the spliced scales of some fish-like ancestor of old, which had scales like those of some of the fossil ganoids. These probably covered over the cartilage girdle and then sunk in. An example of one out of many reptilian structures which appear for a time and then vanish, is found in the procoracoid bone which has apparently much to do with the development of the typical coracoids, but which is absent or reduced to a mere process in the adult bird.* Strangely enough, in the embryo of the common chick the coracoid and scapula fuse together at an early stage, being then in a condition comparable only to that found in the full-grown ostrich. Later this inexplicable fusion is dissolved and the bones complete their development as they began, — two wholly independent structures. they began, — two wholly independent structures. Again, in the embryo of a tern, faint vestiges of teeth The Bird 4 have been observed, instantly bringing to mind that some- what gull-like, toothed bird of old — Icthyornis. what gull-like, toothed bird of old — Icthyornis. The origin and subsequent changes, in the embryo chick, of the vascular system, including the heart, nerves, and arteries, are more intricate than the development of any other system of organs, and for an excellent reason. We know that the frog's egg hatches as a tadpole, which breathes by means of gills and lives, for a considerable time, in the water. The Bird in the Eg We learned in Chapter IV that important parts of the head and sense-organs of birds are derived from metamorphosed gills ; so the inference is that all the changes in the blood-channels, which in the tadpole and frog take place during several months, are in the embryo chick gone through with in a period of a few days. The blood in the heart of a fish is sent from the single ventricle to the gills, and from there it is distributed all over the body. In the gills it passes through the paired series of red fringes and is oxygenated by the water. Now in the chick there are six pairs of these gills, or paired blood-vessels (although not more than three or four are found at one time). The chick breathes by means of a membranous sheet of blood-vessels spread out just beneath the shell, and even the lungs are not brought into use until just before the bird hatches. But strange to say, although there is no water to supply the gill-channels with life- giving oxygen, yet blood actually flows through them, in obedience to the long-forgotten ancestral life-habits — useless these many millions of years. Of all the gill-channels, but three remain in the adult bird. The great aorta, which springs from the heart and . 368. — Pineal eye in Lizard 477 . 368. — Pineal eye in Lizard 477 . 368. — Pineal eye in Lizard The Bird turns to the right (in ourselves, the left-side gill-channel forms this aortic arch), is what is left of the fourth pair of aortic gill-arches, while the two arteries which, in aH higher animals, leads to the right and left lungs, are the sel> same channels which in the creatures of olden time encircbd the sixth pair of gill-bars. F.E. the sixth pair of gill-bars. Although the eye of the bird is far superior to thaf of a fish in seeing abiity, yet in actual structure there is not very nuch difference, except that the bird has gained eyelids, tear^lands, and a few other struc- tures. Fishe-, frogs, lizards, bins, and mammals, tlrough all the ages, Iwe depend- ed on thesJ two eyes and have found them FIG. 369.— Pineal eye in Chick (P. E.). The Bird in the Eg all-sufficirfit; but there are hin# that once, long ago, the ancestors of all the higher animls had a sense- organ, probably of sight, situated, like thafof the mythical Polyphemus, in the centre of the head, In lizards this vestigial organ is sometimes quite well Developed, having a nerve which leads up from the centr of the brain to a kind of translucent, lens-like scale wJch lies among the other scales of the skin, upon the cerre of the forehead. In the long-extinct Ichthyosaurs this^edian eye was prob- F.E. FIG. 369.— Pineal eye in Chick (P. E.). FIG. 369.— Pineal eye in Chick (P. E.). long ago, the ancestors of all the higher animls had a sense- organ, probably of sight, situated, like thafof the mythical Polyphemus, in the centre of the head, In lizards this vestigial organ is sometimes quite well Developed, having a nerve which leads up from the centr of the brain to a kind of translucent, lens-like scale wJch lies among the other scales of the skin, upon the cerre of the forehead. In the long-extinct Ichthyosaurs this^edian eye was prob- The Bird in the Egg 479 ably functional. In an embryo chick of even the third day this organ is remarkably prominent; but although traces of it always remain, yet it fades away to a vestige. Look with a hand-lens at the head of a polywog, and see the whitish dot between the eyes; or when you touch FIG. 370. — Forty-day embryo Ostrich, showing position in the shell. FIG. 370. — Forty-day embryo Ostrich, showing position in the shell. the "soft spot" on the head of a human baby, let it recall the strange third eye which is its cause. And so we might continue to tell of the wonder of embryo life: how up to the sixth day the little being might be mistaken for the embryo of a reptile or a mammal, but from this day onward the bird characteristics become more and more noticeable. On the ninth day feathers The Bird 4 begin to be seen, looking, however, more like tiny cones than anything else (Fig. 11). The muscles and the cartilage skeleton are well defined on the fourteenth day, and about this time the tiny beak with its white egg-tooth is pressed against the membrane of the air-chamber at the large end of the egg. The Bird in the Eg Reptiles also show this tiny bit of sharp lime upon the head, which drops off soon after its function is completed. Not until almost the last day is the mem- brane pierced and the first gasp of air breathed into the little lungs. By an instinctive moving of the head back and forth the shell is filed through and cracked, and the chick rolls out into the world, weak and helpless and for a while absolutely dependent upon warmth and the care of its mother, before it is fit for its future life. (Figs. 18 and 19.) Thus do all wild birds begin life, passing through similar phases within the egg; and although we so often admire a nest full of eggs, yet how seldom do we give thought to the tiny creatures within, — their hearts even at that very minute, perhaps, giving their first fluttering beat! The instant that its eyes have cleared and its shaky legs have gained strength to support its body, the chick begins to use its senses and to store up experiences, taking note of this sound and that taste, learning to fear or to ignore, to flee or to pursue, to call or to remain silent. And thus does the brain of the chick and of all wild nestlings begin to act and its psychological life commences, with intermingled perceptions, instincts, and gleams of intelli- gence. Here belong the making of nests and journeys, be The Bird 4 courtship and songs, the rearing of young birds, the avoid- ing of enemies, the selection of food and suitable haunts, and, lastly, the encountering and overcoming of dangers, — new and wide-spread, — which are now affecting the environ- ment of every creature of this world. Of greater impor- tance than ever before is this adaptation to new con- ditions; since man and his traps and his guns have come upon the scene, upsetting all the world-old order of Nature and slowly, surely, claiming the whole earth for himself. May the naturalists of to-day realize their opportunity and do their best to preserve to us and to posterity what is left to us of wild life ! If not, let us pity the Nature- lover of two hundred years hence! Appendix 4 EVOLUTION ORIGIN OP SPECIES. Charles Darwin. Appleton & Co., New York. Charles Darwin. Appleton & Co., New York. FROM THE GREEKS TO DARWIN. Henry Fairfield Osborn. Macmillan Co., New York. Henry Fa ORGANIC EVOLUTION. Henry Fairfield Osborn. Macmillan Co., New York. ORGANIC EVOLUTION. M. M. Metcalf. Macmillan Co., New York. M. M. Metcalf. Macmillan Co., New York. VARIATION IN ANIMALS AND PLANTS. VARIATION IN ANIMALS AND PLANTS. H. M. Vernon. Henry Holt & Co., New York. VARIATION IN ANIMALS AND PLANTS. H. M. Vernon. Henry Holt & Co., New York. BOOKS FOR THE IDENTIFICATION OF NORTH AMERICAN BIRDS GUIDE TO THE BIRDS OF NEW ENGLAND AND EASTERN NEW YORK. BOOKS FOR THE IDENTIFICATION OF NORTH AMERICAN BIRDS GUIDE TO THE BIRDS OF NEW ENGLAND AND EASTERN NEW YORK. GUIDE TO THE BIRDS OF NEW ENGLAND AND EASTERN NEW YORK. Ralph Hoffmann. Houghton, Mifflin & Co., Boston. Ralph Hoffmann. Houghton, Mifflin & Co HANDBOOK OF BIRDS OP EASTERN NORTH AMERICA. HANDBOOK OF BIRDS OP EASTERN NORTH AMERICA. F. M. Chapman. Appleton & Co., New York. HANDBOOK OF BIRDS OP EASTERN NORTH AMERICA. F. M. Chapman. Appleton & Co., New York. F. M. Chapman. Appleton & Co., New York. HANDBOOK OF BIRDS OP THE WESTERN UNITED STATES. Florence M. Bailey. Houghton, Mifflin & Co., Boston. Florence M. Bailey. Houghton, Mifflin & KEY TO NORTH AMERICAN BIRDS (2 vols.). Elliot Coues. Dana Estes & Co., Boston. KEY TO NORTH AMERICAN BIRDS (2 vols.). Elliot Coues. Dana Estes & Co., Boston. Elliot Coues. Dana Estes & Co., Boston. BIRDS OF NORTH AND MIDDLE AMERICA (3 parts; others to follow). Robert Ridgway. Bulletin of the United States National Museum, No. 50, Washington, D. C. No. 50, Washington, D. C. HISTORY OF NORTH AMERICAN LAND BIRDS (3 vols.). Baird, Brewer, and Ridgway. Little, Brown & Co., Boston. NORTH AMERICAN LAND BIRDS (3 vols.). Baird, Brewer, and Ridgway. Little, Brown & Co., Boston. 483 483 BIRDS IN GENERAL DICTIONARY OF BIRDS. Alfred Newton. A. & C. Black, London. RIVERSIDE NATURAL HISTORY, Vol. IV. Birds. L. Stejneger. Houghton, Mifflin & Co., New York. Abdominal ribs, 79 Abdominal ribs, 79 Abdominal ribs, 79 Adaptation of feet, 361, 362 Adaptation of feet, 361, 362 Adaptative Radiation, 15-18; in war- blers, 361-367 blers, 361-367 Adjutant, head of, 273, 276; hair from neck of, 276 INDEX Figures in heavy-faced type indicate illustrations. B from neck of, 276 ^Epyornis, com. size of egg of, 461 Aftershaft, 36 ^Epyornis, com. size of egg of, 461 Aftershaft, 36 ^Epyornis, com. Aftershaft, 36 ^Epyornis, com. size of egg of, 461 Aftershaft, 36 Back-bone, of Amphioxus, 66; evolu- tion of, 64-70; of shark, 68 Barbs, of Condor's feather, 32 Barbicels, 32, 34 Barbules, 32, 34 Bats, keel of, 83-84; used as food, 158 Beak, see Bill Beaks and Bills, 223-251 Bear, feet of, 102 Bellbird, wattles of, 273 Bill, function of, 223, 224, 250, 251 of Avocet, 237, 239; Archaeopteryx, Back-bone, of Amphioxus, 66; evolu- tion of, 64-70; of shark, 68 Barbs, of Condor's feather, 32 Barbicels, 32, 34 Barbules, 32, 34 Bats, keel of, 83-84; used as food, 158 Beak, see Bill Beaks and Bills, 223-251 Bear, feet of, 102 Bellbird, wattles of, 273 Bill, function of, 223, 224, 250, 251 of Avocet, 237, 239; Archaeopteryx, Aggressive coloration, use of, 308; in Arctic fox, 309; in Cuckoo, 311; in Gyrfalcon, 309, 312; in Ivory Gull, 309, 311; in Penguin, 309, 310; in pickerel, 310; in Snowy Owl, 309, 313 313 Air-sacs, cf. B with respiratory system of insects, 173: extent of, 173; function of, 174, 177; in Prairie Hen, 177 Albatross, wing of, 320, 321, 325, 332 Albinism, 314 Albatross, wing of, 320, 321, 325, 332 Albinism, 314 function of, 223, 224, 250, 251 of Avocet, 237, 239; Archaeopteryx, 226; Cormorant, 227, 228; Cross- bill 24, 249, 245; Crow, 226; Shoveller Duck, 233, 235; Purple Finch, 249; Golden Eagle, 242; Flamingo, 128, 234, 235, 236; Gannet, 227, 228; Boat-billed Heron, 237, 238; Great Blue Heron, 237; Night Heron, 237; Huiabird, 248, 249, 250; Hummingbirds, 244, 245, 246, 247, 248; Ibis, 237, 239; Shell Ibis, 240; Merganser, 233, 234; Nuthatch, 245; Owls, 242; Oyster-catcher, 238, 240; Parrots, 242; Pelican, 228, 229, 230; Crook-billed Plover, 240, 241; American Raven, 225; Black Skim- mer, 231, 232, 236; Snakebird, 228, 229; Dowitcher Snipe, 241; Spoonbill, 220, 236, 239; Stilt, 237; Chimney Swift, 244, 246, 245; Tailor-bird, 245; Tern, 231, 232; Toucans, 243, 244; Triceratops, 485 Alligator, egg of, 429; foot of, 354; nictitating membrane of, 215; re- lation to birds, 9; skull of, 105 Alligator, egg of, 429; foot of, 354; nictitating membrane of, 215; re- lation to birds, 9; skull of, 105 Altricial nestling, 30 Alligator, egg of, 429; foot of, 354; nictitating membrane of, 215; re- lation to birds, 9; skull of, 105 Altricial nestling, 30 Amoeba, 185, 186, 463 Amphioxus, 66; gill-clefts in, 473; notochord of, 66; segments of, 78; trachea of, 169; breathing motions in, 180 Anaximander, 12 Ancestors of birds, 1-18 Aorta, 476 Apoplexy in birds, 202 Apteryx, ody-feathers of, 289; eyes in, 254, 255, 256; sense of touch in, 219 Archseopteryx, as parallel branch, 10; as ancestral type, 10; foot of, 353; general description of, 7; in Berlin Museum, n; in British Museum, 8; probable habits of, 12-13; restora- tion of, 14; tail of, 398, 399, 400, 401 Aristotle, cf. with Darwin, 12 Amphioxus, 66; gill-clefts in, 473; notochord of, 66; segments of, 78; trachea of, 169; breathing motions in, 180 Apoplexy in birds, 202 Apteryx, ody-feathers of, 289; eyes in, 254, 255, 256; sense of touch in, 219 Archseopteryx, as parallel branch, 10; as ancestral type, 10; foot of, 353; general description of, 7; in Berlin Museum, n; in British Museum, 8; probable habits of, 12-13; restora- tion of, 14; tail of, 398, 399, 400, 401 Aristotle, cf. Apoplexy in birds, 202 Apteryx, ody-feathers of, 289; eyes in, 254, 255, 256; sense of touch in, 219 Figures in heavy-faced type indicate illustrations. Artery, 182 Arteries, course of vertebral, 79 Atlas of Jabiru, 72 Auk, wing of Great, 339, 340; wing of razor-billed, 339, 340 Avpcet, bill of, 237, 239 Axis of Jabiru, 72 B MISCELLANEOUS BIRDS OF ESSEX COUNTY, MASSACHUSETTS. C. W. Townsend. Memoir of Nuttall Ornithological Club, No. III. Cambridge, Mass. (A type of a local bird-study ) THE WOODPECKERS. Fannie H. Eckstorm. Houghton, Mifflin & Co., Boston. (A popu- lar study of a single group of birds.) BIRDS OF ESSEX COUNTY, MASSACHUSETTS. C. W. Townsend. Memoir of Nuttall Ornithological Club, No. III. Cambridge, Mass. (A type of a local bird-study ) THE WOODPECKERS. Fannie H. Eckstorm. Houghton, Mifflin & Co., Boston. (A popu- lar study of a single group of birds.) BIRDS OF ESSEX COUNTY, MASSACHUSETTS. C. W. Townsend. Memoir of Nuttall Ornithological Club, No. III. Cambridge, Mass. (A type of a local bird-study ) THE WOODPECKERS. Fannie H. Eckstorm. Houghton, Mifflin & Co., Boston. (A popu- lar study of a single group of birds.) B Coloration, aggressive, see Aggressive coloration; protective, see Protec- tive coloration Columella, of Owl, no, 109 Comb, of Condor, 271; Domestic Cock, 274; Heron, 387, 388; on toe of Chuck-will's-widow, 369 Condor, feathers of, 36; flight of, 324, 326; wattles of, 271 Condyles of skull, 108 Coot, foot of, 387 Coracoid, function of, 84; in man, 85; in reptiles, 84 Cord, spinal, 202 Cormorant, bill of, 227, 228; character of egg-shell of, 460; gullet of, 133; iris of, 256; method of fishing of, 154; tail feathers of, 400 Corpuscles, see Blood Courlan, food of, 153 Cuckoo, aggressive coloration of, 311; colour of eggs of English, 456, 458; foot of, 372 Curassow, Banded, crest of, 261 Crab, used as food, 149 Crane, Crowned, crest of, 264; head of, 252, 265 Demoiselle, crest of, 267, 295 Classification, based on toes, 354, 355 Clavicle, function of, 85; of Hoatzin, 86; Blackbird, down on nestling, 26 Blood, circulation of, 182, 183, 184; compared with amoeba, 185; red corpuscles of, 184, 185; white cor- puscles of, 185 ion, Clavicle, function of, 85; of Hoatzin, 86; in mammals, 86 Claws of foot, 368 Claws of foot, 368 three on wing of, Osprey, 322; Ostrich, 338, 339; Swan, 322 puscles of, 185 Bobolink, cause of colour change in spring, 297 338, 339; Swan, 322 Climate, effect on plumage of, Bob- white, 292, 295; Song Sparrow, 292, 295; South American Pipit, 295, 296; Turkey Vulture, 322; White-throated Sparrow, 291, 294; Wood Thrush, 294 338, 339; Swan, 322 Climate, effect on plumage of, Bob- white, 292, 295; Song Sparrow, 292, 295; South American Pipit, 295, 296; Turkey Vulture, 322; White-throated Sparrow, 291, 294; Wood Thrush, 294 spring, 297 Bob-white, effect of climate on, 293 295; moult of, 43 295; moult of, 43 Body, of Herons, 286; Petrels, 286 Body, of Herons, 286; Petrels, 286 Body-feathers, of Apteryx, 289; Cas- sowary, 288; Snowy Egret, 305, 322; Emeu, 36, 289; Ostrich 236, 288; Scaled Partridge, 289; Pen- guin, 289; Rock-jumper, 288; Snake-bird, 289 Sparrow, 291, 294; Wood Thrush, 294 Cockatoo, feet of, 371; Leadbeater, crest of, 260 Cocoon, as food, 146 Snake-bird, 289 Boltenia, notochord of, 67, 67 Bones, of embryo chick, 473; hollow- ness of, 175, 176; of mammal skull, 103; relation to flight, 176; of skull, 107 Breast-bone, 79-84; evolution of, 79 Breast-bone, 79-84; evolution of, 79 Breast ornament of Wild Turkey, 280 Breast ornament of Wild Turkey, 280 Brain, of bird compared with that of crocodile, 200; with that of Tricera- tops; 200; with that of Walrus. B Coloration, aggressive, see Aggressive coloration; protective, see Protec- tive coloration Columella, of Owl, no, 109 Comb, of Condor, 271; Domestic Cock, 274; Heron, 387, 388; on toe of Chuck-will's-widow, 369 Condor, feathers of, 36; flight of, 324, 326; wattles of, 271 Condyles of skull, 108 Coot, foot of, 387 Coracoid, function of, 84; in man, 85; 486 226; Woodcock, 219, 221, 222; Woodpeckers, 245 Blackbird, down on nestling, 26 Chuck-will's-widow, comb on toe o(, 369 Circulatory system of pigeon, 183 Classification, based on toes, 354, 355 Chuck-will's-widow, comb on toe o(, 369 Circulatory system of pigeon, 183 Classification, based on toes, 354, 355 Clavicle, function of, 85; of Hoatzin, 86; in mammals, 86 Claws of foot, 368 three on wing of, Osprey, 322; Ostrich, 338, 339; Swan, 322 Climate, effect on plumage of, Bob- white, 292, 295; Song Sparrow, 292, 295; South American Pipit, 295, 296; Turkey Vulture, 322; White-throated Sparrow, 291, 294; Wood Thrush, 294 Cockatoo, feet of, 371; Leadbeater, crest of, 260 Cocoon, as food, 146 Cod, eggs of, 428 Collar-bone, see Clavicle Colour, of birds, 287; of young birds, 316, 317; blindness, 301; breast of Bleeding Heart Pigeon, 306, 308 change in moulting of Bald Eagle, 296; Scarlet Tanager, 294, 297; Siberian Black Lark, 295, 297 of eggs, 430 of feathers, 53-61 ; causes of, 54-56; patterns of, 56-60 in mammals, 287; phases, double, 314; relation to haunts, 296-314; use of, 322 . B with Darwin, 12 Archseopteryx, as parallel branch, 10; as ancestral type, 10; foot of, 353; general description of, 7; in Berlin Museum, n; in British Museum, 8; probable habits of, 12-13; restora- tion of, 14; tail of, 398, 399, 400, 401 Aristotle, cf. with Darwin, 12 486 Index Chuck-will's-widow, comb on toe o(, 369 Circulatory system of pigeon, 183 Classification, based on toes, 354, 355 Clavicle, function of, 85; of Hoatzin, 86; in mammals, 86 Claws of foot, 368 three on wing of, Osprey, 322; Ostrich, 338, 339; Swan, 322 Climate, effect on plumage of, Bob- white, 292, 295; Song Sparrow, 292, 295; South American Pipit, 295, 296; Turkey Vulture, 322; White-throated Sparrow, 291, 294; Wood Thrush, 294 Cockatoo, feet of, 371; Leadbeater, crest of, 260 Cocoon, as food, 146 Cod, eggs of, 428 Collar-bone, see Clavicle Colour, of birds, 287; of young birds, 316, 317; blindness, 301; breast of Bleeding Heart Pigeon, 306, 308 change in moulting of Bald Eagle, 296; Scarlet Tanager, 294, 297; Siberian Black Lark, 295, 297 of eggs, 430 of feathers, 53-61 ; causes of, 54-56; patterns of, 56-60 in mammals, 287; phases, double, 314; relation to haunts, 296-314; use of, 322 . B 200; embryo, thirty-six hours old, 472; great size in birds, 199, 200, 201; importance of, 198; nerves of, 202; protection of, 196 protection of, 196 Bulk, birds of largest, 285 Bulk, birds of largest, 285 Bustard, chin-feathers of Great, 267; weight of. 285 weight of. 285 Butterfly, eggs of, 428; torn by birds, 147 weight of. 285 Butterfly, eggs of, 428; torn by birds, 1 Butterfly, eggs of, 428; torn by birds, 147 Buzzard, Black-breasted, feeding on Emeu eggs, 159 Butterfly, eggs of, 428; torn by birds, 147 Buzzard, Black-breasted, feeding on Emeu eggs, 159 Canals, semicircular, 218 Caracara, head of, 271, 272; foot of, 378 Carp, compared with Scaled Partridge, 289, 290 Carrion Hawk, Chimango, food of, 162 Cassowary, body feathers of, 288 eggs of, 436, 453; colour of, 453, 454; character of shell of, 460 helmet of, 275, 288; lack of tail of, 402; tracks of, 396; wing of, 321, 337, 338 Catbird, use of wing in young. 322 Caterpillar, used as food by birds, 146 Cells, of blood, see Blood; of feathers, 34; of muscle, see Muscle Cerebellum, 201 Chewink, foot of, 367 Chicken, stomach glands of, 135 Canals, semicircular, 218 Canals, semicircular, 218 Canals, semicircular, 218 Caracara, head of, 271, 272; foot of, 378 Carp, compared with Scaled Partridge, 289, 290 Carrion Hawk, Chimango, food of, 162 Cassowary, body feathers of, 288 eggs of, 436, 453; colour of, 453, 454; character of shell of, 460 helmet of, 275, 288; lack of tail of, 402; tracks of, 396; wing of, 321, 337, 338 Catbird, use of wing in young. 322 Caterpillar, used as food by birds, 146 Cells, of blood, see Blood; of feathers, 34; of muscle, see Muscle Cerebellum, 201 Chewink, foot of, 367 Chicken, stomach glands of, 135 Index of adult Crested Screamer, 29; Duck Hawk, 27. of Duck, colour of eggs of, 447; oddities of diet of, 160-163; eggs of, 447; food of, 147; crest of Mandarin, 263; nest of Mallard, 447, 448; position of feet in, 382, 389, 390; Side-wheel, see Steamer Duck; bill of Shoveller, 233, 234; gradual loss of flight in Steamer, 337; tail-feathers of, 400; tail of Blue, 406; tail of Pintail, 415 Crane, trachea of, 170, 171 of adult Crested Screamer, 29; Duck Hawk, 27. E Eagle, Bald, iris of eye of, 256; foot of Golden, 374, 376; foot of Harpy, 375; Golden, 242; Harpy, 262, 264; Hyoid of, 114; nictitating membrane of, 214; sight of, 208 Ear, canals of , 218; in owls, 216 217; structure of, 217, 218 Eclipse plumage, 48, 49 Edible birds' nest, see Swiftlet. Eggs, abnormal number laid by African Waxbill, 444; domestic Hen, 444; Flicker, 444 Eagle, Bald, iris of eye of, 256; foot of Golden, 374, 376; foot of Harpy, 375; Golden, 242; Harpy, 262, 264; Hyoid of, 114; nictitating membrane of, 214; sight of, 208 of, 214; sight of, 208 Ear, canals of , 218; in owls, 216 217; structure of, 217, 218 Eclipse plumage, 48, 49 Edible birds' nest, see Swiftlet. Eggs, abnormal number laid by African Waxbill, 444; domestic Hen, 444; Flicker, 444 264, 266; Woodpe Crests, use of, 258 Crests, use of, 258 Crocodile, brain of, 200; gizzard of, 138; gullet of, 134; heart of, 181 of ^Epyornis, 461; alligator, 429; Anna Hummingbird, 443; Archae- opteryx, 13; butterflies, 428; Cali- fornia Partridge, 438; Cassowary, 436, 453 gullet of, 134; heart of, 181 Crop, 127-134; of Caracara, 134;, capa- city of, in Wood Pigeon, 130; ejection of food-pellets from, 132, 133; of English Sparrow, 129; extreme devel- opment of, in Pigeon, 130; in Hoatzin, 130, 131; oil in Petrel's, 131 436, 453 character of surface of, in Casso- wary, 460; Cormorant, 460; Duck, 460; North and South African Ostriches, 460; Tinamou, 460; Mute and Whooping Swans, 460; Woodpeckers, 460 130, 131; oil in Petrel's, 131 Crossbill, bill of, 245, 248, 249 Crossbill, bill of, 245, 248, 249 Cross-fertilization by birds, 144, 145 Cross-fertilization by birds, 144, 145 Crow, brain of, compared with that of Hesperornis, 6; Fish Crow, stealing eggs of herons, 448, 449; foot of, 356; method of feeding on shell-fish, 153; pterylosis of nestling, 39 Cross-fertilization by birds, 144, 145 Crow, brain of, compared with that of Hesperornis, 6; Fish Crow, stealing eggs of herons, 448, 449; foot of, of cod, 428 coloration of eggs, 430; of Casso- wary, 453, 454; causes of, 458, 459; Doves, 446; Ducks, 447: English Cuckoo, 456, 458; Goose, 448; Grouse, 447; Herons, 448; Long- billed Marsh Wren, 446; Murres, 456; Nighthawk, 454, 455; Ostrich, 453, 454; Owls, 451; Parrots, 445; Pelicans, 448; Sea-birds, 455; Short-billed Marsh Wren, 446; Skimmer Black, 454, 455, 456, 459; Tern, 454, 456. E 457; compared with stilt, 452; variability of, 456, 457, 459; Woodpecker, 444 356; method of feeding on shell 153; pterylosis of nestling, 39 356; method of feeding on shell 153; pterylosis of nestling, 39 of Duck, colour of eggs of, 447; oddities of diet of, 160-163; eggs of, 447; food of, 147; crest of Mandarin, 263; nest of Mallard, 447, 448; position of feet in, 382, 389, 390; Side-wheel, see Steamer Duck; bill of Shoveller, 233, 234; gradual loss of flight in Steamer, 337; tail-feathers of, 400; tail of Blue, 406; tail of Pintail, 415 Crane, trachea of, 17 Cranium, see Skull Cranium, see Skull Creeper, Brown, protective coloration of, 303, 304; food of, 150; tail of, 410, 411 Cranium, see Skull Creeper, Brown, protective coloration of, 303, 304; food of, 150; tail of, 410, 411 410, 411 Crest of, California Partridge, 259; Cock-of-the-Rock, 270; Condor, 271; Crowned Crane, 264, 265; Crowned Pigeon, 260, 261; Banded Curassow, 261; Demoiselle Crane, 265, 267; Domestic Cock, 274; Double-crested Pigeon, 270; Eared Pheasant, 268; Harpy Eagle, 262, 264; Hooded Merganser, 263; Hummingbirds, 267; India Peacock, 259; Java Peacock, 258; Kingbird, 260; Ruby-crowned Kinglet, 260; King of Saxony Bird of Paradise, 269, 270; Laughing Thrush, 283; Leadbeater Cockatoo, 260; Mandarin Duck, 263; Night Heron, 259; Plumed Partridge, 259; Six-shafted Bird of Paradise, 267; Snowy Egret, 260; Umbrella-bird, 264, 266; Woodpeckers, 264 Falcon, Peregrine, see Duck Hawk. chick, 22; forty-day Ostrich, 479 gastrula stage of, 466, 468; gill-clefts, 476; hatching of, 480; hatching of egg-tooth of, 480; hatching of Ostrich Chicks, 481; illustrating evolution, 470, 471; limbs of, 97; lung in, 178, 179; method of study- ing, 462, 471; muscles of, 480; muscle-plates of, 69, 70; pineal eye in chick, 478,479; precoracoid, 475; respiration of, 476; ribs, 473, 474; segmentation of Chick, 466, 468; segmentation of frog, 465, 466, 467; sixth-day chick com- pared with reptile and mammal, 479; tarsus of, 99; teeth in tern, 476; third day in egg, 463 Falcon, Peregrine, see Duck Hawk. Family, habits of Warbler, Black-and- white Warbler, 366; Black-throated Green Warbler, 166; Magnolia, 366; Maryland Yellow-throat, 362; Myrtle Warbler, 364; Ovenbird, 366, 375; Pine Warbler, 366; Redstart, 363, 364; Yellow Palm Warbler, 363; Water Thrushes, 364, 365; Worm- eating Warbler, 363 eating Warbler, 363 Fat, of Penguins, 286; Petrels, 286 Feather, aftershaft of, 36; barbicels of, Fat, of Penguins, 286; Petrels, 286 Feather, aftershaft of, 36; barbicels of, 32, 34; barbs of, 34; barbules of, 32, 34; cells of, 34; divisions on wing, 320; growth of Ostrich, 28; model of structure of, 33; pattern on, 58-61; structure of, 31-38 structure of, 31-38 Feathers, arrangement of, 38-40; in tail of Archseopteryx, 398, 399 400, 401; in modern bird, 399, 400 of Cassowary, 338; colour of, 53-61; of Condor, 36, 336; development of, 17-38; downy condition of. 35; of embryo bird, 479, 480; of structure of, 31-38 Feathers, arrangement of, 38-40; in tail of Archseopteryx, 398, 399 400, 401; in modern bird, 399, 400 476; third day in egg, 463 thirty-six hours, 472; brain in, 472; eyes, 472; false vertebrae, 472; thirty-six hours, 472; brain in, 472; eyes, 472; false vertebrae, 472; heart, 472 twelfth-day, 473 thirty-six hours, 472; brain in, 472; eyes, 472; false vertebrae, 472; heart, 472 400, 401; in modern bird, 399, 400 of Cassowary, 338; colour of, 53-61; of Condor, 36, 336; development of, 17-38; downy condition of. 35; of embryo bird, 479, 480; of 400, 401; in modern bird, 399, 400 of Cassowary, 338; colour of, 53-61; of Condor, 36, 336; development of, 17-38; downy condition of. D Darwin, evolutionary theory of, 12 Decorations, use of, 322 Deer, nostrils of, 204; spots in young, 316, 317, tail of, 413 Dermis, 21 Dinosaur, relation to birds, 9; thigh- girdle of, 90; tracks of, 396, 397 Distribution of birds, 361 Dogfish, see Shark Donkey, foot of, 39, 395 Dove, colour of eggs, 446; nest of Mourning, 439; position of eyes in, 252, 253, 254 Down, development of, 23; from head of young Bobolink, 26; magnified from young Song Sparrow, 27; models of development of, 24, 25; Darwin, evolutionary theory of, 12 Decorations, use of, 322 Darwin, evolutionary the Decorations, use of, 322 Deer, nostrils of, 204; spots in young, 316, 317, tail of, 413 Dermis, 21 316, 317, Dermis, 21 Dinosaur, relation to birds, 9; thigh- girdle of, 90; tracks of, 396, 397 Distribution of birds, 361 girdle of, 90; tracks of, 396, 397 Distribution of birds, 361 girdle of, 90; tracks of Distribution of birds, 361 Dogfish, see Shark Dogfish, see Shark Donkey, foot of, 39, 395 Dove, colour of eggs, 457, 459; Woodpecker, 444 comparative size of eggs of Hum- mingbird, 461 Donkey, foot of, 39, 395 Dove, colour of eggs, 446; nest of Mourning, 439; position of eyes in, 252, 253, 254 mingbird, 461 of Emeu, devoured by Buzzard, 159; Fowl, 429, 436 252, 253, 254 Down, development of, 23; from head of young Bobolink, 26; magnified from young Song Sparrow, 27; models of development of, 24, 25; of frog, 428, 465; development of, 465, 466, 467; for embryonic study, 462 466, 467; for embryonic study, 462 of Grebe, 432; Hairy Woodpecker, 445; Index Eggs, of Environment, relation of birds to, 480. D 482 Epiclavicle of fish, 86 ggs, of Hen, development of, 466; for embryonic study, 462; structure of, 464 Epiclavicle of fish, 86 Evolution, of bill of Fla Epiclavicle of fish, 86 Evolution, of bill of Flamingo, 235, 236; bill of Skimmer, 231, 232, 236; breast-bone, 79; Class of birds, 15; colour patterns, 58-61; embryo chick, 470, 471; foot, 353, 358, 361; gill- arches, 114, 115; gizzard, 138; heart, 180, 181; history of theories of, 12; lung, 178, 179, 180; Penguin's wing, 341; ribs, 78; shoulder-girdle, 86, 87; skull, 104-106; tail, 398 399, 400, 402, 403; tree (of birds and reptiles), 10; warblers, 361-367; wings, 91-97 Eye, of Hummingbird, 436; Jellyfish, 427; Junco, 434; Killdeer Plover, 431, 432; Mallard Duck, 447; Mourn- ing Dove, 439; Murre, shape of, number laid by, average bird, 433; 43i number laid by, average bird, 433; Doves, 440; Game-birds, 438; Gulls, 436; Hawks, 440; Hum- mingbirds, 443; Jungle Fowl, 444; Xing Penguin, 435; Moas, 435; .Nighthawk, 442; Ostrich, 433; Owl, 440; Parrots, 441; Peacock Pheasant, 440; Rhea, 433; Sea- birds, 435; Shore-birds, 436; Wad- ing birds, 438; Water-birds, 436, 437, 438 Apteryx, 254, 255, 256; compared with camera, 207, 208; iris of, 210 lashes of Hornbill, 257; Ostrich, 257; Seriema, 257 lids of, 214, 215; Brown Thrasher, with camera, 207, 208; iris of, 210 lashes of Hornbill, 257; Ostrich, 257; Seriema, 257 lids of, 214, 215; Brown Thrasher, 437, 438 relation of number to danger,433-444; relative size compared with bird, 460, 461 of reptiles, 428, salmon, 427; show- ing relation of orders, 452; skate, 428, 429; snake, 429 lids of, 214, 215; Brown Thrasher, 212, 213; Woodcock, 221, 256 mammals, 209, 254; nocturnal birds, 212, 213; Woodcock, 221, 256 mammals, 209, 254; nocturnal birds, 254 pineal eye in, embrvo chick, 478, 479; 460, 461 of reptiles, 428, salmon, 427; show- ing relation of orders, 452; skate, 428, 429; snake, 429 pineal eye in, embrvo chick, 478, 479; child, 479; lizard, 477, 478; polly- wog, 479 position of, in Dove, 252, 253, 254; 428, 429; snake, 429 Egg- tooth, of embryo chick, 480; reptiles, 480 wog, 479 position of, in Dove, 252, 253, 254; Owl, 252, 253, 254 reptiles, 480 Egret, crest of Snowy, 315, 322; neck curves of, 73; use of wing of, 350 Embryo, aorta, 476, 478 development of limbs, 474, 475; of girdles, 475; of three toes, 475 drawing of third-day embryo, 469 feathers of, 479, 480; twelve-day chick, 22; forty-day Ostrich, 479 gastrula stage of, 466, 468; gill-clefts, structure of, 207-210; thirty-six- hour embryo, 472 Expression of face of birds, 252 Expression of face of birds, 252 Expression of face of birds, 252 35; of embryo bird, 479, 480; of twelfth-day, 473 Emeu, body-feathers of, 239; feather of, 36; lack of tail in, 402, 404 Emeu-wren, tail of, 415 twelfth-day, 473 Emeu, body-feathers of, 239; feather of, 36; lack of tail in, 402, 404 Emeu-wren, tail of, 415 Index 489 Feathers (continued), Emeu, 36; of embryo of twelfth- day, 22; moult of, 40-53 number of, in tail, of Archaeopteryx, 400; of Cormorant, 400; of Duck, 400; of Ostrich, 402; of Peafowl, 402; of Fantail Pigeon, 402 Food, affecting structure, 164; indu- cing intoxication, 163; of Apteryx, 147; Buzzard, 159; Courlan, 153; Creepers, 150, Crows, 153; Ducks, 147, 153; Bald Eagle, 148; Gulls, 161; Carrion Hawk, 162; Duck Hawk, 159; New Zealand King- fisher, 161; Locust-birds, 150-152; Osprey, 155; Barn Owl, 158; Bur- rowing Owl, 162; Elf Owl, 158; Strenuous Owl, 158; Oyster-catcher, 153; Kea Parrot, 161; Penguins, 153; Ptarmigan, 144; Raven, 158; Road-runner, 157; Sea-birds, 148; Sea-eagles, 157; Secretary-bird, 157; Red-winged Starlings, 162, 163; Storks, 157; Sugar-birds, 144; Sul- phur Tyrant, 161 ; whales, 148 Food-pellets of Owl, 132, 133 Food, affecting structure, 164; indu- cing intoxication, 163; of Apteryx, 147; Buzzard, 159; Courlan, 153; Creepers, 150, Crows, 153; Ducks, 147, 153; Bald Eagle, 148; Gulls, 161; Carrion Hawk, 162; Duck Hawk, 159; New Zealand King- fisher, 161; Locust-birds, 150-152; Osprey, 155; Barn Owl, 158; Bur- rowing Owl, 162; Elf Owl, 158; Strenuous Owl, 158; Oyster-catcher, 153; Kea Parrot, 161; Penguins, 153; Ptarmigan, 144; Raven, 158; Road-runner, 157; Sea-birds, 148; Sea-eagles, 157; Secretary-bird, 157; Red-winged Starlings, 162, 163; Storks, 157; Sugar-birds, 144; Sul- phur Tyrant, 161 ; whales, 148 Food-pellets of Owl, 132, 133 day, 22; moult of, 40-53 number of, in tail, of Archaeopteryx, 400; of Cormorant, 400; of Duck, umber of, in tail, of Archaeopteryx, 400; of Cormorant, 400; of Duck, 400; of Cormorant, 400; of Duck, 400; of Ostrich, 402; of Peafowl, 400; of Ostrich, 402; of Pe 402; of Fantail Pigeon, 402 402; of Fantail Pigeon, 402 of Ostrich, 35, 336; papillae of nestling, 21 ; powder-downs, 37, 38; ptery- losis, 39; sheaths of, 26; sheaths of young Kingfisher, 29; texture of, 287-289; function of, in Puff-back Shrike, 287; of wing, 320-352; worn-out Hummingbird's breast, 44 worn-out Hummingbird's breast, 44 Feather-ears, of owl, 267, 268 worn-out Hummingbird's breast, 44 Feather-ears, of owl, 267, 268 worn-out Hummingbird's br Feather-ears, of owl, 267, 268 Feather-ears, of owl, 267, 268 Feather-tips, in Bobolink, 297; in Black Lark, 295, 297; in Snow- flake, 297; in English Sparrow, 52, 53; wearing off of, 52, 53, 297 Red-winged Starlings, 162, 163; Storks, 157; Sugar-birds, 144; Sul- phur Tyrant, 161 ; whales, 148 phur Tyrant, 161 ; whales, 14 Food-pellets of Owl, 132, 133 Foot, claws of, 368; tendons 53; wearing off of, 52, 53, 297 Feeding, method of, in Flamingo, 128; in Woodcock, 222 Food-pellets of Owl, 132, 133 Foot, claws of, 368; tendons of, 191, 192; of alligator, 354; Archseop- teryx, 353; bear, 102; of bird cf. with man, 99, 100; Caracara, 378; Casso- wary, 394, 396; Chewink, 367; Chuck- will's- widow, 369; Cockatoo, 371; Coot, 387; Crow, 356; Cuckoo, 372; Donkey, 394, 395; Duck, 382, 389, 390; Golden Eagle, 374, 376; Harpy Eagle, 375; Flam i go, 388; Fly- catchers, 360; Gallinule 385, 386, 387; Grebe, 391; Ruffed Grouse, 380^ 381; Rough-legged Hawk, 389; Heron ,. 387,388; Hummingbirds, 368; Wood Ibis, 102, 385; Kangaroo, 394, 395; Horned Lark, 367; European House Martin, 381: Nuthatch, 359; Oriole, 360; Osprey, 375; Ostrich, 394, 395, 396; Burrowing Owl, 373; Snowy Owl, 375; Owls, 373, 374; Parrots, 372; Passeres, 358, 359; Peacock, 383; Brown Pelican, 355; Penguin, 391, 392; Pheasant, 380; Phalarope, 384; Pipit, 367, 368; Plover, 384; Ptar- migan, 381; Quail, 380; Raven, 356; Rhinoceros-bird, 357; Road-runner, 373; Rock-jumper, 393; Sand-grouse, 383; Semipalmated Sandpiper, 384; Secretary-bird, 378, 379; Snakebird, 391; Swallow, 360; Black-necked Swan, 390; Swift, 368, 369; Vulture, 377; Woodpeckers, 370 Feet, adaptation of, 361, 362; classifi- cation based on, 354, 355; evolu- tion of, 353, 358, 361 tion of, 353, 358, 361 Femur, 98 Fibula, 98 Femur, 98 Fibula, 98 Femur, 98 Fibula, 98 Fighting, method of, in Ruff, 277, 279, 280 Finch, 143; bill of Purple, 249; colour change in Purple, 293 change in Purple, 293 Fins, origin of paired, 96 Fish, used as food, 153; hear change in Purple, 293 Fins, origin of paired, 96 Fins, origin of paired, 96 Fish, used as food, 153; heart of, 181; muscle-flakes of, 78 muscle-flakes of, 78 Fishing, method of, in Cormorants, 154; Frigate-birds, 154; Herons, 156; Kingfishers, 154; Ospreys, 154; Peli- cans, 152; Penguins, 154; Snake- birds, 154; Terns, 154 birds, 154; Terns, 154 Flamingo, bill of, 128, 234, 235; colour change in, 293; foot of, 388; neck of, 73, 281; sternum of, 80; tongue of, 126, 127; trachea of, 168, 169 126, 127; trachea of, 168, 169 Flicker, abnormal number of eggs laid by, 444; hyoid of, 123, 124; protec- tive colouring in young, 302, 303, 304; tongue of, 123, 124 tongue of, 123, 124 Flight, cf. Fox, aggressive coloration in Arctic, 309 with swimming, 327; Condor, 324, 326; Steamer Duck, 337; cer- tain Flycatchers, 324, 326; Gold- finches, 329; Grebes, 341; Gulls, 328; Hummingbirds, 329; Owl Parrot, 333; Pheasant, 323, 324; Crested Screamer, 329; Sparrows, 328; swiftness of, in Pigeon, 350; swiftness of, in Swallow, 352; of Tinamou, 333-336; of Wood- peckers, 329; of Vultures, 328, 330, 33i, 332 377; Woodpeckers, 370 Fowl, egg of, 429, 430; Japanese Long- tailed, 417, 419; number of eggs laid by Jungle, 444; spurs of, 382; tail of Jungle, 418 377; Woodpeckers, 370 Fowl, egg of, 429, 430; Japanese Long- tailed, 417, 419; number of eggs laid by Jungle, 444; spurs of, 382; tail of Jungle, 418 Fox, aggressive coloration in Arctic, 309 33i, 332 Flipper, of Penguin, 341, 342, 343 Flycatcher, feet of, 360; flight of, 324, Framework, 62-102 Framework, 62-102 Framework, 62-102 Frigate-bird, method of feeding of, 154 Frog, egg of, 428; fresh egg of, 462; Flipper, of Penguin, 341, 342, 343 Flycatcher, feet of, 360; flight of, 324, 326; tail of Least, 412 Framework, 62-102 Frigate-bird, method of feeding of, 154 Frog, egg of, 428; fresh egg of, 462; Index 490 490 gastrula of egg of, 466; segmentation of egg ot, 467; used as food, 157 H Hatching of embryo Chick, 480; Os- trich, 481 onic, 480 Limbs, origin of, 96, 97; evolution of, 96, 97; of embryo chick, 474, 475 Lobed toes, of coots, 387; of phalaropes, Ibis, 239, 237; feet of Wood, 385; Indian Wood, beautiful feathers of, 61 ; Wood, resting upon whole foot, 102; Shell, 240 96, 97; of embryo chick, 474, 475 Lobed toes, of coots, 387; of phalaropes, 384 ocust birds, method of feeding, 150- 152; relation of habits to food, 152 Loon, in winter haunts, 6; neck of, 277 Lory, tongue of 125 Ibis, 239, 237; feet of Wood, 385; Indian Wood, beautiful feathers of, 61 ; Wood, resting upon whole foot, 102; Shell, 240 Ichthyornis, lower jaw, 5; restored skeleton of, 3; structure of, 4 Icthyosaurus, eye-plates of, 213 Ilium, 89, 90; function of, 89; of bull- frog, 91 Ingersoll, Ernest, quoted, 433 Insects, use as food of birds, 148-152 Intestines, function of, 141; length in Hummingbird, 140; length in Ostrich, 140; in alligator, 141 Iris of eye, 210; of Bald Eagle, 256; Cormorant, 256; Owl, 256; Puffin, 256; Towhee, 256; Vireo, 256 Ischium, 89, 90 Jabiru, atlas and axis of, 72 Jaguar, standing on toes, 101 Jelly-fish, eggs of, 427 Junco, eggs of, 434; nest of, 434; tail of, 414 K Kangaroo, feet of, 394, 395 Keel, in Flamingo, 80; Hoatzin, re- pressed by crop, 132; model com- paring various, Albatross, Humming- bird, Pigeon, 83; value in classifica- tion of, 82 Kingbird, crest of, 260 Kingfisher, method of fishing of, 154; nest of, 444; New Zealand, food of, 101 Kinglet, Ruby-crowned, crest of, 260 Lacrymal glands, 211 Lamprey, gill-basket of, 112 Lark, foot of horned, 367; tail of meadow, 414; cause of colour change in Siberian black, 295, 297 Lory, tongue of 125 Lungs, evolution of, 178, 179, 180; character of, 174, 175; cf. with those of chameleon, 177, 178 Lyre-bird, tail of, 420, 421 Lyre-bird, tail of, 420, 421 M Mallard, eclipse plumage of, 48. 49; tongue of, 122; trachea of, 168; syrinx drum of, 168, 170 Mammals, used as food, 159 Mankind vs. birds, 18 Maoris, legends of, 13 Martin, feet of European House, 381 Merganser, bill of, 233, 234; crest of Hooded, 263 Mimicry, of Hawk by Cuckoo, 311, 312; Jabiru, atlas and axis of, 72 Jaguar, standing on toes, 101 Jelly-fish, eggs of, 427 Junco, eggs of, 434; nest of, 434; tail of, 414 K Kangaroo, feet of, 394, 395 Keel, in Flamingo, 80; Hoatzin, re- pressed by crop, 132; model com- paring various, Albatross, Humming- bird, Pigeon, 83; value in classifica- tion of, 82 Kingbird, crest of, 260 Kingfisher, method of fishing of, 154; nest of, 444; New Zealand, food of, 101 Kinglet, Ruby-crowned, crest of, 260 G with those of chameleon, 177, 178 Lyre-bird, tail of, 420, 421 M Mallard, eclipse plumage of, 48. 49; tongue of, 122; trachea of, 168; syrinx drum of, 168, 170 Mammals, used as food, 159 Mankind vs. birds, 18 Maoris, legends of, 13 Martin, feet of European House, 381 Merganser, bill of, 233, 234; crest of Hooded, 263 Mimicry, of Hawk by Cuckoo, 311, 312; of Hawk by Hawk, 312 Moa, cf. with Ostrich, 13; number of eggs laid by, 435 Monkeys, toes of, 358 Motmot, protective coloration of, 303; tail of Mexican, 424, 425, 426 Moult, 40-53; of Black Grouse, 48; of Bob-white, 43; of Brown Pelican, 41; causes for, 42-50; of body- feathers of English Sparrow, 52, 53; of wing-feathers of English Sparrow, 46; of feathers, 41; of feather-tips, 52, 53; of Mallard Duck, 47, 48, 49; of mound-builders, 44, 45; of Pen- guin, 51; of Ptarmigan, 48, 50, 51, 52; of reptiles, 40; of Robin, 43; in Thomas, quoted, 2 Hyoid, 111; of eagle, 114 Life in the egg, 462-479; post-embry- onic, 480 K Kangaroo, feet of, 394, 395 Motmot, protective coloration of, 303; tail of Mexican, 424, 425, 426 Kangaroo, feet of, 394, 395 Keel, in Flamingo, 80; Hoatzin, re- pressed by crop, 132; model com- paring various, Albatross, Humming- bird, Pigeon, 83; value in classifica- tion of, 82 Moult, 40-53; of Black Grouse, 48; of Bob-white, 43; of Brown Pelican, 41; causes for, 42-50; of body- feathers of English Sparrow, 52, 53; of wing-feathers of English Sparrow, 46; of feathers, 41; of feather-tips, 52, 53; of Mallard Duck, 47, 48, 49; of mound-builders, 44, 45; of Pen- guin, 51; of Ptarmigan, 48, 50, 51, 52; of reptiles, 40; of Robin, 43; in spring, 45; time of, 42-47 Mouse, used as food, 157 Kingbird, crest of, 260 Kingfisher, method of fishing of, 154; nest of, 444; New Zealand, food of, 101 101 Kinglet, Ruby-crowned, crest of, 260 Kinglet, Ruby-crowned, crest of, 260 Mimicry, of Hawk by Cuckoo, 311, 312; of Hawk by Hawk, 312 Jelly-fish, eggs of, 427 Junco, eggs of, 434; nest of, 434; tail of, 414 K of Hawk by Hawk, 312 Moa, cf. with Ostrich, 13; number of eggs laid by, 435 G G Hawk Duck, food of, 158; head of, 211; feet of Rough-legged, 389; mimicked by Cuckoo, 311, 312; number of eggs laid by, 440; skull of, compared with that of Heron, 199, 200 Gallinule, foot of, 385, 386, 387 Gallinule, foot of, 385, 386, 387 Game-birds number of eggs laid by, 438 Game-birds number of eggs laid by, 438 Gannet, bill of, 227, 228 Game-birds number of eggs laid by, 438 Gannet, bill of, 227, 228 Heads and necks, 252-284 Gannet, bill of, 227, 228 Gastrula, 466, 468; compared with sponge, 469, 470 Heads and necks, 252-284 Head of, Adjutant, 276: Apteryx, 254; 256; Barn Owl, 253; Bell-bird, 273, Caracara, 271, 272; Eared Pheasant, 267, 268; Condor, 271; Crowned Crane, 252, 265; Domestic Cock, 274; Dove, 253; Duck Hawk, 211; Java Peacock, 258; King Vulture, 272, 273; Seriema, 257; Sloth, 210; Wild Turkey, 273, 275 sponge, 469, 470 Gill-arches, of chick, 114; evolution of, 114, 115; ultimate distribution of , in chick, 114, 115 chick, 114, 115 Gill-bars of shark, 113 Gill-bars of shark, 113 Gill-basket of lamprey, 112 Gill-basket of lamprey, 112 Gill-clefts in Amphioxus, 473; embryo chick, 476; fishes, 473 chick, 476; fishes, 473 Giraffe, neck-vertebrae of, compared with those of bird, 73, 75 Girdles, embryonic development of, 475 Heart, beats of that of bird, 182; chambers of, 182, 184; of croco- dile, 181; of fish, 181; of embryo 36 hours old, 472; evolution of, 180, 181; position of, in vertebrates, 181 Helmet of Cassowary, 275, 288; Horn- bill, 278 Hemispheres, cerebral, 200 Hen, abnormal number of eggs laid by domestic, 444 Heart, beats of that of bird, 182; chambers of, 182, 184; of croco- dile, 181; of fish, 181; of embryo 36 hours old, 472; evolution of, 180, 181; position of, in vertebrates, 181 Helmet of Cassowary, 275, 288; Horn- bill, 278 Girdles, embryonic development of, 475 Gizzard, 134-140; change in structure of, 137, 138; function of, 135; of Fruit Pigeons, 138; of Hornbills, 139, 140 Gizzard, 134-140; change in structure of, 137, 138; function of, 135; of Fruit Pigeons, 138; of Hornbills, 139, 140 stones of Cassowary, 136; crocodiles, 138; extinct Moas, 136, 137 of pigeon shown by X-ray, 63 Glottis of nestling Robin, 166; Peli- can, 1 66 Goat-sucker, West African, wing orna- ments of, 349 Goldfinch, flight of, 329; tongue of, 125 181; position of, in vertebrates, 181 Helmet of Cassowary, 275, 288; Horn- bill, 278 140 stones of Cassowary, 136; crocodiles, 138; extinct Moas, 136, 137 of pigeon shown by X-ray, 63 Glottis of nestling Robin, 166; Peli- can, 1 66 Goat-sucker, West African, wing orna- ments of, 349 Goldfinch, flight of, 329; tongue of, 125 stones of Cassowary, 136; crocodiles, 138; extinct Moas, 136, 137 of pigeon shown by X-ray, 63 Hemispheres, cerebral, 200 Hen, abnormal number of eggs laid by domestic, 444 Hen, abnormal number of eggs laid by domestic, 444 of pigeon shown by X-ray, 63 Glottis of nestling Robin, 166; Peli- can, 1 66 domestic, 444 Heron, a still hunter, 156; Boat-billed, 237, 238; body of, 386; cause of downiness in feather of, 35; colony of Great Blue, 450; colour of eggs of, 448; comb on toe of, 387, 388; divisions of feathers in wing of young of, 320; Great Blue, 237; in sleep, 344; Night, 237; Night, crest of, 259; skull of, compared with that of Hawk; 199, 200; standing on toes, 101; tail of Green, 413; use of wing in young Green, 323; wing of Great White, 321 can, 1 66 Goat-sucker, West African, wing orna- ments of, 349 ments of, 349 Goldfinch, flight of, 329; tongue of, 125 Goose, Canada, embryo of, 474; colour Goldfinch, flight of, 329; tongue of, 125 Goose, Canada, embryo of, 474; colour of eggs of, 448; muscle of skin in, 188; sense of hearing in, 216; Spur-wing, wing of, 346, 347; use of wing in young Canada, 322 Crackle, tail of Boat-tailed, 418 Grebe, eggs of, 432; flight of, 341; foot of, 391 Goldfinch, flight of, 329; tongue of, 125 Goose, Canada, embryo of, 474; colour of eggs of, 448; muscle of skin in, 188; sense of hearing in, 216; Spur-wing, wing of, 346, 347; use of wing in young Canada, 322 young Canada, 322 Crackle, tail of Boat-tailed, 418 Crackle, tail of Boat-tailed, 418 Grebe, eggs of, 432; flight of, 341; foot of, 391 of, 391 Grouse, Black, moult of, 48; colour of eggs of, 447; Ruffed, ruff of, 277; Ruffed, use of wing of, 547; toes of Ruffed, 380, 381 Hesperornis, habits and structure, 3-6; restoration of, Frontispiece Hesperornis, habits and structure, 3-6; restoration of, Frontispiece Hoatzin, clavicles of, 86; crop of, 130, 131; keel repressed by crop, 132 Hoatzin, clavicles of, 86; crop of, 130, 131; keel repressed by crop, 132 Ruffed, 380, 381 Guinea Fowl, Vulturine, evolution of colour pattern on wings of, 58, 59, 61 131; keel repressed by crop, 132 Honey Creeper, tongue of, 127, 129 131; keel repressed by crop, 132 Honey Creeper, tongue of, 127, 129 Honey Creeper, tongue of, 127, 129 Hornbill, casque of, 275, 278; eye- lashes of, 257; feeding mate from its gizzard, 137, 138 Honey Creeper, tongue of, 127, 129 Hornbill, casque of, 275, 278; eye- lashes of, 257; feeding mate from its gizzard, 137, 138 Horse, toes of, 358 Gullet, of Cormorant, 133; crocodile, 134 Gull, change in gizzard of, 137, 138; Gull, change in gizzard of, 137, 138; flight of, 328; food of, 161; Her- ring, wing of, 328 its gizzard, 137, 1 Horse, toes of, 358 Hudson, quoted, 329, 333 Huia Bird, bill of, 248, 24 ring, wing of, 328 Ivory, aggressive coloration in, 309, 311; protective coloration in 309, Humidity, effect on plumage of, Bob White, 293, 295; Song Sparrow, 292, 295; White-throated Sparrow, 291, 294; Wood Thrush, 294 3" Laughing, nest of, 437; protective coloration of, 309, 312; number of eggs laid by, 436 295; White-throated S 294; Wood Thrush, 294 Hummingbird, Anna, nest eggs laid by, 436 Gyrfalcon, aggressive coloration in, 309, 294; Wood Thrush, 294 Hummingbird, Anna, nest of, 443; eggs of, 443; bills of, 244, 245, 246, 247, Index 49 248; crests of, 267; eggs of, 436, 461; feet of, 368; flight of, 329; flight of, compared with that of insect, 165; length of intestines in, 140; nest of Ruby-throated, 449; number of eggs laid by, 443; tongue of, 127; wing of, 320, 321, 325; wing-strokes of, 82; worn-out breast-feather of, 44; Huxley, definition of palaeontology, 2; Thomas, quoted, 2 Hyoid, 111; of eagle, 114 Layers of cells in embryo, 470 248; crests of, 267; eggs of, 436, 461; feet of, 368; flight of, 329; flight of, compared with that of insect, 165; length of intestines in, 140; nest of Ruby-throated, 449; number of eggs laid by, 443; tongue of, 127; wing of, 320, 321, 325; wing-strokes of, 82; worn-out breast-feather of, 44; Huxley, definition of palaeontology, 2; Thomas, quoted, 2 Hyoid, 111; of eagle, 114 Layers of cells in embryo, 470 Leg, correlation with neck in Flamingo, 281; in Swan, 282; framework of, 98-102; function of, 98; of Ostrich, 100; skeleton of Ostrich, 99; human, 99; scales of, 368; spurs on, 382, 383; of Bald Eagle, 375; of Cassowary, 394; of Flamingo, 388; of Golden Eagle, 375; of Secretary-bird, 378, 379 Leg, correlation with neck in Flamingo, 281; in Swan, 282; framework of, 98-102; function of, 98; of Ostrich, 100; skeleton of Ostrich, 99; human, 99; scales of, 368; spurs on, 382, 383; of Bald Eagle, 375; of Cassowary, 394; of Flamingo, 388; of Golden Eagle, 375; of Secretary-bird, 378, 379 Life in the egg, 462-479; post-embry- onic, 480 Limbs, origin of, 96, 97; evolution of, 96, 97; of embryo chick, 474, 475 Lobed toes, of coots, 387; of phalaropes, 384 ocust birds, method of feeding, 150- 152; relation of habits to food, 152 Loon, in winter haunts, 6; neck of, 277 Lory, tongue of 125 Lungs, evolution of, 178, 179, 180; character of, 174, 175; cf. N Owl, aggressive coloration of Snowy, 309, 313; cause of downiness in feathers, 35; colour phases of Screech, 314; eggs of Screech, 441; food of Barn, 158; food and feeding habits of Burrowing, 162; food of Elf, 158; food of Strenuous, 158; food-pel- lets ejected by, 132, 133; foot of Burrowing, 373; foot of Snowy, 375; iris of eye of, 256; nest of Burrow- ing, 442; orbit cf Barred, 255; posi- tion of eyes in Barn, 252, 253, 254; sight of Barred, 211 Neck, of Flamingo, 281; Heron, 281; Loon, 277; Snake-bird, 282, 283; Swan, 282 Swan, 282 Neck vertebrae, cf. with Giraffe, 73, 75; cf. with man, 72; cf. with reptile, 73 73 Nerves, action of, 198; cerebral, 202 Nerves, action of, 198; cerebral, 202 Nervous system, 196, 198; of Pigeon, 197; reflex, 197, 198 197; reflex, 197, 198 Nest, of Mourning Dove, 439; Mallard Duck, 447, 448; Laughing Gull, 437; Great Blue Herons, 450; Humming- birds, 448; Anna Hummingbird, 443; Ruby-throated Hummingbird, 449; Junco, 434; Kingfisher, 444; Nighthawk, 455; Ostrich, 453, 454; Burrowing Owl, 442; California Part- ridge, 438; Black Skimmer, 454, 450, 4595 Tern, 453, 456, 457; Hairy Woodpecker, 444, 445 sight of Barred, 211 Owls, bills of, 242; colour of eggs of, 451; foot of, 373, 374; number of eggs laid by, 440 Ovary of fowl, 464 Ovenbird, 365, 366 sight of Barred, 211 Owls, bills of, 242; colour of eggs of, 451; foot of, 373, 374; number of eggs laid by, 440 Ovary of fowl, 464 Ovenbird, 365, 366 eggs laid by, 440 Ovary of fowl, 464 Ovenbird, 365, 366 443; Ruby-throated Hummingbird, 449; Junco, 434; Kingfisher, 444; Nighthawk, 455; Ostrich, 453, 454; Burrowing Owl, 442; California Part- ridge, 438; Black Skimmer, 454, 450, 4595 Tern, 453, 456, 457; Hairy Woodpecker, 444, 445 Ovary of fowl, 464 Ovenbird, 365, 366 Oyster-catcher, bill of, 238, 240; food of, 153 Oyster-catcher, bill of, 238, 240; food of, 153 wing in youn Osteology, 62 Osprey, food of, 155, 156; foot of, 347; method of fishing of, 154; use of wing in young, 322; wing of, 351 Woodpecker, 444, 445 Nictitating membane, of alligator, 215; Eagle, 214; human eye, 215; Brown Thrasher, 213 Woodpecker, 444, 445 Nictitating membane, of alligator, 215; Eagle, 214; human eye, 215; Brown Thrasher, 213 Paradise, King of Saxony Bird of, 276; 269; Six-shafted Bird of, 267, 269, Superb Bird of, 277; Twelve-wired Bird of, 348 Parallelism, in feeding habits, 163, 164 Paradise, King of Saxony Bird of, 276; 269; Six-shafted Bird of, 267, 269, Superb Bird of, 277; Twelve-wired Bird of, 348 Parallelism, in feeding habits, 163, 164 Paramecium, 463; keels, 83, 84 Parrakeet, tail of Grass, 408 Nighthawk, colour of eggs of, 454, 455; nest of, 455; number of eggs laid by, 442; protective coloration in, 296, 301 Parallelism, in feeding habits, 163, 164 Paramecium, 463; keels, 83, 84 Parrakeet, tail of Grass, 408 296, 301 Nostrils, of bird, 204; of daer, 204 Notochord, of Amphioxus, 66; of 296, 301 Nostrils, of bird, 204; of daer, 204 Notochord, of Amphioxus, 66; of Paramecium, 463; keels, 83, Parrakeet, tail of Grass, 408 Parrot, bill of, 242; colour of eggs of, 445; flight of Owl, 333; food of Kea, 161; foot of, 372; number of eggs laid by, 441; use of wing in, 322 Partridge, California, crest of, 259; nest Notochord, of Amphioxus, 66; of Boltenia, 67 Notochord, of Amphioxus, 66; of Boltenia, 67 Boltenia, 67 Nuthatch, bill of, 245; foot of, 359 Boltenia, 67 Nuthatch, bill of, 245; foot of, 359 Nuthatch, bill of, 245; foot of, 359 Nutrition, 116-141 Nuthatch, bill of, Nutrition, 116-141 Nuthatch, bill of, Nutrition, 116-141 441; use of wing in, 322 Partridge, California, crest of, 259; nest and eggs of, 438; Plumed, crest of, 259; Scaled, feathers of, 289, 290 Passeres, foot of, 358, 359 O O Lamprey, gill-basket of, 112 Lark, foot of horned, 367; tail of meadow, 414; cause of colour change in Siberian black, 295, 297 Index 492 492 189; cf. with reptiles, 193, 194; of embryo chick, 480; energy of, in Hummingbird, 83; impressions on bones, 194; pectoral, 190; of skin in Goose, 188; of skin in Penguin, 188; structure of, 193; of wing and breast of Pigeon, 190; of wing in Owl Parrot, 333 479; eyelashes of, 257; leg of, 100: length of intestines, 140; neck and vertebrae of, 69, 71; nest of, 453, 454; number of eggs laid by, 433; pro- tective position of, 306, 307; skele- ton of leg, 99; specific difference in eggs, 460; sternum of, 81; tail of, 402; toes of, 358, 394, 395, 396: wing of, 321, 337, 338, 339 N R Rabbit, tail of, 413 Radius, 92 Ratitae, 82 Raven, bill of American, 225: feeding on ostrich-eggs, 158; foot of, 356 Redstart, life-habits of, 363, 364 Reptiles, causes of colour in, 56; eggs of, 428, 429; trachea of, 169 Respiration of embryo chick, 476 Rhea, number of eggs laid by, 433 Rhinoceros-bird, foot of, 357 Ribs, 77-79; abdominal, of Archaeop- teryx and of reptiles, 79; embryo, 473, 474; function of, 77 79; Hatteria Lizard, 77; Screamer, 77; significance of numerous, 78 Pigment, colour caused by, 54, 55 Pigment, colour caused by, 54, 55 Pipit, effect of climate on, 295, 296; foot of, 367, 368 Raven, bill of American, 225: feeding on ostrich-eggs, 158; foot of, 356 Redstart, life-habits of, 363, 364 foot of, 367, 368 Phalarope, feet of, 384 Pheasant, Argus, colour on ostrich-eggs, 158; foot of, 356 Redstart, life-habits of, 363, 364 Reptiles, causes of colour in, 56; eggs Phalarope, feet of, 384 Pheasant, Argus, colour pattern of, 58; Eared, head of, 267, 268; feet of, 380; flight of, 323, 324; number of eggs laid by Peacock, 440; ruff of Lady Amherst, 277, 278; spurs of Peacock, 383; tail of Peacock, 419, 420; tail of Reeves, 416, 418; wing of, 327 Redstart, life-habits of, 363, 364 Reptiles, causes of colour in, 56; eggs of, 428, 429; trachea of, 169 Respiration of embryo chick, 476 Rhea, number of eggs laid by, 433 Rhinoceros-bird, foot of, 357 Ribs, 77-79; abdominal, of Archaeop- teryx and of reptiles, 79; embryo, 473, 474; function of, 77 79; Hatteria Lizard, 77; Screamer, 77; significance of numerous, 78 tail of Reeves, 416, 418; wing of, 327 Phororachus, skull of, 16; structure of, 13, 14 13, 14 Plantain-eater, green pigment in, 55 Plantain-eater, green pigment in, 55 Ploughshare-bone, 400, 402, 403 Plantain-eater, green pigment in, 55 Ploughshare-bone, 400, 402, 403 Ploughshare-bone, 400, 402, 403 Plover, Crook-billed, 240, 241; feet of, 384; nest and eggs of Killdeer, 431, 432 Pollen, used as food, 144, 145 Powder-down, 38; of Great White Heron, 3? significance of numerous, 78 Road-runner, food of, 157; foot of, 373; tail of, 413 Ploughshare-bone, 400, 402, 403 Plover, Crook-billed, 240, 241; feet of, 384; nest and eggs of Killdeer, 431, 373; tail of, 413 Robin, glottis of nestling, 166; moult of, 43; .spots in young, 316, 317 432 Pollen, used as food, 144, 145 Powder-down, 38; of Great White Heron, 3? Sandgrouse, feet of, 383 Sandpipers, protective coloration in, 299; semipalmated, feet of, 384 O 188; tail of Black-footed, 406; wing of, 321, 341, 342, 343 in, 299; of eggs, 44 Proventriculus, 135 Proventriculus, 135 Ptarmigan, feet of, 381; food of, 144; moult of, 48, 50, 51, 52; protective coloration in, 298, 300, 309 Pterodactyls, 2, 9; weight of, 285 Pterylosis, 39, 40; of nestling Crow, 39 Pubis, 89, 90; in embryo bird, 90 Purlin, iris of eye of, 256 Ptarmigan, feet of, 381; food of, 144; moult of, 48, 50, 51, 52; protective coloration in, 298, 300, 309 406; wing of, 321, 341, 342, 343 Pepper, effect on plumage of, 292 Pterodactyls, 2, 9; weight of, 285 Pterylosis, 39, 40; of nestling Crow, 39 Pubis, 89, 90; in embryo bird, 90 Purlin, iris of eye of, 256 Pepper, effect on plumage of, 292 Petrel; body of, 286; ejecting oil from crop, 131 crop, 131 Pickerel, aggressive coloration in, 310 Pickerel, aggressive coloration in, 310 Pigeon, Blood-breasted, 306, 308; cir- culatory system of, 183; Crowned, crest of, 260, 261 ; Double-crested, 270; extreme development of crop of, 130; gizzard of Fruit, 138; nervous system of, 197; number of eggs laid by, 440; symptoms of flight in, 350, 352; tail of, 402; tail of Fan-tail, 404; wings and breast-muscles of, 190; wing- feather of, 35; X-ray photograph of, 63 R of, 43; .spots in young, 316, 317 Rock-jumper, body-feathers of, 288; feet of, 393 Powder-down, 38; of Great White Heron, Prairie hen, use of air-sacs in, 117 Precocial nestling, 30 Ruff of, Lady Amherst Pheasant, 277, 278; Ruff, 277, 279, 280; Ruffed Grouse, 277; Superb Bird of Paradise, 277 Ruff, feather cloak of, 277, 279, 280 Prairie hen, use of air-sacs Precocial nestling, 30 Precoracoid in embryo, 475 Prairie hen, use of air-sacs in, 117 Precocial nestling, 30 Precoracoid in embryo, 475 Precoracoid in embryo, 475 Primaries, moult of, in Bob- white, 43; English Sparrow, 46; Mallard, 47; Robin, 43 Ruff, feather cloak of, 277, 279, 280 Pigment, colour caused by, 54, 55 O 259; Scaled, feathers of, Passeres, foot of, 358, 359 Peacock, feather of, 20; Ind Peacock, feather of, 20; India, crest of, 259; Java, crest of, 258; ribs of, 78; spurs of, 382, 383; tail of, 422, 423; train of, 422, 423 Peafowl, tail of, 402 Peacock, feather of, 20; India, crest of, 259; Java, crest of, 258; ribs of, 78; spurs of, 382, 383; tail of, 422, 423; train of, 422, 423 Peafowl, tail of, 402 Pectoral girdle, see Shoulder-girdle. train of, 422, 423 Peafowl, tail of, 402 Peafowl, tail of, 402 Osprey, food of, 155, 156; foot of, 347; method of fishing of, 154; use of wing in young, 322; wing of, 351 Pectoral girdle, see Shoulder-girdle. Pelican, bill of, 228, 229, 330; c Pectoral girdle, see Shoulder-girdle. Pelican, bill of, 228, 229, 330; colour of eggs of, 448; downy stage of, 54; foot of, 355; full-grown, 59; glottis of, 166; half-grown, 57; method of fishing of, 152; nestling of Brown, 21; newly hatched, 30; tail of, 408, 409; tongue of, 120, 121 Osteology, 62 Ostrich, body-feathers of, 288; cause of downiness in feather, 35; colour of eggs, 453, 454; compared with Moa, 13; cross-section of wing-bone, 175; eggs of, 436, 453; embryo (40-day), Index migan, 298, 300, 309; Quail, 299, 300; Sandpipers, 299; Sparrows, 299; Black-necked Swan, 305, 306; Sooty Tern, 300, 303; Thayer's experiments in, 299; of eggs, 446-459 Proventriculus, 135 Pelvic girdle, see Thigh-girdle. Pelvic girdle, see Thigh-girdle. Pelvis, compared with that of reptiles, 74; vertebra? in, 74 74; vertebra? in, 74 Penguin, aggressive coloration in. 309, 310; body-leathers of, 289; eggs of, 435; fat of, 280; food of, 153; method of fishing of, 154; moult of, 51; skin- muscles in. Q Quadrate, 109, 110; in reptiles, 111 Quadrate, 109, 110; in reptiles, 111 Quail, feet of, 380; protective colora- tion in, 299, 300 Quezal, sham tail of, 421, 422 Quadrate, 109, 110; in reptiles, 111 Quail, feet of, 380; protective colora- tion in, 299, 300 Quezal, sham tail of, 421, 422 Quadrate, 109, 110; in reptiles, 111 Quail, feet of, 380; protective colora- tion in, 299, 300 tion in, 299, 300 Quezal, sham tail of, 421, 422 Robin, 43 Primaries, of Albatross, 320, 321, 325; Cassowary, 321, 337, 338; Hum- mingbird, 320, 321, 325; Ostriches, 321; Penguins, 321 ^ Salivary glands, original function of, 117, 118; in Swiftlets, 119; Swifts, 119; Woodpeckers, 118 Salmon, eggs of, 427 Sandgrouse, feet of, 383 Sandpipers, protective coloration in, 299; semipalmated, feet of, 384 Salivary glands, original function of, 117, 118; in Swiftlets, 119; Swifts, 119; Woodpeckers, 118 Salmon, eggs of, 427 Sandgrouse, feet of, 383 321; Penguins, 321 ^ Protective coloration, in Brown Creeper, 303, 304; Flickers, 302, 303, 304; Ivory Gull, 309, 311; Laughing Gull, 304, 306; Motmot, 303; Nighthawk, 296, 301; Ostrich, 306, 307; Ptar- Sandgrouse, feet of, 383 Sandpipers, protective coloration in, 299; semipalmated, feet of, 384 Index 494 Sapsucker, tongue of, 124 Snipe, bill of Dowitcher, 241; fighting, see Ruff. Snowshoes of Grouse, 380, 381 Snowflake, colour of change in, 297 Snipe, bill of Dowitcher, 241; fighting, see Ruff. Snowshoes of Grouse, 380, 381 Sapsucker, tongue of, 124 Scales, of carp, 289, 290; on legs of birds, 356, 368 Snowshoes of Grouse, 380, 381 Snowflake, colour of change in, 297 birds, 356, 368 Scapula, 84; position in various ani- mals, 88 Sparrow, albinism in English 314; crop of English, 129; effect of cli- mate on song, 292, 295; effect of humidity on White-throated, 291, 294; flight of, 328; moult of wing- leathers of, 46; moult of body- feathers of, 52, 53; neck of White- throated, 74, protective coloration in, 299; stomach-glands of, 135; tail in moult of, 409; tail of Vesper, 414 mals, 88 Scent-glands, of birds, 286, 287; of mammals, 286 mammals, 286 Screamer, Crested, 330; flight of: 329 Screamer, Crested, 330; flight of: 329 Sea-birds, colour of eggs of, 455 Screamer, Crested, 330; flight of: 329 Sea-birds, colour of eggs of, 455 Sea-birds, colour of egg Sea-eagles, food of, 157 Sea-eagles, food of, 157 Sea-urchins, used as food, 145 Sea-urchins, used as food, 145 Secondaries, number in Albatross, 320, 321, 325; in Hummingbird, 320, 321, 325 325 Secretary-bird, 378, 379; food of, 157 Seeds, 427 Specialization of feet, 358 Seeds, 427 Segmentation of egg, 465-470 Senses, Spoonbill, bill of, 236, 239; use of wing in a, 350 Segmentation of egg, 465-470 Senses, hearing, 215-218; in Geese, 216 sight, 207-215; in Barred Owl, 211; Eagle, 208: Woodcock, 208 smell, 203-206; in Vultures, 205 taste, 218, 219 touch, 219, 222; in Apteryx, 219; Woodcock, 219, 220, 222 Spurs of fowls, 382; of Peacock, 382, 383; Peacock Pheasant, 383; struc- ture of, 382 Squid, used as food, 150, 153 Starling, food of Red-winged, 162, 163; Spurs of fowls, 382; of Peacock, 382, 383; Peacock Pheasant, 383; struc- ture of, 382 Squid, used as food, 150, 153 Starling, food of Red-winged, 162, 163; Wattled, see Locust-bird Wattled, see Locust-bird Sternum, function of, 81; value in classification of, 80; of Flamingo, 80; Ostrich, 81 Stilt, 237 Sternum, function of, 81; value in classification of, 80; of Flamingo, 80; Ostrich, 81 Stilt, 237 Stomach, 134-140; of Chicken (young), 135; English Sparrow, 135; Snake- bird, 139 Storks, food of, 157 Sugar-bird, food of, 144 Sulphur-tyrant, food of, 161 Swallow, food of, 360; tail of Barn, 405, 407 Woodcock, 219, 220, 222 Seriema, 17; eyelashes of, 257; habits of, 15; relation to Phororachus, 15 of, 15; relation to Phororachus, 15 Shark, back-bone of, 68; gill-bars of, 113; skull of, 104; tooth of, 20 of, 15; relation to Phororachus, 15 Shark, back-bone of, 68; gill-bars of, 113; skull of, 104; tooth of, 20 Shore-birds, number of eggs laid by, 436 Shoulder-girdle, 84-88, 85; bones com- posing, 84; evolution of, 86, 87; of fish, 86, 87 of fish, 86, 87 Shrike, Puff-back, feathers in, 287 Sulphur-tyrant, food of, 161 Swallow, food of, 360; tail of Barn, 405, 407 Sulphur-tyrant, food of, 161 Swallow, food of, 360; tail of Barn, 405, 407 Shrike, Puff-back, feathers in, 287 Size, as correlated with distribution, 295 Swan, foot of Black-necked, 390; neck of, 282; protective coloration in Black-necked, 305, 306; sleeping position of, 345; specific difference in egg-shells, 460; trachea of Trumpeter, 170; weight of Trumpeter, 285; wing-bone of Black, 175; use of wing in defence, 346 Swift, bill of Chimney, 244, 245, 246; Skate, egg of, 428, 429 Skate, egg of, 428, 429 Skeleton, of man, leg of, 99; Ostrich, leg of, 99; Pigeon, shown by X-ray photograph, 63; Rooster, compared with contour of body, 65; ways of preparing, 64 preparing, 64 Skimmer, bill of, 231, 232; Black, nest and eggs of, 454, 456, 459; colour of eggs in Black, 454, 455, 456, 459 Skin, layers of, 19; products of, 20 Swift, bill of Chimney, 244, 245, 246; foot of, 368, 369; nest of, 118; tail of Chimney, 410, 412; wing-feathers of, 42 Swiftlets, nest of, 119; use of nests, Swift, bill of Chimney, 244, 245, 246; foot of, 368, 369; nest of, 118; tail of Chimney, 410, 412; wing-feathers of, 42 Swiftlets, nest of, 119; use of nests, of eggs in Black, 454, 455, 456, 459 Skin, layers of, 19; products of, 20 Skin, layers of, 19; products of, 20 Skull, 103-115; bones of, 107; evolu- tion of, 104-106; origin of, 103-104; section through, 201; alligator, 105; Eagle, 106; Hawk, 199, 200; Heron, 199, 200; shark, 104 Sleep, birds in, 344, 355 Sloth, head of, 210 Snail used as food, 148, 453 Snake, egg of, used as food, 157, 429 Snakebird, bill of, 228, 229; body- feathers of, 289; foot of, 391; method of fishing of, 154; neck of, 282, 283; stomach of, 139 Swiftlets, nest of, 119; use of nests, 120 Swim-bladder of fishes, 178, 179 Swim-bladder of fishes, 178, 179 Swimming compared with flight, 327 Swim-bladder of fishes, 178, 179 Swimming compared with flight, 327 Syrinx, drum of Mallard, 168, 170; structure of, 172 Swiftlets, nest of, 119; use of nests, 120 teryx Tail, of Archaeopteryx, 398, 399, 400, 401; evolution of, 398-403; feathers of, 402, 403; lack of, in Emeu, 402, 404; in Cassowary, 402 Tail, of Archaeopteryx, 398, 399, 400, 401; evolution of, 398-403; feathers of, 402, 403; lack of, in Emeu, 402, 404; in Cassowary, 402 Snake, egg of, used as food, 157, 429 Snakebird, bill of, 228, 229; body- feathers of, 289; foot of, 391; method of fishing of, 154; neck of, 282, 283; stomach of, 139 Index 495 Thrush, crest of Laughing, 263; effect of humidity on Wood, 294 Tibia, 98 Thrush, crest of Laughing, 263; effect of humidity on Wood, 294 Tibia, 98 Tail, num number of feathers in Archaeopteryx, 400; Cormorant, 400; Duck, 400; Ostrich, 402; Peafowl, 402; Fan- tail Pigeon, 402 Tinamou, 334; egg-shell of, 460; flight of, 333-336; tail of, 409 tail Pigeon, 402 ornaments of, 414-421; ploughshare- bone of, 400; sham, of Quezal, 421, 422; sh m, of Peacock, 422, 423 of, 333-336; tail of, 409 Toes, of Cassowary, 394, 396; Chuck- will's-widow, 369; Coot, 387; Donkey, 394, 395; embryo, 474; Ballinule, 385,386; Grebe, 391; Ruffed Grouse, 380, 381; Heron, 101; comb on Heron, 387; 388; perching function of hind, 359; horse, 358; Wood Ibis, 385; jaguar, 101; young kangaroo, 394, 395; European House Martin, 381; monkey, 358; number of, 353; Osprey, 374; Ostrich, 358, 394, 395, 396; Snowy Owl, 375; Penguin, 391, 392; Plover, 384; Phalarope, 384; Ptarmigan, 381; Rhinoceros-bird, 357; Sand-grouse, 383; Semipalmated Sandpiper, 384; Snakebird, 391; Vulture, 377; Zygodactyl, 369, 370, 371, 372 422; sh m, of Peacock, 422, 423 vertebrae of Bald Eagle, 403; embryo bird, 403; Ostrich, 402 bird, 403; Ostrich, 402 use of, 405; voluntary decoration of, 424, 425, 425; of creeper, 410, 411; deer, 413; Pin-tail Duck, 415; Blue Duck, 406; Emeu-wren, 415; Least Flycatcher, 412; Japanese long-tailed fowl, 417, 419; Jungle Fowl, 418; Boat-tailed Crackle, 418; Green Heron, 413; Junco, 414; Meadow Lark, 414; Lyre- bird, 420, 421; Mexican Motmot, 424, 425, 425; Murre, 406, 407; Grass Parrakeat, 408; Peacock, 422, 423; Pelican, 408, 409; Black- footed Penguin, 406; Fan-tail Pigeon 404; Peacock Pheasant, 419, 420; Reeves Pheasant, 416, 418; Rabbit, 413; Road-runner, 413; Sparrow in moult, 409; Vesper Sparrow, 414; Barn Swallow, 405, 407; Tern, 407, 409; Chimney Swift, 410, 412; Tinamous, 409; Wagtail, 413; whale, 405; Paradise Whydah- finch, 415, 416; Woodhewers, 410; Woodpeckers, 410, 411, 412 Tongue, of Cockatoo, 125; Ducks and Geese, 120, 121, 122; Flamingo, 126, 127; Flicker, 123, 126; function of fleshy teeth on, 120; Goldfinch, 125; Honey-creeper, 127, 129; Humming- bird, 127; Lory, 125; Owls, Larks, and Swifts, 122; Pelican, 120, 121; Sap- sucker, 124; Toucan, 126; Wood- peckers, 122 peckers, 122 Toucan, bill of, 243, 244; tongue of, 126 Towhee, iris of, 256 Trachea, of amphibian, 169; of cranes, Woodpeckers, 410, 411, 412 Tail-coverts of Quezal, 421, 422; of peacock, 422, 423 Trachea, of amphibian, 169; of cranes, 170, 171; cf. teryx with oesophagus, 167; of duck, 168; of flamingo, 168, 169; of reptiles, 169; of Sparrow, 169; struc- ture, of, 168, 169; of Trumpeter Swan, 170 396, 397 Trachea, of amphibian, 169; of cranes, 170, 171; cf. with oesophagus, 167; of duck, 168; of flamingo, 168, 169; of reptiles, 169; of Sparrow, 169; struc- ture, of, 168, 169; of Trumpeter Swan, 170 396, 397 peacock, 422, 423 Tailor-bird, bill of, 245 peacock, 422, 423 Tailor-bird, bill of, 245 Tailor-bird, bill of, 245 Tanager, colour change in moult of, 294 Tarpon, scale of, 20 Tarsus, 99 Tailor-bird, bill of, 245 Tanager, colour change in moult of, 294 Tracks, of Cassowary, 396; of Dino aur, 396, 397 Triceratops, beak of, 226; brain of, 200 Tarsus, 99 Teeth, in embryo Tern, 476; origin of, 23, 114 23, 114 Temperature, of bird, 186; of man, 186 Tendons, of foot, 191, 192; strength of, Triceratops, beak of, 226; brain of, 200 Turbinal bones, 205 Temperature, of bird, 186; of man, 186 Tendons, of foot, 191, 192; strength of, illustrated, 193; use in perching of, 195 Turkey, breast-ornament of Wild, 280 Turtle, beak of Snapping, 224; egg of, 428, 429 Turkey, breast-ornament of Wild, 280 Turtle, beak of Snapping, 224; egg of, 428, 429 Tern, bill of, 231, 232; colour of eggs of, 454, 456, 457; eggs of Stilt and, 452; method of fishing of, 154; nest of, 453, 456, 457; protective coloration in Sooty, 300, 303; tail of, 407, 409; teeth in embryo, 476; wing of, 352 Thales, 12 Tern, bill of, 231, 232; colour of eggs of, 454, 456, 457; eggs of Stilt and, 452; method of fishing of, 154; nest of, 453, 456, 457; protective coloration in Sooty, 300, 303; tail of, 407, 409; teeth in embryo, 476; wing of, 352 Thales, 12 " Two Bird-Lovsrs in Mexico," quota- tion from, 301 X dactyls, 285 Whale, food of, 148; tail of, compared with that of bird, 405 U Ulna, 2 Umbrella-bird, crest of, 264, 266 Uncinate processes, 77 Ulna, 2 Umbrella-bird, crest of, 264, 266 Uncinate processes, 77 teeth in embryo, 476; wing of, 352 Thales, 12 Thayer, experiments of Abbott, Thigh-bone, see Femur Thigh-bone, see Femur Thigh-girdle, 88-91; bones composing, 88, 89; of embryo bird, cf. X-ray photograph of Pigeon, 63 Y with that of bird, 405 Whydah-finch, tail of Paradise, 415, 416 Whydah-finch, tail of Pa Windpipe, see Trachea U with Dino- saur, 90 Thrasher, eyelids of Brown, 212, 213; nictitating membrane of, 213 Thrasher, eyelids of Brown, 212, 213; nictitating membrane of, 213 Index 496 496 Vertebrae (continued) function of, 196; Jabiru, neck vertebra of, 72; Ostrich, 69, 71; Ostrich-tail, 402 pelvic, of American Flamingo, 76; alligator (young), 76; Bald Eagle, 76 of shark, 68; of tail, 75 Vireo, 144; iris of eye of, 256 Vorticlla, 463, 464 Wing of, Albatross, 320, 321, 325, 332- Great Auk, 339, 340; Racor-billed Auk, 339,340; Cassowary, 321,337, 338; Young Catbird, 322; Condor, 324, 326, 332; Steamer Duck, 337; Snowy Egret, 350; West African Goatsucker, 359; Canada Goose, 346; Young Canada Goose, 322; Spur- winged Goose, 346, 347; Ruffed Grouse,347; Herring Gull, 328; Young Green Heron, 320, 323; Great White Heron, 321; Sleeping Heron, 344; Hummingbird, 320, 321,325; Mallard, 47; Murrelets, 339; Young Osprey, 322, 351; Ostrich, 321,337,338,339; Twelve-wired Bird of Paradise, 348; Parrot, 322; Parrot Owl, 333; Pen- guin, 321, 341, 342, 343; Pheasant, 323, 324, 327; Screamer, 346; Black Skimmers, 324; English Sparrow, 46; Spoonbill, 350; Trumpeter Swan, 346; Tern, 352; Bush Warbler, 347; Wood- cock, 348, 349; Turkey Vulture, 331 Wish-bone, see Clavicle Wing of, Albatross, 320, 321, 325, 332- Great Auk, 339, 340; Racor-billed Auk, 339,340; Cassowary, 321,337, 338; Young Catbird, 322; Condor, 324, 326, 332; Steamer Duck, 337; Snowy Egret, 350; West African Goatsucker, 359; Canada Goose, 346; Young Canada Goose, 322; Spur- winged Goose, 346, 347; Ruffed Grouse,347; Herring Gull, 328; Young Green Heron, 320, 323; Great White Heron, 321; Sleeping Heron, 344; Hummingbird, 320, 321,325; Mallard, 47; Murrelets, 339; Young Osprey, 322, 351; Ostrich, 321,337,338,339; Twelve-wired Bird of Paradise, 348; Parrot, 322; Parrot Owl, 333; Pen- guin, 321, 341, 342, 343; Pheasant, 323, 324, 327; Screamer, 346; Black Skimmers, 324; English Sparrow, 46; Spoonbill, 350; Trumpeter Swan, 346; Tern, 352; Bush Warbler, 347; Wood- cock, 348, 349; Turkey Vulture, 331 Wish-bone, see Clavicle of shark, 68; of tail, 75 Vireo, 144; iris of eye of, 256 Vireo, 144; iris of Vorticlla, 463, 464 Vorticlla, 463, 464 Vulture, colour in young Turkey, 317; flight of, 328, 330, 331, 332; King, head of, 272, 273; sense of sight in, 205; toes of, 377; Turkey, in flight, Y Wing, bones of, 95; change of function in, 337; compared with arm of man, 94; evolution of, 91-97, 319; feather divisions of, 320; framework of, 91- 97; noise of, 345, 347; spurs of, 346, 347; strokes of Hummingbird, 82; use of, in defence, 337 Yellow-throat, life-habits of Maryland, 362 Z W Wading birds, number of eggs laid by, 438 Wagtail, tail of, 413 Walrus, brain of, 200 Wagtail, tail of, 413 Walrus, brain of, 200 Walrus, brain of, 200 Warbler, life-habits of Black-and-white, 366; Black-throated Green, 366; Mag- nolia, 366; Myrtle, 364; Pine, 366; Worm-eating, 163; Yellow Palm, 363 Wish-bone, see Clavicle Woodcock, bill of, 222; eyes of, 221, 256; sense of sight in, 208; sense of touch in, 219, 221, 222; wing-song of, 348 Woodhewer, tail of, 410 Worm-eating, 163; Yellow Palm, 363 Warbler-bush, wings of, 347 Warbler-bush, wings of, 347 Warblers, evolution of, 361-367 Warblers, evolution of, 361-367 Water-birds, number of eggs laid by, 436, 437, 438 Woodhewer, tail of, 410 Woodpecker, tail of 410, 411, 412; nest and eggs of Hairy, 445; char- acter of egg-shell of, 460; colou of eggs of, 444; function of saliva in, 118; tongue of, 122; bill of, 245; crest of, 264; flight of, 329; feet of, 370 Woodhewer, tail of, 410 Woodpecker, tail of 410, 411, 412; nest and eggs of Hairy, 445; char- acter of egg-shell of, 460; colou of eggs of, 444; function of saliva in, 118; tongue of, 122; bill of, 245; crest of, 264; flight of, 329; feet of, 370 436, 437, 438 Wattles, of Bell-bird, 273; Condor, 271; Wild Turkey, 273, 275; King Vulture, 272, 273 272, 273 Waxbill, African, abnormal number of eggs laid by, 444 eggs laid by, 444 Web of toes, of Flamingo, 388; Sea- birds, 389; Semipalmated Sandpiper, 384 Wren, Marsh, colour of eggs of, 446; tail of, 411 Weight of birds, 285, 286; of Ptero- dactyls, 285 tail of, 411 Wrist-bones of bird, 92; of man, 92 with that of bird, 405 Whydah-finch, tail of Paradise, 415, 416 Windpipe, see Trachea THE AMERICAN NATURE SERIES In the hope of doing something towards furnishing a series where the nature-lover can surely find a readable book of high authority, the pub- lishers of the American Science Series have begun the publication of the American Nature Series. It is the intention that in its own way, the new series shall stand on a par with its famous predecessor. The primary object of the new series is to answer the questions which The primary object of the new series is to answer the questions which the contemplation of Nature is constantly arousing in the mind of the unscientific intelligent person. But a collateral object will be to give some intelligent notion ol the "causes of things." intelligent notion ol the "causes of things." While the co-operation of foreign scholars will not be declined, the books will be under the guarantee of American experts, and generally from the American point of view ; and where material crowds space, preference will be given to American facts over others of not more than equal interest. The series will be in six divisions : equal interest. The series will be in six divisions : Yellow-throat, life-habits of Maryland, 362 Z Zygodactyl toes, 369, 370, 371, 372 Zygodactyl toes, 369, 370, 371, 372 III. FUNCTIONS OF NATURE These books will treat of the relation of facts to causes and effect of heredity and the relations of orjranism to environment. 6§x8^ in. These books will treat of the relation of facts to causes and effects — of heredity and the relations of orjranism to environment. 6§x8^ in. Already publisht: These books will treat of the relation of facts to causes and effects — of heredity and the relations of orjranism to environment. 6§x8^ in. of heredity and the rela Already publisht: Already publisht: Already publisht: THE BIRD: ITS FORM AND FUNCTION, by C. W. BEF.BE, Curator of Birds in the N. Y. Zoological Park. 496 pp. $3. 50 net : by mail, 83. 80. Arranged for : Arranged for : THE INSECT : ITS FORM AND FUNCTION, by VERNON L. KELLOGG, Professor in the Leland Stanford Junior University. THE FISH: ITS FORM AND FUNCTION, by H. M. SMITH, of the U. S. Bureau of Fisheries. IV. WORKING WITH NATURE How to propagate, develop, care for and depict the plants and animals. The volumes in this group cover such a range of subjects that it is impracticable to make them of uniform size. How to propagate, develop, care for and depict the plants and animals. The volumes in this group cover such a range of subjects that it is impracticable to make them of uniform size. impracticable to make Already publisht: NATURE AND HEALTH, by EDWARD CURTIS, Professor Emeritus in the College of Physicians and Surgeons. 12mo, $1.25 net; by mail, $1.37. Arranged for : Arranged for : PHOTOGRAPHING NATURE, by E. R. SANBORN, Photographer of the New York Zoological Park. THE FRESHWATER AQUARIUM AND ITS INHABITANTS. A Guide for the Amateur Aquarist, by OTTO EoGELiNoand FREDERICK EHRENBERG. THE SHELLFISH INDUSTRIES, by JAMES L. KELLOGG, Professor in Williams College. CHEMISTRY OF DAILY LIFE, by HENRY P. TALBOT, Professor of Chemistry in the Massachusetts Institute of Technology. DOMESTIC ANIMALS, by WILLIAM H. BREWER, Professor Emeritus in Yale University. 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NMR Structure of Hsp12, a Protein Induced by and Required for Dietary Restriction-Induced Lifespan Extension in Yeast
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Abstract Dietary restriction (DR) extends lifespan in yeast, worms, flies and mammals, suggesting that it may act via conserved processes. However, the downstream mechanisms by which DR increases lifespan remain unclear. We used a gel based proteomic strategy to identify proteins whose expression was induced by DR in yeast and thus may correlate with longevity. One protein up-regulated by DR was Hsp12, a small heat shock protein induced by various manipulations known to retard ageing. Lifespan extension by growth on 0.5% glucose (DR) was abolished in an hsp12D strain, indicating that Hsp12 is essential for the longevity effect of DR. In contrast, deletion of HSP12 had no effect on growth under DR conditions or a variety of environmental stresses, indicating that the effect of Hsp12 on lifespan is not due to increased general stress resistance. Unlike other small heat shock proteins, recombinant Hsp12 displayed negligible in vitro molecular chaperone activity, suggesting that its cellular function does not involve preventing protein aggregation. NMR analysis indicated that Hsp12 is monomeric and intrinsically unfolded in solution, but switches to a 4-helical conformation upon binding to membrane-mimetic SDS micelles. The structure of micelle-bound Hsp12 reported here is consistent with its recently proposed function as a membrane-stabilising ‘lipid chaperone’. Taken together, our data suggest that DR-induced Hsp12 expression contributes to lifespan extension, possibly via membrane alterations. Citation: Herbert AP, Riesen M, Bloxam L, Kosmidou E, Wareing BM, et al. (2012) NMR Structure of Hsp12, a Protein Induced by and Required for Dietary Restriction-Induced Lifespan Extension in Yeast. PLoS ONE 7(7): e41975. doi:10.1371/journal.pone.0041975 Editor: Matt Kaeberlein, University of Washington, United States of America Editor: Matt Kaeberlein, University of Washington, United States of America Received May 13, 2012; Accepted June 26, 2012; Published July 27, 2012 Received May 13, 2012; Accepted June 26, 2012; Published July 27, 2012 rbert et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright:  2012 Herbert et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by a Biotechnology and Biological Sciences Research Council research grant to AM and SRP (Grant Ref ERA16235; funder’s URL http://www.bbsrc.ac.uk/home/home.aspx), a Wellcome Trust project grant to AM and LYL (Grant Ref 090077/Z/09/Z; funder’s URL http://www.wellcome.ac. uk/), and Wellcome Trust Prize Studentships to MR and LB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: amorgan@liverpool.ac.uk . These authors contributed equally to this work. . These authors contributed equally to this work. NMR Structure of Hsp12, a Protein Induced by and Required for Dietary Restriction-Induced Lifespan Extension in Yeast Andrew P. Herbert1,2., Miche`le Riesen1., Leanne Bloxam1, Effie Kosmidou1, Brian M. Wareing1, James R. Johnson1, Marie M. Phelan2, Stephen R. Pennington3, Lu-Yun Lian2, Alan Morgan1* 1 Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom, 2 NMR Centre for Structur Biology, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom, 3 University College Dublin Conway Institute of Biomedical and Biomolecula Research, University College Dublin, Dublin, Ireland PLoS ONE | www.plosone.org Results To validate these protein expression changes, we prepared extracts from yeast containing chromosomally-tagged GFP fusion constructs [18] and performed western blots using a GFP antibody. Examples of proteins selectively induced by DR (GFP-Hxk1) or high osmolarity (GFP-Ctt1) are shown in Fig. S1B. Of the proteins identified as being induced by both interventions, specific bands of the predicted size could not be reproducibly detected for GFP-Rtc3 or –Rgi1; whereas GFP-Eno1 expression was not altered by DR. However, GFP-Hsp12 was confirmed to be induced by both DR and high osmolarity (Fig. S1B). To rule out any artefactual effect of the GFP tag, we raised an antiserum against the N-terminus of Hsp12 and used this in western blots of wild type cells. This revealed a band of the expected size (,12 kDa), which was increased in intensity upon growth in DR and high osmolarity conditions and which was not present in an isogenic hsp12 deletion strain (Fig. S1C), thus confirming the increased expression of Hsp12 under conditions of enhanced longevity. In addition to possessing anti-aggregation properties, small heat shock proteins are often large homo-oligomeric assemblies of folded subunits. To further investigate the possible function of Hsp12, we determined its solution structure using NMR. Recombinant Hsp12 expressed in E. coli was monomeric. The 15N-1H HSQC spectrum showed poor resonance dispersion in the proton dimension, which suggested that Hsp12 is intrinsically disordered in aqueous buffer (Fig. 3A). Recently published circular dichroism studies have shown that Hsp12 gains significant helical content upon binding to lipid or SDS micelles [16], we therefore examined the effect of varying SDS concentrations. The 15N-1H HSQC spectra of Hsp12 showed a dose-dependent increase in dispersion in response to SDS, indicating that Hsp12 adopts a folded conformation upon micelle binding (Fig. 3B). Having determined the optimal SDS concentration for NMR, we then characterised the temperature-dependence of Hsp12 in the presence (Fig. S4A) and absence (Fig. S4B) of SDS. This resulted in linear resonance dispersion until 45uC, above which some resonances deviated from a straight line in the presence of SDS, indicating heat-induced unfolding. These optimised conditions were then used to assign the residues of SDS-bound 15N/13C- labelled Hsp12 (Fig. 3C). To determine if Hsp12 is causally linked to DR-induced lifespan extension, we performed replicative lifespan analysis. This was done by determining the number of daughter cells removed by micromanipulation from individual virgin mother cells [19]. Results To identify proteins that are induced by DR, we analysed extracts from BY4741 yeast cells grown under standard (2% glucose) or DR (0.5% glucose) conditions by 2-D gel electropho- resis. Initial experiments using wide range (pH 3–10) gels revealed no obvious reproducible changes in protein spot abundance, indicating that DR does not cause gross proteomic alterations (Fig. 1A). However, using pH 5.3–6.5 and pH 3–5.6 zoom gels, we could resolve several proteins that were barely detectable in control conditions, yet clearly induced in DR conditions (Fig. 1B, C). Mass spectrometry was used to identify these differentially expressed proteins as Eno1, Hxk1, Hsp12, Rtc3, Rgi1, Sbp1 and Yef3 (Table S1). Various small heat shock proteins have been shown to be ‘holdase’ molecular chaperones that bind to denaturing proteins and prevent their aggregation. To determine if Hsp12 had such activity, we investigated the ability of recombinant purified Hsp12 to prevent aggregation of the model substrate, insulin, using the method of Haslbeck et al. [23]. Addition of DTT reduces the disulphide bonds between the A and B chains of insulin, causing aggregation; whereas in the absence of DTT, insulin remains stable (Fig. S3A). DTT-induced insulin aggregation was greatly reduced by recombinant GST-fusion proteins of the known chaperones, yeast Hsp26 [23] and mammalian cysteine string protein (CSP) [24]; but not by CaBP1s, used as a control for a protein of similar size to Hsp12 with no known or predicted chaperone functions (Fig. S3B). However, GST-Hsp12 was similar to GST-CaBP1s in terms of ability to prevent insulin aggregation. The differences in chaperone activity for GST-Hsp12 and GST- Hsp26 were then assessed in a dose-dependent manner. This revealed that GST-Hsp26 has approximately 100-fold higher anti- aggregation activity than GST-Hsp12 (Fig. S3C), indicating that Hsp12 has very low, if any, intrinsic chaperone activity. ( ) To help pinpoint DR-induced proteins that play a causal role in mediating lifespan extension, as opposed to those whose expression patterns are merely coincidental, we then analysed proteins that were induced by high osmolarity (Fig. S1). Our rationale was based on the observation that lifespan extension by DR and high osmolarity act via a common downstream pathway [17]; and hence the crucial effector proteins are likely to be common to both interventions. Proteins induced by high osmolarity comprised Ctt1, Eno1, Fba1, Hsp12, Hsp26, Hsp31, Lys9, Rtc3, Rgi1 and Oye2 (Table S1). Hsp12 Structure and Function Hsp12 determined here is consistent with its recently proposed function as a membrane-binding ‘lipid chaperone’ [16], which may in turn explain Hsp12’s ability to modulate various phenotypes and cellular functions. Hsp12 determined here is consistent with its recently proposed function as a membrane-binding ‘lipid chaperone’ [16], which may in turn explain Hsp12’s ability to modulate various phenotypes and cellular functions. Stress resistance correlates positively with lifespan in various model organisms and DR may represent a mild stress that extends lifespan via a hormesis-like mechanism [20]. We therefore investigated if deletion of HSP12 reduced resistance to environ- mental stresses. For comparison, we also included sir2 and fob1 deletion strains in this analysis, as deletion of SIR2 and FOB1 is known to decrease and increase replicative lifespan respectively [21,22]. There was no detectable difference between the ability of BY4741 wild type and the deletion mutants to grow under a wide variety of stress conditions, including DR and other stresses that increase Hsp12 expression (Fig. S2). We therefore conclude that Hsp12 does not contribute to general stress resistance. Introduction in increased ribosomal DNA (rDNA) silencing and a consequent reduction in rDNA recombination [6,9,10,11]. An alternative theory is that DR extends lifespan in a Sir2-independent manner by inhibition of the Tor and Sch9 kinase signalling pathways [12,13]. In this latter model, the downstream molecular mechan- isms effecting longevity are not entirely clear, but may include reduced ribosomal protein biogenesis [13,14] as well as reduced rDNA recombination [15]. It is commonly accepted that similar fundamental cellular processes modulate ageing in most eukaryotes [1]. Evidence to support this idea comes from studies of dietary restriction (DR), i.e. underfeeding without malnutrition [2]. DR extends lifespan in most model organisms, including yeast, worms, flies, and mammals [3,4], suggesting that it may act via conserved longevity mechanisms. Studies using the budding yeast, Saccharomyces cerevisiae, have been at the forefront of recent efforts to understand the molecular mechanism of action of DR. Reducing the concentration of glucose in yeast growth media from the standard 2% to 0.5% or below increases both replicative and chronological lifespan in multiple genetic backgrounds and has been suggested to be a model of DR [5,6,7,8]. To shed light on the mechanisms by which DR extends yeast lifespan, we set out to use an unbiased strategy to identify proteins whose expression is induced by DR and thus correlate with longevity. Hsp12, a small heat shock protein whose cellular functions are unclear, was identified by this approach and was found to be essential for lifespan extension by DR. At the molecular level, Hsp12 was found to be unlike other small heat shock proteins, in that it is monomeric and intrinsically unfolded in solution and has negligible in vitro chaperone activity. Upon binding to membrane-mimetic SDS micelles, Hsp12 switches to a 4-helical conformation. The NMR structure of micelle-bound Although the effect of glucose limitation in extending yeast replicative lifespan is not disputed, its mechanism of action remains the subject of considerable debate. One popular model postulates that DR increases lifespan by activating the NAD- dependent histone deacetylase, Sir2, or its homologues, resulting July 2012 | Volume 7 | Issue 7 | e41975 PLoS ONE | www.plosone.org 1 Hsp12 Structure and Function PLoS ONE | www.plosone.org Results Helix III is not as stable as the other four helices, as revealed by the lower number of daNi,i+3, dabi,i+3 connectivities for this helix and more variation in its length compared with the other three helices together with a high RMSD value of 0.465 (Table S2). The experimentally-determined structural data correspond well with prediction using the AGADIR programme [26], which shows that the region between 52–63 has a lower helical propensity compared with the other three helical regions. Extensive analysis of residual dipolar couplings using stretched acrylamide gels revealed no evidence of long-range interactions between the individual helices, indicating that Hsp12 does not form a stably-folded structure. g p y We generated a model of the tertiary structure of Hsp12 using CYANA. The ensemble presented (Fig. 5 and Fig. S5) highlights the flexibility of the a-helices relative to one another. The four a- helices can be more clearly identified in the representative model in Fig. 6A, with the 4th and most C-terminal helix represented in yellow/red. Analysis of the charge distribution reveals each a-helix to be broadly amphipathic, with hydrophobic (green) residues lying on one face and charged (red) residues on the opposite face (Fig. 6 B,C). In addition, the residues flanking each a-helix also tend to be charged. This suggests that hydrophobic residues of Hsp12 insert into the lipidic component of membranes, while the charged (mainly positive) residues interact with negatively charged head groups and project away from the membrane. A Ramachan- dran plot of the data is presented in Fig. S6. Overall, the NMR data indicate that Hsp12 is intrinsically unstructured in aqueous solution, but switches to a dynamic 4-helical conformation upon membrane binding. Figure 2. HSP12 is essential for lifespan extension by DR. (A) The replicative lifespan of the wild type BY4741 strain grown under standard (2% glucose) and DR (0.5% glucose) conditions was de- termined manually on YPD plates by micromanipulation. Mean and median lifespan were 21 and 22, respectively, (n = 55) for 2% glucose; and 31 and 35, respectively, (n = 59) for 0.5% glucose. DR produced a significant increase in lifespan in wild type cells (log-rank test, P,0.001) (B) Mean and median lifespan of the isogenic hsp12D strain was determined as above and found to be 26 and 28, respectively, (n = 39) for 2% glucose; and 25 and 24, respectively, (n = 42) for 0.5% glucose. Results DR did not increase lifespan in hsp12D cells (log-rank test, P 0 5) Figure 2. HSP12 is essential for lifespan extension by DR. (A) The replicative lifespan of the wild type BY4741 strain grown under standard (2% glucose) and DR (0.5% glucose) conditions was de- termined manually on YPD plates by micromanipulation. Mean and median lifespan were 21 and 22, respectively, (n = 55) for 2% glucose; and 31 and 35, respectively, (n = 59) for 0.5% glucose. DR produced a significant increase in lifespan in wild type cells (log-rank test, P,0.001) (B) Mean and median lifespan of the isogenic hsp12D strain was determined as above and found to be 26 and 28, respectively, (n = 39) for 2% glucose; and 25 and 24, respectively, (n = 42) for 0.5% glucose. DR did not increase lifespan in hsp12D cells (log-rank test, P.0.5). doi:10.1371/journal.pone.0041975.g002 Results Wild type BY4741 cells exhibited a mean lifespan of 21 (95% CI: 19–23) on standard (2% glucose) media, which was significantly increased to 31 (95% CI: 28–35) under DR (0.5% glucose) conditions (Fig. 2A). Deletion of HSP12 did not reduce longevity under standard conditions, but rather resulted in a small increase in mean lifespan to 26 (95% CI: 23–29). Strikingly, however, the ability of DR to increase longevity was abolished in the hsp12D strain, which exhibited a mean lifespan of 25 (95% CI: 22–28) under DR conditions (Fig. 2B). These data suggest that the impact of Hsp12 on cellular ageing is complex: the low-level expression observed in standard media has a small negative effect on lifespan, whereas high Hsp12 levels induced by DR are essential for the increase in longevity caused by this intervention. Analysis of the backbone dynamics of Hsp12 in the presence of SDS revealed relatively long T1 relaxation values compared to T2 (Fig. 4 A,C,E), suggesting restricted mobility in the majority of the polypeptide. In contrast, T1 and T2 values were similar in the absence of SDS (Fig. 4 B,D,F), suggesting that the protein is highly dynamic in solution, but is structured on micelles. Consistent with this, analysis of the assigned chemical shifts in Hsp12 using CSI [25] suggested that micelle binding induces the formation of four a-helices (Fig. 4G). These a-helices cover the majority of the polypeptide and comprise residues F9-A16 (Helix I), Q22-A41 PLoS ONE | www.plosone.org July 2012 | Volume 7 | Issue 7 | e41975 PLoS ONE | www.plosone.org 2 Hsp12 Structure and Function PLoS ONE | www.plosone.org July 2012 | Volume 7 | Issue 7 | e41975 July 2012 | Volume 7 | Issue 7 | e41975 3 Hsp12 Structure and Function Figure 1. DR induces expression of a relatively small number of proteins. Wild type BY4741 yeast cells were grown in standard (2% glucose) and DR (0.5% glucose) conditions before lysis and separation of proteins by 2-D electrophoresis. Wide-range (pH 3–10) gels revealed no obvious reproducible differences in protein expression, as illustrated by representative gels shown in panel (A). Narrow pH range gels (pH 3–5.6 and 5.3–6.5) revealed changes in protein spots, which were identified by mass spectrometry. Selected identified proteins are indicated by arrows in panels (B) and (C). doi:10.1371/journal.pone.0041975.g001 doi:10.1371/journal.pone.0041975.g001 (Helix II), V52-G63 (Helix III) and L74-E94 (Helix IV). Discussion The mechanisms by which DR extends yeast replicative lifespan remain unclear and controversial. The data presented here show that DR (via growth in 0.5% glucose) does not induce gross changes in overall protein abundance, but does result in up- regulation of a limited number of proteins. This is consistent with earlier global microarray analysis that revealed changes in mRNA expression for only 133 genes (around 2% of total ORFs) in response to growth in 0.5% glucose [9]. Some of the proteins we found to be upregulated by DR (e.g. Rgi1, Hxk1) are associated with metabolic adaptation from fermentation to respiratory growth; whereas the reason for up-regulation of other proteins (e.g. Hsp12, Rtc3) is less obvious. Both Hsp12 and Rtc3 are also induced by high osmolarity (Fig. S1), which is thought to extend lifespan via the same downstream mechanisms as DR [17], consistent with the idea that DR is a chronic mild stress that increases longevity via hormesis. Indeed, HSP12 mRNA levels are increased in response to diverse environmental stresses, including heat-, osmotic- and oxidative stress [27,28]. HSP12 is also among the top 10 up-regulated genes in response to non-environmental, genetically-mediated impairment of nuclear proteostasis [29] and telomere capping [30]. Figure 2. HSP12 is essential for lifespan extension by DR. (A) The replicative lifespan of the wild type BY4741 strain grown under standard (2% glucose) and DR (0.5% glucose) conditions was de- termined manually on YPD plates by micromanipulation. Mean and median lifespan were 21 and 22, respectively, (n = 55) for 2% glucose; and 31 and 35, respectively, (n = 59) for 0.5% glucose. DR produced a significant increase in lifespan in wild type cells (log-rank test, P,0.001) (B) Mean and median lifespan of the isogenic hsp12D strain was determined as above and found to be 26 and 28, respectively, (n = 39) for 2% glucose; and 25 and 24, respectively, (n = 42) for 0.5% glucose. DR did not increase lifespan in hsp12D cells (log-rank test, P.0.5). Figure 2. HSP12 is essential for lifespan extension by DR. (A) The replicative lifespan of the wild type BY4741 strain grown under standard (2% glucose) and DR (0.5% glucose) conditions was de- termined manually on YPD plates by micromanipulation. Mean and median lifespan were 21 and 22, respectively, (n = 55) for 2% glucose; and 31 and 35, respectively, (n = 59) for 0.5% glucose. Yeast Cell Lysis and Protein Quantification Single yeast colonies were grown in 5 ml of YP media containing the appropriate concentration of glucose overnight at 30uC. The next day, 4 ml of this starter culture was inoculated into 200 ml of YPD media YP media containing the appropriate concentration of glucose and incubated at 30uC C until an OD600 value of 0.6 was obtained. The glucose concentration remaining in the culture media fell by only 0.1% during this period (i.e., from 0.5% to 0.4%), as determined by glucose oxidase assays. At this point, the culture was centrifuged at 8000 g and washed 3 times with distilled water before freezing at 280uC. The equivalent of a 5-ml culture in a frozen pellet was resuspended in 150 ml lysis buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 1% (w/v) DTT, 0.8% (w/v) Pharmalyte, 1 Roche protease inhibitor tablet per 10 ml), transferred to a 2 ml cryogenic vial and subjected to glass bead lysis in a mikrodismembrator for 10 min at 1800 rpm at 4uC. The resulting lysate was cleared by centrifugation at 13000 rpm for 10 min at 4uC. The protein concentration of each lysate was normalised by running samples together on a 1-D mini- gel, staining the gel with Coomassie blue and quantifying the total amount of protein in each track by densitometry. These values were then used to ensure that the same amounts of protein were loaded for each sample to be compared in subsequent 2-D gel electrophoresis or western blotting. In summary, we have shown that Hsp12 is induced by DR, is required for lifespan extension by DR, and have solved the structure of the protein in the free- and micelle-bound state. Although Hsp12 does not have obvious homologues outside of fungi, down-regulation of small heat shock proteins by RNAi also affects ageing in C. elegans [33], consistent with a conserved modulatory role for sHSPs in longevity. Hsp12 displays low-level homology to two mammalian proteins: ageing-associated protein 2 (also known as HIP/ST13; NCBI reference NP_003923) and a- synuclein (NCBI reference P37840). Intriguingly, a-synuclein is also an intrinsically disordered protein in solution that becomes a- helical upon binding to anionic phospholipids and SDS micelles [34]. It is conceivable that these shared properties may have functional implications, as it has been suggested that neuronal a- synuclein organises and stabilises lipids in synaptic vesicle membranes [35]. 2-D Gel Electrophoresis and Protein Identification Yeast lysates were applied at equal protein concentrations to 18 cm IPG strips (GE Healthcare) and allowed to rehydrate for a minimum of 10 hours. The IPG strips were then run on an IEF Multiphor II electrophoresis unit (GE Healthcare) at 20uC for 1 min at 500 V, then for 7 hours at 3500 V. Following this, IPG strips were overlayed on SDS-PAGE gels, sealed using warm agarose, and run using an Ettan Dalt II system (GE Healthcare) at 2.5 W per gel for 30 min and then 20 W per gel at 25uC until the dye front had reached the bottom of the gel. Immediately after SDS-PAGE, the gels were fixed in a solution of 40% v/v MeOH/ 7% (v/v) acetic acid for a minimum of 2 hours and then stained with colloidal Coomassie blue stain (GE Healthcare). Stained gels Discussion All other materials were obtained from Sigma-Aldrich. supplied by Sigma Genosys (Havenhill, UK), genomic DNA isolation kits were from Invitrogen (Paisley, UK); and PCR enzymes/reagents were from Promega (Southampton, UK). Anti- GFP antibody was obtained from AbCam (Cambridge, UK) and custom-generated Hsp12 antiserum was supplied by Genosphere Biotechnologies (Paris, France). Materials for gel electrophoresis were obtained from GE Healthcare or BioRad. All other materials were obtained from Sigma-Aldrich. Hsp26. It therefore seems more likely that Hsp12’s recently discovered function as a membrane stabilising ‘lipid chaperone’ [16] underlies its effect on lifespan. Indeed, Ssd1 has been shown to increase replicative lifespan in a Sir2-independent manner by increasing plasma membrane stability [31]. The transition from unfolded Hsp12 in solution to a dynamic 4-helical lipid-bound conformation observed here may similarly contribute to mem- brane stability under DR conditions, which in turn may be functionally relevant to increasing lifespan. However, it remains possible that unfolded Hsp12 in the nucleus and cytosol performs membrane-binding-independent functions that impact on replica- tive lifespan during DR. Yeast Strains Deletion strains with the appropriate ORF replaced by the KanMX4 cassette in the BY4741 background (MATa his3D1 leu2D0 met15 D0 ura3D0) [36] were used for gel electrophoresis, lifespan analysis and stress assays and were obtained from Invitrogen (Paisley, UK). GFP-labelled strains in the BY4741 background were used to validate mass spectrometry identifica- tions and were obtained from Invitrogen. All mutants were confirmed by PCR using gene-specific and KanMX primers. p g The structure of Hsp12 bound to SDS micelles described here provides insight into how Hsp12 may interact with cellular membranes. This involves a switch from an intrinsically disordered conformation in solution to a structured, 4-helical lipid-bound conformation, as recently proposed [16]. Our extensive NMR analysis using stretched acrylamide gels failed to reveal interactions between the four a-helices of Hsp12, suggesting a dynamic structure whereby each a-helix is free to move independently within a 2-dimensional lipid bilayer. The lack of a stable folded tertiary structure could be due to the observed instability of helix 3. While our work was being prepared for publication, the NMR structure of micelle-bound Hsp12 was independently reported [32]. This shows a similar 4-helical conformation in the presence of SDS, although some differences are apparent in the secondary structural models of the individual helices (F9-K18, Y25-G42, Q54-S59 and D76-V100 versus F9-A16, Q22-A41, V52-G63 and L74-E94 in our study). We propose that the dynamic nature of Hsp12’s four a-helices we report here could be important for its membrane stabilising function [16], as this would enable simultaneous linkage of several distinct membrane sub-domains via a single protein tether. Yeast Cell Lysis and Protein Quantification It is tempting to speculate that enhanced membrane stabilisation by functional homologues of Hsp12 may be relevant to DR-induced longevity in higher organisms. Discussion DR produced a significant increase in lifespan in wild type cells (log-rank test, P,0.001) (B) Mean and median lifespan of the isogenic hsp12D strain was determined as above and found to be 26 and 28, respectively, (n = 39) for 2% glucose; and 25 and 24, respectively, (n = 42) for 0.5% glucose. DR did not increase lifespan in hsp12D cells (log-rank test, P.0.5). Despite the strong induction of HSP12 gene expression in response to diverse stresses, deletion of HSP12 does not generally affect sensitivity to these stresses. Significantly, however, we found that lifespan extension by growth on 0.5% glucose was abolished in an hsp12D strain, indicating that Hsp12 is essential for the longevity effect of DR. What mechanism(s) could explain this action of Hsp12 on lifespan? One tempting explanation is that, like other small heat shock proteins, Hsp12 prevents the aggregation of a variety of cellular proteins during DR-induced stress. However, we found that Hsp12 has negligible anti-aggregation properties in vitro, in contrast to well established ‘holdase’ co-chaperones such as doi:10.1371/journal.pone.0041975.g002 July 2012 | Volume 7 | Issue 7 | e41975 4 PLoS ONE | www.plosone.org Hsp12 Structure and Function PLoS ONE | www.plosone.org 5 July PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41975 PLoS ONE | www.plosone.org July 2012 | Volume 7 | Issue 7 | e41975 PLoS ONE | www.plosone.org 5 Hsp12 Structure and Function Figure 3. Hsp12 is unstructured in solution, but folds in the presence of SDS. (A) 1H-15N HSQC spectrum of Hsp12 in aqueous solution at 298 K. The spectrum shows only sharp peaks with random coil shifts indicating the absence of any structured regions. (B) 1H-15N HSQC spectrum of Hsp12 at 303 K in the presence of increasing concentrations of SDS (0, 1, 2, 5, 8 mM Red -. Blue). SDS causes a considerable increase in the amount of chemical shift dispersion implying increased levels of folded material/regions. (C) Assigned 1H-15N HSQC spectrum of Hsp12 at 318 K in the presence of 100 mM SDS. doi:10.1371/journal.pone.0041975.g003 p doi:10.1371/journal.pone.0041975.g003 supplied by Sigma Genosys (Havenhill, UK), genomic DNA isolation kits were from Invitrogen (Paisley, UK); and PCR enzymes/reagents were from Promega (Southampton, UK). Anti- GFP antibody was obtained from AbCam (Cambridge, UK) and custom-generated Hsp12 antiserum was supplied by Genosphere Biotechnologies (Paris, France). Materials for gel electrophoresis were obtained from GE Healthcare or BioRad. Materials and Methods Chemicals and Reagents July 2012 | Volume 7 | Issue 7 | e41975 Chemicals and Reagents Chemicals and Reagents Materials for yeast culture were obtained from Sigma-Aldrich (Poole, UK) and Foremedium (Norwich, UK). PCR primers were g Materials for yeast culture were obtained from Sigma-Aldrich (Poole, UK) and Foremedium (Norwich, UK). PCR primers were PLoS ONE | www.plosone.org July 2012 | Volume 7 | Issue 7 | e41975 6 Hsp12 Structure and Function PLoS ONE | www.plosone.org 7 July 2012 | PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e41975 7 PLoS ONE | www.plosone.org Hsp12 Structure and Function Hsp12 Structure and Function Figure 4. Backbone dynamics and chemical shift-based secondary structure of Hsp12. T1, T2 and T1/T2 relaxation values are shown for Hsp12 in the presence (A,C,E) and absence (B,D,F) of 100 mM SDS at 318 K. T1 and T2 relaxation times for micelle-bound (A,C) Hsp12 show significant variation; contrasting with the similar relaxation values observed for free Hsp12 (B,D). Micelle-bound Hsp12 (E) shows grouped variations in the T1/T2 values ranging from approximately 1.5 to 14, indicating a wide range of mobility and a clear differentiation of secondary structure elements; whereas the free form (F) shows consistent values of around 2, indicating a completely unstructured protein. (G) The assigned chemical shifts at 318 K in 100 mM SDS expressed as deviation from random coil are shown aligned with the primary sequence and the positions of the a-helices. doi:10.1371/journal.pone.0041975.g004 Western Blotting were imaged with a GS-710 Imaging Densitometer (BioRad) and protein spot changes analysed by eye and using PD Quest software (BioRad). Protein spots of interest were excised from gels, dried under vacuum, then re-hydrated in 25 mM NH4CO3 containing 5 ng/ml trypsin overnight at 37uC. Tryptic peptides were resuspended 1:1 in matrix solution (10 mg/ml HCCA in 50% (v/v) ethanol, 50% (v/v) acetonitrile, 0.001% (v/v) trifluoroacetic acid) and 1 ml of this mixture (25% of the total digest) was spiked with 50 fmol of ACTH peptide, loaded onto a MALDI target (Waters/Micromass Massprep workstation) and analysed on a MALDI-TOF mass spectrometer (Waters/Micromass M@ LDI). Tryptic peptides were identified by peptide mass fingerprint matching using MASCOT (UniProt release 2.6) allowing for 1 missed cleavage with a mass accuracy of 0.25 Da. were imaged with a GS-710 Imaging Densitometer (BioRad) and protein spot changes analysed by eye and using PD Quest software (BioRad). Protein spots of interest were excised from gels, dried under vacuum, then re-hydrated in 25 mM NH4CO3 containing 5 ng/ml trypsin overnight at 37uC. Tryptic peptides were resuspended 1:1 in matrix solution (10 mg/ml HCCA in 50% (v/v) ethanol, 50% (v/v) acetonitrile, 0.001% (v/v) trifluoroacetic acid) and 1 ml of this mixture (25% of the total digest) was spiked with 50 fmol of ACTH peptide, loaded onto a MALDI target (Waters/Micromass Massprep workstation) and analysed on a MALDI-TOF mass spectrometer (Waters/Micromass M@ LDI). Tryptic peptides were identified by peptide mass fingerprint matching using MASCOT (UniProt release 2.6) allowing for 1 missed cleavage with a mass accuracy of 0.25 Da. Yeast lysates prepared as above were run on 1-D SDS-PAGE gels and transferred to nitrocellulose. Blots were probed with either anti-GFP antibody (AbCam, Cambridge, UK) or with custom- generated antiserum (Genosphere Biotechnologies, Paris, France) raised against a synthetic peptide corresponding to the N-terminal 14 residues of Hsp12 with an additional cysteine for conjugation (sequence: MSDAGRKGFGEKASC). Blots were visualised by enhanced chemiluminescence and imaged with a BioRad Chemi- Doc XRS imager (BioRad). Lifespan Analysis This was performed as described previously [19]. Briefly, strains were grown at 30uC until they reached an OD600 of 0.6–1.0. One Figure 5. Ensemble of structures calculated for micelle-bound Hsp12 overlaid on each of the four helices. Ensemble of twenty structures overlaid on helices I (A), II (B), III (C) and IV (D). No long-range interactions were detected and so the helices appear free to move independently with no overall fold being evident. doi:10.1371/journal.pone.0041975.g005 Figure 5. Ensemble of structures calculated for micelle-bound Hsp12 overlaid on each of the four helices. Ensemble of twenty structures overlaid on helices I (A), II (B), III (C) and IV (D). No long-range interactions were detected and so the helices appear free to move independently with no overall fold being evident. doi:10.1371/journal.pone.0041975.g005 PLoS ONE | www.plosone.org July 2012 | Volume 7 | Issue 7 | e41975 8 Hsp12 Structure and Function Figure 6. Helical properties of micelle-bound Hsp12. (A) The four a-helices are represented as ribbons and colour coded from the N-termin (blue) to the C-terminus (red) in a representative structure. (B,C) Analysis of charge distribution with hydrophobic residues labelled green a charged residues labelled red in both ribbon (B) and surface (C) representation, illustrating the amphipathic nature of Hsp12. Structures we generated using Chimera. doi:10.1371/journal.pone.0041975.g006 PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e419 Figure 6. Helical properties of micelle-bound Hsp12. (A) The four a-helices are represented as ribbons and colour coded from the N-terminus (blue) to the C-terminus (red) in a representative structure. (B,C) Analysis of charge distribution with hydrophobic residues labelled green and charged residues labelled red in both ribbon (B) and surface (C) representation, illustrating the amphipathic nature of Hsp12. Structures were generated using Chimera. doi:10.1371/journal.pone.0041975.g006 Figure 6. Helical properties of micelle-bound Hsp12. (A) The four a-helices are represented as ribbons and colour coded from the N-terminu (blue) to the C-terminus (red) in a representative structure. (B,C) Analysis of charge distribution with hydrophobic residues labelled green an charged residues labelled red in both ribbon (B) and surface (C) representation, illustrating the amphipathic nature of Hsp12. Structures we generated using Chimera. doi:10.1371/journal.pone.0041975.g006 Figure 6. Helical properties of micelle-bound Hsp12. (A) The four a-helices are represented as ribbons and colour coded from the N-terminus (blue) to the C-terminus (red) in a representative structure. PLoS ONE | www.plosone.org Aggregation Assay This was based on the method of Haslbeck et al. [23]. Insulin was added to a final concentration of 45 mM in 40 mM HEPES- KOH, pH 7.5 in a 96-well plate in the presence or absence of recombinant proteins. Aggregation was induced by the addition of 1.5 ml of 1 M DTT in a final volume of 100 ml, or by adding 1.5 ml of water as a no-aggregation control. Measurements were taken in an Emax microplate reader (Molecular Devices, Sunnyvale, USA) at 405 nm every 10 min for 3 hours, then one final reading was obtained the next day after agitating the plate to disperse any large aggregates. The anti-aggregation efficiency of each recombinant protein was calculated as follows. For each individual assay, the highest absorbance reading for the aggrega- tion control (insulin + DTT) was assumed to reflect total aggregation of insulin (26 mg per well) and set as 100%. Anti- NMR Methods NMR samples were prepared in 10 mM PO4 32, 40 mM NaCl, 2 mM NaN3, pH 6.5 in 90% (v/v) H2O/10% (v/v) D2O or 100% D2O, at a final protein concentration of approximately 450 mM. Hsp12 samples in the presence of SDS were prepared using the same buffer with the addition of 100 mM SDS. Spectra in the absence and presence of SDS were acquired at, respectively, 303 K and 318 K, on Bruker Avance III 600 and 800 MHz spectrometers equipped with cryogenic triple resonance probes. All NMR spectra were processed with TopSpin (Bruker) and analysed using the CCPN Analysis package [38]. Sequence- specific backbone and side-chain resonance assignment of Hsp12 was made using 3-D HNCA, HN(CA)CB, HN(CO)CA, HNCO, CBCA(CO)NH, HBHANH, HBHA(CO)NH and HCCH- TOCSY experiments. Assignment of aromatic side-chain residues was made using 2-D [1H-13C] HSQC and homonuclear 1H NOESY and TOCSY spectra. Interproton distance restraints were obtained from NOEs derived from 3-D 15N- and 13C-edited NOESY-HSQC experiments. The secondary structure for the Hsp12 protein ensemble was determined using the STRIDE algorithm [39]. The basis for secondary structure is a combination of backbone torsion angle and location of hydrogen bonds. Furthermore, using the ensemble for secondary structure de- termination enabled the location of the helices to be identified based on twenty structures rather than one. RMSD values for the ensemble were determined by global rmsd fit using MOLMOL [40] which determines the average pairwise alignment RMSD for all pairs in the ensemble. IPAP 1H 15N RDC data was collected unaligned and aligned using the stretched gel method [41] on an 800 MHz spectrometer equipped with TCI cryoprobe. For each sample a 600 ml preparation of gel mix (4–8% acrylamide), containing a final concentration of 100–200 mM protein, was used and quadrupole splitting measured to estimate the degree of alignment. RDCs were collected on a 4.0% acrylamide gel of initial 6 mm diameter compressed into a 4.6 mm diameter tube with quadrupole splitting of 1.5 Hz containing 150 mM 15N Hsp12 and buffer conditions: 100 mM SDS, 10mM PO4 32, 40 mM NaCl, pH 4.5. Couplings were measured using CCPN analysis and a purpose-built Perl script, before direct incorporation into structure calculation using CYANA [42]. Coordinates have been deposited in the RCSB protein databank (PDB accession number: 4AXP) and the Biological Magnetic Resonance Bank (BMRB accession number: 18523). Recombinant Proteins GST-Hsp12 and GST-Hsp26 were constructed using the Invitrogen Gateway cloning protocol. Primers for amplifying Hsp12 and 26 from yeast genomic DNA were designed from the ORF sequences of the required genes fused with the correspond- ing attB1 and attB2 primer sequences. The destination vector used for N-terminal GST-fusion, pG-GEX6p-B1, was made in-house. Recombinant GST-fusion proteins were expressed in BL21 (DE3) E. coli, lysed using a One Shot cell disrupter (Constant Systems, Daventry, UK) and purified using glutathione-Sepharose as previously described [37]. p y For NMR studies, the ORF of Hsp12 was codon optimised for E. coli expression and then synthesised de novo by GeneArt (Invitrogen). The resulting construct was then cloned into the pE- SumoProKan vector (Invitrogen) and expressed in BL21 (DE3) E. coli in M9 medium containing 1 g/L 15NH4Cl as the sole nitrogen source, with either 4 g/L 12C-glucose or 13C-glucose as the sole carbon source, to produce, respectively, [U-15N]- or [U-15N,13C]- Hsp12. Recombinant His-SUMO-tagged Hsp12 was purified using HisTrap immobilised metal affinity columns (GE Health- care) via imidazole elution. The SUMO-specific protease, ULP1, was used to cleave Hsp12 (with no vector-derived residues) from the His-SUMO tag, which was removed by passage through a second immobilised metal affinity column. The resulting Hsp12 protein was then dialysed against 10 mM KHPO3, 40 mM NaCl, pH 6.5 and concentrated prior to use in NMR. Stress Assays Single colonies were grown overnight in 5 ml liquid YP media containing 2% (w/v) glucose. The next morning, cultures were diluted to OD600 = 1 in sterile H2O and then serially diluted ten- fold five times in sterile H2O. The serial dilutions were then spotted with a replica plater onto YP media containing the indicated stressors. Plates were incubated at 30uC for 2 to 4 days and then imaged with a BioRad ChemiDoc XRS imager (BioRad). Hsp12 Structure and Function Hsp12 Structure and Function microlitre of culture was streaked onto plates and left at 30uC for 1–2 h. After this time, cell doublets were moved to uninhabited regions of the plate. When these budded again, (newly formed) virgin yeast cells were removed by micromanipulation to a new location. All future buds produced by these daughter cells were micromanipulated away and catalogued. The plates were in- cubated at 30uC during working hours, and moved to 4uC overnight. Lifespan was defined as number of daughter cells removed from the mother cell. All lifespans were observed at least twice. Statistical significance was assessed using the log rank test and deemed significant at P,0.05. aggregation activity was then calculated by subtracting the aggregated material in each condition from 100%, converting to mg insulin and then dividing by the respective amount of each GST-tagged protein to determine the anti-aggregation properties per mg recombinant protein. Lifespan Analysis (B,C) Analysis of charge distribution with hydrophobic residues labelled green and charged residues labelled red in both ribbon (B) and surface (C) representation, illustrating the amphipathic nature of Hsp12. Structures were generated using Chimera. doi:10.1371/journal.pone.0041975.g006 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org July 2012 | Volume 7 | Issue 7 | e41975 9 PLoS ONE | www.plosone.org References 1. Guarente L, Kenyon C (2000) Genetic pathways that regulate ageing in model organisms. Nature 408: 255–262. 14. Steffen KK, MacKay VL, Kerr EO, Tsuchiya M, Hu D, et al. (2008) Yeast life span extension by depletion of 60s ribosomal subunits is mediated by Gcn4. Cell 133: 292–302. 2. Bishop NA, Guarente L (2007) Genetic links between diet and lifespan: shared mechanisms from yeast to humans. Nat Rev Genet 8: 835–844. 15. Riesen M, Morgan A (2009) Calorie restriction reduces rDNA recombination independently of rDNA silencing. Aging Cell 8: 624–632. 3. Partridge L, Pletcher SD, Mair W (2005) Dietary restriction, mortality trajectories, risk and damage. Mech Ageing Dev 126: 35–41. 16. Welker S, Rudolph B, Frenzel E, Hagn F, Liebisch G, et al. (2010) Hsp12 is an intrinsically unstructured stress protein that folds upon membrane association and modulates membrane function. Mol Cell 39: 507–520. 4. Merry BJ (2005) Dietary restriction in rodents–delayed or retarded ageing? Mech Ageing Dev 126: 951–959. 17. Kaeberlein M, Andalis AA, Fink GR, Guarente L (2002) High osmolarity extends life span in Saccharomyces cerevisiae by a mechanism related to calorie restriction. Mol Cell Biol 22: 8056–8066. 5. Jiang JC, Jaruga E, Repnevskaya MV, Jazwinski SM (2000) An intervention resembling caloric restriction prolongs life span and retards aging in yeast. Faseb J 14: 2135–2137. 18. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, et al. (2003) Global analysis of protein localization in budding yeast. Nature 425: 686–691. 6. Lin S-J, Defossez P-A, Guarente L (2000) Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289: 2126–2128. 19. Mankouri HW, Morgan A (2001) The DNA helicase activity of yeast Sgs1p is essential for normal lifespan but not for resistance to topoisomerase inhibitors. Mechanisms of Ageing and Development 122: 1107–1120. 7. Smith DL, McClure JM, Matecic M, Smith JS (2007) Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins. Aging Cell 6: 649–662. 20. Gems D, Partridge L (2008) Stress-response hormesis and aging: ‘‘that which does not kill us makes us stronger’’. Cell Metab 7: 200–203. 8. Fabrizio P, Gattazzo C, Battistella L, Wei M, Cheng C, et al. (2005) Sir2 blocks extreme life-span extension. Cell 123: 655–667. 21. Kaeberlein M, McVey M, Guarente L (1999) The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes and Development 13: 2570–2580. 9. Acknowledgments We thank Dr Lee Haynes (University of Liverpool, UK) for the gift of GST-CaBP1s. We thank Dr Lee Haynes (University of Liverpool, UK) for the gift of GST-CaBP1s. Figure S4 Temperature optimisation of Hsp12. 1H-15N HSQC spectrum of Hsp12 in the presence of 100 mM SDS (A) or in aqueous solution (B) at different temperatures (298, 303, 308, 313, 318, 323 K, Blue -. Red). Increases in temperature are associated with a sharpening of peaks, indicating that HSP12 does not undergo significant unfolding even up to temperatures of 323 K. (TIF) Figure S5 Twenty structures calculated for micelle- bound Hsp12 tiled individually. Structures were generated using chimera. (TIF) Figure S2 HSP12 is not required for general stress resistance. Overnight cultures of BY4741 wild type and deletion strains were serially diluted and then spotted with a replica plater onto YPD plates containing 2% glucose at 30uC unless indicated otherwise. Plates were incubated at 30uC for 2 to 4 days and then imaged in a BioRad Universal Hood II Imager (BioRad). (TIF) Figure S6 Ramachandran plot. The percentage of ordered residues in the presence of 100 mM SDS at 45uC was 80.9% in most favoured regions, 18.1% in additionally allowed regions, 0.6% in generously allowed regions and 0.4% in disallowed regions. (TIF) Figure S3 Recombinant Hsp12 has negligible in vitro chaperone activity. (A) 45 mM insulin was supplemented with 1.5 ml water (open squares) or 1 M DTT (black squares) and aggregation over time at room temperature was measured at A405 in a microplate reader. (B) Aggregation assays were performed as above in the presence or absence of the indicated GST-fusion proteins. Anti-aggregation activity is shown as the amount of insulin in mg which is prevented from aggregation by 1 mg of recombinant protein. Data shown are pooled from multiple experiments (n = 7 for GST-Hsp12; n = 8 for GST-Hsp26; n = 4 for GST-CSP; n = 4 for GST-CaBP1s). The difference between GST-Hsp12 and GST-CaBP1s was deemed significant at P,0.05 using a Student’s t-test. (C) Dose-response curves of recombinant GST-Hsp12 and GST-Hsp26. GST-Hsp26 greatly reduces insulin aggregation, whereas GST-Hsp12 has mimimal effect. (TIF) Table S1 Mass spectrometry identifications of proteins induced by dietary restriction and high osmolarity. Information from the Saccharomyces Genome Database (SGD) is presented along with peptide mass fingerprinting data obtained for each identified protein. (XLS) Table S2 Average RMSD values for Hsp12 helices. RMSD values calculated from the mean CYANA coordinates for helices I-IV are shown. (DOC) Author Contributions Conceived and designed the experiments: AM SRP AH MR LYL. Performed the experiments: AH MR LB EK BMW. Analyzed the data: AM SRP AH MR LYL. Contributed reagents/materials/analysis tools: JRJ MMP. Wrote the paper: AM. Supporting Information Figure S1 Hsp12 is induced by multiple lifespan- extending interventions. (A) Wild type BY4741 yeast cells were grown in standard (2% glucose) and high osmolarity (20% glucose) conditions before lysis and separation of proteins by 2-D electrophoresis on narrow pH range gels (pH 3.5–6 and 5.3–6.5). Selected spot changes identified by mass spectrometry are indicated by arrows. (B) S288c yeast cells expressing chromoso- mally GFP-tagged fusions of selected proteins identified as being induced by DR or high osmolarity were grown in 0.5%, 2% and 20% glucose, separated by 1-D SDS-PAGE and western blotted with anti-GFP antiserum. (C) Wild type BY4741 yeast grown in 0.5%, 2% and 20% glucose (left panel); or wild type and isogenic PLoS ONE | www.plosone.org July 2012 | Volume 7 | Issue 7 | e41975 10 Hsp12 Structure and Function hsp12D strains grown in 0.05%, 0.5% and 2% glucose (right panel) were separated by 1-D SDS-PAGE and western blotted with anti- Hsp12 antiserum. (TIF) hsp12D strains grown in 0.05%, 0.5% and 2% glucose (right panel) were separated by 1-D SDS-PAGE and western blotted with anti- Hsp12 antiserum. (TIF) Hsp12 Structure and Function 27. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, et al. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11: 4241–4257. 35. Madine J, Doig AJ, Middleton DA (2006) A study of the regional effects of alpha-synuclein on the organization and stability of phospholipid bilayers. Biochemistry 45: 5783–5792. 36. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. 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(2008) A genome wide analysis of the response to uncapped telomeres in budding yeast reveals a novel role for the NAD+ biosynthetic gene BNA2 in chromosome end protection. Genome Biol 9: R146. 38. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, et al. (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59: 687–696. 39. Heinig M, Frishman D (2004) STRIDE: a web server for secondary structure assignment from known atomic coordinates of proteins. Nucleic Acids Research 32: W500–502. 31. Kaeberlein M, Andalis AA, Liszt GB, Fink GR, Guarente L (2004) Saccharomyces cerevisiae SSD1-V confers longevity by a Sir2p-independent mechanism. Genetics 166: 1661–1672. 40. Koradi R, Billeter M, Wuthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph 14: 51–55, 29–32. 32. Singarapu KK, Tonelli M, Chow DC, Frederick RO, Westler WM, et al. (2011) Structural characterization of Hsp12, the heat shock protein from Saccharo- myces cerevisiae, in aqueous solution where it is intrinsically disordered and in detergent micelles where it is locally alpha-helical. J Biol Chem 286: 43447– 43453. 41. References Lin SJ, Kaeberlein M, Andalis AA, Sturtz LA, Defossez PA, et al. (2002) Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418: 344–348. 22. Defossez P-A, Prusty R, Kaeberlein M, Lin S-J, Ferrigno P, et al. (1999) Elimination of replication block protein Fob1 extends the life span of yeast mother cells. Molecular Cell 3: 447–455. 10. Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Sinclair DA (2003) Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 423: 181–185. 23. Haslbeck M, Walke S, Stromer T, Ehrnsperger M, White HE, et al. (1999) Hsp26: a temperature-regulated chaperone. Embo J 18: 6744–6751. 11. 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Monitoring Glacier Calving using Underwater Sound Abstract. Climate shifts are particularly conspicuous in the Arctic. Satellite and terrestrial observations show significant in- creases in the melting and breakup of Arctic tidewater glaciers and their influence on sea level rise. Increasing melt rates are creating an urgency to better understand the link between atmospheric and oceanic conditions and glacier frontal ablation through iceberg calving and melting. Elucidating this link requires a combination of short and long-time scale measurements of terminus activity. Recent work has demonstrated the potential of using underwater sound to quantify the time and scale 5 of calving events to yield integrated estimates of ice mass loss (Glowacki and Deane, 2020). Here, we present estimates of Abstract. Climate shifts are particularly conspicuous in the Arctic. Satellite and terrestrial observations show significant in- creases in the melting and breakup of Arctic tidewater glaciers and their influence on sea level rise. Increasing melt rates are creating an urgency to better understand the link between atmospheric and oceanic conditions and glacier frontal ablation through iceberg calving and melting. Elucidating this link requires a combination of short and long-time scale measurements of terminus activity. Recent work has demonstrated the potential of using underwater sound to quantify the time and scale 5 of calving events to yield integrated estimates of ice mass loss (Glowacki and Deane, 2020). Here, we present estimates of subaerial calving flux using underwater sound recorded at Hansbreen, Svalbard in September 2013 combined with an algo- rithm for the automatic detection of calving events. The method is compared with ice calving volumes estimated from geodetic measurements of the movement of the glacier terminus and an analysis of satellite images. The total volume of above-water 5 10 calving during the 26 days of acoustical observation is estimated to be 1.7 ± 0.7 × 107 m3, whereas the subaerial calving flux 10 estimated by traditional methods is 7 ± 2 × 106 m3. The results suggest that passive cryoacoustics is a viable technique for long-term monitoring of mass loss from marine-terminating glaciers. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Monitoring Glacier Calving using Underwater Sound Jarosław T˛egowski1,*, Oskar Glowacki2,*, Michał Ciepły3, Małgorzata Błaszczyk3, Jacek Jania3, Mateusz Moskalik2, Philippe Blondel4, and Grant B. Deane5 1Institute of Oceanography, University of Gdansk, Gdynia, Poland 2Institute of Geophysics, Polish Academy of Sciences, Warsaw, Poland 3Faculty of Natural Sciences, Institute of Earth Sciences, University of Silesia in Katowice, Sosnowiec, Poland 4Department of Physics, University of Bath, Bath, UK 5Scripps Institution of Oceanography, UCSD, La Jolla, California, USA *These authors contributed equally to this work. Correspondence: Oskar Glowacki (oglowacki@igf.edu.pl), Grant B. Deane (gdeane@ucsd.edu) 1 Introduction The loss in the mass of the Greenland ice sheet, which increased from 41 ± 17 Gt yr−1 in 1990–2000 to 286 ± 20 Gt yr−1 in 2010-2018 (Mouginot et al., 2019), is of great concern. Greenland’s contribution to sea level rise by the end of the 21st century 15 is estimated to be 90 ± 50 and 32 ± 17 mm for RCP8.5 and RCP2.6 greenhouse gas concentration scenarios, respectively (Goelzer et al., 2020); these estimates exclude peripheral glaciers and ice caps. Moreover, recent studies have shown that the accelerated break-up and melting of Antarctica – the largest reservoir of fresh water on Earth – will be responsible for a sea level rise of at least 0.5 cm yr−1 by 2100 (Paolo et al., 2015; DeConto et al., 2021). Despite these concerning numbers, the loss of land-based ice in the coming decades could be under-predicted. For example, Greenland and Antarctic ice sheets likely have 20 tipping points at around 1.5 −2.0◦C of a mean global temperature increase compared to the pre-industrial era (Pattyn et al., 2018; Pattyn and Morlighem, 2020; Noël et al., 2021). The severe consequences of sea level rise and current uncertainties in 1 1 Calving is a complex process that depends on a broad range of factors, including but not limited to (i) changes in glacier surface velocity along the flow line (longitudinal stretching), (ii) submarine melting of the terminus (undercutting), and (iii) geometry of the glacier-bay system (van der Veen, 2002; Benn et al., 2007b). This complexity makes the formulation of a general model of how calving changes in response to warming conditions a challenging task that requires long-term, temporally-resolved data sets (e.g., van der Veen, 2002; Benn et al., 2007a). 40 Passive cryoacoustics, the use of ambient sounds to study ice-ocean interactions in polar regions, has the potential to provide the long-term, temporally-resolved time series required for calving model development and monitoring the long-term stability of glaciers and ice sheets (see discussion in Glowacki and Deane, 2020), provided that the geophysical signals of relevance can be recovered from the properties of the sound. The field of ambient noise oceanography is well-established and has been used to recover signals such as wind speed and precipitation, for example. The use of passive acoustics in polar regions is a 45 relatively new field, still under development (Glowacki et al., 2015; Pettit et al., 2015; Deane et al., 2019; Yun et al., 2021; Podolskiy et al., 2022). A recent milestone has been the demonstration that the mass of a calving iceberg can be estimated from its underwater impact noise with the sea surface (Glowacki and Deane, 2020). The method exploits the observed power law relationship impact noise with the sea surface (Glowacki and Deane, 2020). The method exploits the observed power law relationship between the kinetic energy of a falling ice block immediately prior to impact and the resulting sound energy produced. The 50 kinetic energy of an impacting block can be estimated from the sound energy radiated provided that propagation losses between the block and the recording location can be estimated. Because the drop height of icebergs is limited by the vertical extent of the terminus, the estimate of impact energy can then be converted into a block mass using what Glowacki and Deane (2020) call a "mass-weighted average drop height", ˆh. between the kinetic energy of a falling ice block immediately prior to impact and the resulting sound energy produced. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. models of glacial retreat suggest that long-term monitoring of glacial stability is both important and urgent (Straneo et al., 2019). There are many challenges to the long-term monitoring of glaciers and ice shelves. Polar regions are remote and plunged 25 into darkness for up to 6 months a year. Moreover, capturing the episodic character of ice breakup usually requires sub-second temporal resolution over year-long time scales. Despite a suite of current techniques, including satellite technology (Smith et al., 2020; Podgórski and P˛etlicki, 2020), camera observations (Medrzycka et al., 2016; How et al., 2019), terrestrial laser scanning (P˛etlicki and Kinnard, 2016; Podgórski et al., 2018), ground-based radar imaging (Xie et al., 2018; Walter et al., 25 2020), sonar scans (Sugiyama et al., 2019; Sutherland et al., 2019), and seismic records (Köhler et al., 2016; Podolskiy and 30 Walter, 2016), continuous and long-term observations of the scale and rapidity of ice loss in logistically challenging polar regions are still urgently required (Straneo et al., 2013). There are 3 major processes driving mass loss from marine-terminating glaciers: surface melting, submarine melting, and calving, which is the mechanical loss of ice from the edges of glaciers or ice shelves (Benn et al., 2007b). Calving and 2020), sonar scans (Sugiyama et al., 2019; Sutherland et al., 2019), and seismic records (Köhler et al., 2016; Podolskiy and 30 Walter, 2016), continuous and long-term observations of the scale and rapidity of ice loss in logistically challenging polar regions are still urgently required (Straneo et al., 2013). There are 3 major processes driving mass loss from marine-terminating glaciers: surface melting, submarine melting, and calving, which is the mechanical loss of ice from the edges of glaciers or ice shelves (Benn et al., 2007b). Calving and regions are still urgently required (Straneo et al., 2013). There are 3 major processes driving mass loss from marine-terminating glaciers: surface melting, submarine melting, and calving, which is the mechanical loss of ice from the edges of glaciers or ice shelves (Benn et al., 2007b). Calving and submarine melting - the two mechanisms of frontal ablation - are very difficult to quantify separately (Truffer and Motyka, 35 2016). 2 Study Site The site for this study is Hansbreen, a retreating, grounded, polythermal tidewater glacier terminating in Hornsund Fjord, 60 Svalbard. Its calving mode, believed to be driven by melt undercutting and many relatively small calving events from above the water surface (P˛etlicki et al., 2015), provides a challenging test case for camera and satellite estimates of mass flux. A schematic of the study site is shown in Fig. 1. Hansbreen covers an area of around 54 km2, is more than 15 km long (Błaszczyk et al., 2013), and has a 1.5 km-wide active calving front with an average height of around 40 m (Błaszczyk et al., 2009, 2021), which is approximately 30–40% of the total ice thickness. The glacier surface flow is dominated by basal motion 65 in the ablation area (Vieli et al., 2004) and the mean annual flow velocity near the terminus and its calving flux is estimated to be 177 my−1 and 35×106 m3y−1, respectively (Błaszczyk et al., 2019). Both glacial behavior (P˛etlicki et al., 2015; Błaszczyk et al., 2021) and the propagation of calving sounds (Glowacki et al., 2016) are sensitive to the space and time-varying thermohaline structure of water masses in the bay, which were characterized with CTD casts. The water temperature and salinity in the center of the bay ranged from -1.8 °C to more than 2.0 °C and from 70 30 PSU to almost 35 PSU during 2015 and 2016 (Moskalik et al., 2018). Sound propagation also depends on water depth, which drops to almost 100 m along a transect parallel to the glacier terminus (see Fig. 1A). The morphology of the bay varies greatly, with numerous moraines and flat areas created during the retreat of Hansbreen (Moskalik et al., 2018). with CTD casts. The water temperature and salinity in the center of the bay ranged from -1.8 °C to more than 2.0 °C and from 70 30 PSU to almost 35 PSU during 2015 and 2016 (Moskalik et al., 2018). Sound propagation also depends on water depth, which drops to almost 100 m along a transect parallel to the glacier terminus (see Fig. 1A). The morphology of the bay varies greatly, with numerous moraines and flat areas created during the retreat of Hansbreen (Moskalik et al., 2018). https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. events and the removal of contaminating signals, such as iceberg disintegration. Acoustic estimates of the ice volume loss are compared with concurrent camera and satellite observations. events and the removal of contaminating signals, such as iceberg disintegration. Acoustic estimates of the ice volume loss are compared with concurrent camera and satellite observations. The 50 kinetic energy of an impacting block can be estimated from the sound energy radiated provided that propagation losses between the block and the recording location can be estimated. Because the drop height of icebergs is limited by the vertical extent of the terminus, the estimate of impact energy can then be converted into a block mass using what Glowacki and Deane (2020) call a "mass-weighted average drop height", ˆh. Here we demonstrate the use of passive cryoacoustics to monitor the subaerial calving flux from a tidewater glacier in 55 southwestern Spitsbergen over 26 days of continuous measurements using algorithms for the automatic detection of calving Here we demonstrate the use of passive cryoacoustics to monitor the subaerial calving flux from a tidewater glacier in 55 southwestern Spitsbergen over 26 days of continuous measurements using algorithms for the automatic detection of calving 2 2 3 Automatic Detection of Calving Events Glowacki and Deane (2020) have shown that the mass of individual icebergs can be estimated from the sound they produce 75 when they impact the sea surface. In that study, 169 subaerial calving events from Hansbreen were manually identified in time-lapse photography and simultaneous recordings of the ambient sound, both made roughly 1 km from the glacier terminus. Despite significant variability in the total sound energy produced by an impact event for icebergs of comparable size, Monte Carlo simulations of ensemble time-series show that accurate estimates of subaerial calving flux can be made if sufficient events are averaged (40 events for 20% standard error for Hansbreen). 80 events are averaged (40 events for 20% standard error for Hansbreen). 80 The manual identification of calving events using time-lapse photography and underwater sound is accurate but too la- bor intensive to be seriously considered for long-term data sets. This issue is addressed here by developing algorithms for the automatic detection of ice impact noise in the ambient sound recordings and rejection of interfering events, such as the disintegration of icebergs in the bay to obtain accurate estimates of ice loss from the terminus of Hansbreen due to calving. The manual identification of calving events using time-lapse photography and underwater sound is accurate but too la- bor intensive to be seriously considered for long-term data sets. This issue is addressed here by developing algorithms for the automatic detection of ice impact noise in the ambient sound recordings and rejection of interfering events, such as the disintegration of icebergs in the bay to obtain accurate estimates of ice loss from the terminus of Hansbreen due to calving. The automatic detection of calving events is based on the observation that the ambient sound field in a glacial bay is 85 composed of two distinct categories of sound sources: 1. sources that radiate noise with statistical properties that are stationary on the time-scale of a calving event (such as melting glacier ice, for example) and 2. transient sources, like calving, superposed The automatic detection of calving events is based on the observation that the ambient sound field in a glacial bay is 85 composed of two distinct categories of sound sources: 1. sources that radiate noise with statistical properties that are stationary on the time-scale of a calving event (such as melting glacier ice, for example) and 2. transient sources, like calving, superposed 3 3 The sound power, Pb, between a lower and upper frequency band, f0 = 30 Hz to f1 = 100 Hz is then computed 95 using The signal processing scheme for event detection works as follows. A 10 minute segment of ambient sound is analyzed into a spectrogram. The sound power, Pb, between a lower and upper frequency band, f0 = 30 Hz to f1 = 100 Hz is then computed 95 using Pb = B(Ω) ⊗  M(Θ) ⊗ f1 Z f0 Pxxdf  , (1) Pb = B(Ω) ⊗  M(Θ) ⊗ f1 Z f0 Pxxdf  , (1) where Pxx is the power spectral density estimate for the noise segment and the operators M(Θ) and B(Ω) are the filters described below. The selection of frequency limits is motivated by previous analyses of the calving noise presented in Glowacki and Deane (2020) and Glowacki (2020) (see Appendix A: Materials and Methods for more details). The integral in equation 100 1 is calculated using the trapezoidal rule. The filter M(Θ) is a median filter of order Θ = 7 and is useful for eliminating loud, impulsive noise sources that are too short to be calving noise. A boxcar filter B(Ω) of length Ω= 32 points is applied to further reduce the effects of impulsive noise sources and random variations in noise power. and Deane (2020) and Glowacki (2020) (see Appendix A: Materials and Methods for more details). The integral in equation 100 1 is calculated using the trapezoidal rule. The filter M(Θ) is a median filter of order Θ = 7 and is useful for eliminating loud, impulsive noise sources that are too short to be calving noise. A boxcar filter B(Ω) of length Ω= 32 points is applied to further reduce the effects of impulsive noise sources and random variations in noise power. A baseline power for the noise in the absence of calving, Pbase, is taken to be the median of the probability density distri- bution of Pb. A threshold of detection, Pthres, is then selected to be 1.5 times the largest value of Pb with relative likelihood 105 A baseline power for the noise in the absence of calving, Pbase, is taken to be the median of the probability density distri- bution of Pb. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. on this stationary background. During periods when noise in the bay is dominated by ice melting and calving, the signal during a 10-minute data segment has the form of a wandering baseline punctuated at random intervals by hill-shaped increases as l i t Th l l d i i f th b li i ti d t h i th t f i lti th 90 on this stationary background. During periods when noise in the bay is dominated by ice melting and calving, the signal during a 10-minute data segment has the form of a wandering baseline punctuated at random intervals by hill-shaped increases as calving events occur. The level and noisiness of the baseline varies over time due to changes in the rate of ice melting on the 90 glacier terminus, proximity to the hydrophone of icebergs drifting in the bay, and variable acoustic propagation through the bay. These varying conditions are unpredictable and require the selection of a baseline level and event detection threshold that is adaptable on this stationary background. During periods when noise in the bay is dominated by ice melting and calving, the signal during a 10-minute data segment has the form of a wandering baseline punctuated at random intervals by hill-shaped increases as calving events occur. The level and noisiness of the baseline varies over time due to changes in the rate of ice melting on the 90 glacier terminus, proximity to the hydrophone of icebergs drifting in the bay, and variable acoustic propagation through the bay. These varying conditions are unpredictable and require the selection of a baseline level and event detection threshold that is adaptable. calving events occur. The level and noisiness of the baseline varies over time due to changes in the rate of ice melting on the 90 glacier terminus, proximity to the hydrophone of icebergs drifting in the bay, and variable acoustic propagation through the bay. These varying conditions are unpredictable and require the selection of a baseline level and event detection threshold that is adaptable. 90 The signal processing scheme for event detection works as follows. A 10-minute segment of ambient sound is analyzed into a spectrogram. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. A threshold of detection, Pthres, is then selected to be 1.5 times the largest value of Pb with relative likelihood 105 ϕ(Pthres) > ϕ(Pbase)e−β, (2) where β = 5. This constant plays a central role in detector performance and is referred to as the ’detection factor’ (see Appendix A: Materials and Methods). ϕ(Pthres) > ϕ(Pbase)e−β, (2) ϕ( thres) ϕ( base) , ( ) where β = 5. This constant plays a central role in detector performance and is referred to as the ’detection factor’ (see Appendix A: Materials and Methods). where β = 5. This constant plays a central role in detector performance and is referred to as the ’detection factor’ (see Appendix A: Materials and Methods). Calving events are identified as continuous periods of time when Pb exceeds Pthres. The detection algorithm is based on the 110 idea that calving events are relatively rare within 10-minute intervals and periods of time containing many successive events will drive the algorithm to select only the most energetic of them (see Appendix A: Materials and Methods). Calving events are identified as continuous periods of time when Pb exceeds Pthres. The detection algorithm is based on the 110 idea that calving events are relatively rare within 10-minute intervals and periods of time containing many successive events will drive the algorithm to select only the most energetic of them (see Appendix A: Materials and Methods). 110 An example of the detector output is shown in Fig. 2. Figure 2A shows 1 minute of ambient sound energy containing a calving event, presented in dB as a function of frequency and time. Figure 2B shows the output from the band-pass and median filters, the baseline level, the threshold level, and detection of the calving event. The detector was run on the 26 days of 115 recordings, resulting in the detection of 4258 events (see Appendix A: Materials and Methods for a discussion of how sensitive the algorithm is to the choice of detection factor). The primary output of the event detector are the event start and end times, t0 and t1. 4 4 4 Interfering Event Removal The problem remains of identifying events that were detected as calving but which are not. Visual inspection of noise spec- 120 trograms combined with listening to the corresponding sound recordings revealed two major sources of interfering sounds: (1) the disintegration of icebergs close to the acoustic buoy, which sounds like a calving event because the iceberg sheds blocks of ice into the water as it disintegrates, and (2) water movement around the hydrophone (flow noise). Other interfering events include calving from the terminus with highly-energetic contacts between the falling iceberg and the terminus, icebergs collid- ing with other ice pieces that occupy the sea surface along the terminus, and calving events involving ice block rotation in the 125 air, resulting in large contact area between the iceberg and sea surface leading to atypical sound production. Although all these events represent a challenge for the technique, the most concerning are the flow noise and disintegrating icebergs, which can appear to be very large calving events. The interfering events must be removed from the calving inventory before the ice mass loss is estimated. To deal with the misclassification problem, we take two steps. ing with other ice pieces that occupy the sea surface along the terminus, and calving events involving ice block rotation in the 125 air, resulting in large contact area between the iceberg and sea surface leading to atypical sound production. Although all these events represent a challenge for the technique, the most concerning are the flow noise and disintegrating icebergs, which can appear to be very large calving events. The interfering events must be removed from the calving inventory before the ice mass loss is estimated. To deal with the misclassification problem, we take two steps. First, we take advantage of the fact that submarine melting of glacier ice also generates underwater noise due to the impulsive 130 release of pressurized air bubbles into the water (Urick, 1971; Deane et al., 2014; Pettit et al., 2015). Glowacki et al. (2018) have demonstrated that the melt noise has an α-stable distribution. Importantly, the characteristic exponent of the α-stable distribution indicates proximity to a melting source. When the melting iceberg is close to the receiver, the underwater noise at frequencies 1—10 kHz is more impulsive and the exponent α deviates from 2. 4 Interfering Event Removal Here, we assume the following: if α < 1.95 for First, we take advantage of the fact that submarine melting of glacier ice also generates underwater noise due to the impulsive 130 release of pressurized air bubbles into the water (Urick, 1971; Deane et al., 2014; Pettit et al., 2015). Glowacki et al. (2018) have demonstrated that the melt noise has an α-stable distribution. Importantly, the characteristic exponent of the α-stable distribution indicates proximity to a melting source. When the melting iceberg is close to the receiver, the underwater noise at frequencies 1—10 kHz is more impulsive and the exponent α deviates from 2. Here, we assume the following: if α < 1.95 for First, we take advantage of the fact that submarine melting of glacier ice also generates underwater noise due to the impulsive 130 release of pressurized air bubbles into the water (Urick, 1971; Deane et al., 2014; Pettit et al., 2015). Glowacki et al. (2018) have demonstrated that the melt noise has an α-stable distribution. Importantly, the characteristic exponent of the α-stable distribution indicates proximity to a melting source. When the melting iceberg is close to the receiver, the underwater noise at frequencies 1—10 kHz is more impulsive and the exponent α deviates from 2. Here, we assume the following: if α < 1.95 for the noise segment immediately preceding the calving noise, there is a high likelihood that the corresponding event detected by 135 the automated algorithm is in fact an iceberg disintegrating close to the hydrophone. Such events are discarded. In this case, 875 events were removed from the inventory. Second the noise segments are occasionally contaminated with flow noise which originates from water turbulence advected the noise segment immediately preceding the calving noise, there is a high likelihood that the corresponding event detected by 135 the automated algorithm is in fact an iceberg disintegrating close to the hydrophone. Such events are discarded. In this case, 875 events were removed from the inventory. Second, the noise segments are occasionally contaminated with flow noise, which originates from water turbulence advected past the hydrophone and is most pronounced at frequencies below ∼50 Hz. An overlap in frequency between the calving 875 events were removed from the inventory. Second, the noise segments are occasionally contaminated with flow noise, which originates from water turbulence advected past the hydrophone and is most pronounced at frequencies below ∼50 Hz. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. The total number of submarine calving events in the present inventory is not expected to be large. Reported statistics of submarine calving from other study sites show that such events are typically much less common than subaerial calving (Minowa et al., 2018; Köhler et al., 2019; How et al., 2019). Glowacki (2022) estimated that submarine calving events at Hansbreen are 8–10 times less frequent than subaerial events. It must be noted, however, that the low rate of occurrence of submarine 155 calving is offset by the fact that submarine events often produce the biggest icebergs (Warren et al., 1995; Motyka, 1997; O’Neel et al., 2007; Glowacki, 2022). Consequently, attributing this component of the total calving flux to subaerial events may introduce an error disproportionately large relative to their number. In response to this problem, a new semi-automatic method for distinguishing submarine and subaerial calving events was recently proposed (Glowacki, 2022). However, there is no way at the present time to classify calving styles from sound recordings using fully automated methods. The time and 160 frequency structure of the underwater noise from submarine and subaerial calving differs significantly (Glowacki, 2022); for example, the underwater noise from submarine calving typically has longer duration, with individual sound-source mechanisms separated by the quiescent periods. As a result, a universal calving detector should (i) take into account the differences in time and frequency structure of the calving noise and (ii) be validated with high-frequency time-lapse images synchronized with long-term acoustic recordings; this task is beyond the scope of the present study. Keeping this limitation in mind, the section 165 that follows will explain how the subaerial calving flux can be estimated acoustically, with an assumption that all detected events are subaerial. 4 Interfering Event Removal An overlap in frequency between the calving detector band (30–100 Hz) and the flow noise (2–50 Hz) can result in false classifications of the episodes of intense water flow 140 around the hydrophone as calving events. To deal with this issue, we discard all events for which the ratio between the average power spectral density at 2–30 Hz and 50–1000 Hz is higher than or equal to 1. This procedure is based on the observation that calving noise extends into the 50–1000 Hz band, whereas flow noise is most pronounced at frequencies below 30 Hz. Using these criteria, 328 misclassifications were identified and manually validated by listening to the selected noise segments and visual inspections of spectrograms. In total, 1128 events were discarded due to the fact that 75 events were identified as both 145 flow noise and iceberg disintegration. One issue remains, which is the fact that some fraction of the event signals found come from submarine calving events. Submarine calving events are a problem because the parameters in the power law relationship between the energy of noise production and kinetic energy of block impact are for subaerial events and will not apply to submarine events (Glowacki and Deane, 2020). Thus any submarine events included in the calving inventory will not have their volume calculated correctly. 150 Moreover, even if their volume were calculated correctly, they are not detected by the camera observations for this study. Deane, 2020). Thus any submarine events included in the calving inventory will not have their volume calculated correctly. 150 Moreover, even if their volume were calculated correctly, they are not detected by the camera observations for this study. 5 5 5 Estimating Ice Volume Loss Following event detection, the next step is to compute the total noise energy radiated by the calving event at a standard reference distance of 1 m, Eac,imp. This step requires the calculation of the time and frequency integrated energy of the calving noise. 170 The energy of the impact sound radiated by a falling ice block can be expressed in terms of the observed sound energy by accounting for propagation effects and the addition of background noise using: Eac,imp = 4π ρwcw f1 Z f0     t0+∆t Z t0 Pxx dt − t0 Z t0−∆t Pxx dt  × 10 −T L(f) 10  df, (3) (3) (3) where ρw is the water density, cw is the sound speed, f0 = 30 Hz and f1 = 100 Hz are lower and upper frequency limits, t0 is the start time of the calving event determined from the event detector, ∆t = t1 −t0 is the calving signal duration, where t1 5 where ρw is the water density, cw is the sound speed, f0 = 30 Hz and f1 = 100 Hz are lower and upper frequency limits, t0 where ρw is the water density, cw is the sound speed, f0 30 Hz and f1 100 Hz are lower and upper frequency limits, t0 is the start time of the calving event determined from the event detector, ∆t = t1 −t0 is the calving signal duration, where t1 175 is the end time of the calving signal, TL is the frequency-dependent transmission loss between the hydrophone and the point of ice block impact (in decibels). The acoustic signal is integrated over the surface area of a unit sphere (4π) to obtain total noise energy in joules. The two integrals over time, R t0+∆t t0 Pxx dt and R t0 t0−∆t Pxx dt, are the frequency spectral densities of the calving noise plus background noise, and background noise alone, respectively. The transmission loss is calculated using the standard numerical code RAM (Collins, 1993) (see Appendix A: Materials and Methods for further details). 180 is the start time of the calving event determined from the event detector, ∆t = t1 −t0 is the calving signal duration, where t1 175 is the end time of the calving signal, TL is the frequency-dependent transmission loss between the hydrophone and the point of ice block impact (in decibels). https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. In the next step, the kinetic energy of the falling block of ice, Eimp, is calculated from the total impact sound energy radiated into the bay, Eac,imp, using the power law relationship: Eimp = γEκ ac,imp + ζ, (4) where γ = 1.45 × 109, κ = 0.18 and ζ = −2.66 × 109 are constants determined using the calibration dataset collected in 2016 (4) Eimp = γEκ ac,imp + ζ, Eimp = γEκ ac,imp + ζ, where γ = 1.45 × 109, κ = 0.18 and ζ = −2.66 × 109 are constants determined using the calibration dataset collected in 2016 (Glowacki and Deane (2020); see Appendix A: Materials and Methods for details). Finally, the iceberg volume is calculated as 185 (Glowacki and Deane (2020); see Appendix A: Materials and Methods for details). Finally, the iceberg volume is calculated as 185 (Glowacki and Deane (2020); see Appendix A: Materials and Methods for details). Finally, 5 V = M ρi = Eimp ρigˆh , (5) V = M ρi = Eimp ρigˆh , (5) (5) V = ρi = p ρigˆh , where M is the iceberg mass, ρi = 917 kg m−3 is assumed ice density, g = 9.81 m s−2 is the acc where M is the iceberg mass, ρi = 917 kg m−3 is assumed ice density, g = 9.81 m s−2 is the acceleration due to gravity and ˆh 20 7 i h i h d d h i h i d f h i f H b (Gl ki d D 2020) 6 Results 190 The cumulative subaerial ice volume loss over the 26 days of observation is 1.7±0.7×107 m3 from the acoustic measurements and 7±2×106 m3 from satellite data combined with stake measurements of the glacier velocity. The discrepancy may be due to several reasons. First, any submarine calving events are treated as subaerial in the acoustic analysis. Consequently, the ice volume loss from acoustics is almost certainly overestimated. This issue has been addressed recently by 205 Glowacki (2022) and can be incorporated into a long-term analysis once a method is developed for the automatic detection of submarine calving. Second, the power law relationship in Eq. 4 is based on the calibration dataset that does not cover the full range of impact noise energies observed in 2013 data (see Fig. A2 and discussion in Appendix A: Materials and Methods). Third, the location of the hydrophone – far from the terminus and in shallow water – was unfavorable because the propagation Consequently, the ice volume loss from acoustics is almost certainly overestimated. This issue has been addressed recently by 205 Glowacki (2022) and can be incorporated into a long-term analysis once a method is developed for the automatic detection of submarine calving. Second, the power law relationship in Eq. 4 is based on the calibration dataset that does not cover the full range of impact noise energies observed in 2013 data (see Fig. A2 and discussion in Appendix A: Materials and Methods). Third, the location of the hydrophone – far from the terminus and in shallow water – was unfavorable because the propagation Consequently, the ice volume loss from acoustics is almost certainly overestimated. This issue has been addressed recently by 205 Glowacki (2022) and can be incorporated into a long-term analysis once a method is developed for the automatic detection of submarine calving. Second, the power law relationship in Eq. 4 is based on the calibration dataset that does not cover the full range of impact noise energies observed in 2013 data (see Fig. A2 and discussion in Appendix A: Materials and Methods). Third, the location of the hydrophone – far from the terminus and in shallow water – was unfavorable because the propagation conditions of the calving noise are very different for different segments of the terminus (see the variable bathymetry along the 210 black dashed lines in Fig. 5 Estimating Ice Volume Loss The acoustic signal is integrated over the surface area of a unit sphere (4π) to obtain total noise energy in joules. The two integrals over time, R t0+∆t t0 Pxx dt and R t0 t0−∆t Pxx dt, are the frequency spectral densities of the calving noise plus background noise, and background noise alone, respectively. The transmission loss is calculated using the standard numerical code RAM (Collins, 1993) (see Appendix A: Materials and Methods for further details). 180 6 6 Results 190 Figure 3 shows a comparison of two time series: (i) subaerial calving flux derived from iceberg impact noise and (ii) terminus area loss estimated from camera observations over the 26 days of analysis (see Appendix A: Materials and Methods). The ice volume loss was calculated every 3 hours, which is the sampling time of the camera system. Both data streams show variability between samples and between each other. This is to be expected; Glowacki and Deane (2020) demonstrated that the acoustic Figure 3 shows a comparison of two time series: (i) subaerial calving flux derived from iceberg impact noise and (ii) terminus area loss estimated from camera observations over the 26 days of analysis (see Appendix A: Materials and Methods). The ice volume loss was calculated every 3 hours, which is the sampling time of the camera system. Both data streams show variability between samples and between each other. This is to be expected; Glowacki and Deane (2020) demonstrated that the acoustic method is accurate if sufficient calving events are averaged but significant variability is observed in the signal between blocks 195 of comparable volume. Moreover, camera observations are sensitive not only to weather conditions but also to the location of calving events due to the irregular shape of the terminus and oblique angle of view (see Fig. 1B). However, there is a clear relationship between the acoustic measurements of subaerial calving fluxes and the time-lapse observation of the terminus area loss, despite the limitations of both methods. The Pearson’s correlation coefficient between the two data streams smoothed with a 1-day running average is 0.6. 200 The question now is: how do the acoustic measurements of the total subaerial calving flux compare to results obtained with more traditional methods? The cumulative subaerial ice volume loss over the 26 days of observation is 1.7±0.7×107 m3 from the acoustic measurements and 7±2×106 m3 from satellite data combined with stake measurements of the glacier velocity. The discrepancy may be due to several reasons. First, any submarine calving events are treated as subaerial in the acoustic analysis. with a 1-day running average is 0.6. 200 The question now is: how do the acoustic measurements of the total subaerial calving flux compare to results obtained with more traditional methods? https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. (Glowacki and Deane, 2020). Finally, stake measurements of the ice velocity that were used to calculate subaerial calving flux from satellite images are from August 2013, which is a month before the acoustic data were collected. It is likely that Hansbreen accelerated in September due to heavy rainfall events; such ‘autumn acceleration’ has been reported for other tidewater glaciers in Svalbard (e.g., Luckman et al., 2015; Schellenberger et al., 2015). Moreover, stake measurements do not capture ice velocity 5 variations along the glacier terminus. The results of the experiment presented here are encouraging, given the limitations listed above and completely independent nature of the methods used to make the measurements. (Glowacki and Deane, 2020). Finally, stake measurements of the ice velocity that were used to calculate subaerial calving flux from satellite images are from August 2013, which is a month before the acoustic data were collected. It is likely that Hansbreen accelerated in September due to heavy rainfall events; such ‘autumn acceleration’ has been reported for other tidewater glaciers in Svalbard (e.g., Luckman et al., 2015; Schellenberger et al., 2015). Moreover, stake measurements do not capture ice velocity 5 variations along the glacier terminus. The results of the experiment presented here are encouraging, given the limitations listed above and completely independent nature of the methods used to make the measurements. 215 6 Results 190 1A), which has a large effect on the transmission losses of low-frequency sounds in a glacial bay 7 7 7 Long-term Monitoring of Subaerial Calving Fluxes The study of Glowacki and Deane (2020) demonstrated the potential for underwater ambient sound in glacial terminal bays to provide estimates of subaerial calving flux. Guided by this new opportunity, an impact-to-noise conversion model has been 220 applied here to a roughly 1-month long continuous recording of ambient sound in the bay of Hansbreen in Svalbard to es- timate subaerial calving flux. Good agreement is found between acoustic measurements of ice volume loss and the camera observations of the terminus area loss. The cumulative subarial calving flux estimated using the acoustic approach is higher than the ice loss derived using a pair of satellite images combined with stake measurements of the glacier velocity. Clearly, the acoustic method requires further improvements, but the results are encouraging, especially as acoustic recorders are increas- 225 ingly deployed in the Arctic for other studies, such as studies of biodiversity or human impacts like shipping, and they can be harnessed to also provide measurements of any neighboring glaciers. This study reinforces the idea that passive underwater acoustics offers a new and viable method for monitoring mass loss from marine-terminating glaciers and ice shelves. The development of algorithms for the automated detection of calving events and the removal of interfering events, such as disintegrating icebergs and flow noise, creates the possibility of monitoring the stability of tidewater glacier termini and 230 ice shelves over long time scales. We envision a scenario where a number of relatively inexpensive autonomous underwa- ter recording systems are deployed over year-long intervals around the termini of selected glaciers on a recurrent schedule. The analysis of the resulting signals would eventually allow glacial stability to be monitored over decadal and longer time scales. Concurrent measurements of relevant environmental drivers, such as water temperature, insolation and precipitation for example, could provide important insights into the processes controlling terminus ablation. 235 as disintegrating icebergs and flow noise, creates the possibility of monitoring the stability of tidewater glacier termini and 230 ice shelves over long time scales. We envision a scenario where a number of relatively inexpensive autonomous underwa- ter recording systems are deployed over year-long intervals around the termini of selected glaciers on a recurrent schedule. The analysis of the resulting signals would eventually allow glacial stability to be monitored over decadal and longer time scales. 7 Long-term Monitoring of Subaerial Calving Fluxes Concurrent measurements of relevant environmental drivers, such as water temperature, insolation and precipitation for example, could provide important insights into the processes controlling terminus ablation. 235 Personal experience making long-term recordings in the Arctic has taught us that not all deployed recording systems are recovered. It is therefore essential to keep the unit cost of recording systems low to allow for as much redundancy as possible within a fixed budget. Power consumption and data storage are two important drivers of cost. Requirements for both of these factors can be reduced by recording ambient sound on a fixed schedule or recording whenever the sound level exceeds a preset example, could provide important insights into the processes controlling terminus ablation. 235 Personal experience making long-term recordings in the Arctic has taught us that not all deployed recording systems are recovered. It is therefore essential to keep the unit cost of recording systems low to allow for as much redundancy as possible within a fixed budget. Power consumption and data storage are two important drivers of cost. Requirements for both of these factors can be reduced by recording ambient sound on a fixed schedule or recording whenever the sound level exceeds a preset threshold. These two possibilities will now be discussed. 240 threshold. These two possibilities will now be discussed. 240 Figure 4 shows the effect of recording coverage on the estimated total number of calving events (A) and cumulative ice volume loss (B) over the study period. A continuous acoustic record corresponds to ∆= 0%. Periods of active recording change from 1 hour per day (≈4%) to full coverage. Final estimates are derived proportionally, i.e. values obtained for 10% and 20% coverage are multiplied by 10 and 5, respectively. The specific hours of recordings during a day are selected repeatedly threshold. These two possibilities will now be discussed. 240 Figure 4 shows the effect of recording coverage on the estimated total number of calving events (A) and cumulative ice volume loss (B) over the study period. A continuous acoustic record corresponds to ∆= 0%. Periods of active recording change from 1 hour per day (≈4%) to full coverage. Final estimates are derived proportionally, i.e. values obtained for 10% and 20% coverage are multiplied by 10 and 5, respectively. The specific hours of recordings during a day are selected repeatedly threshold. These two possibilities will now be discussed. Exploiting this fact through the use of event-driven recording systemsi could result in significant power savings for autonomous recorders. 255 7 Long-term Monitoring of Subaerial Calving Fluxes 240 Figure 4 shows the effect of recording coverage on the estimated total number of calving events (A) and cumulative ice volume loss (B) over the study period. A continuous acoustic record corresponds to ∆= 0%. Periods of active recording change from 1 hour per day (≈4%) to full coverage. Final estimates are derived proportionally, i.e. values obtained for 10% and 20% coverage are multiplied by 10 and 5, respectively. The specific hours of recordings during a day are selected repeatedly 8 8 Concluding Remarks This first automated analysis of underwater sound recorded near the terminus of a tidewater glacier, compared with camera and satellite observations, demonstrates the feasibility of using cryoacoustics to monitor subaerial calving fluxes over extended periods. Challenges remain. The method has been verified for Hansbreen, a glacier of a scale common to Spitsbergen, but the glaciers in Greenland can be factors of 10 or more larger in both horizontal and vertical scales. Moreover, calving mode and 260 propagation conditions will vary between glaciers and there will be variability in levels of ice coverage in the terminus bay, which depend on calving rate, circulatory flow (long, narrow fjords are more prone to melange buildup), and sea ice coverage. Some or all of these factors may need to be accounted for when applying cryoacoustics to quantify subaerial calving flux between tidewater glaciers. Cryoacoustics meets some of the important requirements for long-term monitoring of subaerial calving fluxes. The high 265 rate of signal acquisition ensures that every calving event can be detected, provided its signal is above the detection threshold and below the rejection threshold. Although the uncertainty in the volume estimate for a single event is high, the total volume estimate becomes precise if a sufficient number of events are accumulated (Glowacki and Deane, 2020). With current tech- nological limitations, it is not possible to take time-lapse photographs with high enough frame rates to ensure that all events are captured. Moreover, cryoacoustics is insensitive to lighting conditions and relatively insensitive to weather conditions. For 270 example, the dominant spectral component of noise generated by rain lies well above the ’calving band’ (Pumphrey and Crum, 1988). Both cryoacoustics and cryoseismology provide temporally-resolved, continuous records of calving activity that are ex- pected to be relatively insensitive to changing environmental conditions (Podolskiy and Walter, 2016; Deane et al., 2019). are captured. Moreover, cryoacoustics is insensitive to lighting conditions and relatively insensitive to weather conditions. For 270 example, the dominant spectral component of noise generated by rain lies well above the ’calving band’ (Pumphrey and Crum, 1988). Both cryoacoustics and cryoseismology provide temporally-resolved, continuous records of calving activity that are ex- pected to be relatively insensitive to changing environmental conditions (Podolskiy and Walter, 2016; Deane et al., 2019). https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. using all possible combinations, creating a distribution of outputs for the chosen value of coverage. The event count is much 245 less sensitive to the recording schedule than the subaerial calving flux. Nevertheless, one standard deviation of the estimated ice volume loss at 30% coverage is within 10% of the reference value. This result demonstrates the possibility of significant reduction in recording time (by 70%) with relatively low increase in the resulting error. using all possible combinations, creating a distribution of outputs for the chosen value of coverage. The event count is much 245 less sensitive to the recording schedule than the subaerial calving flux. Nevertheless, one standard deviation of the estimated ice volume loss at 30% coverage is within 10% of the reference value. This result demonstrates the possibility of significant reduction in recording time (by 70%) with relatively low increase in the resulting error. Figure 5 shows the percentage of time occupied by calving noise during the experiment and the acoustic estimate of daily calving rate. On average, calving events were active for only about 1% of the time; this corresponds to around 120 events per 250 day. However, we expect that the calving detector may split single calving events into several detections. This is especially likely in the case of free falls of highly disintegrated icebergs. Therefore, we assume that the real daily calving rate is likely lower than the acoustic estimate. Although calving activity changes significantly with seasons and geographical location, it is still expected to be a small fraction of the total record. Exploiting this fact through the use of event-driven recording systems could result in significant power savings for autonomous recorders. 255 calving rate. On average, calving events were active for only about 1% of the time; this corresponds to around 120 events per 250 day. However, we expect that the calving detector may split single calving events into several detections. This is especially likely in the case of free falls of highly disintegrated icebergs. Therefore, we assume that the real daily calving rate is likely lower than the acoustic estimate. Although calving activity changes significantly with seasons and geographical location, it is still expected to be a small fraction of the total record. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. out the burden of a glacier-by-glacier calibration. Moreover, it may be possible to extract other geophysical signals of interest, such as terminus melt rate, from cryoacoustic records (Pettit et al., 2015; Deane et al., 2019). Cryoacoustics, combined with other remote sensing methods, such as photogrammetry, cryoseismology and satellite obser- vations, can form the core of a long-term monitoring system for subaerial calving fluxes from glaciers and ice shelves. 280 Data availability. The acoustic data used in this study are available upon request from J.T. (jaroslaw.tegowski@ug.edu.pl) Data availability. The acoustic data used in this study are available upon request from J.T. (jaroslaw.tegowski@ug.edu.pl) A1 Acoustic and Camera Observations Nevertheless, the image analysis provides useful information about the overall calving activity at Hansbreen during the 295 study period. frame. Nevertheless, the image analysis provides useful information about the overall calving activity at Hansbreen during the 295 study period. 8 Concluding Remarks Cryoacoustics has the additional benefit that the physics of sound production, which stems from the entrainment of bubbles 275 around falling blocks of ice and collective oscillations of bubble plumes (Glowacki, 2020), is expected to apply broadly across calving glaciers in polar regions. If true, this will enable subaerial calving flux to be extracted from cryoacoustic signals with- Cryoacoustics has the additional benefit that the physics of sound production, which stems from the entrainment of bubbles 275 around falling blocks of ice and collective oscillations of bubble plumes (Glowacki, 2020), is expected to apply broadly across calving glaciers in polar regions. If true, this will enable subaerial calving flux to be extracted from cryoacoustic signals with- Cryoacoustics has the additional benefit that the physics of sound production, which stems from the entrainment of bubbles 275 around falling blocks of ice and collective oscillations of bubble plumes (Glowacki, 2020), is expected to apply broadly across calving glaciers in polar regions. If true, this will enable subaerial calving flux to be extracted from cryoacoustic signals with- 9 A2 Satellite Observations The volume of ice lost from Hansbreen through subaerial calving was estimated from satellite imagery and GPS observations of the glacier surface movement. subaerial calving flux is estimated using Q = ¯Uice + UfrontLH, where Q is the volumetric The volume of ice lost from Hansbreen through subaerial calving was estimated from satellite imagery and GPS observations of the glacier surface movement. subaerial calving flux is estimated using Q = ¯Uice + UfrontLH, where Q is the volumetric flux of icebergs, ¯Uice is the mean ice flow velocity, Ufront is the front retreat/advance velocity, and L and H respectively are 300 the cliff length and height above sea level. The length of Hansbreen’s active cliff and the average front retreat per day have been extracted from two multispectral Landsat 8 satellite images (2013-08-24 and 2013-09-25). Cliff height above sea level was determined in 2015 using a Riegl VZ-6000 high resolution 3D laser scanner. The glacier surface velocity was estimated using a mass balance stake mounted near the calving front (designated GPS in Fig. A1). Changes in stake position were measured with precise dGPS in August 2013. The subaerial calving flux estimated over the 26 days of acoustic measurements 305 of the glacier surface movement. subaerial calving flux is estimated using Q = Uice + UfrontLH, where Q is the volumetric flux of icebergs, ¯Uice is the mean ice flow velocity, Ufront is the front retreat/advance velocity, and L and H respectively are 300 the cliff length and height above sea level. The length of Hansbreen’s active cliff and the average front retreat per day have been extracted from two multispectral Landsat 8 satellite images (2013-08-24 and 2013-09-25). Cliff height above sea level was determined in 2015 using a Riegl VZ-6000 high resolution 3D laser scanner. The glacier surface velocity was estimated using a mass balance stake mounted near the calving front (designated GPS in Fig. A1). Changes in stake position were flux of icebergs, ¯Uice is the mean ice flow velocity, Ufront is the front retreat/advance velocity, and L and H respectively are 300 the cliff length and height above sea level. The length of Hansbreen’s active cliff and the average front retreat per day have been extracted from two multispectral Landsat 8 satellite images (2013-08-24 and 2013-09-25). Cliff height above sea level was determined in 2015 using a Riegl VZ-6000 high resolution 3D laser scanner. A1 Acoustic and Camera Observations Acoustic and camera data were collected around Hansbreen from 5 – 30 September, 2013. Noise recordings were made with a 285 High Tech. Inc. HTI-96-MIN omnidirectional hydrophone mounted on the seafloor at a depth of 22 m and approximately 1.95 km from the glacier terminus. Data were sampled at a frequency of 32 kHz with 16 bits of dynamic range. Photographs of the terminus were taken every 3 hours using a Canon D1000 time-lapse camera located 160 m above sea level and 0.8 to 2 km from the glacier terminus (see Fig. 1). Acoustic and camera data were collected around Hansbreen from 5 – 30 September, 2013. Noise recordings were made with a 285 High Tech. Inc. HTI-96-MIN omnidirectional hydrophone mounted on the seafloor at a depth of 22 m and approximately 1.95 km from the glacier terminus. Data were sampled at a frequency of 32 kHz with 16 bits of dynamic range. Photographs of the terminus were taken every 3 hours using a Canon D1000 time-lapse camera located 160 m above sea level and 0.8 to 2 km from the glacier terminus (see Fig. 1). The images were analyzed using a differences technique to estimate the area of ice lost from the cliff face through calving 290 when lighting and weather conditions permitted. Changes at the calving front associated with calving events were found by comparing two consecutive time-lapse images. The terminus area loss was then estimated using known ice cliff heights at given locations. This methodology is sensitive to the variability of the terminus shape along its edge due to the oblique angle of the camera view. Moreover, we are aware that the glacier can calve more than once at the same location during a 3-hour time The images were analyzed using a differences technique to estimate the area of ice lost from the cliff face through calving 290 when lighting and weather conditions permitted. Changes at the calving front associated with calving events were found by comparing two consecutive time-lapse images. The terminus area loss was then estimated using known ice cliff heights at given locations. This methodology is sensitive to the variability of the terminus shape along its edge due to the oblique angle of the camera view. Moreover, we are aware that the glacier can calve more than once at the same location during a 3-hour time frame. A3 Calving Impact Energy Estimation The calving impact energy was estimated from the noise energy using a power-law relationship model shown in Eq. 4. The parameters of the model are based on the calibration dataset presented in Glowacki and Deane (2020) that includes time- 315 lapse images and underwater noise recordings of 169 calving events observed in 2016 at Hansbreen. However, the power-law model proposed by Glowacki and Deane (2020) was improved through the re-analysis of the calibration dataset. The following changes/modifications were applied: The calving impact energy was estimated from the noise energy using a power-law relationship model shown in Eq. 4. The parameters of the model are based on the calibration dataset presented in Glowacki and Deane (2020) that includes time- 315 lapse images and underwater noise recordings of 169 calving events observed in 2016 at Hansbreen. However, the power-law model proposed by Glowacki and Deane (2020) was improved through the re-analysis of the calibration dataset. The following changes/modifications were applied: 1. Subsequent to publication, it was discovered that the acoustic recordings contained a small DC offset, which created a minor bias in the event energy estimates. This issue has been corrected here by running the acoustic recordings through a (30 - 100) Hz band-pass filter, the lower frequency of which is roughly equal to the low-frequency cut-off of the waveguide comprised of the sea surface and seafloor of the terminus bay. 2. The Bellhop ray-tracing model was used in Glowacki and Deane (2020) to calculate the loss of acoustic energy between the iceberg/water impact locations and the hydrophone. Here, the transmission loss of the calving noise was calculated using the parabolic equation model RAM (Collins, 1993) that is more recommended for low-frequency problems (Jensen et al., 2011). The grain size of the sediments and source depth were set to ϕ = 5 and Zs = 5 m, respectively. The attenuation, sound speed and density of the sediments were calculated from the grain size using formulas proposed by Hamilton (1972) and Hamilton and Bachman (1982). Moreover, in the present study, transmission losses were evaluated for a whole range of frequencies between 30 and 100 Hz, while a single nominal source frequency of 50 Hz was used in Glowacki and Deane (2020). 330 3. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. the glacier surface velocity field was estimated using offset tracking from TerraSAR satellite radar images (2012-12-15 and 2012-12-26). subaerial calving flux was then estimated within 40 m sections of the glacier front to allow for variations in velocity and cliff height (see the grey profile in Fig. A1). The total volume of icebergs calved during the observation period is estimated to be (6±1)×106 m3. It should be borne in mind, however, that TerraSAR images were collected in December 2012 0 and not during the study period. Nevertheless, the results show good agreement with subaerial calving flux estimated using stake measurements. the glacier surface velocity field was estimated using offset tracking from TerraSAR satellite radar images (2012-12-15 and 2012-12-26). subaerial calving flux was then estimated within 40 m sections of the glacier front to allow for variations in velocity and cliff height (see the grey profile in Fig. A1). The total volume of icebergs calved during the observation period is estimated to be (6±1)×106 m3. It should be borne in mind, however, that TerraSAR images were collected in December 2012 0 and not during the study period. Nevertheless, the results show good agreement with subaerial calving flux estimated using stake measurements. A2 Satellite Observations The glacier surface velocity was estimated using a mass balance stake mounted near the calving front (designated GPS in Fig. A1). Changes in stake position were measured with precise dGPS in August 2013. The subaerial calving flux estimated over the 26 days of acoustic measurements 305 is (7 ± 2) × 106 m3, i.e. ca. 0.27 × 106 m3 d−1. This is the result used for comparison with the acoustic data. As a crosscheck, 10 Leaving aside the causes, the lack of the highest impact noise energies in the calibration dataset results in extrapolation of the power-law model in the analysis of data collected in 2013; unfortunately, the extrapolation applies to calving events that contribute the most to the subaerial calving flux. This is one possible explanation 355 of the discrepancy between calving fluxes derived from traditional methods and the acoustic technique. The improvement of the power-law model in Eq. 4 requires long-term acoustic measurements close to different glaciers combined with measurements of ice block volumes with optical techniques (e.g., time-lapse photography, terrestrial laser scanning). Collecting calibration data that would include a whole range of styles and magnitudes of calving events is critical for the usefulness of the acoustic technique in the monitoring of subaerial calving fluxes. 360 the extrapolation applies to calving events that contribute the most to the subaerial calving flux. This is one possible explanation 355 of the discrepancy between calving fluxes derived from traditional methods and the acoustic technique. The improvement of the power-law model in Eq. 4 requires long-term acoustic measurements close to different glaciers combined with measurements of ice block volumes with optical techniques (e.g., time-lapse photography, terrestrial laser scanning). Collecting calibration data that would include a whole range of styles and magnitudes of calving events is critical for the usefulness of the acoustic technique in the monitoring of subaerial calving fluxes. 360 scanning). Collecting calibration data that would include a whole range of styles and magnitudes of calving events is critical for the usefulness of the acoustic technique in the monitoring of subaerial calving fluxes. 360 A3 Calving Impact Energy Estimation In Glowacki and Deane (2020), the parameters of the power-law relationship between the impact energy and noise energy were estimated by performing a linear least-mean-squares analysis of log-transformed variables; the impact noise energy was used as a dependent variable to estimate the impact-to-noise conversion coefficients. Here, the block impact energy was used as a dependent variable to minimize the error in the subaerial calving flux. Moreover, the coefficients of the power-law relationship were estimated using a non-linear regression model and non-transformed variables (function fitnlm in Matlab). 335 Problems remain. Most importantly, the calibration dataset is limited to 169 calving events and certainly do not represent the full range of possible values of the impact noise energy at Hansbreen. As a consequence, the power-law model – that is based 11 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Second, the 2016 data do not include calving events with Eac,imp > 4000 joules either. The lack of the largest events in the calibration dataset may be due to several reasons: (i) Hansbreen may have been in different melting regimes in 2013 and 2016, which caused differences in calving styles (e.g., differences in the water depth at the terminus, glacier surface velocity, water content, etc.), (ii) co-occurrence of large-scale calving events and bad weather/lighting conditions that prevented estimation of 350 impact energy from time-lapse images in the calibration dataset, (iii) underestimated transmission loss for the acoustic data collected in 2013 because of the different location of the hydrophone compared to 2016 leading to a different propagation path (note the shallow sill close to the recorder in Fig. 1A). Leaving aside the causes, the lack of the highest impact noise energies in the calibration dataset results in extrapolation of the power-law model in the analysis of data collected in 2013; unfortunately, the extrapolation applies to calving events that contribute the most to the subaerial calving flux. This is one possible explanation 355 f h di b l i fl d i d f di i l h d d h i h i content, etc.), (ii) co-occurrence of large-scale calving events and bad weather/lighting conditions that prevented estimation of 350 impact energy from time-lapse images in the calibration dataset, (iii) underestimated transmission loss for the acoustic data collected in 2013 because of the different location of the hydrophone compared to 2016 leading to a different propagation path (note the shallow sill close to the recorder in Fig. 1A). Leaving aside the causes, the lack of the highest impact noise energies in the calibration dataset results in extrapolation of the power-law model in the analysis of data collected in 2013; unfortunately, content, etc.), (ii) co-occurrence of large-scale calving events and bad weather/lighting conditions that prevented estimation of 350 impact energy from time-lapse images in the calibration dataset, (iii) underestimated transmission loss for the acoustic data collected in 2013 because of the different location of the hydrophone compared to 2016 leading to a different propagation path (note the shallow sill close to the recorder in Fig. 1A). A4 Calving Event Detection Algorithm The calving event detection algorithm depends on the selection of an event detection factor, β, and a frequency band over which the ambient sound is filtered before integration over time. Figure A3 shows a summary of how the number of detected calving events varies with the detection factor β; interfering events were not removed in this analysis. As might be expected, the total number of events decreases rapidly with increasing 365 detection factor because increasing β results in the exclusion of the lowest-energy events. The results presented in Fig. 3 were generated using β = 5, which was found to be an optimal selection in terms of limiting detection of non-calving events and minimizing the loss of true calving events. The selection of β consisted of the visual analysis of noise spectrograms and audible inspection of sound recordings performed for a number of detections. g y g β; g events were not removed in this analysis. As might be expected, the total number of events decreases rapidly with increasing 365 detection factor because increasing β results in the exclusion of the lowest-energy events. The results presented in Fig. 3 were generated using β = 5, which was found to be an optimal selection in terms of limiting detection of non-calving events and minimizing the loss of true calving events. The selection of β consisted of the visual analysis of noise spectrograms and audible inspection of sound recordings performed for a number of detections. The detection factor selected here for the terminal bay of Hansbreen is likely not the optimal detection factor for other 370 environments or geometries for the terminus and recording station. This is because the detector performance is sensitive to the The detection factor selected here for the terminal bay of Hansbreen is likely not the optimal detection factor for other 370 environments or geometries for the terminus and recording station. This is because the detector performance is sensitive to the 12 The calving detector in the present form has two major weaknesses: (1) subaerial and submarine calving events cannot be distinguished and treated separately and (2) certainly there are still some unexplored interfering events that either cause false detections or periodically lower the signal-to-noise ratio, which makes calving detection more difficult (i.e., some calving events remain undetected). The latter baseline noise level. 385 Here we use a simple but robust algorithm for the detection of subaerial calving events. The calving detector in the present form has two major weaknesses: (1) subaerial and submarine calving events cannot be distinguished and treated separately and (2) certainly there are still some unexplored interfering events that either cause false detections or periodically lower the signal-to-noise ratio, which makes calving detection more difficult (i.e., some calving events remain undetected). The latter baseline noise level. 385 Here we use a simple but robust algorithm for the detection of subaerial calving events. The calving detector in the present form has two major weaknesses: (1) subaerial and submarine calving events cannot be distinguished and treated separately and (2) certainly there are still some unexplored interfering events that either cause false detections or periodically lower the signal-to-noise ratio, which makes calving detection more difficult (i.e., some calving events remain undetected). The latter issue can be partly addressed with the use of more sophisticated detection techniques. See, for example, recent work in the 390 cryoseismology community by Carr et al. (2020) and Köhler et al. (2022). However, we speculate that better understanding of different (non-calving) sound-source mechanisms and their underwater acoustic signatures is required before more sophis- ticated algorithms are implemented. Examples may include but are not limited to interactions between floating growlers and icebergs, ice fracturing events, water outflows from subglacial conduits, coastline landslides, and activity of marine mammals. issue can be partly addressed with the use of more sophisticated detection techniques. See, for example, recent work in the 390 cryoseismology community by Carr et al. (2020) and Köhler et al. (2022). However, we speculate that better understanding of different (non-calving) sound-source mechanisms and their underwater acoustic signatures is required before more sophis- ticated algorithms are implemented. Examples may include but are not limited to interactions between floating growlers and icebergs, ice fracturing events, water outflows from subglacial conduits, coastline landslides, and activity of marine mammals. Most of these “interfering” noise sources can be investigated with meteorological and oceanographic measurements, high- 395 frequency photographic images, laser scans, or radar scans synchronized with acoustic recordings. For example, Deane et al. (2014) reported that interactions of surface gravity waves with underside of ice ledges at the periphery of icebergs are sources of underwater noise emission below 500 Hz. Some sound-source mechanisms, like underwater fracturing events, require novel observational techniques or scaled laboratory experiments. Most of these “interfering” noise sources can be investigated with meteorological and oceanographic measurements, high- 395 frequency photographic images, laser scans, or radar scans synchronized with acoustic recordings. For example, Deane et al. (2014) reported that interactions of surface gravity waves with underside of ice ledges at the periphery of icebergs are sources of underwater noise emission below 500 Hz. Some sound-source mechanisms, like underwater fracturing events, require novel observational techniques or scaled laboratory experiments. The problem remains with detection of submarine events. An inclusion of submarine events would be beneficial for two 400 reasons: (i) to find out how frequent these events are and (ii) to provide more accurate estimates of subaerial calving fluxes by rejecting submarine events from the analysis. Moreover, it should not be ruled out that the methodology for estimating submarine calving fluxes from sound recordings made in glacial bays could be developed. If so, it would be possible to derive both subaerial and submarine calving fluxes using acoustic techniques. Recent work by Glowacki (2022) demonstrated that The problem remains with detection of submarine events. An inclusion of submarine events would be beneficial for two 400 reasons: (i) to find out how frequent these events are and (ii) to provide more accurate estimates of subaerial calving fluxes by rejecting submarine events from the analysis. Moreover, it should not be ruled out that the methodology for estimating submarine calving fluxes from sound recordings made in glacial bays could be developed. If so, it would be possible to derive both subaerial and submarine calving fluxes using acoustic techniques. Recent work by Glowacki (2022) demonstrated that the two calving modes can be acoustically distinguished using parameters of the log-normal distribution of the calving noise 405 combined with calving signal duration. A semi-automatic detection of start and end times of calving noise was used. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. signal-to-noise ratio at the hydrophone location and activity of other sources, such as nearby icebergs, which will vary from site to site. It is noted in the main text that the event detection algorithm is based on the assumption that a good estimate for the background noise is the median noise level in the detector frequency band. This assumption holds well when calving events are 375 relatively infrequent within an observational interval selected for analysis (10 minutes here) but may bias the detector toward the exclusion of low energy events if too many events occur within the interval. The validity of the assumption was tested by computing the background noise level iteratively: detected events are removed from the record and a baseline noise level recomputed in successive passes through the data segment. The results of this analysis for change in number of events detected, total acoustic energy at the receiver and summed event durations are shown in Fig. A4. The total percentage change in acoustic 380 energy of all detected events is less than 3%, which is not a significant source of error given other, larger uncertainties in the analysis, such as hydrophone calibration and propagation loss. The percentage change in number of events is similarly minor. There is a larger impact on the duration of events because of two effects: 1. increasing the baseline noise level increases the detection threshold and decreases event duration and 2. the division of a single event into 2 or more events is sensitive to the total acoustic energy at the receiver and summed event durations are shown in Fig. A4. The total percentage change in acoustic 380 energy of all detected events is less than 3%, which is not a significant source of error given other, larger uncertainties in the analysis, such as hydrophone calibration and propagation loss. The percentage change in number of events is similarly minor. There is a larger impact on the duration of events because of two effects: 1. increasing the baseline noise level increases the detection threshold and decreases event duration and 2. the division of a single event into 2 or more events is sensitive to the baseline noise level. 385 Here we use a simple but robust algorithm for the detection of subaerial calving events. is co-architect of the sound inversion methodology and detection algorithm, participated 415 in field campaign, and participated in manuscript preparation. p p p p p p pi p g p p pi p g and participated in manuscript preparation. G.B.D. is co-architect of the sound inversion methodology and detection algorithm, participated 415 in field campaign, and participated in manuscript preparation. and participated in manuscript preparation. G.B.D. is co-architect of the sound inversion methodology and detection algorithm, participated 415 in field campaign, and participated in manuscript preparation. and participated in manuscript preparation. G.B.D. is co-architect of the sound inversion methodology and detection algorithm, participated 415 in field campaign, and participated in manuscript preparation. However, the two calving modes can be acoustically distinguished using parameters of the log-normal distribution of the calving noise 405 combined with calving signal duration. A semi-automatic detection of start and end times of calving noise was used. However, 13 Acknowledgements. This work has been supported by the National Science Centre, Poland (grants 2011/03/B/ST10/04275 and 2021/43/D/ST10/0 Ministry of Science and Higher Education of Poland (grant 1621/MOB/V/2017/0 and subsidy for the Institute of Geophysics, Polish Academy of Sciences), U.S. Office of Naval Research, Ocean Acoustics Division (grant N00014-17-1-2633), USA National Science Foundation Office 420 of Polar Programs (grant OPP-1748265), and Research Council of Norway (Arctic Field Grant, RIS ID: 6133). We gratefully acknowledge the support of the staff at the Polish Polar Research Station in Hornsund (PPS). CTD data were collected under the oceanographic monitoring of the PPS (available at https://dataportal.igf.edu.pl/). TerraSAR-X data provided by German Aerospace Center (DLR, project LAN2787). Landsat-8 data made available by the United States Geological Survey (USGS). https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. the problem remains of how to automatically select t0 and t1 for submarine calving events; this would require long-term calibration dataset of synchronized high-frequency time-lapse images and noise recordings. Such data are not available for calving events observed in 2013. Consequently, the analysis of submarine events was out of the scope of this study. the problem remains of how to automatically select t0 and t1 for submarine calving events; this would require long-term calibration dataset of synchronized high-frequency time-lapse images and noise recordings. Such data are not available for calving events observed in 2013. Consequently, the analysis of submarine events was out of the scope of this study. Author contributions. J.T. conceived the project, led the 2013 field expedition to Svalbard and participated in data analysis and interpre- 410 tation, and participated in manuscript preparation. O.G. is co-architect of the sound inversion methodology, performed data analysis and interpretation, and participated in manuscript preparation. M.C. collected and processed the photographic data of terminus ablation. M.B. collected and processed satellite and stick data of glacier velocity and ice mass loss. J.J. provided scientific oversight into data analysis and participated in manuscript preparation. M.M. participated in the field campaigns and provided CTD data. P.B. participated in field campaigns Author contributions. J.T. conceived the project, led the 2013 field expedition to Svalbard and participated in data analysis and interpre- 410 tation, and participated in manuscript preparation. O.G. is co-architect of the sound inversion methodology, performed data analysis and interpretation, and participated in manuscript preparation. M.C. collected and processed the photographic data of terminus ablation. M.B. collected and processed satellite and stick data of glacier velocity and ice mass loss. J.J. provided scientific oversight into data analysis and participated in manuscript preparation. M.M. participated in the field campaigns and provided CTD data. P.B. participated in field campaigns and participated in manuscript preparation. G.B.D. is co-architect of the sound inversion methodology and detection algorithm, participated 415 in field campaign, and participated in manuscript preparation. collected and processed satellite and stick data of glacier velocity and ice mass loss. J.J. provided scientific oversight into data analysis and participated in manuscript preparation. M.M. participated in the field campaigns and provided CTD data. P.B. participated in field campaigns and participated in manuscript preparation. G.B.D. References 425 Benn, D. I., Hulton, N. R., and Mottram, R. 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P., and Vieli, A.: Calving event size measurements and statistics of Eqip Sermia, Greenland, from terrestrial radar interferometry, The Cryosphere, 14, 1051–1066, https://doi.org/10.5194/tc-14-1051-2020, 2020. W C R Gl N F H i S Wi h V K A R d Ri A Ch i i f id l i Gl i S Warren, C. R., Glasser, N. F., Harrison, S., Winchester, V., Kerr, A. R., and Rivera, A.: Characteristics of tide-water calving at Glaciar San Rafael, Chile, Journal of Glaciology, 41, 273–289, https://doi.org/10.3189/S0022143000016178, 1995. 555 Rafael, Chile, Journal of Glaciology, 41, 273–289, https://doi.org/10.3189/S0022143000016178, 1995. 555 Xie, S., Dixon, T. H., Voytenko, D., Deng, F., and Holland, D. M.: Grounding line migration through the calving season at Jakobshavn Isbræ, Greenland, observed with terrestrial radar interferometry, The Cryosphere, 12, 1387–1400, https://doi.org/10.5194/tc-12-1387-2018, 2018. Yun, S., Lee, W. S., Dziak, R. P., Roche, L., Matsumoto, H., Lau, T.-K., Sremba, A., Mellinger, D. K., Haxel, J. H., Kang, S.-G., Hong, J. K., and Park, Y.: Quantifying Soundscapes in the Ross Sea, Antarctica Using Long-Term Autonomous Hydroacoustic Monitoring Systems, Yun, S., Lee, W. S., Dziak, R. P., Roche, L., Matsumoto, H., Lau, T.-K., Sremba, A., Mellinger, D. K., Haxel, J. H., Kang, S.-G., Hong, J. K., and Park, Y.: Quantifying Soundscapes in the Ross Sea, Antarctica Using Long-Term Autonomous Hydroacoustic Monitoring Systems, Frontiers in Marine Science, 8, https://doi.org/10.3389/fmars.2021.703411, 2021. 560 and Park, Y.: Quantifying Soundscapes in the Ross Sea, Antarctica Using Long-Term Autonomous Hydroacoustic Monitoring Systems, Frontiers in Marine Science, 8, https://doi.org/10.3389/fmars.2021.703411, 2021. 560 Frontiers in Marine Science, 8, https://doi.org/10.3389/fmars.2021.703411, 2021. 560 Frontiers in Marine Science, 8, https://doi.org/10.3389/fmars.2021.703411, 2021. 560 18 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 1. (A) Map of the study site and (B) example image of the Hansbreen terminus. Locations of the time-lapse camera and the acoustic buoy are marked with yellow and white font, respectively. The black dashed lines show different propagation transects that were used for estimating the noise transmission loss. Landsat 8 satellite data collected on 11 September 2013, courtesy of the US Geological Survey, Department of the Interior. Bathymetric data provided by (1) the Norwegian Hydrographic Service under the permit no. 13/G722, issued to the Institute of Geophysics Polish Academy of Sciences, and (2) the Faculty of Natural Sciences, Institute of Earth Sciences, University of Silesia in Katowice, Sosnowiec, Poland (Błaszczyk et al., 2021). Figure 1. (A) Map of the study site and (B) example image of the Hansbreen terminus. Locations of the time-lapse camera and the acoustic buoy are marked with yellow and white font, respectively. The black dashed lines show different propagation transects that were used for estimating the noise transmission loss. Landsat 8 satellite data collected on 11 September 2013, courtesy of the US Geological Survey, Department of the Interior. Bathymetric data provided by (1) the Norwegian Hydrographic Service under the permit no. 13/G722, issued to the Institute of Geophysics Polish Academy of Sciences, and (2) the Faculty of Natural Sciences, Institute of Earth Sciences, University of Silesia in Katowice, Sosnowiec, Poland (Błaszczyk et al., 2021). 19 Figure 2. Calving detector explained. Top: A spectrogram of the sound produced by a calving event within the background noise radiated by the melting terminus of Hansbreen. The frequencies f0 and f1 on the right hand side of the plot denote the lower and upper limits of the detection frequencies (see text for details). Bottom: Noise power from the spectrogram above, integrated from f0 to f1 and filtered. The calving event is detected when the noise power exceeds the power threshold, Pthres, as annotated by t0 and t1. The threshold power is based th b li P hi h i d t i d f t ti ti l l i f th d ( t t f d t il ) https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 2. Calving detector explained. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Top: A spectrogram of the sound produced by a calving event within the background noise radiated by the melting terminus of Hansbreen. The frequencies f0 and f1 on the right hand side of the plot denote the lower and upper limits of the detection frequencies (see text for details). Bottom: Noise power from the spectrogram above, integrated from f0 to f1 and filtered. The calving event is detected when the noise power exceeds the power threshold, Pthres, as annotated by t0 and t1. The threshold power is based on the baseline power, Pbase, which is determined from a statistical analysis of the sound (see text for details). 20 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 3. A comparison of ice volume loss derived from iceberg impact noise and terminus area loss from camera observations. The ice volume loss was calculated every 3 hours, which is the sampling time of the camera system. The thick lines show the two data streams smoothed with 1-day running average. Figure 3. A comparison of ice volume loss derived from iceberg impact noise and terminus area loss from camera observations. The ice volume loss was calculated every 3 hours, which is the sampling time of the camera system. The thick lines show the two data streams smoothed with 1-day running average. 21 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 4. The effect of sampling coverage on acoustic estimates of (A) event count and (B) ice volume loss. Recording periods range from 1 hour per day to continuous. The reference value of ∆= 0% is for full coverage. Figure 4. The effect of sampling coverage on acoustic estimates of (A) event count and (B) ice volume loss. Recording periods range from 1 hour per day to continuous. The reference value of ∆= 0% is for full coverage. 22 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 5. (blue) Percentage of time occupied by calving noise over the study period and (orange) the acoustic estimate of daily calving rate. Figure 5. (blue) Percentage of time occupied by calving noise over the study period and (orange) the acoustic estimate of daily calving rate. Figure 5. (blue) Percentage of time occupied by calving noise over the study period and (orange) the acoustic estimate of daily calving rate. Figure 5. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. (blue) Percentage of time occupied by calving noise over the study period and (orange) the acoustic estimate of daily calving rate. 23 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A1. Data used in the subaerial calving flux calculations. Horizontal velocity map from offset tracking on repeat TerraSAR satellite radar images (2012-12-15 and 2012-12-26), retreat of Hansbreen cliff and localization of stake position with dGPS measurements between August 5 and August 24, 2013. The grey profile with dots shows division of the ice cliff into 40 m lengths, along which precise calculation of subaerial calving flux were made. Figure A1. Data used in the subaerial calving flux calculations. Horizontal velocity map from offset tracking on repeat TerraSAR satellite radar images (2012-12-15 and 2012-12-26), retreat of Hansbreen cliff and localization of stake position with dGPS measurements between August 5 and August 24, 2013. The grey profile with dots shows division of the ice cliff into 40 m lengths, along which precise calculation of subaerial calving flux were made. 24 24 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A2. (A) Histograms and (B) empirical cumulative distribution functions (ECDFs) of log-transformed impact noise energies calculated for calibration data (2016; orange) and acoustic measurements used in this study (2013; blue). Figure A2. (A) Histograms and (B) empirical cumulative distribution functions (ECDFs) of log-transformed impact noise energies calculated for calibration data (2016; orange) and acoustic measurements used in this study (2013; blue). 25 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A3. Relationship between detection factor β and the number of detected calving events. Interfering events are not removed in this analysis. Figure A3. Relationship between detection factor β and the number of detected calving events. Interfering events are not removed analysis. Figure A3. Relationship between detection factor β and the number of detected calving events. Interfering events are not removed in this analysis. Figure A3. Relationship between detection factor β and the number of detected calving events. Interfering events are not removed in this analysis. 26 https://doi.org/10.5194/egusphere-2023-115 Preprint. Discussion started: 9 February 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A4. Change in number of events detected (N), total acoustic energy at the receiver (E) and summed event durations (D) versus iteration number for the background noise estimate. Figure A4. Change in number of events detected (N), total acoustic energy at the receiver (E) and summed event durations (D) versus iteration number for the background noise estimate. Figure A4. Change in number of events detected (N), total acoustic energy at the receiver (E) and summed event durations (D) versus iteration number for the background noise estimate. 27 27
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2. Unsere Studie: Design und Methoden
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2. Unsere Studie: Design und Methoden Die diesem Buch zu Grunde liegende empirische Untersuchung hatte ein spezifisch aufgefächertes qualitatives Design (siehe Abbildung 1 ), wobei Expert:inneninterviews und Organisationsfallstudien die wesentlichen Stützpfeiler waren. Zu Beginn stand die theoretische Auseinandersetzung mit den Rahmenbedingungen, unter denen organisierte Interaktionsarbeit in den von uns betrachteten Branchen gegenwärtig (auch schon vor Corona) stattfindet. Überprüft und ergänzt wurde diese durch 15 Expert:innengespräche (teils mit zwei Personen) (siehe Kap. 3.1und 3.5) (vgl. Gläser/Laudel 2010; Bogner/ Littig/Menz 2009, 2014; Kaiser 2014). Diese wurden 2020 mit 21 Branchenkenner:innen aus Verbänden und Gewerkschaften geführt. Themen waren u.a. die Sichtweisen auf Dynamiken interner Steuerung (Management), ferner wahrgenommene Einflüsse externer Steuerung (Politik, Markt) und schließlich in der Branche beobachtete Unsicherheiten sowie der emotionale Umgang damit. Für die Sektoren Weiterbildung und JBH wurde zusätzlich eine Fokusgruppendiskussion mit Expert:innen geführt, da diese Branche unter den für das Forschungsprojekt relevanten Kontextgesichtspunkten wenig beforscht ist. Ein wesentlicher Baustein des Projekts waren Organisationsfallstudien1 in vier ausgewählten gemeinnützigen Einrichtungen in deutschen Großstädten (mit variierenden Profilen und einer gewissen regionalen Varianz von Nord-, Süd- und Ostdeutschland), die schon seit längerem in diesem Segment tätig sind (weitere Charakteristiken siehe Kap. 3.2) und überwiegend in 2021 erhoben wurden. Unser Sample bestand jeweils aus einer Organisation in den Sub-Sektoren, also: je eine Organisation im Bereich der Jugendberufshilfe, 1 Sie orientiert sich an geläufigen Ansätzen der ›Case-Study‹-Forschung und fokussiert komplexe Strukturzusammenhänge und Prozessverläufe, wobei Kontextbezug, Offenheit und Multiperspektivität wesentliche Gütemaßstäbe darstellen (vgl. Pflüger/ Pongratz/Trinczek 2017). 26 Organisierte Zerrissenheit der Weiterbildung für Ältere, der stationären und der ambulanten Pflege. Jede Organisationsfallstudie basierte auf Einzelinterviews (in wenigen Fällen auch Interviews mit zwei Personen gleichzeitig), einer Gruppendiskussion sowie Dokumentenanalysen. Pro Einrichtung wurden sechs bis elf ein- bis zweieinhalbstündige problemzentrierte Interviews mit narrativen Anteilen durchgeführt (vgl. Witzel 1985; zu dieser Kombination Scheibelhofer 2008), dabei ging es in einem ersten Teil um das Erleben des beruflichen Alltags und eventueller Herausforderungen (in Bezug auf Rahmenbedingungen, Management, Organisation und Nutzer:innen), in dem zweiten Teil wurde dann stärker auf emotionale Belastungen fokussiert. Insgesamt haben wir in dieser Weise 36 Personen interviewt. Befragt wurden Beschäftigte unterschiedlicher Qualifikationsstufen und Funktionsbereiche, Leitungskräfte (auf der unteren, mittleren und höchsten Managementebene) und Personalvertreter:innen.2 Hinzu kam für jede Einrichtung eine etwa zweistündige Gruppendiskussion (vgl. Bohnsack/Przyborski/Schäffer 2010; Lamnek 2006; Kühn/Koschel 2018) mit vier bis acht Betriebsangehörigen, in deren Mittelpunkt das Thema Solidarität auf den Ebenen Betrieb (inklusive der Erfahrungen mit Nutzer:innen), Branche und Gesellschaft bzw. Wohlfahrtsstaat stand. Mit Ausnahme der organisationsinternen Gruppendiskussion in der Jugendberufshilfe wurde in den anderen Fallstudien mit diesem Instrument vor allem das mittlere Management erreicht. Durch unseren gewählten Feldzugang über die Geschäftsleitung der jeweiligen Einrichtungen haben diese auch die Befragten für die Einzelgespräche und die Gruppendiskussion ausgewählt, unter Beachtung unserer Maßgabe, dass wir verschiedene Funktionsbereiche befragen wollten. Dabei waren die Organisationen auch darauf angewiesen, dass Beschäftigte sich dafür bereit erklärten. Die Gesamtzahl der einzeln oder in 2 So wurden im Pflegesektor Pflegehelferinnen, examinierte Pflegefachkräfte, Betreuungskräfte, Hauswirtschafts- und Reinigungspersonal befragt. Befragte in Leitungsfunktionen waren Teamleitungen, Pflegedienstleitungen, Heimleitungen und oberste Geschäftsführungen. Im Weiterbildungssektor befragten wir gewerbliche Ausbilder:innen in Berufsausbildungsmaßnahmen, Job-Coaches und akademisch qualifizierte Sozialpädagog:innen. Befragte in Leitungsfunktionen waren hier Bereichsleitungen (z.B. für Jugendliche oder Erwachsene), Standortleitungen und Geschäftsführungen von Zweigstellen. Der Frauenanteil im gesamten Sample der Fallstudien liegt bei 75 %. Im Sample waren alle Altersgruppen vertreten. Bei der Darstellung der Befunde wird, wo es relevant erschien, auf deren Zugehörigkeit jeweils hingewiesen. Vertreten waren ebenfalls Personen mit Migrationsgeschichte. 2. Unsere Studie: Design und Methoden Gruppendiskussionen Befragten aller vier Fallstudien beträgt insgesamt 56 Personen. Zu den Organisationsfallstudien gehörte auch die Sammlung und Sichtung schriftlicher Artefakte, die einer Dokumentenanalyse (vgl. Wolff 2008) unterzogen wurden. Dabei haben wir die Organisationen um interne Dokumente gebeten – etwa Geschäftsberichte oder Tätigkeits- und Qualitäts(management)berichte – und öffentlich zugängliche Daten (z.B. Internetauftritt, Zeitungen, Flyer) gesammelt. Abbildung 1: Methodisches Design Eigene Darstellung 27 28 Organisierte Zerrissenheit Das mannigfaltige gewonnene Material aus den Organisationsfallstudien wurde im Rekurs auf die qualitative Inhaltsanalyse (vgl. Mayring 2015) ausgewertet. Dies diente dazu, Informationen zu ordnen bzw. zu sichern und das Gesamtmaterial besser überblicken zu können. Manifeste Aussagen wurden paraphrasiert und mithilfe eines Softwareprogramms kodiert und inhaltsanalytisch ausgewertet, was schließlich in Interviewportraits für jedes Einzelinterview mündete, in denen die von den Befragten dargestellten Themen zusammengefasst sowie besonders prägnante Zitatstellen eingefügt wurden (siehe Befunde in Kap. 3.2 und 3.3). Durch diese intensive Auseinandersetzung mit dem Material war es möglich, relevante Schlüsselstellen vor allem in den Einzelinterviews zu identifizieren, welche dann rekonstruktiv/interpretierend analysiert wurden, um sich so einen besseren Zugang zum latenten emotionalen Gehalt zu erschließen und Zusammenhänge zu Solidaritätskonstrukten im oben genannten Verständnis herausarbeiten zu können (siehe exemplarische Feinanalysen in Kap. 3.4). Ging es in der Inhaltsanalyse noch darum, was genau gesagt wurde, untersuchten wir angelehnt an rekonstruktive Verfahren für einzelne Passagen sequenzanalytisch, was die sprachlichen Ausgestaltungen über latente Sinnkonstruktionen verraten. Dabei orientierten wir uns an den Grundprinzipien rekonstruktiv-hermeneutischer Analyse, welche im integrativen Basisverfahren nach Kruse (2015) beschrieben sind; Stellen, die besonders aufschlussreich für das emotionale Erleben wirkten, wurden also einer mikroskopischen Feinanalyse unterzogen (vgl. ebd., S. 475ff.; auch Kleres 2011, 2015 in Bezug auf Emotionen). Nach diesen Tiefenanalysen wird der Blick in die Breite der untersuchten Branchen auf Basis der Erkenntnisse aus den Expert:innengesprächen gerichtet (siehe Kap. 3.5). Dies dient der Einordnung und Plausibilisierung der Befunde aus den Organisationsfallstudien.
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Ein Mentoring-Programm mit Coaching-Anteilen für die Ausbildung von Lehrpersonen
Beiträge zur Lehrerinnen- und Lehrerbildung
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Niggli, Alois Ein Mentoring-Programm mit Coaching-Anteilen für die Ausbildung von Lehrpersonen Beiträge zur Lehrerbildung 19 (2001) 2, S. 244-250 Quellenangabe/ Reference: Niggli, Alois: Ein Mentoring-Programm mit Coaching-Anteilen für die Ausbildung von Lehrpersonen In: Beiträge zur Lehrerbildung 19 (2001) 2, S. 244-250 - URN: urn:nbn:de:0111-pedocs-134580 - DOI: 10.25656/01:13458 https://nbn-resolving.org/urn:nbn:de:0111-pedocs-134580 https://doi.org/10.25656/01:13458 in Kooperation mit / in cooperation with: http://www.bzl-online.ch Nutzungsbedingungen Terms of use Gewährt wird ein nicht exklusives, nicht übertragbares, persönliches und beschränktes Recht auf Nutzung dieses Dokuments. Dieses Dokument ist ausschließlich für den persönlichen, nicht-kommerziellen Gebrauch bestimmt. Die Nutzung stellt keine Übertragung des Eigentumsrechts an diesem Dokument dar und gilt vorbehaltlich der folgenden Einschränkungen: Auf sämtlichen Kopien dieses Dokuments müssen alle Urheberrechtshinweise und sonstigen Hinweise auf gesetzlichen Schutz beibehalten werden. Sie dürfen dieses Dokument nicht in irgendeiner Weise abändern, noch dürfen Sie dieses Dokument für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, aufführen, vertreiben oder anderweitig nutzen. Mit der Verwendung dieses Dokuments erkennen Sie die Nutzungsbedingungen an. We grant a non-exclusive, non-transferable, individual and limited right to using this document. This document is solely intended for your personal, non-commercial use. Use of this document does not include any transfer of property rights and it is conditional to the following limitations: All of the copies of this documents must retain all copyright information and other information regarding legal protection. You are not allowed to alter this document in any way, to copy it for public or commercial purposes, to exhibit the document in public, to perform, distribute or otherwise use the document in public. By using this particular document, you accept the above-stated conditions of use. Kontakt / Contact: peDOCS DIPF | Leibniz-Institut für Bildungsforschung und Bildungsinformation Informationszentrum (IZ) Bildung E-Mail: pedocs@dipf.de Internet: www.pedocs.de 244 Beiträge zur Lehrerbildung, 19 (2), 2001 Ein Mentoring-Programm mit Coaching-Anteilen für die Ausbildung von Lehrpersonen Alois Niggli In einer professionsorientierten Ausbildung für künftige Lehrkräfte ist der subjektive Austausch von Wissen grundlegend, weil gesicherte Handlungsanleitungen nicht vermittelt werden können. Mentorpersonen fällt deshalb die Aufgabe zu, entsprechende Förderungsbeziehungen aufzubauen. Andererseits gelten sie auch als Teil des Systems, das mittels Mentoring-Programmen seine Interessen implementieren will. Diese beiden präskriptiven Ausrichtungen konfligieren nicht selten und lassen sich wohl nie optimal gegeneinander ausbalancieren. Mit dem folgenden Modell wird der Anspruch vertreten, zum Umgang mit dieser Spannung einen Beitrag zu leisten. Vorgeschlagen werden drei strategische Bearbeitungsbereiche. Die Entwicklung einer beruflichen Handlungskompetenz ist ein schwieriges Unterfangen, wenn man jeweils nie genau wissen kann, ~b man nun richtig oder falsch gehandelt hat. Alle, die mit Kindern und Jugendlichen umgehen, wissen, dass Unterrichts- und Erziehungsarbeit jedoch nie sicheren Erfolg zeitigt. Jede Lehre realisiert sich in Form einer Beziehungspraxis. Sachliche Ziele lassen sich infolgedessen immer nur tendenziell erreichen (vgl. Combe, 1997, S. 10.). Studierende in Lehrerbildungsgängen haben nicht selten Schwierigkeiten, diese komplexe Situation zu akzeptieren. Legitimerweise verlangen sie von ihrer Ausbildung Gewissheit. Sie möchten die Aufgaben, vor die sie im institutionellen Kontext von Schule gestellt werden, erfolgreich bewältigen können. Wenn berufliches Wissen jedoch nie sicheren Erfolg verbürgt, dann kann professionelles Handeln nicht durch deduktive Anwendung von erworbenem Fachwissen der wissenschaftlichen Disziplin gekennzeichnet sein. Die Lehrerbildung ist grundsätzlich nicht in der Lage, die eingeforderte Sicherheit zu vermitteln. Aus diesem Grunde sollte Lernen wenn immer möglich reflexiv und situiert arrangiert werden. Interne Regulationen müssen bedeutsam werden. Mentoringl ist eine Möglichkeit, diesem Anspruch nahe zu kommen. In untenstehender Abbildung (vgl. Abb. 1) wird ein sogenanntes 3-Wege-Modell zum Mentoring vorgestellt, das den genannten Grundvoraussetzungen Rechnung trägt. Im theoretischen Kontext einer- Ausbildung zur Profession wird auf Seiten der Lernenden eine wissensorientierte Kompetenz- und eine Steuerungsebene der Person unterschieden. Auf beiden Ebenen begleiten Mentorinnen und Mentoren Lernende in ihrer Entwicklung. Diese Begleitung erfolgt im Rahmen einer förderlichen Beziehung. Auf der Kompetenzebene entsteht eine professionelle Entwicklung aus der Auseinandersetzung mit dem erwähnten Nicht-Perfektsein, wenn dieses nicht nur wahrgenommen, sondern im Rahmen der in der Ausbildung vertretenen Kompentenzprofile auch als bearbeitbar erlebt wird (s. Bauer, 1998, S. 355). Mentorinnen und Mentoren unterstützen diese Bearbeitbarkeit. Dabei müssen sie auch das Kompetenzpmfil der ' Mentor (oder Mentes) ist in der griechischen Mythologie der Freund des Odysseus. Er kümmert sich während der Irrfahrten des Königs um seinen Sohn Telemach, und wird zur emotionalen und intellektuellen Leitfigur des Jünglings. In dieser Eigenschaft wird er ein Vorbild für Erzieher und zum 'Vater' des modernen 'Mentoring' in Wirtschaft und Wissenschaft (Strasser & Schliesselberger, 2000, S. 13). Begleiten und beraten in der berufspraktischen Ausbildung http://www.bzl-online.ch 245 Organisation glaubwürdig verkörpern. Sie können nicht einfach unterstützen, was Lernende für richtig halten. Im Modell werden zwei Wissensbereiche unterschieden, die gemeinsam bearbeitet werden können: der Tätigkeitsbereich ('knowing how'), in welchem Können manifest wird und das Erklärungswissen ('knowing what') (vgl. auch Heitzmann & Messner, 2001, S. 14). Den Entwicklungsaufgaben in diesen zwei Wissensbereichen werden im Rahmen eines Mentoring spezifische Förderungsmodi (als Gesprächstypen) zugeordnet, nämlich (a) das Feedback erweiternde Praxisgespräch und (b) das reflexive Praxisgespräch. Die beiden Wissensformen bedingen jeweils eine andere Behandlung, weil Erklärungswissen erst in Können transformiert werden muss. Wahrnehmung des Unterrichtsgeschehens (Spontanfeedback) Weg 1 I erweiterndes Praxisgespräch I Weg 2 kraxisgespracn Kompetenzebene der Unterrichtssituation ) I( I Personale Steuerungsebene Abbildung 1: Modell des 3-Wege Mentoring (3WM) Diese Entwicklung hat andererseits aber auch über den Aufbau eines beruflichen Selbst2 zu erfolgen. Unter dem beruflichen Selbst wird in Anlehnung an Bauer (1998, S. 344) ein Bewusstsein verstanden, das die persönlichen Entwicklungsaufgaben steuert, die jedem künftigen Pädagogen gestellt sind. Auf dieser Ebene setzen Lernende ihre Ziele selbst. Sie sind Gegenstand der Selbstwahrnehmung, denn durch Dieser Begriff mag zwar wenig präzise sein. Allerdings eröffnet er Anschluss an Entwicklungsaufgaben, welche die Person in ihrem So-Sein betreffen und nicht lediglich einzelne berufliche Kompetenzen kennzeichnen. 246 Beiträge zur Lehrerbildung, 19 (2), 2001 die erlebten Unsicherheiten werden sie in ihrem bemflichen Selbstverständnis ständig herausgefordert. Lernende müssen sich neue Ziele setzen, ihr Wissen erneuern, generell ihre Entwicklung zur Profession steuern. Im vorliegenden Mentoringkonzept wird diese Selbststeuerung durch Coaching begleitet. Durch diese Unterstützung soll das individuelle Potential bewusst und zugänglich gemacht werden, damit derldie Coachee aktiv und möglichst erfolgreich die ausbildungsbezogenen Herausfordemngen bewältigen kann. Coaching wird im vorliegenden Modell auf diese eher non-direktive Beziehung eingeschränkt. Eine Mentorin oder ein Mentor muss sich darüber im Klaren sein, auf welchem Weg Lernende jeweils begleitet werden sollen. Weg I : Entwicklung im Tätigkeitsbereich Aufgmnd der unsicheren Resultate erziehenschen Handelns sind angehende Lehrkräfte in ihrer Ausbildung auf Hinweise über die Wirkung ihrer unterrichtlichen Tätigkeit in hohem Masse angewiesen. Diese Informationen sind für ihre Selbsteinschätzung und vor allem für ihre Selbstwirksamkeit (Hertramph & Henmann, 1999) von nicht zu unterschätzender Bedeutung. Der Lehrer(innen)bemf zeichnet sich generell durch ein hohes Mass an Selbstdefinition bei gleichzeitig institutionell bedingter 'Rückmeldearmut' aus (Schaefers & Koch, 2000, S. 614). Referenzpunkt auf Weg 1 ist deshalb die konkret wahrnehmbare Untemchtstätigkeit. Sie wird als 'knowinghow' (Ryle, 1969) gesehen. Wegleitend sind praxiswirksame (im weiteren Sinne technische) Regeln, die auf intensiven Erfahmngen beruhen. Dieses Handlungswissen ist für eine aussenstehende Beobachter-Mentorperson nie in seiner Gesamtheit, sondern lediglich an der Oberfläche als 'Skill' wahrnehmbar. Dabei handelt es sich um Basishandlungen, die sich im Kontext eines Ganzen ereignen, von dem sie selber Teil sind. Je nach Kontextbezug kann man eher geschlossene oder offenere Skills unterscheiden. Geschlossene Skills haben wenig Kontext. Sie sind algorithmisch beschreib- und lernbar (z.B.: 'Einen verständlichen Kurzvortrag halten'). Offene, komplexe Skills haben mehr Kontext (z. B.: Kooperativen Unterricht adäquat einsetzen). Sie beinhalten unterschiedliche Absichten, die unterschiedliche Funktionen erfüllen können (s. Tomlinson, 1998, S. 15). Die Behandlung von 'Skill-Ausschnitten' der beobachtbaren Untemchtstätigkeit kann in der Ausbildungspraxis über konkretes Feedback erfolgen. Von Feedbackerweiterung kann deshalb gesprochen werden, weil Lehrpersonen über ihre Selbstwahmehmung des Untemchts immer auch spontanem Feedback ausgesetzt sind. Die Rückmeldung der Mentorperson orientiert sich darüber hinaus jedoch an vereinbarten praxiswirksamen Standards. Vereinbart sind Standards deshalb, weil Wirksamkeit nicht 'objektiv' begründbar ist. Sie unterscheiden sich in ihrer Konkretheit, je nachdem ob offene oder eher geschlossene Skills beobachtet werden sollen. Mit ihrem Feedback sollten Mentorpersonen zu allererst versuchen, das Können der Lernenden herauszuschälen. Übereinstimmende Wahrnehmungen können dann zu mehr Gewissheit über erfolgreiche oder gegebenenfalls über zu verändernde Strategien verhelfen. Diskrepanzen bedürfen der anschliessenden Klämng. Diese kann in einem reflexiven Praxisgespräch herbeigeführt werden. Begleiten und beraten in der berufspraktischen Ausbildung Die Befolgung bestimmter Regeln im Rahmen des 'knowing how' kann darüber hinaus manchmal intuitiv geschehen. Das obgenannte Untemchtsverhalten kann dann die Form eines impliziten 'tacit knowledge' (Neuweg, 1999) haben, dessen Hebung ins Bewusstsein (s. Weg 2) als 'knowing what' besonderer Anstrengung bedarf. Dies ist eine Voraussetzung, dass Verändemngen überhaupt erst in Angriff genommen werden. Weg 2: Entwicklung des Erklärungswissens ' Referenzpunkt auf Weg 2 ist das Wissen, das mehr oder weniger losgelöst von der abgelaufenen Tätigkeit existiert. Geht es um dieses Hintergmndwissen, dann muss ein Gespräch anders stmktunert sein, als wenn man sich über die Wahrnehmung konkreter Tätigkeiten einigt. Nach der Terminologie von Ryle (1969) handelt es sich bei diesem Hintergmndwissen um 'knowing what' (Erklämngswissen über die Praxis). Dieses Erklämngswissen kann aus unterschiedlichen Quellen stammen (vgl. Shulman, 1986, 1987). Es umfasst u.a. sowohl nomologisches Wissen der wissenschaftlichen Disziplinen wie auch das professionelle Wissen kompetenter Lehrpersonen. Das zugeordnete Reflexive Praxisgespräch zielt auf verständigung über das Erklärungswissen. Reflexion soll zu einer Ausdifferenziemng des Hintergmndwissens beitragen. Es hat sich als praktikabel erwiesen, entsprechende Gespräche wie folgt zu stmkturieren (vgi. Abb. 2). 1. Beschreibung I Beschreibuna der .- . I Informieren - - - - 2. Analyse 1 Konfrontieren 3. Synthese 1 4. Optionen 1 Rekonstruieren I Schlussfol~erun- klären Ereignisse auf die man sich bezieht festlegen I Mentoriin und Mentee 1 I Mentee Mentoriin I Mentoriin und Mentee i. d. R. Mentee - Abbildung 2: Phasen im Reflexiven Praxisgespräch Vorerst wird die wahrgenommene Realität von beiden Partnern deskriptiv dargestellt. Die Mentoridder Mentor gibt anschliessend Anstösse zur Klärung von Hintergmndwissen (z.B. 'Welche Vorannahmen haben Sie zum Vorwissen der Lernenden getroffen?). Die Realität wird dabei unter dem Anspmch untersucht, selbst neues Erklämngswissen zu generieren. Inspirationen der Mentorperson beim Konfrontieren können Offerten sein, die von den Handelnden angenommen oder auch verworfen werden können. Die Geltung entsprechender Erklärungsansätze ist allein in der Intersubjektivität der Verständigung begründet. In der Synthesephase wird das Hintergrundwissen allenfalls neu stmkturiert (z. B. 'Gibt es Dinge, die Sie anders sehen als zu Beginn des Gesprächs?'). Erst dann werden mögliche neue Zielsetzungen vereinbart. Dies ist deshalb notwendig, weil Reflexion allein für die bemfliche Entwicklung nicht hinreichend ist. Praxiswirksam wird das erzeugte Wissen erst im Kontext der Beiträge zur Lehrerbildung, 19 (Z), 2001 248 konkreten Unterrichtstätigkeit. Darüber sollen die Lernenden jedoch Feedback erhalten und nicht nur reflektieren müssen. Weg 3: Entwicklung des Professionellen Selbst Bei den oben beschriebenen Wissenskomplexen handelt es sich um funktionale Anforderungen, die notwendig sind, um einen Beruf kompetent auszuführen. Daneben ist eine Lehrkraft aber quasi immer auch auf einer persönlichen Steuerungsebene, in ihrem beruflichen Selbst herausgefordert (vgl. Bauer, 1998). Es geht dabei nicht primär um Qualifizierung, sondern um Subjektwerdung. Konkret bedeutet dies U. a.: - sich als Veränderungsprojekt zu verstehen und entsprechend zu handeln, - eigenes Lernen reflektiert zu begleiten, - Lernanlässe selbst festzustellen, zu steuern und in Gang zu setzen. Diese Selbstbeobachtung hat überprüfende Funktion. Sie sollte nicht dazu führen, sich auf Unzulänglichkeiten zu fixieren. Ansätze, die sich unter dem Sammelbegriff 'Coaching' etabliert haben, können diesen Prozess begleiten und unterstützen. Das zugeordnete Coaching-Gespräch fördert somit das Verständnis 'über mich selbst'. Dieser Erkenntnisprozess wird in zwei Schleifen strukturiert (vgl. Abb. 3). Tatsachen Situations- Hintergründe Ausgangslagel Problem Ziele, Prinzipien Alternativen notwendigen Bewusstheit und Erkenntnis für seinelihre Situation einen "guten" Entscheid treffen Diagnose Entscheidung Begleiten und beraten in d e r berufspraktischen Ausbildung 249 sen zu strukturieren. Sein wichtigstes Arbeitsinstrument findet der Coach in der Fragetechnik der offenen Frage. Können Lehrerbildnerinnen und Lehrerbildner die berufliche Entwicklung Studierender auf den vorgeschlagenen drei Wegen jedoch überhaupt fördern? Diese Frage zielt in letzter Konsequenz auf die Effizienz der im Modell vertretenen Massnahmen. In diesem Problemkontext hat die theoretische Arbeit von Vygotsky in den letzten Jahren zunehmende Beachtung gefunden (s. Reiman, 1999). Der Prozess der kognitiven Konstruktion vollzieht sich nach Vygotskys Annahmen nicht von selbst. Notwendig sind soziale Interaktionen. Entwicklung resultiert damit aus ständigen Anforderungen der Gesellschaft an das Individuum. Aus diesen Grundannahmen leitet sich ab, dass Mentorpersonen eine aktive Rolle zu spielen haben. Kompetente ExpertenPersonen können die Entwicklung fördern, indem sie angemessene Anforderungen stellen. Mit dem vorgeschlagenen 3-Wege-Modell lassen sich diese Anforderungen bereichsspezifisch differenzieren und moderieren. Die Herausforderung für Ausbildnerinnen und Ausbildner wird deshalb darin zu sehen sein, auf den drei vorgeschlagenen Wegen Unterstützung bei der Bewältigung kognitiver Verunsicherung einerseits.zu gewähren, andererseits kognitives Ungleichgewicht aber auch auszulösen. Das angemessene Pendeln zwischen diesen Polen könnte letzten Endes eine Kunst sein, die hohe diagnostische Sensibilität voraussetzt. Es ist eingebettet in ein Coaching, das ein Bemühen unterstützt, das der Selbstentwicklung Gestalt geben soll. Feedback annehmen, reflektieren, sich beruflich entwickeln zu müssen stellt für Lernende immer auch eine Zumutung dar, nämlich die Zumutung, 'die Welt, meine Tatigkeiten und mich selbst anders zu sehen und zu deuten als ich es gerne möchte' (Reichenbach, 2000, S. 803). Mentorinnen und Mentoren begleiten die Lernenden dabei als kritische Freunde. Literatur Konsequenzen Abbildung 3: Erkenntnisprozessim Coaching (Furter, 2000) Der Erkenntnisprozess geht von einer aktuellen Frage-/Problemstellung in der Gegenwart aus und verläuft in einer 'Schleife' in die Vergangenheit, um die gewordene Situation und deren Ursachen zu verstehen. Der Weg kehrt zum/zur Coachee als Entscheiderlin zurück: die Konklusion ist nun in seinendihrem Bewusstsein und für ihnlsie relevant. Der Prozess der Entscheidungsfindung nimmt seinen Ausgang bei Zielen und Prinzipien, die derldie Coachee festlegt und macht jetzt eine 'Schleife' in die Zukunft: wie könnten die künftige(n) Situation(en) aussehen, und welches wären die Konsequenzen? Der Weg kehrt dann wiederum zundzur Coachee zurück, derldie schliesslich eine Entscheidung trifft. Die Aufgabe des Coachs besteht nun darin, denldie Coachee durch diese Schlaufen zu begleiten und dabei die verschiedenen Pha- Bauer, K. 0. (1998). 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